Carbonated hydroxyapatites precipitated in the presence of Ti

Hydroxyapatites (HA) were prepared by precipitation from an aqueous solution with Ti4+ (0-2500 microg/g) and with carbonate (0.8-4.0%) at pH 7.0. The uptake of Ti was found to be 75% of the original amounts. Stoichiometric ratios of Ca/P (1.67) were found for low carbonate samples. X-ray diffraction and IR spectroscopy have shown that samples have structural data characteristic for HA. Heat treatment and thermogravimetric analysis (20-900 degrees C) have shown carbonate decomposition enhanced by the presence of Ti and no transformation of the HA structure. It was also found that 0.2 mol of adsorbed and 0.6-0.8 mol of crystalline water are released from the samples during heating. Transmission electron microscopy revealed the formation of plate like crystals which increase in size with increase of carbonate content. Samples with high carbonate and high Ti content have irregular shape and are sensitive to electron beam irradiation as opposed to non-doped samples. Ti appears to have a destabilizing effect on HA. The incorporation of Ti in HA and the biological relevance of Ti in bone crystals is discussed.     

Fluoride and carbonate incorporation into hydroxyapatite under condition of cyclic pH variation

Fluoride and carbonate ions, which are present in the inorganic phase of bone, enamel, and dentine, are known to play an important and opposite role in the process of recrystallization. We have investigated the incorporation of fluoride and carbonate ions into hydroxyapatite structure under conditions of cyclic pH fluctuation and the effect of these incorporations on the conversion of hydroxyapatite into β-tricalcium phosphate after heat treatment. Fluoro-hydroxyapatite has been obtained as unique crystalline phase up to 20 fluoride at. %. The degree of substitution of fluoride for hydroxyl does not depend on the extent of carbonate incorporated into the apatite structure. On the other hand, the carbonate incorporation into the apatite structure seems to be hindered by the contemporary presence of fluoride. Both fluoride and carbonate exhibit a stabilizing effect on the apatite structure, as far as its conversion into β-tricalcium phosphate is concerned. The order of efficiency in stabilizing the apatite structure is F⁻ > F⁻ + CO₃²⁻ > C0₃²⁻.     

Acid catalyzed transesterification as a route to poly(3-hydroxybutyrate-co-epsilon-caprolactone) copolymers from their homopolymers

Copolymers of (R)-3-hydroxybutyric acid (HB) and epsilon-caprolactone (CL) with a composition ranging from 28 to 81 mol % of HB were synthesized by transesterification of the corresponding homopolymers in solution in the presence of 4-toluenesulfonic acid. The copolyesters were characterized with regard to their molecular weights, thermal properties, molar compositions, and average block length of repeating units by gel permeation chromatography (GPC), differential scanning calorimetry, (1)H NMR, and (13)C NMR, respectively. Random and microblock copolymers could be obtained depending on experimental conditions, with weight-average molecular weights of up to 20,000. The glass transition temperature decreased from 2 to -42 degrees C as the CL content was increased from 0 to 72 mol %. The melting temperature (T(m)) of the PCL phase decreased from 70 to 46 degrees C as the HB content changed from 0 to 47 mol %, while the T(m) of the PHB phase decreased from 177 degrees C to 163 degrees C as the CL content changed from 0 to 72 mol %. Matrix-assisted laser desorption ionization time-of-flight mass spectra of GPC fractionated samples allowed us to ascertain that copolymers rich in HB units have mostly hydroxyl and carboxyl end groups, while copolymers rich in CL units have mostly tosyl and carboxyl end groups.     

Thermodynamics of statherin adsorption onto hydroxyapatite

Statherin is a salivary protein that inhibits the nucleation and growth of hydroxyapatite crystals in the supersaturated environment of the oral cavity. The thermodynamics of adsorption of statherin onto hydroxyapatite crystals have been characterized here by isothermal titration calorimetry and equilibrium adsorption isotherm analysis. At 25 degrees C, statherin adsorption is characterized by an exothermic enthalpy of approximately 3 kcal/mol that diminishes to zero at approximately 25% surface coverage. The initial heat of statherin adsorption increases with temperature, displaying a positive heat capacity change of 194 +/- 7 cal K(-)(1) mol(-)(1) at 25 degrees C. The heat of adsorption during this initial phase is strongly dependent on the buffer species, and from the differential heats of buffer ionization, it can be calculated that approximately one proton is taken up by the crystal or protein upon adsorption. The free energy of adsorption is dominated at all coverages by a large positive entropy (>or=23 cal K(-)(1) mol(-)(1)), which may be partially due to the loss of organized water that hydrates the protein and the mineral surface prior to adsorption. These results are interpreted using a two-site model for adsorption of statherin onto the hydroxyapatite crystals.     

