Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Apparent association constants for E. coli ribosomal proteins S4, S7, S8, S15, S17 and S20 binding to 16S RNA.

We have previously reported the development of a technique utilizing nitrocellulose filters, which rapidly separates ribosomal protein-ribosomal RNA complexes from unbound protein. We have used this technique to obtain binding data for the association of proteins S4, S7, S8, S15, S17, and S20 with 16S RNA. With the exception of protein S17, the association behavior for each of these proteins exhibits a single binding site with a unique binding constant. The apparent association constants have been calculated and have been found to have a range from 1.6 x 10(6) M-1 for protein S7 to 7.1 x 10(7) M-1 for protein S17. The Scatchard plot for the protein S17 binding data is biphasic, suggesting that within the RNA population two different binding sites exist, each with a different apparent association constant.     

Roles for beta(pat-3) integrins in development and function of Caenorhabditis elegans muscles and gonads

Heterodimeric integrin receptors for extracellular matrix (ECM) play vital roles in bidirectional signaling during tissue development, organization, remodeling, and repair. The beta integrin subunit cytoplasmic domain is essential for transmission of many of these signals and overexpression of an unpaired beta tail in cultured cells inhibits endogenous integrins. Unlike vertebrates, which have at least nine beta subunit genes, the nematode Caenorhabditis elegans expresses only one beta subunit (betapat-3), and a null mutation in this gene causes embryonic lethality. To determine the functions of integrins during larval development and in adult tissues, we have taken a dominant negative approach by expression of an HA-betatail transgene composed of a hemagglutinin (HA) epitope tag extracellular domain connected to the betapat-3 transmembrane and cytoplasmic domains. Expression of this transgene in muscle and gonad, major sites of integrin expression, caused a variety of phenotypes dependent on the level of transgene expression. Abnormalities in body wall and sex muscles led to uncoordinated movement and egg-laying defects. Significant anomalies in migration and pathfinding were caused by tissue-specific expression of HA-betatail in the distal tip cells (DTC), the cells that direct gonad morphogenesis. A pat-3 gene with Tyr to Phe mutations in the cytoplasmic domain was able to rescue pat-3 null animals but also showed DTC migration defects. These results show that betapat-3 plays important roles in post-embryonic organogenesis and tissue function.     

Basement membranes: Putting up the barriers

The basement membrane is a highly organized extracellular matrix with adhesive and barrier functions. Assembly of this matrix uses two types of cell surface receptor, integrins and dystroglycan, to coordinate formation of a polygonal network of laminin, a major basement membrane protein.     

Organogenesis: cutting to the chase

Gonad morphogenesis in Caenorhabditis elegans requires two secreted proteases. Recent studies show that alterations of the extracellular matrix component fibulin-1 rescue gonadogenesis in the absence of these proteases. This finding is a critical step toward understanding the role of extracellular matrix in organogenesis.     

Distinct activation states of alpha5beta1 integrin show differential binding to RGD and synergy domains of fibronectin

alpha5beta1 integrin can occupy several distinct conformational states which support different strengths of binding to fibronectin [García, A. J., et al. (1998) J. Biol. Chem. 273, 34710-34715]. Using a model system in which specific activating monoclonal antibodies were used to achieve uniform activated states, the binding of alpha5beta1 to full-length wild-type fibronectin and mutants of fibronectin in the defined RGD and PHSRN synergy sites was analyzed using a novel method that measures the strength of the coupling between integrin and its ligand. Neither TS2/16- nor AG89-activated alpha5beta1 showed significant mechanical coupling to RGD-deleted fibronectin. However, peptide competition assays demonstrated a 6-fold difference in the binding affinities of these two states for RGD. The mutant synergy site reduced the AG89 (low)-activated state to background levels, but the TS2/16-activated state still retained approximately 30% of the wild-type activity. Thus, these two active binding states of alpha5beta1 interact differently with both the RGD and synergy domains. The failure of the AG89-activated state to show mechanical coupling to either the RGD or synergy domain mutants was unexpected and implies that the RGD domain itself does not contribute significant mechanical strength to the alpha5beta1-fibronectin interaction. The lack of RGD alone to support alpha5beta1 coupling was further confirmed using a synthetic polymer presenting multiple copies of the RGD loop. These results suggest a model in which the RGD domain serves to activate and align the alpha5beta1-fibronectin interface, and the synergy site provides the mechanical strength to the bond.     

