Statistical characterization of piezoelectric coefficient d23 in cow bone.

In a newly developed, highly sensitive dilatometer we applied pulsatile electric fields to five dry bone samples cut from mid-tibial sections within a 90 degrees angle from the rear to front axis. Samples of five cows were studied. We measured the piezoelectric coefficient d23 establishing its mean and confidence interval for the first time. An analysis of variance detected a significant difference of the coefficient between animals (P < 0.01) but not between samples (P = 0.5). Between animals the coefficient ranged from 9.6 x 10(-14) to 27.1 x 10(-14) C/N. It can no longer be assumed that piezoelectricity is an inherent property of bone, constant between animals.     

Piezoelectricity in tendon and bone

We confirm that the electrical signals produced by samples of dried bone and tendon bent in the cantilever mode are d.c. square waves in response to step loading. The signals are similar to those produced by piezoelectric mineral crystals also tested, but with an important exception: the sign of the voltage reverses on end-for-end rotation of the mineral samples but does not change for bone and tendon. We deduce a theoretical expression for the expected signal using the standard theory of piezoelectricity and the contiuum theory of elasticity. The derived expression correctly describes the experimental results for the mineral samples but fails for bone and tendon.

We then extend the standard theory of piezoelectricity by including a term relating the polarization to the gradient of the stress via a tensor of rank four. This extension leads to agreement with experiment.     

The effect of hydrocortisone on neutrophil functional activity

The male (CBA X C57BL) FI mice received 125 mg of hydrocortisone per kg body weight intraperitoneally. The functional activity of neutrophils has been evaluated by means of nitroblue terazolium test (NBT-test) values taken before or after heat-killed S. marcescens cell stimulation in vitro by 2, 12, 24 h 3, 7 or 14 days post hormonal treatment. Throughout the 1st day after hydrocortisone administration the NBT-test values taken prior to as well as post microbial neutrophil stimulation were steadily increased. This effect could be seen as early as 2 h post hormone administration and it was linked with growing leukopenia and total decrease of blood granulocyte content. By the 3rd day the same very values turned up to become normal. The NBT-test values after microbial stimulation of neutrophils were 1.7 or 2.4 lower after hydrocortisone had been added to blood in vitro in a dose 3.5 X 10(-6) M or 7 X 10(-5) M.     

Induced piezoelectricity in isotropic biomaterial.

Isotropic material can be made to exhibit piezoelectric effects by the application of a constant electric field. For insulators, the piezoelectric strain constant is proportional to the applied electric field and for semiconductors, an additional out-of-phase component of piezoelectricity is proportional to the electric current density in the sample. The two induced coefficients are proportional to the strain-dependent dielectric constant (depsilon/dS + epsilon) and resistivity (drho/dS - rho), respectively. The latter is more important at frequencies such that rhoepsilonomega less than 1, often the case in biopolymers.Signals from induced piezoelectricity in nature may be larger than those from true piezoelectricity.     

The thermal depolymerization of porcine submaxillary mucin

The time dependence of the molecular weight, radius of gyration, and hydrodynamic size distribution for porcine submaxillary mucin (PSM) in solution have been studied using static and dynamic light scattering. The weight average molecular weight (Mw) of PSM in 6 M guanidine HCl, pH 7, is initially 3 X 10(6) and decreases with time in three phases: rapidly from 3-2 X 10(6), less rapidly from 2-0.9 X 10(6), and slowly below 0.9 X 10(6). The rates of decrease are much greater at pH 2. The energy of activation associated with each phase is 20 kcal/mol, which is similar to that reported for peptide bond cleavage at an aspartic acid residue. Addition of mercaptoethanol to PSM in 6 M guanidine HCl leads to a rapid decrease in Mw to 0.9 X 10(6), followed by a very slow further decrease. These results suggest that native PSM consists of subunits (Mw = 0.9 X 10(6] that are linked by disulfide bonds to form dimers (Mw = 2 X 10(6] and then higher aggregates. This cross-linking appears to occur at unglycosylated regions of the protein core, which are believed to be richer in aspartic acid than the rest of the molecule.     