Changes in acetylcholine concentration, miniature end-plate potentials and synaptic vesicles in frog neuromuscular preparations during lanthanum treatment

ACh content and synaptic ultrastructure were compared in neuromuscular preparations (sartorius muscle of Rana esculenta) incubated in control saline and in saline containing 1 mM LaCl3. ACh concentrations remained constant for 6 hr in control preparations. La3+ caused a 38% depletion of ACh within the first 30 min with subsequent recovery to 120% of control values within 3-4 hr. Recovery was prevented by hemicholinium-3. At 23 degrees C La3+ caused complete loss of synaptic vesicles: no depletion was seen at 4 degrees C. Initially MEPP frequency increased 300- to 700-fold (23 degrees C), then declined. Mean vesicle diameter did not change, but SD increased. As the frequency of MEPPs declined, the percentage of s-MEPPs greatly increased. La3+ had a postsynaptic effect which increased the amplitudes of both s-MEPPs and bell-MEPPs within a few seconds. The s-MEPP mean did not change during the course of La3+ treatment although the bell-MEPP mean usually decreased. How the decrease in synaptic vesicles, decrease in MEPP frequencies, and changes in ACh levels relate to changes in the percentage of different classes of quanta is discussed.     

Controlling membrane cholesterol content. A role for polyunsaturated (docosahexaenoate) phospholipids

The molecular organization of cholesterol in 1,2-didocosahexaenoylphosphatidylcholine (22:6-22:6PC) and 1-stearoyl-2-docosahexaenoylphosphatidylcholine (18:0-22:6PC) bilayers was investigated. Using low- and wide-angle X-ray diffraction (XRD), we determined that the solubility of the sterol at 20 degrees C was 11 +/- 3 mol % in 22:6-22:6PC vs 55 +/- 3 mol % in 18:0-22:6PC bilayers. Solubility in the dipolyunsaturated membrane rose to 17 +/- 3 mol % at 40 degrees C, while in the saturated-polyunsaturated membrane there was no change within experimental uncertainty. We compared the molecular orientation of [3alpha-(2)H(1)]cholesterol incorporated into 22:6-22:6PC bilayers to its solubility limit and into 18:0-22:6PC bilayers to a comparable concentration (10 mol %) in solid-state (2)H NMR experiments. The sterol possessed a tilt angle alpha(0) = 24 degrees +/- 1 degrees in 22:6-22:6PC that was independent of temperature over a range from 20 to 40 degrees C. In contrast, the value was alpha(0) = 21 degrees +/- 1 degrees in 18:0-22:6 bilayers at 20 degrees C and increased to alpha(0) = 24 degrees +/- 1 degrees at 40 degrees C. We attribute the low solubility of cholesterol in 22:6-22:6PC membranes to steric incompatibility between the rigid steroid moiety and the highly disordered docosahexaenoic acid (DHA) chain, which has the potential to promote lateral heterogeneity within DHA-rich membranes. Considering 22:6-22:6PC to be the most unsaturated phospholipid found in vivo, this model membrane study provides a point of reference for elucidating the role of sterol-lipid interactions in controlling local compositional organization. Our results form the basis for a model that is consistent with cholesterol's ability to modulate the activity of certain neural transmembrane proteins.     

Interactions of triglycerides with phospholipids: incorporation into the bilayer structure and formation of emulsions

