Charles Y. C. Pak

Ionic interaction with bone mineral I. Evidence for an isoionic calcium exchange with hydroxyapatite

Abstract Exchange of calcium with hydroxyapatite crystals in aqueous suspension was measured with 45Ca. The results were analyzed by fitting to a curve characterized by four exponential terms, of the form: Isotope uptake (or release) = σ i=I i=4A i e −α i t Total exchange was also calculated from measurements of non-isotopic calcium. Isotopic steady state was reached by 65 h. Uptake and release were characterized by similar A and α values. Release in 1 h was the same whether uptake had been carried out for 1 or 65 h. This, with the high values of α1 and α2, indicates that early exchange is quantitatively accounted for largely by the contribution of two ‘fast’ compartments. As further evidence for exchange, the uptake of isotope by hydroxyapatite was completely reversible by a wash-out procedure. Release was largely prevented by omission of Ca2+ from solution, whether or not they were replaced with Na+; this indicates that calcium uptake is an isoionic exchange process.

Ionic interation with bone mineral: II. The control of Ca2+ and PO43−exchange by univalent cation-Ca2+ substitution at the hydroxyapatite crystal surface

The effect of Mg2+, citrate3−, F− and SO42− on the exchange of45Ca2+ and32PO43− with the mineral phase of bone (ethylenediamine-extracted) was determined under physiological conditions. Mg2+ substituted for Ca2+ at the adherent liquid film (hydration shell), while citrate3− displaced PO43− from both the adherent liquid film and the crystal surface. These bone-seeking elements thus increased the apparent solubility of bone mineral. In contrast, F− markedly reduced the concentration of Ca2+ and PO43− in both the ambient solution and the adherent liquid film, probably by inducing the formation of a CaF2 boundary layer at the crystal surface. There was a net uptake of Ca2+ and PO43− by the solids phase in the presence of Mg2+ and F−, but not in citrate3− solutions. SO42− did not significantly affect the solubility, dissolution, or growth of bone mineral.

Intermediates in influenza virus PR/8 haemagglutinin-induced membrane fusion.

The fusion kinetics with erythrocyte ghosts of two influenza A virus strains, A/Aichi/2/68 (X:31) and A/PR/8/34 (PR/8), were compared and correlated with the kinetics of haemagglutinin (HA) conformational change. Previously it had been shown that X:31 fuses with liposomes or erythrocytes at 4 degrees C, pH 5 after a lag time of 5 to 10 min whereas PR/8 displayed no fusion with liposomes at that temperature. We have confirmed the absence of cold fusion by PR/8 with erythrocyte ghosts. In contrast to X:31, PR/8 could not be committed to fuse at neutral pH and 37 degrees C by a preincubation at low pH and 4 degrees C. To examine whether the lack of commitment and cold fusion were due to a failure of PR/8 HA to undergo conformational changes at low temperature and pH, we analysed susceptibility of HA to proteinase K digestion, liposome binding to the virus, and immunoprecipitations of HA with conformation-specific antibodies. Although there was little binding of PR/8 to liposomes at 4 degrees C and pH 5, we did observe exposure of the fusion peptide. This study reveals a low temperature intermediate in membrane fusion exhibited by the HA of influenza virus strain PR/8, which involves low pH-induced conformational changes including exposure of the fusion peptide with little interaction of HA with the target membrane.

Enhancement of renal excretion of zinc by hydrochlorothiazide

Abstract The effect of hydrochlorothiazide (50 mg twice a day orally for 5 days) on the metabolism of zinc was examined in four patients with idiopathic hypercalciuria, one with idiopathic osteoporosis, one with normocalciuric nephrolithiasis, one normal volunteer, and one with normocalcemic primary hyperparathyroidism. Urinary zinc progressively increased until the peak value, at approximately twice the mean of control values, was reached on the fifth day of treatment with hydrochlorothiazide. After treatment was stopped, it gradually decreased towards control values. Serum concentration of zinc increased slightly or did not change during treatment. In contrast, furosemide, triamterene, mercaptomerin, or oral “sodium loading” did not affect renal excretion of zinc.

The effect of polypeptide hormones on lipid monolayers. II. The effect of insulin analogues, vasopressin, oxytocin, thyrocalcitonin, adrenocorticotropin, and 3′,5′-cyclic AMP on the uptake of Ca2+ by monomolecular films of monooctadecyl phosphate

Abstract 1. 1. Like insulin, some analogues of insulin inhibited the adsorption of Ca2+ at a monooctadecyl phosphate monolayer; the inhibition was, in all cases, less than the inhibition by intact insulin. 2. 2. Facilitation of release of Ca2+ from the monolayer by insulin required that the insulin molecule be intact, as release was facilitated only very slightly when des-A1-glycine-des-B1-phenylalanine1-insulin or insulin B-chain-S-sulfonate replaced insulin, and did not occur at all in the absence of alanine in position B30. 3. 3. Vasopressin and oxytocin inhibited Ca2+ adsorption. In addition, vasopressin facilitated Ca2+ release. 4. 4. Thyrocalcitonin profoundly inhibited Ca2+ adsorption but facilitated only slightly its release. 5. 5. 3′,5′-cyclic AMP facilitated slightly the adsorption of Ca2+. 6. 6. It is suggested that the biphasic action of insulin and vasopressin on Ca2+ uptake depends upon the concurrence of a 6-membered disulfide ring at or near the N-terminus of the molecule, and a proline followed by a positively charged amino acid (arginine or lysine) in the two positions preceding the C-terminal amino acid.