Obesity and thyroid function. 2. The effect of prolonged caloric restriction on Achilles tendon reflex values.

The results obtained are indicative of changes in thyroid function during prolonged caloric restriction. Achilles tendon reflex values were studied in obese individuals under reducing treatment. More pronounced weight loss during the first three months was associated with a shortening of Achilles tendon reflex values, or its values remained unchanged. From the sixth month on a growing percentage of obese patients in our group displayed prolongation of Achilles tendon reflex values coupled with a decrease in weight showed a statistically significant prolongation of Achilles tendon reflex values. Thyreoglobulin given at this stage normalised Achilles tendon values and reinduced weight decrease.     

Interactions of manganese(II) and small ligands with Busycon canaliculatum hemocyanin

Cyanide (5 X 10(-3) M) and thioacetamide (5 X 10(-3) M) increase the P50 values (P02 required for 50% oxygenation) of hemocyanin by 100%, respectively. Using an ion-exchange method involving 14CN-, we have found that cyanide forms a 1:1 complex with hemocyanin in the concentration range examined: Kf = 2.3 X Mw M-1 at room temperature, where Kf is association constant and Mw is molecular weight of hemocyanin. This strong binding of cyanide to hemocyanin is to be expected from the effect of this ion on the oxygenation of hemocyanin. The effects of manganese(II) ion and fluoride on the oxygenation of hemocyanin are found to be weak. The nmr measurements, however, suggest that manganese(II) ion does have some interactions with the active site of hemocyanin.     

Characterization of water in unfertilized and fertilized sea urchin eggs

The water in unfertilized and fertilized sea urchin eggs was characterized with a proton nuclear magnetic resonance (NMR) titration method assuming fast proton diffusion (FPD) between water compartments. This method involves stepwise dehydration with sequential T1 relaxation time and water content determinations. The results analyzed by the FPD model give evidence of intracellular water compartments with three different correlation times: 6 X 10(-12) sec (bulk water), 1 X 10(-10) sec (structured water) and about 2 X 10(-9) sec (bound water). Fertilization is accompanied by a substantial increase in bulk water (from 111 to 414 g H2O per 100 g dry mass) and by a decrease in the water of hydration (from 128 g to 56 g per 100 g dry mass). This study shows that 54% of the water in the unfertilized sea urchin egg has motional properties different from bulk water and that this percentage decreases dramatically shortly after fertilization. Most of the change in T1 relaxation rate observed at fertilization can be accounted for by uptake of bulk water associated with elevation of the fertilization membrane.     

Enzyme kinetics in solvents of increased viscosity. Dynamic aspects of carbonic anhydrase catalysis

The dependence of enzymatic catalysis on diffusion rates in solution was examined with regard to high specific activity carbonic anhydrase (CA II) by varying the viscosity of the reaction medium with added glycerol, sucrose, and ficoll (a copolymer of sucrose and epichlorohydrin). Responses of the Michaelis-Menten parameters associated with CO2 hydration and HCO3- dehydration were deduced and analyzed by utilizing a spectrophotometric stopped-flow technique. It was found that both kcatHCO3 (= 3.9 X 10(5) s-1 at pH 5.90) and kcatCO2 (= 1.2 X 10(5) s-1 at pH 5.90 and 8.6 X 10(5) s-1 at pH 8.80) steadily decreased with the addition of monomeric viscogen while both KmHC03- (= 20 mM at pH 5.90) and KmCO2 (= 18 mM at pH 5.90 and 13 mM at pH 8.80) remained independent of viscosity, within experimental error. These results indicate that some kind of proton-transfer-related event is primarily responsible for the observed rate decrease. The three polyhydroxy cosolutes exhibited significant differences with regard to the magnitude of the viscosity effect on the kcat of the enzyme, with glycerol affecting the largest decrease, sucrose affecting a moderate one, and ficoll having virtually no effect. The discrepancy between glycerol and sucrose could be largely reconciled by correcting for diffusion-unrelated effects as estimated from rate studies of considerably slower CA II catalyzed acetaldehyde hydration and p-nitrophenyl acetate hydrolysis. Ficoll, however, was found to be unsuitable as a viscogenic probe because it failed to appreciably decrease the mobilities of smaller ions (as deduced from electrolytic conductance measurements) despite its capacity to greatly increase the macroscopic viscosity of the medium. Our best estimates indicate that this reaction comes within ca. 30% of the diffusion limit at 0.890 cP and 25 degrees C for both CO2 hydration and HCO3- dehydration reactions. However, it is reasonable to expect this value to be considerably higher in the natural environment of the enzyme because of the relatively high viscosities attained in the interior of erythrocytes.     