Interactions of carbonyl 13C-enriched triacylglycerols (TG) with phospholipid bilayers [egg phosphatidylcholine (PC), dipalmitoylphosphatidylcholine (DPPC), and an ether-linked phosphatidylcholine] were studied by 13C NMR spectroscopy. Up to 3 mol % triolein (TO) or tripalmitin (TP) was incorporated into DPPC vesicles by cosonication of the TG and DPPC at approximately 50 degrees C. NMR studies were carried out in a temperature range (30-50 degrees C) in which pure TO is a liquid whereas pure TP is a solid. In spectra of DPPC vesicles with TG at 40-50 degrees C, both TO and TP had narrow carbonyl resonances, indicative of rapid motions, and chemical shifts indicative of H bonding of the TG carbonyls with solvent (H2O) at the aqueous interfaces of the vesicle bilayer. Below the phase transition temperature of the DPPC/TG vesicles (approximately 36 degrees C), most phospholipid peaks broadened markedly. In DPPC vesicles with TP, the TP carbonyl peaks broadened beyond detection below the transition, whereas in vesicles with TO, the TO carbonyl peaks showed little change in line width or chemical shift and no change in the integrated intensity. Thus, in the gel phase, TP solidified with DPPC, whereas TO was fluid and remained oriented at the aqueous interfaces. Egg PC vesicles incorporated up to 2 mol % TP at 35 degrees C; the TP carbonyl peaks had line-width and chemical shift values similar to those for TP (or TO) in liquid-crystalline DPPC. TO incorporated into ether-linked PC had properties very similar to TO in ester-linked PC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine diphosphate ribose transferase from baby-hamster kidney cells (BHK-21/C13). Characterization of the reaction and product.

Some properties of ADP-ribose transferase, and its reaction product, from BHK-21/C13 cells are described. Enzyme activity was found almost exclusively in nuclei (90%), with the remaining 10% located in the cytosolic fraction. The nuclear enzyme is chromatin-bound and requires bivalent cations, preferably Mg2+, a pH of 8.0 and a temperature of 25 degrees C for optimal activity. Chromatin preparations incorporated radioactivity from [14C]NAD+ into acid-insoluble material for about 60 min. Kinetics for substrate NAD+ utilization were not of Michaelis--Menten type; biphasic kinetics were shown from a double-reciprocal plot (1/reaction velocity against 1/[NAD+]) and from a 'Hofstee' plot (reaction velocity/[NAD+] against reaction velocity). The transferase is unstable in the absence of Mg2+ ions. It is inhibited by thymidine, nicotinamide and nicotinamide analogues, but not by ATP, which stimulates it at concentrations of 5 mM and above. The enzyme requires thiol groups for activity; it is readily inhibited by N-ethylmaleimide at 0.5 mM. The product of the reaction is stable under acid conditions at temperatures up to 25 degrees C, but it is hydrolysed by HClO4 at 70 degrees C. It is resistant to NaOH, but is cleaved from its attachment to protein with alkali into trichloroacetic acid-insoluble and -soluble components. On the basis of Cs2SO4- density-gradient analysis under denaturing conditions (gradients included urea and guanidinium hydrochloride), and analysis of the reaction product directly on hydroxyapatite, we conclude that most of the radioactive ADP-ribose residues are firmly bound to protein, presumably in covalent linkage. Hydroxyapatite-chromatographic analysis of ADP-ribose residues released from protein by alkaline digestion showed a spectrum of molecular sizes including mono-, oligo- and poly-(ADP-ribose), when chromatin was incubated initially with [14C]NAD+ for 10 min and then for a further 30 min after addition of excess non-radioactive NAD+, only about 10% of the radioactive mono-(ADP-ribose) could be 'chased' into longer-chain molecules. Hydroxyapatite analysis was also used to show that, whereas all ADP-ribose residues were released from protein with NaOH, only 50% of them were susceptible to hydroxylamine. These hydroxylamine-sensitive residues included all size classes, although mono-(ADP-ribose) predominated. Finally, there was an approximately equal distribution of ADP-ribose incorporated into HCl-soluble proteins (including the histones) and HCl-insoluble proteins (including the non-histone proteins) when chromatin was incubated with NAD+ up to 0.5 mM, but at higher NAD+ concentrations more ADP-ribose was incorporated into the HCl-soluble fraction (82% at 4.0 mM-NAD+).     

Bilayer interactions of ether- and ester-linked phospholipids: dihexadecyl- and dipalmitoylphosphatidylcholines

