A new model to simulate the elastic properties of mineralized collagen fibril

Bone, because of its hierarchical composite structure, exhibits an excellent combination of stiffness and toughness, which is due substantially to the structural order and deformation at the smaller length scales. Here, we focus on the mineralized collagen fibril, consisting of hydroxyapatite plates with nanometric dimensions aligned within a protein matrix, and emphasize the relationship between the structure and elastic properties of a mineralized collagen fibril. We create two- and three-dimensional representative volume elements to represent the structure of the fibril and evaluate the importance of the parameters defining its structure and properties of the constituent mineral and collagen phase. Elastic stiffnesses are calculated by the finite element method and compared with experimental data obtained by synchrotron X-ray diffraction. The computational results match the experimental data well, and provide insight into the role of the phases and morphology on the elastic deformation characteristics. Also, the effects of water, imperfections in the mineral phase and mineral content outside the mineralized collagen fibril upon its elastic properties are discussed.     

Chemical modification of arginine residues of lung galaptin and fibronectin. Effects on fibroblast binding.

Lung galaptin bound to lung fibroblasts with a Kd of 190 nM, and this binding could be inhibited by 20 mM-lactose. Selective modifications of the arginine residues of galaptin with cyclohexane-1,2-dione did not change its lectin activity or its binding to fibroblasts. By contrast, modification of the arginine residues of plasma fibronectin resulted in a marked diminution of protein-fibroblast binding. Selective modification of arginine residues may provide a useful probe for -Arg-Gly-Asp-Xaa cell-binding sequences of proteins.     

Evidence against proton gradient formation being the cause of chlorophyll fluorescence quenching by N-methylphenazonium methosulfate.

In strong illumination, 3-(3, 4-dichlorophenyl)-1,1-dimethylurea (DCMU)-poisoned chloroplasts exhibit a high yield of chlorophyll fluorescence while P-700 turnover, proton uptake, and phosphorylation are inhibited and a pH gradient is undectectable. When 10muM N-methylphenazonium methosulfate (PMS) is included, the fluorescence yield in light is substantially reduced, and when 100 muM ascorbate is also included, the yield is diminished approximately to the level in darkness. Only very slight increases in P-700 turnover and proton uptake (but no detectable pH gradient) accompany the fluorescence yield decline. When 10muM PMS and 15 mM ascorbate are added to poisoned chloroplasts (the oxygen concentration being greatly reduced), P-700 turnover, proton uptake, the pH gradient and phosphorylation all reach high levels. In this case, the yield of chlorophyll fluorescence is low and is the same in both light and dark. Further addition of an uncoupler eliminates proton uptake, the pH gradient and phosphorylation but does not significantly elevate the fluorescence yield. From these observations we suggest that, in DCMU-poisoned chloroplasts, the fluorescence quenching with PMS occurrs by a mechanism unrelated to the generation of a phosphyorylation potential. With chloroplasts unpoisoned by DCMU, PMS quenches fluorescence and considerably stimulates proton uptake, the pH gradient and phosphorylation. However, in this case, PMS serves to restore net electron transport.     

The salmonid elastic fibril. An investigation of some chemical and physical parameters.

Elastic fibrils were isolated, as electron microscopically homogeneous preparations, from salmon (Salmo salar) and trout (Salmo gairdneri) bulbus arteriosus by extraction of other tissue components with guanidinium hydrochloride. The preparations exhibited compositions widely at variance with that of bovine elastin, the differences including both the overall concentration and the relative proportions of the crosslinks. Absorption and fluorescence spectroscopy ruled out the presence of tyrosine-derived crosslinks. The wide-angle X-ray diffraction pattern of the salmonid preparations showed broad reflections corresponding to spacings of 9.8, 4.5, and 2.2 Å, similar to bovine elastin. The mechanical behavior of the salmon preparation was characterized by a linear response to stress, with minimal hysteresis, a Young's modulus of 5.5 × 10⁵ N m^?2, and a breaking strain of 1.5.     

