Intermediate Domain Research Articles

Seeing GroEL at 6 A resolution by single particle electron cryomicroscopy.

We present a reconstruction of native GroEL by electron cryomicroscopy (cryo-EM) and single particle analysis at 6 A resolution. alpha helices are clearly visible and beta sheet density is also visible at this resolution. While the overall conformation of this structure is quite consistent with the published X-ray data, a measurable shift in the positions of three alpha helices in the intermediate domain is observed, not consistent with any of the 7 monomeric structures in the Protein Data Bank model (1OEL). In addition, there is evidence for slight rearrangement or flexibility in parts of the apical domain. The 6 A resolution cryo-EM GroEL structure clearly demonstrates the veracity and expanding scope of cryo-EM and the single particle reconstruction technique for macromolecular machines.

Transition from the paramagnetic to the antiferromagnetic state actualized inFeBr2

The magnetic ordering process of the antiferromagnetFeBr2 has been studied by measuring low-field dc-magnetization, ac-susceptibility and the Mössbauerspectrum. All quantities measured show anomalous behaviour around the Néel temperatureTN = 14.2 K. We proposedefining Tp (>TN) by the temperature where the magnetization is maximum. Because it isTp,not TN, that signifies the onset of magnetic ordering. We indicate thatFeBr2 transforms from the paramagnetic to antiferromagnetic (AF) state through anintermediate AF domain state which exists in a certain narrow temperature regionΔT just belowTp. From thefact that ΔT is sample-dependent, we have inferred that the stacking fault of the hexagonalc-layers plays an important role in the formation of the intermediate AF domain state.

Receptor interacting protein is ubiquitinated by cellular inhibitor of apoptosis proteins (c-IAP1 and c-IAP2) in vitro

Receptor interacting protein (RIP) is recruited to tumor necrosis factor-α receptor 1 (TNFR1) complex upon stimulation and plays a crucial role in the receptor-mediated NF-κB activation. Among the components of the TNFR1 complex are proteins that possess ubiquitin-protein isopeptide ligase (E3) activities, such as TNFR1-associated factor 2 (TRAF2), cellular inhibitor of apoptosis proteins (c-IAPs) namely, c-IAP1 and c-IAP2. Here, we showed that ectopically expressed RIP is ubiquitinated, and either the intermediate or death domain of RIP is required for this modification. Expression of c-IAP1 and c-IAP2 decreased the steady-state level of RIP, which was blocked by inhibition of the 26S proteasome. RIP degradation requires intact c-IAP2 containing the RING domain. Our in vitro ubiquitination assay revealed that while TRAF2 had no effect, both c-IAP1 and c-IAP2-mediated RIP ubiquitination with similar efficiency, indicating that c-IAPs can function as E3 toward RIP.

The magnetoacoustic anomaly in Fe 3BO 6

The measurements of active sound attenuation in the vicinity of spontaneous first-order phase transition in an orthorombic weak ferromagnet Fe 3BO 6 were carried out. A decrease in the attenuation of active sound passed across a sample at a point of phase transition was observed. The theory of coupled magnetoacoustic waves in Fe 3BO 6 was created. It is shown that the decrease of attenuation at the point of phase transition can be explained by taking into account the intermediate domain structure in the temperature region, where two magnetic phases coexist.

Development of local orthorhombic fabrics within a simple-shear dominated sinistral transpression zone: the Arronches sheared gneisses (Iberian Massif, Portugal)