In vitro deposition of hydroxyapatite on cortical bone collagen stimulated by deformation-induced piezoelectricity

In the present work, we have studied the effect of the piezoelectricity of elastically deformed cortical bone collagen on surface using a biomimetic approach. The mineralization process induced as a consequence of the piezoelectricity effect was evaluated using scanning electron microscopy (SEM), thermally stimulated depolarization current (TSDC), and differential scanning calorimetry (DSC). SEM micrographs showed that mineralization occurred predominantly over the compressed side of bone collagen, due to the effect of piezoelectricity, when the sample was immersed in the simulated body fluid (SBF) in a cell-free system. The TSDC method was used to examine the complex collagen dielectric response. The dielectric spectra of deformed and undeformed collagen samples with different hydration levels were compared and correlated with the mineralization process followed by SEM. The dielectric measurements showed that the mineralization induced significant changes in the dielectric spectra of the deformed sample. DSC and TSDC results demonstrated a reduction of the collagen glass transition as the mineralization process advanced. The combined use of SEM, TSDC, and DSC showed that, even without osteoblasts present, the piezoelectric dipoles produced by deformed collagen can produce the precipitation of hydroxyapatite by electrochemical means, without a catalytic converter as occurs in classical biomimetic deposition.     

Physical properties of the dimyristoylphosphatidylcholine vesicle and of complexes formed by its interaction with apolipoprotein C-III.

The structure of a single bilayer vesicle of dimyristoylphosphatidylcholine has been characterized by sedimentation, densimetry, and light-scattering measurements. The molecular weight, partial specific volume, Stokes radius, and degree by hydration were found to be 2.68 X 10(6), 0.972 cm3/g, 125 A, and 0.86 g/g, respectively. From these quantities, a spherically symmetrical model has been derived that features a phospholipid bilayer 35.5 A thick and a hydration shell 9.3 A thick. This particle was shown to bind apolipoprotein C-III (apoC-III) up to 0.08 g/g without loss of its original vesicular structure. At protein-lipid ratios in excess of 0.08 g/g, sedimentation, gel chromatography, and light-scattering measurement indicated a dramatic decrease in Stokes radius and molecular weight. The sedimentation data showed these parameters to become constant at protein-lipid ratios in excess of 0.25 g/g. In this region, the Stokes radius and molecular weight were found to be approximately 80 A and 442 000, respectively. Within the constraints of these values and other data, several models for this complex are discussed.     