Endogenous ligands of rat lung beta-galactoside-binding protein (galaptin) isolated by affinity chromatography on carboxyamidomethylated-galaptin-Sepharose.

Rat lung beta-galactoside-binding protein (galaptin) is developmentally regulated during postnatal lung development. In common with other vertebrate galaptins, it is very labile when purified and dependent on the presence of exogenous thiol reagents. Reaction of rat lung galaptin with iodoacetamide resulted in a stable active carboxyamidomethylated galaptin that could be coupled to Sepharose. The resultant affinity matrix bound asialoglycoproteins, and these could be quantitatively eluted with disaccharide haptens. The carboxyamidomethylated-galaptin-Sepharose affinity matrix was used to search for endogenous ligands in 13-day-rat lung. Cytosolic fractions of developing rat lung contained no moieties that could be specifically eluted with disaccharide hapten. Only when membranous fractions were extracted with 1% Triton were glycoproteins solubilized that bound to the affinity matrix and could be specifically eluted with disaccharide hapten. The eluted glycoproteins were potent inhibitors of galaptin binding to asialo-orosomucoid. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis identified these glycoproteins as being of high Mr, with three components of Mr 160000-200000 and a smaller component of Mr 75000. This is the first evidence for specific membrane-associated glycoproteins being the ligands of rat lung galaptin.     

Evidence against the involvement of DNA synthesis in phytochrome-mediated photomorphogenesis

In order to clarify the relationship between photomorphogenesis and DNA replication we investigated the effect of continuous far-red or white light on the synthesis of DNA in the cotyledons and the hypocotyl of mustard seedlings between 36 and 108 h after sowing. The total DNA content of the cotyledons (about 2.2 pg cell^-1) did not significantly change during this period although long-term labeling experiments revealed newly synthesized DNA of nuclear, plastid, and mitochondrial origin. Light had no detectable effect on total DNA content and on the labeling of either DNA fraction. Histoautoradiography indicated that nuclear DNA synthesis was exclusively localized in dividing stomatal cells and in sieve tube companion cells undergoing endopolyploidization. The DNA content of the hypocotyl increased continuously but likewise showed no detectable effect of light. It is concluded that cell growth and differentiation during photomorphogenesis is independent of DNA synthesis.Abbreviation DABA 3,5-diaminobenzoic acid     

Contribution of tree structures in the lung to lung elastic recoil

The direct contribution of forces in tree structures in the lung to lung recoil pressure and changes in recoil pressure induced by alterations of the forces are analyzed. The analysis distinguishes the contributions of axial and circumferential tensions in the trees and indicates that only axial tensions directly contribute to static recoil. This contribution is derived from analysis of the axial forces transmitted across a random plane transecting the lung. The change in recoil pressure induced by changes in axial tension is similarly derived. Alterations of circumferential tensions in the trees indirectly change recoil by causing nonuniform deformations of the surrounding lung parenchyma, and a continuum elasticity solution for the stress induced by the deformations is derived. Sample calculations are presented for the airway tree based on available data on airway morphometric and mechanical properties. The increase in recoil pressure accompanying increases in axial and circumferential tensions with contraction of airway smooth muscle is also analyzed. The calculations indicate that axial stresses in the airway tree out to bronchioles directly contribute only a small fraction of the static recoil pressure. However, it is found that contraction of smooth muscle in these airways can increase recoil pressure appreciably (10-20%), mainly by the deformation of the parenchyma with increases in circumferential tension in smaller airways. The results indicate that the geometric and mechanical properties of the airway tree are such that only peripheral elements of the tree can substantially affect the elastic properties of the lung. The possible contributions of vascular trees for which data on mechanical and morphometric properties are more limited are also discussed.     