Abstract The Coimbra-Cordoba shear zone (Iberian Massif), characterized by simple-shear dominated sinistral transpression, exposes several outcrops of strongly sheared peralkaline gneisses surrounded by mica schists and amphibolites. These gneisses are included in the Arronches Tectonic Unit, a thick unit of mylonitic rocks with a steep foliation and an associated gently plunging stretching lineation parallel to the fold axes. Strain partitioning is testified by widely spaced anastomosing shear bands around less-strained domains and by the existence of different shearing domains ranging from relatively ‘less-strained’ and coarse-grained mylonites to highly strained and fine-grained ultramylonites. Three shearing domains defined by textural and structural changes resulted from progressive deformation and increasing strain, which leads to increased mylonitization of gneisses. This is revealed by the increased modal percentage of the matrix and the decreased percentage of porphyroclasts, accompanied by evolution from orthorhombic to monoclinic fabrics: Conjugate Shearing Domain ( CSD ), Intermediate Sinistral Domain ( ISD ), and Sinistral Domain ( SD ). This contribution shows that in a simple-shear sinistral dominated transpression zone with a well-developed and widespread monoclinic fabric, it is possible to find mechanical conditions to produce local orthorhombic fabrics. In the Arronches gneisses a local strain regime exists in apparent contradiction with the bulk deformation regime.

RICK activates a NF-kappaB-dependent anti-human cytomegalovirus response.

The adapter kinase receptor interacting protein-like interacting caspase-like apoptosis regulatory protein kinase (RICK, also called RIP2 and CARDIAK) was found to be elevated at both the protein and RNA levels during human cytomegalovirus (HCMV) replication, suggesting either that the virus may require RICK for replication or that RICK is part of an unsuccessful host attempt to inhibit HCMV replication. It is demonstrated here that forced expression of RICK in either a kinase active or inactive form activates nuclear factor (NF)-kappaB by means of its intermediate domain and potently blocks HCMV replication in human fibroblasts. Importantly, NF-kappaB activation, which exerted a modestly positive effect on the early phase of infection, clearly had a strongly negative impact during later viral steps. A stable inhibitor of NF-kappaB (IkappaB) reverses the RICK inhibitory effect, and activation of NF-kappaB by IkappaB kinase beta expression is inhibitory to HCMV, demonstrating that NF-kappaB activation is part of a potent anti-HCMV response. Supernatant transfer experiments identified interferon-beta as a downstream component of the RICK inhibitory pathway. RICK expression was found to synergize with HCMV infection in the induction of interferon-beta expression. This study identifies an endogenous RICK-activated, NF-kappaB- and interferon-beta-dependent antiviral pathway that is either inhibited or faulty under normal HCMV replication conditions; efforts to bolster this pathway may lead to novel anti-viral approaches.

Domain Motions in GroEL upon Binding of an Oligopeptide

GroEL assists protein folding by preventing the interaction of partially folded molecules with other non-native proteins. It binds them, sequesters them, and then releases them so that they can fold in an ATP-driven cycle. Previous studies have also shown that protein substrates, GroES, and oligopeptides bind to partially overlapped sites on the apical domain surfaces of GroEL. In this study, we have determined the crystal structure at 3.0 Å resolution of a symmetric (GroEL–peptide) 14 complex. The binding of each of these small 12 amino acid residue peptides to GroEL involves interactions between three adjacent apical domains of GroEL. Each peptide interacts primarily with a single GroEL subunit. Residues R231 and R268 from adjacent subunits isolate each substrate-binding pocket, and prevent bound substrates from sliding into adjacent binding pockets. As a consequence of peptide binding, domains rotate and inter-domain interactions are greatly enhanced. The direction of rotation of the apical domain of each GroEL subunit is opposite to that of its intermediate domain. Viewed from outside, the apical domains rotate clockwise within one GroEL ring, while the ATP-induced apical domain rotation is counter-clockwise.

Dynamics and scaling of polymer networks: Vicsek fractals and hydrodynamic interactions

In previous works we have analysed the scaling behavior of model fractal polymers both in the presence of hydrodynamic interactions (HI, Zimm-model) as well as when such interactions are missing (Rouse-model). While in Rouse-type approaches scaling holds both for very high and very low frequencies, as well as in the intermediate domain, for Sierpinski-type lattices scaling does not hold in the Zimm-case in the intermediate frequency regime. In this paper we consider Vicsek-fractals which are tree-like (belonging, as the dendrimers, to hyperbranched structures), and we analyse their dynamics, both under Rouse- and under Zimm-conditions. By this we get additional insight into the problem, since the Vicsek fractals are (distinct from Sierpinski-type networks) devoid of loops. Remarkably we find that Vicsek fractals scale rather well, even when taking HI into account. We perform our analysis based on quite large Vicsek-fractals, especially in the Rouse case, where we can determine their eigenvalues through a very efficient iterative procedure.