Properties of liver microsomal cholesterol 5,6-oxide hydrolase

Cholestane 3 beta,5 alpha, 6 beta-triol has been identified as the exclusive product formed on hydration of cholesterol 5,6 alpha- and 5,6 beta-oxide catalyzed by cholesterol oxide hydrolase in liver microsomes obtained from five mammalian species. Highest activities were present in microsomes from rats and humans. Both acid- and base-catalyzed hydrolysis of the two epoxides also produce this product, presumably due to preference for pseudo-axial opening of the oxirane ring to form product with a trans-AB ring junction. Although the beta-oxide is more reactive than the alpha-oxide upon acid-catalyzed hydration, the alpha-oxide is a 4.5-fold better substrate than the beta-oxide as indicated by values of Vmax/Km. The kinetic parameters Vmax and Km for the reaction catalyzed by rat liver microsomes are 1.68 +/- 0.15 X 10(-7) M min-1 and 10.6 +/- 1.5 microM for the alpha-oxide and 1.32 +/- 0.11 X 10(-7) M min-1 and 37.2 +/- 5.5 microM for the beta-oxide at 0.35 mg protein/ml, pH 7.4, 6.35% (v/v) CH3CN, and 37 degrees C. Several imino compounds are competitive inhibitors for the enzyme from rat liver. The most effective of these is 5,6 alpha-iminocholestanol (Ki = 0.085 microM) which was known to be a good inhibitor from previous studies. Inhibition by aziridines is consistent with the participation of acid catalysis in the mechanism of action of the enzyme. Cholesterol oxide hydrolase is a distinct enzyme from oxidosqualene cyclase as well as microsomal epoxide hydrolase (EC 3.3.2.3) and the recently reported mouse hepatic microsomal epoxide hydrolase that catalyzes the hydration of trans-stilbene oxide.     

Thermal stabilization of collagen molecules in bone tissue

Differential thermal calorimetry (DSC) analysis of partially dehydrated bovine bone, demineralized bone and bovine tendon collagen was performed up to 300 °C to determine factors influencing stability of mineralized collagen in bone tissue. Two endothermal regions were recognized. The first, attributed to denaturation of collagen triple helix, was hydration dependent and had a peak at 155–165 °C in bone, 118–137 °C in tendon and 131–136 °C in demineralized bone. The second region extended from 245 to 290 °C in bone and from 200 to 280 °C in tendon and was connected with melting and decomposition of collagen. Differences in thermodynamic parameters between cortical and trabecular bone tissue were stated. Activation energy of collagen unfolding in native bone tissue increased with dehydration of the bone. From the results of the present study we conclude that dehydrated bone collagen is thermally very stable both in native and in demineralized bone. Presence of mineral additionally stabilizes bone tissue.     

Influence of tendon slack on electromechanical delay in the human medial gastrocnemius in vivo.

The purpose of this study was to clarify the influence of muscle-tendon complex stretch on electromechanical delay (EMD) in terms of the extent of tendon slack in the human medial gastrocnemius (MG). EMD and MG tendon length were measured at each of five ankle joint angles (-30, -20, -10, 0, and 5 degrees : positive values for dorsiflexion) using percutaneous electrical stimulation and ultrasonography, respectively. The extent of MG tendon slack was calculated as MG tendon length shortening, standardized with MG tendon slack length obtained at the joint angle (-16 degrees +/- 5 degrees ) where the passive ankle joint torque was zero. EMD at -30 degrees (19.2 +/-2.2 ms) and -20 degrees (17.2 +/- 1.3 ms) was significantly greater than that at -10 degrees (16.0 +/-2.3 ms), 0 degrees (15.0 +/-1.4 ms), and 5 degrees (14.8 +/-1.4 ms), and at 0 and 5 degrees, respectively. The relative EMD, normalized with the maximal EMD for each subject, decreased dependent on the extent of decrease in MG tendon slack. There were no significant differences in EMD among the joint angles (-10, 0, and 5 degrees ) where MG tendon slack was taken up. These results suggest that the extent of tendon slack is an important factor for determining EMD.     