Effects of cellulose phosphate and sodium phosphate on formation product and activity product of brushite in urine

Abstract The crystal nidus of brushite (nidus of renal stones containing calcium) cannot form when the activity product (state of saturation) of brushite in urine is less than the formation product (the lowest state of supersaturation supporting nucleation). Treatment measures should therefore be directed at reducing the activity product or increasing the formation product. The response to treatment with cellulose phosphate and with sodium phosphate was evaluated according to this criterion. Cellulose phosphate decreased the activity product while slightly increasing or not significantly affecting the formation product. Sodium phosphate increased both the activity product and the formation product of brushite. The formation product increased largely as the result of the increase in urinary pyrophosphate. In most patients, the increase in the activity product during treatment with sodium phosphate was fully compensated by the increase in the formation product.

Transient domains induced by influenza haemagglutinin during membrane fusion

During low pH-induced fusion of influenza virus with erythrocytes we have observed differential dispersion of viral lipid and haemagglutinin (HA) into the erythrocyte membrane, and viral RNA into the erythrocyte using fluorescence video microscopy. The movement of both viral lipid and HA from virus to cell was restricted during the initial stages of fusion relative to free diffusion. This indicates the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation. Fluorescence anisotropy of phospholipid analogues incorporated into the viral membrane decreased when the pH was lowered to levels required for optimum fusion. This indicates that the restricted motion of viral membrane components was not due to rigidification of membrane lipids. The movement of HA from the fusion site was also assessed by photosensitized labelling by means of a fluorescent substrate (NBD-taurine) passing through the band 3 sialoglycoprotein (the erythrocyte anion transporter). We also examined the flow of lipid and aqueous markers during fusion of HA-expressing cells with labelled erythrocytes. During this cell-cell fusion, movement of lipid between fusing membranes begins before the fusion pore is wide enough to allow diffusion of aqueous molecules (M(r) > 500). The data indicate that HA is capable of creating domains in the membrane and controlling continuity of aqueous compartments which are bounded by such domains.

Renal Stones of Calcium Phosphate Physicochemical Basis for Their Formation

Summary The activity product of Ca2+ and HPO4 2− was calculated for the urine before and after incubation with brushite (CaHPO4 · 2H2O). This permitted an estimation of the degree of saturation of urine in terms of Ca2+ and HPO4 2−. The urine of patients with idiopathic hypercalciuria was supersaturated with respect to brushite at all pH. However, the urine of normocalciuric subjects with stone was supersaturated only at high urinary pH.

The effect of polypeptide hormones on lipid monolayers. III. The effect of insulin, vasopressin, oxytocin, albumin, and prostaglandin E1 on the specific resistance to the evaporation of water through monomolecular films of monoöctadecyl phosphate, stearic acid, and stearyl alcohol

Abstract The hypothesis that insulin and vasopressin increase the permeability of cells to water and water-soluble substances by decreasing the adsorption of Ca 2+ at the outer surface of the plasma membrane was tested in a model system. Changes in the specific resistance to the evaporation of water through monooctadecyl phosphate (mOP) monolayers were compared with simultaneous changes in Ca 2+ adsorption. Insulin and vasopressin cause a decrease in the resistance (or, alternatively, increase the transport of water) which can be accounted for only in part by the inhibition of Ca 2+ adsorption. The action is specific for these hormones and dependent on interaction between the hormone and monolayer molecules.

Effect of X31 influenza virus fusion on phosphatidylserine asymmetry in erythrocytes

Influenza virus fusion is mediated by its fusion protein, hemagglutinin (HA). HA undergoes a low pH dependent conformational change that results in insertion into the cell membrane bilayer, formation of a fusion pore, and merging of membrane lipids and establishment of cytoplasmic continuity. Erythrocytes, which can serve as targets of influenza virus fusion, display an asymmetric transbilayer arrangement of their phospholipids. The effect of influenza virus fusion on erythrocyte phosphatidylserine asymmetry was determined. Influenza virus were bound to erythrocytes containing the fluorescent membrane probe NBD-PS in the inner leaflet. Induction of fusion by exposure to a low pH environment resulted in movement of PS to the outer leaflet of the cell as well as hemolysis. Insertion of the fusion protein into erythrocytes and subsequent fusion can be distinguished from hemolysis by examining the interaction of a soluble form of HA (BHA) with cells and by monitoring viral fusion at low temperatures. No hemolysis was observed under either condition. BHA binding and insertion into cells did not affect the asymmetry of PS. Incubation of influenza virus fusion at pH 5, 0 degrees C resulted in complete fusion but no outward movement of PS was observed. These findings suggest the viral fusion pore does not involve a rearrangement of the transbilayer phospholipid organization of the target membrane.