Mixed phospholipid systems of ether-linked 1,2-dihexadecylphosphatidylcholine (DHPC) and ester-linked 1,2-dipalmitoylphosphatidylcholine (DPPC) have been studied by differential scanning calorimetry and X-ray diffraction. At maximum hydration (60 wt % water), DHPC shows three reversible transitions: a main (chain melting) transition, TM = 44.2 degrees C; a pretransition, TP = 36.2 degrees C; and a subtransition, TS = 5.5 degrees C. DPPC shows two reversible transitions: TM = 41.3 degrees C and TP = 36.5 degrees C. TM decreases linearly from 44.2 to 41.3 degrees C as DPPC is incorporated into DHPC bilayers; TP exhibits eutectic behavior, decreasing sharply to reach 23.3 degrees C at 40.4 mol % DPPC and then increasing over the range 40-100 mol % DPPC; TS remains constant at 4-5 degrees C and is not observed at greater than 20 mol % DPPC. At 50 degrees C, X-ray diffraction shows a liquid-crystalline bilayer L alpha phase at all DHPC:DPPC mole ratios. At 22 degrees C, DHPC shows an interdigitated bilayer gel L beta phase (bilayer periodicity d = 47.0 A) into which approximately 30 mol % DPPC can be incorporated. Above 30 mol % DPPC, a noninterdigitated gel L beta' phase (d = 64-66 A) is observed. Thus, at T greater than TM, DHPC and DPPC are miscible in all proportions in an L alpha bilayer phase. In contrast, a composition-dependent gel----gel transition between interdigitated and noninterdigitated bilayers is observed at T less than TP, and this leads to eutectic behavior of the DHPC/DPPC system.     

Insulin binding and degradation studies on erythrocytes at different temperatures

Insulin binding of human erythrocytes has been investigated between 0 and 37 degrees C using porcine 125I-insulin/unlabeled porcine insulin and mono [125I] (Tyr-A14)biosynthetic human insulin/ unlabeled biosynthetic human insulin, respectively. Either system exhibited a regular thermodynamical behavior between 0 and 22 degrees C, giving unitary free-energy changes of about -58/ -59 kJ/mol, unitary entropy changes of about +55/ +70 J/K per mol and a reaction heat of -43.1/ -38.3 kJ/mol. From 22 up to 37 degrees C an irregular thermodynamical behavior could be observed, which can be partially explained by an increased insulin degradation during incubation and an additional time-dependent binding of the degradation products.     

Novel lipase-catalyzed ring-opening copolymerization of lactide and trimethylene carbonate forming poly(ester carbonate)s.

Poly(lactide-co-trimethylene carbonate)s were prepared by the lipase-catalyzed ring-opening copolymerization of lactide and trimethylene carbonate having carbonate content from 0 to 100%. Their thermal properties and enzymatic degradability were measured. The L,L-, D,D- and D,L-lactides were copolymerized with trimethylene carbonate by porcine pancreatic lipase to produce random copolymers having molecular weights of up to 21000. The glass transition temperature (Tg of the copolymer was dependent on the carbonate content and the Tg values linearly decreased from 35 degrees C (polylactide) to -8 degrees C [poly(trimethylene carbonate)]. Among the lipases tested, the porcine pancreatic lipase and proteinase K showed biodegradability towards poly(ester-carbonate)s.     

Microcalorimetric measurement of the enthalpy of binding of rabbit skeletal myosin subfragment 1 and heavy meromyosin to F-actin

The heat of binding of rabbit skeletal myosin subfragment 1 (myosin-S1) and heavy meromyosin (HMM) to F-actin has been measured by batch calorimetry. Proton release measurements in unbuffered solutions indicate that less than 0.1 mol of protons is absorbed or released per mol of myosin head bound to actin. Hence, the measured heats are approximately equal to the enthalpy of myosin-S1 and HMM binding to actin. The enthalpy of binding of myosin-S1 to actin was +22 +/- 3 and +27 +/- 5 kJ/mol of myosin-S1 in two series of experiments at 12 degrees C and +26 +/- 5 kJ/mol of myosin-S1 at 0 degrees C, indicating that delta Cp for this reaction in the range of 0-12 degrees C is small (-80 J/mol/K). The enthalpy of binding of HMM to actin at 12 degrees C was found to be +26 +/- 1 kJ/mol of myosin head. The enthalpies determined here and the equilibrium constants obtained from the literature for measurements at 20 degrees C under identical solvent conditions were used to estimate the entropy of the association of myosin S1 and HMM with F-actin: +235 J/mol/K for myosin-S1 and +190 J/mol of myosin head/K for HMM. Thermodynamic parameters of the interaction of myosin-S1 with actin and ADP or AMP-PNP can be evaluated using the enthalpy of association of myosin-S1 with actin determined here, together with literature values for the equilibrium constants and enthalpies of binding of these nucleotides to myosin-S1. The calculated enthalpies of binding of ADP or AMP-PNP to actomyosin-S1 are small and negative.     

