Composition and structure of the pericellular environment. Physiological function and chemical composition of pericellular proteoglycan (an evolutionary view).

Connective tissue cells exist in a meshwork of insoluble fibres, the interstices of which are filled with soluble, high molecular mass, anionic material of a predominantly carbohydrate nature. The interactions of fibres with the interfibrillar material are central to the discussion of connective tissue physiology. As with all soluble polymers, the interfibrillar polyanion tends to "swell' and the tangled mass of chains offers considerable resistance to penetration by the large insoluble fibres. The consequent pressure to "inflate' the fibrous network is important in giving elasticity to cartilage, transparency to cornea, etc. Branched structures (of proteoglycans) and straight-chain forms (of hyaluronate) are compared for their ability to fulfil these functions. Apart from their physical ("non-specific') roles proteoglycans and glycosaminoglycans are able to interact physicochemically with, for example, collagen in ways which show considerable specificity, and which presumably are important in the laying down of the fibrous network as well as in maintaining its mechanical integrity. It is proposed that the role played by radiation, particularly as mediated via the hydrated electron (eaq) was dominant in the pre- and post-biotic evolution of pericellular environments.     

Bovine brain phosphatidylinositol transfer protein. Effects of pH, ionic strength and lipid composition on transfer activity

Phosphatidylinositol and phosphatidylcholine are transferred between bilayer membranes in the presence of a specific phosphatidylinositol transfer protein isolated from bovine brain. The effects of pH, ionic strength and lipid composition on the rate of transfer of these phospholipids between small unilamellar vesicles have been investigated. At low ionic strength, phosphatidylinositol transfer between vesicles prepared from phosphatidylcholine and 5 mol% phosphatidylinositol was maximal at about pH 5 and moderately dependent on hydrogen ion concentration in more alkaline regions. A similar dependence on pH was noted for phosphatidylcholine transfer between membranes containing phosphatidylcholine or mixtures of phosphatidylcholine and 5 mol% phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine or stearylamine. The rate of transfer between anionic vesicles was somewhat higher than that between neutral or cationic vesicles. At higher ionic strength the transfer reactions in neutral and alkaline regions were less sensitive to pH. Phospholipid transfers between vesicles containing 5 mol% of anionic lipid increased sharply as ionic strength decreased below 0.1. In contrast, phosphatidylcholine transfer between membranes which contained only zwitterionic phospholipids or 5 mol% stearylamine was unaffected by variations of ionic strength. Irrespective of the lipid composition of membranes, pH affected both the apparent Km and Vmax, while ionic strength generally affected the apparent Vmax. These results indicate a significant role of electrostatic interactions in the phospholipid transfer catalyzed by phosphatidylinositol transfer protein.     

Collagen V localizes to pericellular sites during tendon collagen fibrillogenesis

During tendon development collagen fibrillogenesis occurs in extracellular micro-domains defined by the tenocytes. This permits cellular regulation of the extracellular steps involved in the tissue-specific matrix assembly required for function. The hypothesis tested here is that collagen V associates with the tenocyte surface where it functions in regulation of collagen assembly and cell-directed fibril deposition. The in vitro and in vivo data demonstrate that collagen V is a quantitatively minor component of the tendon. It is preferentially localized on the tenocyte surface as distinct foci in tendons and in cell culture. In vitro data indicate that this interaction with the tenocyte is not HSPG GAG-dependent. Collagen V is present as the mature, processed form, is absent from the media, and is a significant part of the detergent-insoluble cell layer, presumably as part of a membrane-associated complex. In contrast, procollagen I is not efficiently processed and is found predominantly in the culture media. Our data suggest that the regulatory role of collagen V requires collagen V to occupy a different cellular niche from the structural collagen I. In monolayer cultures, the conversion to the tissue form of collagen V and its deposition with the cell layer suggest efficient engagement of procollagen V with pericellular receptors and processing enzymes. The secretion of collagen I into the media and inefficient processing of procollagen I suggest reduced accessibility to these pericellular molecules due to disengagement from the cell surface. This all points to differential spatial localization of collagen V as a mechanism to optimize its regulatory roles during the cell-surface directed steps in tendon collagen fibril assembly.     