Collagen crosslinking and cartilage glycosaminoglycan composition in normal and scoliotic chickens

The amounts of lysine-derived crosslinks in collagens from tendon, cartilage, intervertebral disc, and bone and changes in the composition of sternal cartilage glycosaminoglycans were estimated in two lines of chickens, a control-isogenic line and a line that develops scoliosis. In the scoliotic line, scoliosis first appears at 3-4 weeks and progressively increases in severity and incidence so that 90% of the birds express the lesion by week 10. We have reported previously that cartilage, tendon, and bone collagens from scoliotic birds are more soluble than corresponding collagens from normal birds. Herein, collagen crosslinking and altered proteoglycan metabolism are examined as possible mechanisms for the differences in collagen solubility. At 1 week of age there were fewer reducible crosslinking amino acids (hydroxylysinonorleucine, dihydroxylysinonorleucine, and lysinonorleucine) in collagens from sternal cartilage and tendon in the scoliotic line than in the isogenic line. However, by week 3 and at weeks 5 or 7 values were similar in both groups. The amounts of hydroxypyridinium in vertebral bone and intervertebral disc collagen were also similar in both groups of birds. Consequently, differences in collagen crosslinking do not appear to be a persistent developmental defect underlying the expression of scoliosis in the model. However, differences were observed in cartilage proteoglycans and glycosaminoglycans from the scoliotic line that were not present in cartilage from the isogenic line. The average molecular weight of the uronide-containing glycosaminoglycans was 30% less in the scoliotic line than in the isogenic line, i.e., 12,000 compared to 18,000. The size distribution of cartilage proteoglycans from the scoliotic line also differed from that of proteoglycans from the isogenic line.(ABSTRACT TRUNCATED AT 250 WORDS)     

The nature of protein dipole moments: experimental and calculated permanent dipole of alpha-chymotrypsin

The electric dichroism of alpha-chymotrypsin has been measured in buffers of various pH values and ion compositions. The stationary dichroism obtained as a function of the electric field strength is not compatible with an induced dipole mechanism and clearly shows that alpha-chymotrypsin is associated with a substantial permanent dipole moment. After correction for the internal directing electric field according to a sphere model, the dipole moment is 1.6 X 10(-27) C m at pH 8.3 (corresponding to 480 D). This value decreases with decreasing pH (to 1.2 X 10(-27) C m at pH 4.2), but is almost independent of the monovalent salt concentration in the range from 2 to 12 mM and of Mg2+ addition up to 1 mM. The assignment of the permanent dipole moment is confirmed by analysis of the dichroism rise curves. The dichroism decay time constants of (31 +/- 1) ns at 2 degrees C can be represented by a spherical model with a radius of 25-26 A, which is consistent with the known X-ray structure. The limiting linear dichroism is slightly dependent on the buffer composition and demonstrates subtle variations of the protein structure. As a complement to the experimental results, electric and hydrodynamic parameters of alpha-chymotrypsin have been calculated according to the known X-ray structure. Bead model simulations provide the center of diffusion, which is used to calculate dipole moments according to the equilibrium charge distribution evaluated from standard pK values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stabilizing influence of calcium and magnesium ions on the decameric structure of chiton hemocyanins