Binding of Clostridium botulinum type C neurotoxin to rat brain synaptosomes

The binding of Clostridium botulinum type C neurotoxin to rat brain synaptosomes was determined by the use of 125I-neurotoxin. The binding was independent of the incubation temperature (0 degrees C and 37 degrees C) and was equilibrated in 10 min. The dose dependent of 125I-toxin binding to synaptosomes at 0 degrees C showed that there were two kinds of toxin receptors on the synaptosomal membrane; the association constants and maximum binding values were 1.05 x 10(10 M-1, 5.25 x 10(-13) mol/mg of synaptosomal protein and 5.00 x 10(6) M-1, 5.00 x 10(-12) mol/mg of synaptosomal protein, respectively. When the incubation of toxin with synaptosomes was continued at 37 degrees C after 125I-toxin had been pre-incubated with synaptosomes at 0 degrees C for 10 min, the displacement of labeled toxin by the addition of excess amounts of unlabeled toxin decreased slightly with increasing incubation time, and finally 0.4% of the bound 125I-toxin was not displaced from synaptosomes. The binding of 125I-toxin to synaptosomes was inhibited by anti-heavy chain IgG and a monoclonal antibody which neutralized toxin and recognized heavy chain. These results suggest that the binding sites of toxin to synaptosomes are localized on heavy chain and a small amount of the bound toxin is incorporated into the synaptosomal membrane or synaptosomes.     

Thermodynamic analysis of the binding of component enzymes in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

The peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2, EC 2.3.1.12) binds tightly but mutually exclusively to dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4) and pyruvate decarboxylase (E1, EC 1.2.4.1) in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Isothermal titration calorimetry (ITC) experiments demonstrated that the enthalpies of binding (DeltaH degrees ) of both E3 and E1 with the PSBD varied with salt concentration, temperature, pH, and buffer composition. There is little significant difference in the free energies of binding (DeltaG degrees = -12.6 kcal/mol for E3 and = -12.9 kcal/mol for E1 at pH 7.4 and 25 degrees C). However, the association with E3 was characterized by a small, unfavorable enthalpy change (DeltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TDeltaS degrees = +14.8 kcal/mol), whereas that with E1 was accompanied by a favorable enthalpy change (DeltaH degrees = -8.4 kcal/mol) and a less positive entropy change (TDeltaS degrees = +4.5 kcal/mol). Values of DeltaC(p) of -316 cal/molK and -470 cal/molK were obtained for the binding of E3 and E1, respectively. The value for E3 was not compatible with the DeltaC(p) calculated from the nonpolar surface area buried in the crystal structure of the E3-PSBD complex. In this instance, a large negative DeltaC(p) is not indicative of a classical hydrophobic interaction. In differential scanning calorimetry experiments, the midpoint melting temperature (T(m)) of E3 increased from 91 degrees C to 97.1 degrees C when it was bound to PSBD, and that of E1 increased from 65.2 degrees C to 70.0 degrees C. These high T(m) values eliminate unfolding as a major source of the anomalous DeltaC(p) effects at the temperatures (10-37 degrees C) used for the ITC experiments.     

Calcium transport in brain synaptosomes during depolarization. The role of potential-dependent channels and Na+/Ca2+ metabolism

The contribution of Ca2+ channels and Na+/Ca2+ exchange to Ca2+ uptake in rat brain synaptosomes upon long- (t greater than or equal to 30 s) and short-term (t less than 30 s) depolarization by high K+ was studied by measuring the 45Ca content and free Ca2+ concentration (from Quin-2 fluorescence). At 37 degrees C, the system responsible for the K+-stimulated uptake of 45Ca (t greater than or equal to 30 s) and the Na+/Ca+ exchanger are characterized by a similar concentration dependence of external Ca2+ (Ca0(2+] and K0+ as well as by an equal sensitivity to verapamil (Ki = approximately 20-40 microM) and La2+ (Ki = approximately 50 microM). These data and the results from predepolarization suggest that the 45Ca entry into synaptosomes at t greater than or equal to 30 s is due to the activation of Na+/Ca+ exchange caused by its electrogenic component, while the insignificant contribution of Ca2+ channels can be accounted for by their inactivation. At low temperatures (2-4 degrees C) which decelerate the inactivation, the initial phase of 45Ca uptake is fully provided for by Ca2+ channels, showing a lower (as compared to the exchanger) affinity for Ca0(2+) (K0.5 greater than 1 mM)m a greater sensitivity to La3+ (Ki = approximately 0.2-0.3 microM) and verapamil (Ki = approximately 2-3 microM); these channels are fully inactivated by predepolarization with K0+, ouabain and batrachotoxin. The Ca2+ channels can be related to T-type channels, since they are not blocked by nicardipine and niphedipine.     