A telomere-binding protein (TRF2/MTBP) from mouse nuclear matrix with motives of an intermediate filament-type rod domain.

In previous work, we identified a telomeric DNA-binding protein (termed telomere-membrane binding protein, MTBP) in the envelope of the frog oocyte nucleus and raised antibodies against it. Here we present immunological evidence which suggests strongly that MTBP is identical with the vertebrate telomeric DNA-binding protein TRF2 (telomere-repeat factor 2). MTBP/TRF2 possesses motif which resembles rod domain characteristic of intermediate filament (IF) proteins as shown by immunological cross-reactivity with characteristic antibodies, as well as amino acid sequence homology. Anti-MTBP antibodies recognised a protein of the same M, as TRF2 in extracts of mouse nuclei and nuclear matrix as shown by ion-exchange chromatography, gel shift assays, and Western blots. This mouse MTBP analogue forms more stable complexes with the vertebrate telomeric DNA fragment (T(2)AG(3))(135) than with the corresponding fragment from Tetrahymena (T(2)G(4))(130). Proteins in each of these complexes are recognised by anti-MTBP antibody. In situ hybridization with the vertebrate telomeric DNA sequence (T(2)AG(3))(135) and immunofluorescence with anti-MTBP antibody had shown earlier that these are co-localised in the nucleus of mouse cells, and here MTBP is shown to be associated with the residual membrane of hepatocyte nuclei using Western blotting and immunofluorescence. Some immunofluorescence signal from MTBP is localized at chromosome extremities on metaphase plates from mouse cell culture, but the main signal is seen in patches scattered around the chromosomes which were identified as remnants of the nuclear envelope by double labelling with antibodies against lamin B. These observations suggest that MTBP/TRF2 is a good candidate for the attachment of telomeres to the nuclear envelope in somatic cells.

Generalized Taylor dispersion in suspensions of gyrotactic swimming micro-organisms

We calculate the average swimming velocity and dispersion rate characterizing the transport of swimming gyrotactic micro-organisms suspended in homogeneous (simple) shear. These are requisite effective phenomenological coefficients for the macroscale continuum modelling of bioconvection and related collective-dynamics phenomena. The swimming cells are modelled as rigid axisymmetric dipolar particles subject to stochastic Brownian rotations. Calculations are effected via application of the generalized Taylor dispersion scheme. Attention is focused on finite (as opposed to weak) shear. Results indicate that the largest transverse average swimming velocities (essential to gyrotactic focusing) appear shortly after transition from the ‘tumbling’ mode of motion to cells swimming in the equilibrium direction. At sufficiently large shear rates, dispersivity is not monotonically decreasing with external-field intensity. Exceptional dispersion rates which are unique to non-spherical cells appear in the ‘intermediate domain’ of external fields. These are rationalized in terms of the corresponding deterministic problem (i.e. in the absence of diffusion) when cell rotary motion is governed by the simultaneous coexistence of multiple stable attractors.

Elastic waves in non-Newtonian (Maxwell) fluid-saturated porous media

This paper studies the elastic waves in non-Newtonian (Maxwell) fluid-saturated porous media with the nonzero boundary slip velocity for pore size distribution. The coefficient bF m (ω) that measures the deviation from Poiseuille flow friction in such media is presented. Based on this coefficient, we investigate the properties of elastic waves by calculating their phase velocities and attenuation coefficients as functions of frequency and the behaviour of the dynamic permeability. The study shows that the pore size distribution removes oscillations in all physical quantities in the non-Newtonian regime. Consideration of the nonzero boundary slip effect in non-Newtonian (Maxwell) fluid-saturated porous media results in (a) an overall increase of the dynamic permeability, (b) an increase of phase velocities of fast Biot waves and shear waves except in the low frequency domain and an overall increase of phase velocity of slow Biot waves and (c) an overall increase of the attenuation of three Biot waves in the intermediate frequency domain except in the deeply non-Newtonian regime. The study also shows that the attenuation coefficient of slow Biot waves is small in the deeply non-Newtonian regime at higher frequency, which encourages us to detect slow Biot waves in oil-saturated porous rock.

