Properties of an Ezrin Mutant Defective in F-actin Binding

Ezrin, radixin and moesin are a family of proteins that provide a link between the plasma membrane and the cortical actin cytoskeleton. The regulated targeting of ezrin to the plasma membrane and its association with cortical F-actin are more than likely functions necessary for a number of cellular processes, such as cell adhesion, motility, morphogenesis and cell signalling. The interaction with F-actin was originally mapped to the last 34 residues of ezrin, which correspond to the last three helices (αB, αC and αD) of the C-terminal tail. We set out to identify and mutate the ezrin/F-actin binding site in order to pinpoint the role of F-actin interaction in morphological processes as well as signal transduction. We report here the generation of an ezrin mutant defective in F-actin binding. We identified four actin-binding residues, T576, K577, R579 and I580, that form a contiguous patch on the surface of the last helix, αD. Interestingly, mutagenesis of R579 also eliminated the interaction of band four-point one, ezrin, radixin, moesin homology domains (FERM) and the C-terminal tail domain, identifying a hotspot of the FERM/tail interaction. In vivo expression of the ezrin mutant defective in F-actin binding and FERM/tail interaction (R579A) altered the normal cell surface structure dramatically and inhibited cell migration. Further, we showed that ezrin/F-actin binding is required for the receptor tyrosine kinase signal transfer to the Ras/MAP kinase signalling pathway. Taken together, these observations highlight the importance of ezrin/F-actin function in the development of dynamic membrane/actin structures critical for cell shape and motility, as well as signal transduction.     

DEFENSE EVOLUTION IN THE GRACILARIACEAE (RHODOPHYTA): SUBSTRATE‐REGULATED OXIDATION OF AGAR OLIGOSACCHARIDES IS MORE ANCIENT THAN THE OLIGOAGAR‐ACTIVATED OXIDATIVE BURST1

Combined phylogenetic, physiological, and biochemical approaches revealed that differences in defense‐related responses among 17 species belonging to the Gracilariaceae were consistent with their evolutionary history. An oxidative burst response resulting from activation of NADPH oxidase was always observed in two of the subgenera of Gracilaria sensu lato (Gracilaria, Hydropuntia), but not in Gracilariopsis and in species related to Gracilaria chilensis (“chilensis” clade). On the other hand, all species examined except Gracilaria tenuistipitata var. liui and Gracilariopsis longissima responded with up‐regulation of agar oligosaccharide oxidase to an challenge with agar oligosaccharides. As indicated by pharmacological experiments conducted with Gracilaria chilensis and Gracilaria sp. “dura,” the up‐regulation of agar oligosaccharide oxidase involved an NAD(P)H‐dependent signaling pathway, but not kinase activity. By contrast, the activation of NADPH oxidase requires protein phosphorylation. Both responses are therefore independent, and the agar oligosaccharide‐activated oxidative burst evolved after the capacity to oxidize agar oligosaccharide, probably providing additional defensive capacity to the most recently differentiated clades of Gracilariaceae. As demonstrated with Gracilaria gracilis, Gracilaria dura, and Gracilariopsis longissima, the different responses to agar oligosaccharides allow for a fast and nondestructive distinction among different clades of gracilarioids that are morphologically convergent. Based upon sequences of the chloroplast‐encoded rbcL gene, this study suggests that at least some of the samples from NW America recorded as Gs. lemanaeiformis are probably Gs. chorda. Moreover, previous records of Gracilaria conferta from Israel are shown to be based upon misidentification of Gracilaria sp. “dura,” a species that belongs to the Hydropuntia subgenus.     

Post-fire regeneration in a Mediterranean pine forest with historically low fire frequency

Species of Mediterranean vegetation are known to regenerate directly after fire. The phenomenon of autosuccession (direct regeneration) has been found to be often combined with an increase of species richness during the first years after fire due to the high abundance of short-lived herbaceous plants facilitated by plentiful nutrients and light. The high degree of vegetation resilience, which is expressed in terms of autosuccession, has been explained by the selective pressure of fire in historic times. According to existing palaeoecological data, however, the Pinus halepensis forests in the Ricote Mountains (Province of Murcia, SE Spain) did not experience substantial fire impact before the presence of man nor are they especially fire-prone today. Therefore, we studied post-fire regeneration to find out if direct succession is present or if species from pre-fire vegetation are absent during the post-fire regeneration stages. Patterns of succession were deduced from observations made in sample plots on sites of a known regeneration age as well as in adjacent unburnt areas. The results of the vegetation analyses, including a Detrended Correspondence Analysis, indicate that Pinus halepensis forest regeneration after fire resembles autosuccession. As regards the presence of woody species, there is a high percentage similarity on north (83%) and south (70%) facing slopes during the first year after fire vs. reference areas which is due, for example, to direct regeneration of the resprouting Quercus coccifera or seeders like Pinus halepensis or Fumana laevipes. However, if herbaceous species are included in the comparison, the similarity on north-facing sites decreases (to 53%) with the presence of additional species, mainly ruderals like Anagallis arvensis or Reseda phyteuma, and even woody species on the burnt plots. This effect indicates “enhanced autosuccession”, which was not found on south-facing sites where overall species richness was very high irrespective of the impact of fire. Locally we found limited regeneration of some species, for example Pinus halepensis at high altitudes (1000 m), even 22 years after fire. As we assume that historical fires did not play an important role in the area and direct succession is present nevertheless, our results support the theory that autosuccession is not a process restricted to fire-prone areas. Fire has been only one of several selective forces since human settlement that probably led to a set of species pre-adapted against recurrent disturbance.     