Studies on (Na+ + K+)-activated ATPase. XXXVIII. A 100 000 molecular weight protein as the low-energy phosphorylated intermediate of the enzyme.

Phosphorylation of NaI-treated bovine brain cortex microsomes by inorganic phosphate in the presence of Mg2+ and ouabain has been studied at 0 degrees C (pH 7.4) and 20 degrees C (pH 7.0). Nearly maximal (90%) and half-maximal phosphorylation are achieved at 20 degrees C within 2 min with 50--155 and 5.6--17 muM 32Pi, respectively, and at 0 degrees C within 75 s with 300--600 and 33--66 muM 32Pi, respectively. Maximal phosphorylation yields 146 pmol 32P - mg-1 protein. Without ouabain (20 degrees C, pH 7.0) less than 25% of the incorporation observed in the presence of ouabain is reached. Preincubation of the native microsomes with Mg2+ and K+, in order to decompose possibly present high-energy phosphoryl-bonds prior to ouabain treatment, does not affect the maximal phosphate incorporation. This indicates that the inorganic phosphate incorporation is not due to an exchange with high-energy phosphoryl-bonds, which might have been preserved in the microsomal preparations. Phosphorylation of the native microsomes by ATP in the presence of Mg2+ and Na+ reaches 90 and 50% maximal levels within 15--30 s at 0 degrees C and pH 7.4 at concentrations of [gamma-32P]ATP of 5--32 and 0.5--3.5 muM, respectively. The maximal phosphorylation level is 149 pmol 32P-mg-1 protein, equal to that of ouabain-treated microsomes phosphorylated by inorganic phosphate. Both inorganic phosphate and ATP phosphorylate on site per active enzyme subunit of 135 000 molecular weight. From the equilibrium constants for the phosphorylation of ouabain-treated microsomes by inorganic phosphate at 0 degrees C and 20 degrees C standard free-energy changes of --5.4 and --6.8 kcal/mol, respectively, are calculated. These values yield a standard enthalpy change of 14 kcal/mol and an entropy change of 70 cal/mol - degree K. This characterizes the reaction as a process driven by an entropy change. The intermediate formed by phosphorylation with Pi has maximal stability at acidic pH, as is the case for the intermediate formed with ATP. Solubilization in sodium dodecyl sulfate stabilizes the phosphoryl-bond in the pH range of 4--7. The non-solubilized preparation has optimal stability at pH 2--4, the level of which is equal to that of detergent-solubilized intermediate. Sodium dodecyl sulfate gel electrophoresis of the microsomes at pH 3, following incorporation of 32Pi yields 11 protein bands, only one of which (mol. wt 100 000--106 000) carries the radioactive label. This protein has the same molecular weight as the protein, which is phosphorylated by ATP in the presence of Mg2+ and Na+.     

The electrophoretic properties and aggregation of mouse lymphoma cells, chinese-hamster fibroblasts and a somatic-cell hybrid.

1. The electrophoretic mobilities of a mouse lymphoma cell, a Chinese-hamster fibroblast and a somatic-cell hybrid (also fibroblastic), produced by fusion of the hamster cell and a mouse lymphoma cell, were measured at 25 degrees C over a range of pH, concentration of Ca2+ ions and concentration of La3+ ions. 2. All the cells have pI at pH3.5. 3. Ca2+ ions decrease the mobilities and zeta potentials of the cells to zero in the range 1-100mM. 4. La3+ ions lower the mobilities and zeta potentials in the range 10 muM-1 mM, and the cells become positively charged above 1 mM. 5. The data are consistent with specific adsorption of La3+ ions on approx. 2 X 10(14) sites/m2 of cell surface with a free energy of approx. -37kJ/mol. 6. The effects of Ca2+, La3+ and ionic strength on the extent of aggregation of the cells and of neuraminidase-treated cells were studied. 7. Ca2+ ions do not markedly increase aggregation, whereas La3+ ions gave rise to extensive aggregation in the range 10 muM-1 mM, corresponding to the region of La3+ adsorption. 8. Both fibroblastic cell lines are aggregated at high ionic strength. 9. The fibroblastic cells have larger amounts of trypsin-sensitive carbohydrate than does the lymphoma cell; the possible role of this material in cellular aggregation is discussed.     

