A cellular tensegrity model to analyse the structural viscoelasticity of the cytoskeleton

This study describes the viscoelastic properties of a refined cellular-tensegrity model composed of six rigid bars connected to a continuous network of 24 viscoelastic pre-stretched cables (Voigt bodies) in order to analyse the role of the cytoskeleton spatial rearrangement on the viscoelastic response of living adherent cells. This structural contribution was determined from the relationships between the global viscoelastic properties of the tensegrity model, i.e., normalized viscosity modulus (eta(*)), normalized elasticity modulus (E(*)), and the physical properties of the constitutive elements, i.e., their normalized length (L(*)) and normalized initial internal tension (T(*)). We used a numerical method to simulate the deformation of the structure in response to different types of loading, while varying by several orders of magnitude L(*) and T(*). The numerical results obtained reveal that eta(*) remains almost independent of changes in T(*) (eta(*) proportional, variant T(*+0.1)), whereas E(*) increases with approximately the square root of the internal tension T(*) (from E(*) proportional, variant T(*+0.3) to E(*) proportional, variant T(*+0.7)). Moreover, structural viscosity eta(*) and elasticity E(*) are both inversely proportional to the square of the size of the structure (eta(*) proportional, variant L(*-2) and E(*) proportional, variant L(*-2)). These structural properties appear consistent with cytoskeleton (CSK) mechanical properties measured experimentally by various methods which are specific to the CSK micromanipulation in living adherent cells. Present results suggest, for the first time, that the effect of structural rearrangement of CSK elements on global CSK behavior is characterized by a faster cellular mechanical response relatively to the CSK element response, which thus contributes to the solidification process observed in adherent cells. In extending to the viscoelastic properties the analysis of the mechanical response of the cellular 30-element tensegrity model, the present study contributes to the understanding of recent results on the cellular-dynamic response and allows to reunify the scattered data reported for the viscoelastic properties of living adherent cells.     

Multiple transmembrane amino acid requirements suggest a highly specific interaction between the bovine papillomavirus E5 oncoprotein and the platelet-derived growth factor beta receptor

The bovine papillomavirus E5 protein activates the cellular platelet-derived growth factor beta receptor (PDGFbetaR) tyrosine kinase in a ligand-independent manner. Evidence suggests that the small transmembrane E5 protein homodimerizes and physically interacts with the transmembrane domain of the PDGFbetaR, thereby inducing constitutive dimerization and activation of this receptor. Amino acids in the receptor previously found to be required for the PDGFbetaR-E5 interaction are a transmembrane Thr513 and a juxtamembrane Lys499. Here, we sought to determine if these are the only two receptor amino acids required for an interaction with the E5 protein. Substitution of large portions of the PDGFbetaR transmembrane domain indicated that additional amino acids in both the amino and carboxyl halves of the receptor transmembrane domain are required for a productive interaction with the E5 protein. Indeed, individual amino acid substitutions in the receptor transmembrane domain identified roles for the extracellular proximal transmembrane residues in the interaction. These data suggest that multiple amino acids within the transmembrane domain of the PDGFbetaR are required for a stable interaction with the E5 protein. These may be involved in direct protein-protein contacts or may support the proper transmembrane alpha-helical conformation for optimal positioning of the primary amino acid requirements.     

The CXCR3 binding chemokine IP-10/CXCL10: structure and receptor interactions

The structure of IP-10 was solved by NMR spectroscopy and represents the first structure from the class of agonists toward the receptor CXCR3. CXCR3 binding chemokines are unique in their ability to bind receptors from both the CC and CXC classes of chemokine receptors. An unusual structural feature of IP-10 was identified that may provide the basis for the ability of IP-10 to bind both CXCR3 and CCR3. The surface of IP-10 that interacts with the N-terminus of CXCR3 was defined by monitoring changes in the NMR spectrum of IP-10 upon addition of a CXCR3 N-terminal peptide. These studies indicated that the interaction involves a hydrophobic cleft, formed by the N-loop and 40s-loop region of IP-10, similar to the interaction surface observed for other chemokines such as IL-8. An additional region of interaction was observed that consists of a hydrophobic cleft formed by the N-terminus of IP-10 and 30s-loop of IP-10.     

Axial progenitors with extensive potency are localised to the mouse chordoneural hinge

Elongation of the mouse anteroposterior axis depends on a small population of progenitors initially located in the primitive streak and later in the tail bud. Gene expression and lineage tracing have shown that there are many features common to these progenitor tissues throughout axial elongation. However, the identity and location of the progenitors is unclear. We show by lineage tracing that the descendants of 8.5 d.p.c. node and anterior primitive streak which remain in the tail bud are located in distinct territories: (1) ventral node descendants are located in the widened posterior end of the notochord; and (2) descendants of anterior streak are located in both the tail bud mesoderm, and in the posterior end of the neurectoderm. We show that cells from the posterior neurectoderm are fated to give rise to mesoderm even after posterior neuropore closure. The posterior end of the notochord, together with the ventral neurectoderm above it, is thus topologically equivalent to the chordoneural hinge region defined in Xenopus and chick. A stem cell model has been proposed for progenitors of two of the axial tissues, the myotome and spinal cord. Because it was possible that labelled cells in the tail bud represented stem cells, tail bud mesoderm and chordoneural hinge were grafted to 8.5 d.p.c. primitive streak to compare their developmental potency. This revealed that cells from the bulk of the tail bud mesoderm are disadvantaged in such heterochronic grafts from incorporating into the axis and even when they do so, they tend to contribute to short stretches of somites suggesting that tail bud mesoderm is restricted in potency. By contrast, cells from the chordoneural hinge of up to 12.5 d.p.c. embryos contribute efficiently to regions of the axis formed after grafting to 8.5 d.p.c. embryos, and also repopulate the tail bud. These cells were additionally capable of serial passage through three successive generations of embryos in culture without apparent loss of potency. This potential for self-renewal in chordoneural hinge cells strongly suggests that stem cells are located in this region.     