Charged hybrid block copolymer-lipid-cholesterol vesicles: pH,ionic environment,and composition dependence of phase transitions

The impacts of pH, salt concentration (expressed as Debye length), and composition on the phase behavior of hybrid block copolymer-lipid-cholesterol bilayers incorporating carboxyl-terminated poly(butadiene)-block-poly(ethylene oxide) copolymer (PBdPEO1800^(?)) or/and non-carboxyl-terminated PBdPEO (PBdPEO1800 or/and PBdPEO950), egg sphingomyelin (egg SM), and cholesterol were examined using fluorescence spectroscopy of laurdan. Laurdan emission spectra were decomposed into three lognormal curves as functions of energy. The ratio of the area of the mid-energy peak to the sum of the areas of all three peaks was evaluated as vesicles were cooled, yielding temperature breakpoint values (T_break) expected to be within the range of the phase transition temperature. T_break values displayed dependence on pH, Debye length, and vesicle composition consistent with an electrostatic repulsion contribution to vesicle phase behavior. Increased pH and Debye length, for which a greater dissociated fraction of PBdPEO1800^(?) and a greater energy of electrostatic repulsion would be expected, resulted in T_break values as much as 10 °C less than at low pH or short Debye lengths. Additionally, at Debye lengths comparable to those at physiologically relevant ionic strength, T_break at pH 5.9 was observed to be slightly higher than at pH 7.0 for vesicles containing 50 mol% PBdPEO1800^(?). Electrostatic effects observed for hybrid vesicles incorporating significant amounts of carboxyl-terminated polymer may have the ability to drive phase separation in response to pH drops—such as those observed after endocytosis—in physiologically relevant conditions, suggesting the utility of such materials for drug delivery.     

Influence of ionic composition,buffering agent,and pH on the histochemical demonstration of myofibrillar actomyosin ATPase

Summary The influence of the composition of the preincubation medium on the histochemical demonstration of myofibrillar actomyosin ATPase, including a variety of carboxylic acid and non-carboxylic acid buffering compounds and neutral salts, was studied. In inorganic salt-free systems the rate of the activation of type I fibers and inactivation of type II fibers was accelerated when the carboxylic acids had longer chain length or multiple carobxyl groups. Of these factors, the number of carboxyl groups was dominant with a 100 mM citrate buffer producing a sharp differentiation between fiber types. In contrast, the time course of the response was exceptionally long in an acetate buffer. The time course of the ATPase reaction was also modified by other buffers at pH 4.60. The most notable were an ascorbate — glycine buffer system which produced little or no deviation from the alkaline preincubation staining pattern after prolonged preincubation and a pyrophosphate system which produced a rapid change. Neutral salts in the preincubation medium accelerated the time course of the inactivation — activation process with the order for the halogen salts of K⁺ being F⁻<Cl⁻<Br⁻<I⁻, which is a progression by molecular weight. The only sequence for cations on the myofibrillar actomyosin ATPase was Li⁺< Na⁺<K⁺. The response to salts was concentration dependent. An interaction existed between buffering compound, type of salt, and pH. These experiments demonstrate that the histochemical differentiation of fiber types by the myofibrillar actomyosin ATPase reaction depends upon a modification of some component(s) of the myofibrillar complex that can be influenced by a number of factors.     

pH titrations of molluscan paramyosin at two different ionic strengths.