Disruption of a Calmodulin Central Helix-like Region of 10-Formyltetrahydrofolate Dehydrogenase Impairs Its Dehydrogenase Activity by Uncoupling the Functional Domains

10-Formyltetrahydrofolate dehydrogenase (FDH) is composed of three domains and possesses three catalytic activities but has only two catalytic centers. The amino-terminal domain (residue 1-310) bears 10-formyltetrahydrofolate hydrolase activity, the carboxyl-terminal domain (residue 420-902) bears an aldehyde dehydrogenase activity, and the full-length FDH produces 10-formyltetrahydrofolate dehydrogenase activity. The intermediate linker (residues 311-419) connecting the two catalytic domains does not contribute directly to the enzyme catalytic centers but is crucial for 10-formyltetrahydrofolate dehydrogenase activity. We have identified a region within the intermediate domain (residues 384-405) that shows sequence similarity to the central helix of calmodulin. Deletion of either the entire putative helix or the central part of the helix or replacement of the six residues within the central part with alanines resulted in total loss of the 10-formyltetrahydrofolate dehydrogenase activity, whereas the full hydrolase and aldehyde dehydrogenase activities were retained. Alanine-scanning mutagenesis revealed that neither of the six residues alone is required for FDH activity. Analysis of the predicted secondary structures and circular dichroic and fluorescence spectroscopy studies of the intermediate domain expressed as a separate protein showed that this region is likely to consist of two alpha-helices connected by a flexible loop. Our results suggest that flexibility within the putative helix is important for FDH function and could be a point for regulation of the enzyme.

Thermal and back-action noises in dual-sphere gravitational-wave detectors

We study the sensitivity limits of a broadband gravitational-waves detector based on dual resonators such as nested spheres. We determine both the thermal and back-action noises when the resonators displacements are read-out with an optomechanical sensor. We analyze the contributions of all mechanical modes, using a new method to deal with the force-displacement transfer functions in the intermediate frequency domain between the two gravitational-waves sensitive modes associated with each resonator. This method gives an accurate estimate of the mechanical response, together with an evaluation of the estimate error. We show that very high sensitivities can be reached on a wide frequency band for realistic parameters in the case of a dual-sphere detector.

Synthesis of ceria based ion conducting mesoporous membranes by soft-chemistry

Ce0.9Gd0.1O1.95 (CGO) nanophase materials and related porous supported membranes were synthesized by a soft-chemistry route. One of the membranes was post-impregnated with Pt nanoparticles. The CGO average crystallite size is in the range 9–22 nm and the specific surface area varies from 43.4 to 6.7 m2/g in the intermediate temperature domain between 500 and 700 °C. The gas permeation properties of Pt-free and Pt post-impregnated CGO porous membranes were studied up to 500 °C. Although, the intrinsic bulk ionic conduction properties of the CGO material cannot be expected to contribute to oxygen transport at such a low temperature, attractive surface diffusion effects have been evidenced, which were clearly enhanced by a dispersion of Pt particles. The observed inversion of O2/N2 selectivity (α>1) compared to the Knudsen selectivity αK(O2/N2)=0.935 was attributed to the effect of the nanophase CGO material. Insertion of Pt nanoparticles increased oxygen surface diffusion at low temperature independently of the CGO effect. The barrier effect of the membranes and the induced solid/gas contact time were considered as key parameters to develop these porous nanophase ceramic membranes.