Properties of the oxidation of exogenous NADH and NADPH by plant mitochondria. Evidence against a phosphatase or a nicotinamide nucleotide transhydrogenase being responsible for NADPH oxidation

(1) The optimum pH for the oxidation of exogenous NADH by mitochondria from both Jerusalem artichoke (Helianthus tuberosus) tubers and Arum maculatum spadices was 7.0–7.1. NADPH oxidation had a lower optimum pH of 6.6 in Arum and 6.0 in Jerusalem artichoke mitochondria. In both types of mitochondria the rates of NADH and NADPH oxidation were identical below pH 6.0–5.5. (2) It is shown conclusively that neither a phosphatase converting NADPH to NADH nor a nicotinamide nucleotide transhydrogenase was involved in the oxidation of NADPH by these mitochondria. (3) Palmitoyl-CoA, an inhibitor of transhydrogenase activity in mammalian mitochondria, inhibits both NADH and NADPH oxidation by plant mitochondria with a K_i of about 10 μM. (4) It is concluded that the known properties of NAD(P)H oxidation are best explained by assuming the presence of a second dehydrogenase specific for NADPH. At low pH, electron flow from the two dehydrogenases to oxygen shares a common rate-limiting step.     

Cultured epidermis influences the fibril organization of purified type I collagen gels

Purified type I collagen gel used as culture substrate was composed of unstriated fibrils. Before culture, gel fragments were coated with culture medium with or without fetal calf serum (FCS+ coated or FCS- coated gels). Each gel fragment was apposed to a fragment of frog skin at the medium/air interface in Trowell culture chamber. After 7 days at 20 degrees C, the coated gels were covered with newly formed epidermis containing fibronectin localized around the keratinocytes, whose morphology was considerably modified. Fibroblast-shaped keratinocytes were localized in the anterior zone of the newly formed epidermis on FCS+ gels. The long axis of the cells was parallel to the gel surface, where numerous unstriated fibrils were located. Polyhedral keratinocytes were located in the posterior zone on FCS+ gels or the anterior and posterior zones on FCS- gels with the long axis perpendicular to the gel surface. Numerous cross-striated fibrils were found under the cultured keratinocytes in the vicinity of the basal filipodia. This model is useful for the study of collagen gel reorganization by keratinocytes.     

Human splenic galaptin: carbohydrate-binding specificity and characterization of the combining site.

A galactose-binding lectin (galaptin) from human spleen has been purified to homogeneity by affinity chromatography on asialofetuin-Sepharose. The carbohydrate-binding specificity of galaptin has been investigated by analyzing the binding of galaptin to asialofetuin in the presence of putative inhibitors. An enzyme-linked immunosorbent assay (ELISA) was developed that involved adsorption of asialofetuin to microtiter plates. Galaptin bound to asialofetuin was detected with polyclonal rabbit anti-galaptin serum followed by goat anti-rabbit IgG-peroxidase conjugate. The concentrations of inhibitors giving 50% inhibition of galaptin binding relative to controls were graphically determined and normalized relative to galactose or lactose. These analyses revealed that galaptin has a combining site at least as large as a disaccharide. The disaccharides having non-reducing-terminal beta-galactosyl residues linked (1,3), (1,4), and (1,6) to Glc or GlcNAc are better inhibitors than free Gal. GalNAc, either free or glycosidically linked, appears to have no affinity for the lectin. The nitrophenyl galactosides are better inhibitors than methyl galactosides, indicating the occurrence of hydrophobic interactions. The data indicate that OH groups at C-4 and C-6 of Gal and the OH at C-3 of GlcNAc in Gal beta(1,4)GlcNAc are important for lectin sugar interaction. Our data support the hypothesis that endogenous receptors for galaptin are most likely lactosaminoglycan moieties.     