Characterization of Cah,a calcium-binding and heat-extractable autotransporter protein of enterohaemorrhagic Escherichia coli

We have identified and characterized a protein of enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7 that shares homology with antigen 43 and AIDA-I of E. coli. The gene encoding this protein consists of a 2850 bp open reading frame and was named cah for calcium binding antigen 43 homologue. The prototype EHEC strain EDL933 possesses identical duplicate copies of cah (cah1 and cah2), which showed 100% identity at the nucleotide level. We showed that E. coli K-12 containing the recombinant cah gene produced two proteins, an approximately 80 kDa outer membrane protein and a 43.0 kDa heat-extractable protein. The Cah protein contains a predicted 52-amino-acid extended signal sequence found in several autotransporter proteins, and N-terminal sequencing data indicated that the 43.0 kDa passenger protein was derived from cleavage of the signal sequence from alanine at position 53. Phenotypes such as autoaggregation and change in bacterial shape were observed when a recombinant plasmid containing the cah gene was introduced into a laboratory E. coli strain, and these phenotypes were eliminated upon mutation of the cah gene. The passenger domain contains six domains found in calcium-binding proteins, and the recombinant Cah passenger protein bound 45Ca2+. In E. coli O157:H7, Cah is a heat-extractable protein, the expression of which is induced in minimal essential media and under divalent ion-depleting conditions; it also participates in the formation of biofilms. Our results provide insight into the expression, secretion and preliminary features of the calcium-binding Cah autotransporter protein of EHEC O157:H7.     

Molecular mobility of elastin: effect of molecular architecture

The thermal and dielectric properties of elastin and two soluble derivatives (kappa-elastin and derived elastin peptides from enzymatic elastolysis) were investigated in the freeze-dried state in a wide temperature range (from -180 to +220 degrees C). The glass transition of these amorphous proteins was studied by differential scanning calorimetry (DSC). The dielectric relaxations of both proteins were followed by thermally stimulated currents (TSC), an isochronal dielectric spectrometry running at variable temperature, analogous to a low-frequency spectroscopy (10(-3)-10(-2) Hz) and by dynamic dielectric spectroscopy (DDS), performed isothermally with the frequency varying from 10(-2) to 3 x 10(6) Hz. The combination of TSC and DDS experiments and the determination of the activation parameters of the relaxation times inform about the molecular mobility of the proteins, both in the glassy state and in the liquid state. Major differences between the relaxation behavior of elastin and its soluble derivatives have been discussed and correlated with the molecular architecture of the proteins.     

Impairment of macrophage survival by NaCl: implications for early pulmonary inflammation in cystic fibrosis

Inflammation, characterized by the presence of proinflammatory chemokines and neutrophils, is a hallmark of early airway disease in infants with cystic fibrosis (CF), although the underlying mechanisms remain poorly defined. In this study, we evaluated the role of NaCl and the ensuing hyperosmolar effect on tumor necrosis factor (TNF)-alpha signaling and apoptosis in macrophages. Incubation of mouse macrophages with NaCl activated p38(mapk) and the p46(jnk) and p54(jnk) c-jun NH(2)-terminal kinase isoforms, but not p42(mapk/erk2) or Akt. Similar results were obtained with sorbitol, suggesting a general response to hyperosmolarity. Strikingly, the activation of p42(mapk/erk2) and Akt by TNF-alpha was also inhibited in the presence of NaCl. Because the activation of p42(mapk/erk2) and Akt has been associated with survival responses, we investigated the effect of NaCl on macrophage apoptosis. The results indicated a synergistic increase in apoptosis when macrophages were exposed to TNF-alpha in the presence of NaCl compared with stimulation with TNF-alpha alone or NaCl alone. Furthermore, pharmacological inhibition of p42(mapk/erk2) and Akt mimicked the effect of NaCl. Collectively, these findings indicate that modest elevations in NaCl differentially regulate the activation of mitogen-activated protein kinases and Akt and potentiate macrophage apoptosis. We speculate that augmentation of macrophage apoptosis in CF airways may result in decreased clearance of neutrophils and in deficiencies in the elimination of common CF pathogens.     

The effectiveness of web-based, multimedia tutorials for teaching methods of human body composition analysis

This study examined the effectiveness of a series of Web-based, multimedia tutorials on methods of human body composition analysis. Tutorials were developed around four body composition topics: hydrodensitometry (underwater weighing), dual-energy X-ray absorptiometry, bioelectrical impedance analysis, and total body electrical conductivity. Thirty-two students enrolled in the course were randomly assigned to learn the material through either the Web-based tutorials only ("Computer"), a traditional lecture format ("Lecture"), or lectures supplemented with Web-based tutorials ("Both"). All students were administered a validated pretest before randomization and an identical posttest at the completion of the course. The reliability of the test was 0.84. The mean score changes from pretest to posttest were not significantly different among the groups (65.4 plus minus 17.31, 78.82 plus minus 21.50, and 76 plus minus 21.22 for the Computer, Both, and Lecture groups, respectively). Additionally, a Likert-type assessment found equally positive attitudes toward all three formats. The results indicate that Web-based tutorials are as effective as the traditional lecture format for teaching these topics.