Synthesis, crystal structure, magnetic properties and electrochemical behaviour of the mixed valence compound [Cu(CN)3Cu(C3H10N2)2]

The binuclear mixed valence copper(I/II) compound [Cu^I(CN)₃Cu^II(tn)₂] (1) (tn = propane-1,3-diamine) and its acetonitrile adduct [Cu^I(CN)₃Cu^II(tn)₂] · 2MeCN (2) have been synthesized. Complex 1 crystallizes triclinic, space group , a = 8.117(2) Å, b = 8.389(2) Å, c = 11.920(2) Å, α = 108.728(3)°, β = 100.024(3)°, γ = 104.888(4)°, Z = 2, and compound 2 monoclinic, space group P2₁/m, a = 8.752(2) Å, b = 13.243(3) Å, c = 9.549(2) Å, β = 114.678(4)°, Z = 2. In both crystal structures, the binuclear [Cu^I(CN)₃Cu^II(tn)₂] complex with slightly different bonding geometries is formed. One of the three nitrogen atoms of a Cu^I(CN)₃ moiety is coordinated to Cu(II) at the apex of a square-pyramid with two chelating ligands tn on its base. The shortest intramolecular Cu^II?Cu^II distance in 1 is 5.640(7) Å. The EPR behaviour of 1 has been investigated at room temperature and at 77 K. The magnetic properties were measured in the temperature range 1.8-300 K.     

Rapid disease development in scrapie-infected mice deficient for CD40 ligand

The inhibition of CD40-CD40L interaction-mediated signalling was suggested as a therapeutic strategy for the treatment of Alzheimer's disease. Conversely, CD40-deficient neurons were reported to be more vulnerable to stress associated with ageing as well as nerve growth factor-beta and serum withdrawal. We studied the scrapie infection of CD40L-deficient (CD40L(-/-)) mice to see whether ablation of the CD40L gene would be beneficial or detrimental in this model of a neurodegenerative amyloidosis. CD40L(-/-) mice died on average 40 days earlier than wild-type control mice and exhibited a more pronounced vacuolation of the neuropil and an increased microglia activation. The experimental model indicates that a deficiency for CD40L is highly detrimental in prion diseases and reinforces the neuroprotective function of intact CD40-CD40L interactions. The stimulation of neuroprotective pathways may represent a possibility to delay therapeutically the disease onset in prion infections of the central nervous system.     

The architecture of chicken chromosome territories changes during differentiation

Background  

Between cell divisions the chromatin fiber of each chromosome is restricted to a subvolume of the interphase cell nucleus called chromosome territory. The internal organization of these chromosome territories is still largely unknown.     

Potency estimation of vaccine batches remains unaffected by anesthesia for intracerebral injection

Potency testing of rabies and whole-cell pertussis vaccine batches is still performed by an intracerebral (i.c.) challenge test, in conformity with international regulatory requirements. For the i.c. injection, the use of anesthesia is strongly recommended to alleviate the severe pain induced by the procedure. Today, anesthesia is not consistently mentioned in regulatory requirements, in contrast to the times when the potency tests were developed. The introduction of anesthesia is hampered, due to the lack of data on a hypothetical impact of anesthesia on potency estimation. Here, we show the comparative analysis of the extensive batch release data set of a rabies vaccine for human use that was tested in two laboratories of which only one applied anesthesia. In essence, we find that the mean batch test results were similar to each other, demonstrating that anesthesia for i.c. injection does not interfere with potency estimation. Consequently, we recommend the update of regulatory requirements and protocols and support the implementation of anesthesia for i.c. injection.     

Separating Fusion from Rivalry

Visual fusion is the process in which differing but compatible binocular information is transformed into a unified percept. Even though this is at the basis of binocular vision, the underlying neural processes are, as yet, poorly understood. In our study we therefore aimed to investigate neural correlates of visual fusion. To this end, we presented binocularly compatible, fusible (BF), and incompatible, rivaling (BR) stimuli, as well as an intermediate stimulus type containing both binocularly fusible and monocular, incompatible elements (BFR). Comparing BFR stimuli with BF and BR stimuli, respectively, we were able to disentangle brain responses associated with either visual fusion or rivalry. By means of functional magnetic resonance imaging, we measured brain responses to these stimulus classes in the visual cortex, and investigated them in detail at various retinal eccentricities. Compared with BF stimuli, the response to BFR stimuli was elevated in visual cortical areas V1 and V2, but not in V3 and V4 – implying that the response to monocular stimulus features decreased from V1 to V4. Compared to BR stimuli, the response to BFR stimuli decreased with increasing eccentricity, specifically within V3 and V4. Taken together, it seems that although the processing of exclusively monocular information decreases from V1 to V4, the processing of binocularly fused information increases from earlier to later visual areas. Our findings suggest the presence of an inhibitory neural mechanism which, depending on the presence of fusion, acts differently on the processing of monocular information.