The stabilizing effects of Ca2+ and Mg2+ ions on the decameric structure of hemocyanins from two representative chitons, Stenoplax conspicua and Mopalia muscosa were investigated by light-scattering molecular weight measurements, ultracentrifugation, absorbance, and circular dichroism methods. The dissociation profiles at any given pH resulting from the decrease in divalent ion concentration, investigated at a fixed protein concentration of 0.1 g.liter-1, could be fitted by a decamer-to-dimer-to monomer scheme of subunit dissociation. The initial decline in the light-scattering molecular weight curves required one or two apparent binding sites per hemocyanin dimer formed as intermediate dissociation product, with apparent dissociation constants (kD,2) for Ca2+ ions of 0.7 to 7 X 10(-4) M, not very different from the value of 2.5 X 10(-4) M obtained by Makino by equilibrium dialysis for the hemocyanin of the opistobranch, Dolabella auricularia. The binding of Mg2+ ion to S. conspicua and M. muscosa hemocyanins appears to be both weaker than the binding of Ca2+ and more pH dependent, with kD,2 values ranging from the 3 X 10(-4) to 4 X 10(-2) M at pH 8.5 to 9.5. The dissociation the decameric hemocyanin species (sedimentation coefficient ca. 60 S) is also observed in the ultracentrifugation with the initial appearance of 18-20 S dimers, followed by a shift in equilibrium to monomeric species of lower sedimentation rates of 11-12 S as the divalent ion concentration is reduced below 1 X 10(-4) M Ca2+ and Mg2+. The dissociation of dimers to monomers in the second step of the reaction is characterized by one or two binding sites per subunit and a somewhat stronger affinity for divalent ions, indicated by apparent dissociation constants (kD,1) of 0.7 X 10(-4) to 3 X 10(-3) M. Circular dichroism and absorbance measurements at 222 and 346 nm suggest no significant changes in the conformation of the hemocyanin subunits produced by the different stages of subunit dissociation.     

Bone marrow-derived matrix metalloproteinase-9 is associated with fibrous adhesion formation after murine flexor tendon injury

The pathogenesis of adhesions following primary tendon repair is poorly understood, but is thought to involve dysregulation of matrix metalloproteinases (Mmps). We have previously demonstrated that Mmp9 gene expression is increased during the inflammatory phase following murine flexor digitorum (FDL) tendon repair in association with increased adhesions. To further investigate the role of Mmp9, the cellular, molecular, and biomechanical features of healing were examined in WT and Mmp9(-/-) mice using the FDL tendon repair model. Adhesions persisted in WT, but were reduced in Mmp9(-/-) mice by 21 days without any decrease in strength. Deletion of Mmp9 resulted in accelerated expression of neo-tendon associated genes, Gdf5 and Smad8, and delayed expression of collagen I and collagen III. Furthermore, WT bone marrow cells (GFP(+)) migrated specifically to the tendon repair site. Transplanting myeloablated Mmp9(-/-) mice with WT marrow cells resulted in greater adhesions than observed in Mmp9(-/-) mice and similar to those seen in WT mice. These studies show that Mmp9 is primarily derived from bone marrow cells that migrate to the repair site, and mediates adhesion formation in injured tendons. Mmp9 is a potential target to limit adhesion formation in tendon healing.     

Impact of electrical cable insulating oil on the mineralisation of [1-(14)C]glucose in soil

Subsurface high voltage electric cables are commonly insulated using dodecylbenzene in combination with mineral oil. This work assessed the impact of increasing concentrations of cable insulating oil (0-10% dry weight) on soil microbial respiration as determined by mineralisation of [1-(14)C]glucose (11 microg C g(-1) soil). Acute impact was assessed from 0 days to 21 days, and chronic impact was assessed after 300 days. This study found that cable insulating oil increased respiratory activity of soil microflora. The extent of impact was found to depend on both oil concentration and the length of oil-soil contact time. Following acute exposure (21-days oil-soil contact time), it was found that oil concentrations up to 1% promoted a significant (P<0.05) increase in the extent of [1-(14)C]glucose mineralisation to (14)CO(2) relative to the control. In contrast, higher concentrations of cable insulating oil (5% and 10%) promoted no significant (P0.05) increase in the extent of [1-(14)C]glucose mineralisation to (14)CO(2) relative to the control. Following chronic exposure (300-days oil-soil contact time), the extent of mineralisation was greater at all oil concentrations applied relative to the control. For oil concentrations up to and including 1%, there was a decrease in the extent of elevation in mineralisation relative to the values after 21-days exposure. At higher oil concentrations, namely 5% and 10%, the extent of elevation in mineralisation was comparable with that after 21-days oil-soil contact time. We suggest that the increase in mineralisation of glucose indicates that cable insulating oil is a readily available carbon source to the carbon-limited soil microflora.