Fibronectin matrix composition and organization can regulate cell migration during amphibian development

Fibronectin (FN) is an adhesive extracellular matrix component that is essential for vertebrate development. It forms a fibrillar matrix at the cell surface which controls cell morphology, migration, proliferation, and other important cellular processes. To address specific functions of FN matrix structure during early vertebrate development, we introduced normal and mutant recombinant FNs (recFNs) into the blastocoel cavity of embryos of the amphibian Pleurodeles waltl. Here we show that a native recFN FN(A-B-) as well as recFNs with specific mutations in the cell-binding domain, FN(RGD-) and FN(syn-), or in a FN-binding region, FNDeltaIII(1), are assembled into fibrillar matrix. A recFN (FNDeltaIII(1-7)) that forms a structurally distinct matrix in cultured cells was assembled into aggregates at the cell periphery and was able to inhibit assembly of endogenous amphibian FN matrix in a dose-dependent manner. Cell adhesion, spreading, and migration were perturbed in vitro and in vivo on chimeric matrices containing FN(RGD-), FN(syn-), or FNDeltaIII(1-7) co-assembled with amphibian FN. Developmentally, this perturbation resulted in defects in mesoderm patterning and inhibition of gastrulation. These results indicate that FN matrix fibrillar structure and composition are important determinants of cell adhesion and migration during development.     

Modeling <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> metabolism: from genome to metabolic fluxes

Background  

Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. In general, most of the recent work on N. meningitidis focuses on potential antigens and their functions, immunogenicity, and pathogenicity mechanisms. Very little work has been carried out on Neisseria primary metabolism over the past 25 years.     

Simulation of human atherosclerotic femoral plaque tissue: the influence of plaque material model on numerical results

Background

Due to the limited number of experimental studies that mechanically characterise human atherosclerotic plaque tissue from the femoral arteries, a recent trend has emerged in current literature whereby one set of material data based on aortic plaque tissue is employed to numerically represent diseased femoral artery tissue. This study aims to generate novel vessel-appropriate material models for femoral plaque tissue and assess the influence of using material models based on experimental data generated from aortic plaque testing to represent diseased femoral arterial tissue.

Methods

Novel material models based on experimental data generated from testing of atherosclerotic femoral artery tissue are developed and a computational analysis of the revascularisation of a quarter model idealised diseased femoral artery from a 90% diameter stenosis to a 10% diameter stenosis is performed using these novel material models. The simulation is also performed using material models based on experimental data obtained from aortic plaque testing in order to examine the effect of employing vessel appropriate material models versus those currently employed in literature to represent femoral plaque tissue.

Results

Simulations that employ material models based on atherosclerotic aortic tissue exhibit much higher maximum principal stresses within the plaque than simulations that employ material models based on atherosclerotic femoral tissue. Specifically, employing a material model based on calcified aortic tissue, instead of one based on heavily calcified femoral tissue, to represent diseased femoral arterial vessels results in a 487 fold increase in maximum principal stress within the plaque at a depth of 0.8 mm from the lumen.

Conclusions

Large differences are induced on numerical results as a consequence of employing material models based on aortic plaque, in place of material models based on femoral plaque, to represent a diseased femoral vessel. Due to these large discrepancies, future studies should seek to employ vessel-appropriate material models to simulate the response of diseased femoral tissue in order to obtain the most accurate numerical results.