Flexibility of the Ure2 prion domain is important for amyloid fibril formation

Ure2, the protein determinant of the Saccharomyces cerevisiae prion [URE3], has a natively disordered N-terminal domain that is important for prion formation in vivo and amyloid formation in vitro; the globular C-domain has a glutathione transferase-like fold. In the present study, we swapped the position of the N- and C-terminal regions, with or without an intervening peptide linker, to create the Ure2 variants CLN-Ure2 and CN-Ure2 respectively. The native structural content and stability of the variants were the same as wild-type Ure2, as indicated by enzymatic activity, far-UV CD analysis and equilibrium denaturation. CLN-Ure2 was able to form amyloid-like fibrils, but with a significantly longer lag time than wild-type Ure2; and the two proteins were unable to cross-seed. Under the same conditions, CN-Ure2 showed limited ability to form fibrils, but this was improved after addition of 0.03?M guanidinium chloride. As for wild-type Ure2, allosteric enzyme activity was observed in fibrils of CLN-Ure2 and CN-Ure2, consistent with retention of the native-like dimeric structure of the C-domains within the fibrils. Proteolytically digested fibrils of CLN-Ure2 and CN-Ure2 showed the same residual fibril core morphology as wild-type Ure2. The results suggest that the position of the prion domain affects the ability of Ure2 to form fibrils primarily due to effects on its flexibility.     

Evidence against an involvement of cyclic nucleotides in the induction of betacyanin synthesis by cytokinins.

1. A wide range of purine bases, nucleosides and cyclic nucleotides were shown to induce betacyanin synthesis in Amaranthus seedlings. 2. The induction of pigment by benzyladenine, dibutyryl cyclic AMP or cyclic AMP was not potentiated by aminophylline. Aminophylline was shown to inhibit Amaranthus cyclic AMP phosphodiesterase activity. 4. Incubation of seedlings with aminophylline inhibited the conversion of 6-[G--3H]benzyladenine into presumed 9- and 7-glucosylbenzyladenine. 5. Induction of betacyanin synthesis by 6-benzyladenine or by exposure to red light was not accompanied by changes in the total cyclic AMP content in seedlings. 6. It is concluded that the inducers tested act as cytokinin analogues; no evidence was obtained to support cyclic AMP as an intermediate in the induction process.     

Evidence for a highly elastic shell-core organization of cochlear outer hair cells by local membrane indentation

Cochlear outer hair cells (OHCs) are thought to play an essential role in the high sensitivity and sharp frequency selectivity of the hearing organ by generating forces that amplify the vibrations of this organ at frequencies up to several tens of kHz. This tuning process depends on the mechanical properties of the cochlear partition, which OHC activity has been proposed to modulate on a cycle-by-cycle basis. OHCs have a specialized shell-core ultrastructure believed to be important for the mechanics of these cells and for their unique electromotility properties. Here we use atomic force microscopy to investigate the mechanical properties of isolated living OHCs and to show that indentation mechanics of their membrane is consistent with a shell-core organization. Indentations of OHCs are also found to be highly nonhysteretic at deformation rates of more than 40 microm/s, which suggests the OHC lateral wall is a highly elastic structure, with little viscous dissipation, as would appear to be required in view of the very rapid changes in shape and mechanics OHCs are believed to undergo in vivo.     

Evidence against direct involvement of cyclic GMP or cyclic AMP in bacterial chemotactic signaling.

Defects in phosphotransferase chemotaxis in cya and cpd mutants previously cited as evidence of a cyclic GMP or cyclic AMP intermediate in signal transduction were not reproduced in a study of chemotaxis in Escherichia coli and Salmonella typhimurium. In cya mutants, which lack adenylate cyclase, the addition of cyclic AMP was required for synthesis of proteins that were necessary for phosphotransferase transport and chemotaxis. However, the induced cells retained normal phosphotransferase chemotaxis after cyclic AMP was removed. Phosphotransferase chemotaxis was normal in a cpd mutant of S. typhimurium that has elevated levels of cyclic GMP and cyclic AMP. S. typhimurium crr mutants are deficient in enzyme III glucose, which is a component of the glucose transport system, and a regulator of adenylate cyclase. After preincubation with cyclic AMP, the crr mutants were deficient in enzyme II glucose-mediated transport and chemotaxis, but other chemotactic responses were normal. It is concluded that cyclic GMP does not determine the frequency of tumbling and is probably not a component of the transduction pathway. The only known role of cyclic AMP is in the synthesis of some proteins that are subject to catabolite repression.