Acid- and calcium-induced structural changes in phosphatidylethanolamine membranes stabilized by cholesteryl hemisuccinate

The membrane stabilization effect of cholesteryl hemisuccinate (CHEMS) and the sensitivity of the CHEMS-phosphatidylethanolamine membranes to protons and calcium ions were studied by differential scanning calorimetry, freeze-fracture electron microscopy, and 31P NMR. (1) At neutral pH, the addition of 8 mol % CHEMS to transesterified egg phosphatidylethanolamine (TPE) raised the lamellar-hexagonal transition temperature of TPE by 11 degrees C. Stable bilayer vesicles were formed when the incorporated CHEMS exceeded 20 mol %. (2) At a pH below 5.5, the protonation of CHEMS enhanced the formation of the hexagonal phase (HII) of TPE. At 25 mol % CHEMS the bilayer-hexagonal transition temperature was lowered by 30 degrees C at pH 4.5. (3) The endothermic acid-induced hexagonal hexagonal transition of TPE-CHEMS was suppressed at 35 mol % CHEMS. However, 31P NMR and electron microscopy indicated that a lamellar-hexagonal transition still occurred at this composition. (4) The main transition of TPE was not affected by the protonation of the incorporated CHEMS, indicating that no macroscopic phase separation occurred in TPE-CHEMS mixtures at low pH. (5) In contrast to the HII-promoting effect of H+, the neutralization of the negative charge on TPE-CHEMS by Ca2+ resulted in aggregates that remained in the lamellar structure even at the hexagonal transition temperature of TPE. It is suggested that calcium might form a complex between CHEMS in apposed bilayers. These results are related to the possible biological function of acidic cholesterol esters in biomembranes.     

X-ray diffraction and calorimetric study of N-lignoceryl sphingomyelin membranes.

Differential scanning calorimetry and x-ray diffraction have been used to investigate hydrated multibilayers of N-lignoceryl sphingomyelin (C24:0-SM) in the hydration range 0-75 wt % H2O. Anhydrous C24:0-SM exhibits a single endothermic transition at 81.3 degrees C (delta H = 3.6 kcal/mol). At low hydration (12.1 wt % H2O), three different endothermic transitions are observed: low-temperature transition (T1) at 39.4 degrees C (transition enthalpy (delta H1) = 2.8 kcal/mol), intermediate-temperature transition (T2) at 45.5 degrees C, and high-temperature transition (T3) at 51.3 degrees C (combined transition enthalpy (delta H2 + 3) = 5.03 kcal/mol). On increasing hydration, all three transition temperatures of C24:0-SM decrease slightly to reach limiting values of 36.7 degrees C (T1), 44.4 degrees C (T2), and 48.4 degrees C (T3) at approximately 20 wt % H2O. At 22 degrees C (below T1), x-ray diffraction of C24:0-SM at different hydration levels shows two wide-angle reflections, a sharp one at 1/4.2 A-1 and a more diffuse one at 1/4.0 A-1 together with lamellar reflections corresponding to bilayer periodicities increasing from d = 65.4 A to a limiting value of 71.1 A. Electron density profiles show a constant bilayer thickness dp-p approximately 50 A. In contrast, at 40 degrees C (between T1 and T2) a single sharp wide-angle reflection at approximately 1/4.2 A-1 is observed. The lamellar reflections correspond to a larger bilayer periodicity (increasing from d = 69.3-80.2 A) and there is some increase in dp-p (52-56 A) with hydration.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA base sequence homologies among strains of Streptococcus sanguis.

DNA was isolated from 19 strains and substrains of Streptococcus sanguis and analysed for guanine plus cytosine (GC) contents and base sequence homologies. Three groups could be discerned: group 1 strains had 40-8 to 42-8 mol % GC; group 2, 42-7 to 44-0 mol % GC; group 3, 43-8 to 46-4 mol % GC. DNA homologies between groups 1 and 3 were 40 to 60% at 67 degrees C and 40% at 72 degrees C. The homologies of group 2 towards groups 1 and 3 were much lower. Strains in groups 1 and 3 hydrolysed arginine and aesculin and fermented inulin; group 2 strains did not. Groups 1 and 3 could be considered subspecies of S. sanguis. Group 2 should not be considered S. sanguis.