Paramyosin extracted from the adductor muscle of Mercenaria mercenaria, the chowder clam, was titrated both in 0.3 M KCl and in 1 mM KCl. Both the presumed native form of the molecule, acid-R-paramyosin, and a slightly degraded form, beta-paramyosin, were studied. Titrations of both types of paramyosin were similar in 1 mM k+, except that the native paramyosin is more highly charged at pH 3.2 than beta-paramyosin, as postulated previously (DeLaney and Krause, 1976, Macromolecules, 9:455), and that more groups titrate on the native molecule than on beta-paramyosin, both between pH 3.2 and 3.3 and between pH 3.2 and 10. Titrations in 0.30 M KCl, unlike those in 1 mM K, depended on starting pH; long term exposure to alkali solutions during dialysis, previously shown to cause partial dephosphorylation of paramyosin (Cooley et al., 1979, J. Biol. Chem., 254:2195), apparently also leads to a change in intermolecular interactions sufficient to cause changes in the titration curves in 0.30 M KCl but not in 1 mM K+.     

Measurement of pH to quantify urease activity.

The rate of change of pH caused by the hydrolysis of urea was measured for urease solutions of 18 different concentrations. Concentrations were converted into an activity, A (measured in IU/cm3), by using a titrimetric method to assay the urease sample. For activities in the range 0.6-38 IU/cm3, A was related to the initial rate of change of pH, (dpH/dt)0, (measured in s-1) by the empirical relationship: A = 549(dpH/dt)0-1423(dpH/dt)2(0). Values of (dpH/dt)0 were sensitive to changes in the urease activity of about 0.6 IU/cm3.     

Intracellular pH and ionic channels in the Loligo vulgaris giant axon.

Squid giant axons were used to investigate the reversible effects of intracellular pH(pHi) on the kinetic properties of ionic channels. The pharmacologically separated K+ and Na+ currents were measured under: (a) internal perfusion, (b) enzymatic Pronase treatment, and (c) continuous estimate of periaxonal ion accumulation. Variation of internal pH from 4.8 to 11 resulted in: (a) a decrease of steady-state sodium inactivation at positive potentials similar to the effect of the proteolytic enzyme Pronase, (b) a shift of the h infinity (E) curve toward depolarizing voltages, and (c) a decrease of the time constant of inactivation for potentials below -20 mV (an increase above). A plot of the steady-state sodium conductance at E = +40 mV as a function of pHi suggests that two groups with pKa 10.4 and 5.6 affect respectively the inactivation gate and the rate constants for the transition from the inactivated to the second open state (h2) (Chandler and Meves, 1970b). The voltage shifts of the kinetic parameters predicted by the Gouy-Chapman-Stern theory are well satisfied at high pHi and less at low. Once corrected for voltage shifts, the forward rate constants for channel opening were found to be slowed with the acidity of the internal or external solution.     

Measurement of the pH of frozen buffer solutions by using pH indicators.

A method was established to estimate the pH change of several buffers solutions on freezing by using a combination of pH indicators. Among more than 30 buffers solutions examined, almost half exhibited a pH change in the temperature range between freezing point and 220 degrees K; the results were tabulated. Glycerol was found to suppress the pH changes because of its "salt buffer" effect.     

Base composition analysis of oligonucleotides containing apurinic sites.

Upon base composition analysis, oligonucleotides which are labeled at the 3'-terminus with fluorescein or biotin generate an additional, late eluting peak in the HPLC chromatogram. Investigation of this effect revealed the haptens acted as apurinic sites, and phosphodiesterase cleavage of the phosphate bond between the upstream nucleotide and apurinic site is inhibited. Extension of this work with a base-stable apurinic site inserted into all possible junctures of 5'-TGAC-3' tetramers showed this to be a general effect. As a consequence of this work, acid-catalyzed depurination resulting in apurinic sites can be monitored in oligonucleotide synthesis.     