Twoendothelin 1effectors,hand2andbapx1,pattern ventral pharyngeal cartilage and the jaw joint

A conserved endothelin 1 signaling pathway patterns the jaw and other pharyngeal skeletal elements in mice, chicks and zebrafish. In zebrafish, endothelin 1 (edn1 or sucker) is required for formation of ventral cartilages and joints in the anterior pharyngeal arches of young larvae. Here we present genetic analyses in the zebrafish of two edn1 downstream targets, the bHLH transcription factor Hand2 and the homeobox transcription factor Bapx1, that mediate dorsoventral (DV) patterning in the anterior pharyngeal arches. First we show that edn1-expressing cells in the first (mandibular) and second (hyoid) pharyngeal arch primordia are located most ventrally and surrounded by hand2-expressing cells. Next we show that along the DV axis of the early first arch primordia, bapx1 is expressed in an intermediate domain, which later marks the jaw joint, and this expression requires edn1 function. bapx1 function is required for formation of the jaw joint, the joint-associated retroarticular process of Meckel's cartilage, and the retroarticular bone. Jaw joint expression of chd and gdf5 also requires bapx1 function. Similar to edn1, hand2 is required for ventral pharyngeal cartilage formation. However, the early ventral arch edn1-dependent expression of five genes (dlx3, EphA3, gsc, msxe and msxb) are all present in hand2 mutants. Further, msxe and msxb are upregulated in hand2 mutant ventral arches. Slightly later, an edn1-dependent ventral first arch expression domain of gsc is absent in hand2 mutants, providing a common downstream target of edn1 and hand2. In hand2 mutants, bapx1 expression is present at the joint region, and expanded ventrally. In addition, expression of eng2, normally restricted to first arch dorsal mesoderm, expands ventrally in hand2 and edn1 mutants. Thus, ventral pharyngeal specification involves repression of dorsal and intermediate (joint region) fates. Together our results reveal two critical edn1 effectors that pattern the vertebrate jaw: hand2 specifies ventral pharyngeal cartilage of the lower jaw and bapx1 specifies the jaw joint.

Production of aqueous colloidal dispersions of carbon nanotubes

Stable homogeneous dispersions of carbon nanotubes (CNTs) have been prepared by using sodium dodecyl sulfate (SDS) as dispersing agent. To our knowledge, it is the first report to quantitatively characterize colloidal stability of the dispersions by UV–vis spectrophometric measurements. When the sediment time reaches 500 h, the supernatant CNT concentration drops as much as 50% for the bare CNT suspension, compared to 15% with the addition of SDS. Furthermore, after 150 h, no precipitation is found for CNT/SDS dispersions, exhibiting an extreme stability. Zeta potential, auger electron microscopy, and FTIR analysis are employed to investigate the adsorption mechanism in detail. It has been concluded that the surfactant containing a single straight-chain hydrophobic segment and a terminal hydrophilic segment can modify the CNTs–suspending medium interface and prevent aggregation over long periods. The morphology of the CNT dispersions is observed with optical microscopy. An intermediate domain of homogeneously dispersed nanotubes exhibits an optimum at 0.5 wt% CNTs and 2.0 wt% SDS.

Inhibition of interleukin 1 receptor/Toll-like receptor signaling through the alternatively spliced, short form of MyD88 is due to its failure to recruit IRAK-4.

Toll-like receptors (TLRs) and members of the proinflammatory interleukin 1 receptor (IL-1R) family are dependent on the presence of MyD88 for efficient signal transduction. The bipartite nature of MyD88 (N-terminal death domain [DD] and COOH-terminal Toll/IL-1 receptor [TIR] domain) allows it to link the TIR domain of IL-1R/TLR with the DD of the Ser/Thr kinase termed IL-1R–associated kinase (IRAK)-1. This triggers IRAK-1 phosphorylation and in turn the activation of multiple signaling cascades such as activation of the transcription factor nuclear factor (NF)-κB. In contrast, expression of MyD88 short (MyD88s), an alternatively spliced form of MyD88 that lacks only the short intermediate domain separating the DD and TIR domains, leads to a shutdown of IL-1/lipopolysaccharide-induced NF-κB activation. Here, we provide the molecular explanation for this difference. MyD88 but not MyD88s strongly interacts with IRAK-4, a newly identified kinase essential for IL-1R/TLR signaling. In the presence of MyD88s, IRAK-1 is not phosphorylated and neither activates NF-κB nor is ubiquitinated. Thus, MyD88s acts as a negative regulator of IL-1R/TLR/MyD88-triggered signals, leading to a transcriptionally controlled negative regulation of innate immune responses.