Cartilage electromechanics--I. Electrokinetic transduction and the effects of electrolyte pH and ionic strength

Articular cartilage contains a high fixed charge density under physiological conditions associated primarily with the ionized proteoglycan molecules of the extracellular matrix. Oscillatory compression of cartilage using physiological loads produces electrical potentials that have been shown previously to be the result of an electrokinetic (streaming) transduction mechanism. We have now observed two additional electromechanical phenomena not previously seen in cartilage or other soft tissues: 'streaming current' and 'current-generated stress'. Sinusoidal mechanical compression induced a sinusoidal streaming current density through cartilage disks when the Ag/AgCl electrodes that were used to compress the cartilage were shorted together externally. Conversely, a sinusoidal current density applied to the tissue generated a sinusoidal mechanical stress within the tissue. Both these phenomena were found to be consistent with the same electrokinetic transduction mechanism responsible for the streaming potential. Changes in the measured streaming potential response that resulted from modification of bath ionic strength and pH have provided additional insights into the molecular origins of these transduction processes. Finally, we have now observed streaming potentials in living cartilage maintained in organ culture, as well as in previously frozen tissue.     

Low pH value of pericellular medium as a factor limiting cell proliferation in dense cultures

An attempt was made to determine the role of metabolic acidification of pericellular medium in regulating cell proliferation. A method of measuring the pH of pericellular medium at a distance exceeding a Debye radius of 5-10 A from the cell surface (pHp) was developed. The values of pHp and pH measured in the medium at a distance of greater than 1 cm from cells (pHm) were found to differ, depending on the cell population density. At a density of at least 7 X 10(5) cells/cm2 (maximum saturation density) and at pHm 7.4-7.6, pHp reached a value of approximately 6.5. It was found that pHm 6.5 was unfavorable for cell proliferation in sparse cultures, where pHm and pHp were equal. Based on these findings, low pHp as revealed in the present work using dense cultures at optimal pHm can be considered to be a limiting factor for cell multiplication.     

The protein-mediated transfer of phosphatidylcholine between membranes. The effect of membrane lipid composition and ionic composition of the medium.

The phosphatidylcholine exchange protein from bovine liver catalyzes the transfer of phosphatidylcholine between rat liver mitochondria and sonicated liposomes. The effect of changes in the liposomal lipid composition and ionic composition of the medium on the transfer have been determined. In addition, it has been determined how these changes affected the electrophoretic mobility i.e. the surface charge of the membrane particles involved. Transfer was inhibited by the incorporation of negatively charged phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol into the phosphatidylcholine-containing vesicles; zwitterionic phosphatidyl-ethanolamine had much less of an inhibitory effect while positively charged stearylamine stimulated. The cation Mg2+ and, to a lesser extent, K+ overcame the inhibitory effect exerted by phosphatidic acid, in that concentration range where these ions neutralized the negative surface charge most effectively. Under conditions where Mg2+ and K+ affected the membrane surface charge relatively little inhibition was observed. In measuring the protein-mediated transfer between a monolayer and vesicles consisting of only phosphatidylcholine, cations inhibited the transfer in the order La3+ greater than Mg2+ larger than or equal to Ca2+ greater than K+ = Na+. Inhibition was not related to the ionic strength, and very likely reflects an interference of these cations with an electrostatic interaction between the exchange protein and the polar head group of phosphatidylcholine.     

Electrostatic interactions at charged lipid membranes. Measurement of surface pH with fluorescent lipoid pH indicators.

The 5-dimethylaminonapthalene-1-sulfonyl (dansyl) chromophore attached to the polar head groups of lipids has been used as a fluorescent lipoid pH indicator to evaluate the interfacial pH in lipid-water lamellar systems prepared from negatively charged lipids. The pH in the vicinity of the charged lipid bilayers is different from the pH of the bulk aqueous phase and the difference is a function of the electrolyte concentration in the aqueous phase and of the lipid packing in the bilayer. At a fixed electrolyte concentration in the aqueous phase, the observed interfacial pH is 0.6 to 0.7 pH units lower above the thermal phase transition of the lipid than it is below this temperature. A quantitative interpretation of the results is given on the basis of the Gouy-Chapman theory. The results indicate that the dansyl chromophore is located in front of the charged surface and its distance from this surface increases with a decrease in lipid packing.