Modelling Zinc-binding Proteins with GADGET: Genetic Algorithm and Distance Geometry for Exploring Topology

A novel combination of optimization methods (Genetic Algorithm with Distance Geometry) has been developed and shown to find near-optimal solutions to a set of imposed structural constraints. With this modelling tool (GADGET), the fold-space of a variety of small zinc-binding proteins was investigated under the constraints required to form a zinc-binding site (or pair of sites). Analysis of the results concentrated on the ring-finger domain as the “classic” zinc-finger domains were too constrained to provide much topological variety, whilst the TFIIH domain (which has large unstructured loops) did not behave well. The intermediate ring-finger domain, however, was found to adopt a variety of different folds, many of which had near-optimal scores under the fitness function employed in GADGET (forming good secondary structures and zinc-coordination). Although the native fold was dominant amongst the solutions, the discovery of good alternate folds shows that even the eight residues constrained to form two zinc-binding sites was not sufficient to uniquely determine the native fold. Despite this, the fold-space of 48 theoretically possible folds was greatly reduced with just six topologies found in significant numbers.

A direct requirement for Hedgehog signaling for normal specification of all ventral progenitor domains in the presumptive mammalian spinal cord

The hedgehog signaling pathway organizes the developing ventral neural tube by establishing distinct neural progenitor fates along the dorsoventral axis. Smoothened (Smo) is essential for all Hedgehog (Hh) signaling, and genetic inactivation of Smo cells autonomously blocks the ability of cells to transduce the Hh signal. Using a chimeric approach, we examined the behavior of Smo null mutant neural progenitor cells in the developing vertebrate spinal cord, and we show that direct Hh signaling is essential for the specification of all ventral progenitor populations. Further, Hh signaling extends into the dorsal half of the spinal cord including the intermediate Dbx expression domain. Surprisingly, in the absence of Sonic hedgehog (Shh), we observe the presence of a Smo-dependent Hh signaling activity operating in the ventral half of the spinal cord that most likely reflects Indian hedgehog (Ihh) signaling originating from the underlying gut endoderm. Comparative studies of Shh, Smo, and Gli3 single and compound mutants reveal that Hh signaling acts in part to specify neural cell identity by counteracting the repressive action of Gli3 on p0, p1, p2, and pMN formation. However, whereas these cell identities are restored in Gli3/Smo compound mutants, correct stratification of the rescued ventral cell types is lost. Thus, Hh signaling is essential for organizing ventral cell pattern, possibly through the control of differential cell affinities.

Active crustal fragmentation along the Scotia–Antarctic plate boundary east of the South Orkney Microcontinent (Antarctica)

The structure of the Scotia–Antarctic plate boundary is poorly known east of the South Orkney Microcontinent. New multichannel seismic profiles, together with magnetic, gravity and swath bathymetry data obtained during the SCAN97 cruise, show a complex relief of raised blocks and elongated depressions that may reach more than 6000 m in depth. These depressions develop in relation with extensional active structures and constitute an uncommon feature in the oceans, where most of the trenches are formed in subduction contexts. The main crustal elements of the area include the oceanic crust of the Scotia Plate, the Discovery Bank composed of continental crust, a tectonic domain with intermediate features between continental and oceanic crusts that includes the Southern Bank, and the oceanic crust of the northern Weddell Sea, representing the Antarctic Plate. The Intermediate Domain was probably developed during the Late Cenozoic subduction of the Weddell Sea oceanic crust below the Discovery Bank. The fault zone associated with the plate boundary is characterized at present by sinistral transcurrent and transtensional slips, which develop a NE–SW elongated deep pull-apart basin with extreme crustal thinning and mantle uplift. The complex bathymetry and structure of the plate boundary are consequences of the presence of continental and intermediate crusts – where the deformations are concentrated – between the two stable oceanic domains. The location of a major part of the plate boundaries around the Scotia Arc is probably determined by the position of the continental and intermediate crustal fragments surrounded by oceanic crust, due to the differential behavior experienced during deformation.