Adsorption of cations to phosphatidylinositol 4,5-bisphosphate

We investigated the binding of physiologically and pharmacologically relevant ions to the phosphoinositides by making 31P NMR, electrophoretic mobility, surface potential, and calcium activity measurements. We studied the binding of protons to phosphatidylinositol 4,5-bisphosphate (PIP2) by measuring the effect of pH on the chemical shifts of the 31P NMR signals from the two monoester phosphate groups of PIP2. We studied the binding of potassium, calcium, magnesium, spermine, and gentamicin ions to the phosphoinositides by measuring the effect of these cations on the electrophoretic mobility of multilamellar vesicles formed from mixtures of phosphatidylcholine (PC) and either phosphatidylinositol, phosphatidylinositol 4-phosphate, or PIP2; the adsorption of these cations depends on the surface potential of the membrane and can be described qualitatively by combining the Gouy-Chapman theory with Langmuir adsorption isotherms. Monovalent anionic phospholipids, such as phosphatidylserine and phosphatidylinositol, produce a negative electrostatic potential at the cytoplasmic surface of plasma membranes of erythrocytes, platelets, and other cells. When the electrostatic potential at the surface of a PC/PIP2 bilayer membrane is -30 mV and the aqueous phase contains 0.1 M KCl at pH 7.0, PIP2 binds about one hydrogen and one potassium ion and has a net charge of about -3. Our mobility, surface potential, and electrode measurements suggest that a negligible fraction of the PIP2 molecules in a cell bind calcium ions, but a significant fraction may bind magnesium and spermine ions.     

Kinetic and Physical Studies of Cell Death Induced By Chemotherapeutic Agents Or Hyperthermia

The kinetics of three physical parameters: cell density, relative cytoplasmic viscosity and DNA stability to denaturation have been measured during the period preceding cell death induced by hyperthermia, methylprednisolone and a series of cancer chemotherapeutic agents. This series of measurements employed cultured human lymphoblastoid cells as an experimental system to establish the changes that can be observed in the early stages of cell death, prior to applying such measurements to tissue biopsies from solid human tumours. Cell death, induced by hyperthermia up to 43°C, methylprednisolone, vincristine, 5-fluorouracil, BCNU and melphalan, showed essentially identical and reproducible changes corresponding to those which characterize programmed cell death (apoptosis). Such changes could also be observed following hyperthermia above 43°C, but reproducibility was poor and increasing damage to the cell membranes was evident. In cells treated with adriamycin or methotrexate, cell sub-populations showing an increase in cell density were not detected. Measurements of DNA stability were readily performed by flow cytofluorometry thus allowing rapid quantitation of the fraction of cells in the early stages of cell death. Modified flow cytometric instrumentation would further allow measurement of cytoplastic viscosity as an additional parameter to indicate entry into programmed cell death. This suggests that these measurements could readily be applied to cell suspensions derived from tumour tissue biopsies for a more accurate assessment of tumour growth rate, and to allow monitoring of response to therapy in sequential tumour biopsies.     

Large divalent cations and electrostatic potentials adjacent to membranes. A theoretical calculation

We have extended the Gouy-Chapman theory of the electrostatic diffuse double layer by considering the finite size of divalent cations in the aqueous phase adjacent to a charged surface. The divalent cations are modeled as either two point charges connected by an infinitely thin, rigid "rod" or two noninteracting point charges connected by an infinitely thin, flexible "string." We use the extended theory to predict the effects of a cation of length 10 A (1 nm) on the zeta and surface potentials of phospholipid bilayer membranes. The predictions of the rod and string models are similar to one another but differ markedly from the predictions of the Gouy-Chapman theory. Specifically, the extended model predicts that a large divalent cation will have a smaller effect on the potential adjacent to a negatively charged bilayer membrane than a point divalent cation, that the magnitude of this discrepancy will decrease as the Debye length increases, and that a large divalent cation will produce a negative zeta potential on a membrane formed from zwitterionic lipids. These predictions agree qualitatively with the experimental results obtained with the large divalent cation hexamethonium. We discuss the biological relevance of our calculations in the context of the interaction of cationic drugs with receptor sites on cell membranes.     

13C NMR Analysis of some simple tetrahydroisoquinolines

¹³C NMR resonances of 15 simple tetrahydroisoquinolines have been assigned on the basis of chemical shift theory, ¹³C-¹H coupling constants     

Structural studies of an Inv (1, −2) kappa light chain

A Bence Jones protein with phenotype Inv (1, –2) was isolated from the urine of a patient with multiple myeloma. Inv typing of the patient's relatives established the presence of anInv ¹ allele in the kindred, and that the patient's genotype wasInv ¹/Inv ³. Hence, the absence of Inv (2) in the Bence Jones protein was shown to be genetic and not an artifact caused by the disease. The tryptic peptide-containing residues 191 through 194 were isolated and shown to be composed of Leu, Tyr, Ala, Cys, with Leu at the amino end. Hence, the residue at 191 is the same as that present in Inv (1, 2) Bence Jones proteins. More detailed study of the tryptic peptides established that residue 153 is Val rather than Ala as in all other K chains thus far studied. The primary sequence: Ala¹⁵³, Leu¹⁹¹ determines Inv (1, 2); Ala¹⁵³, Val¹⁹¹ determines Inv (3); and Val¹⁵³, Leu¹⁹¹ determines Inv (1). The Val¹⁵³, Val¹⁹¹ sequence has not been observed. It may correspond to Inv (–). These data are strikingly similar to the data for the Kern and Oz isotypes (changes at 154 and 191, respectively) in the chain. As in the case of theK chain, only three of the four possible combinations have been observed. The implications of this parallelism and of crystallographic findings on chains, reported by others, are discussed.     

Effects of a Single Histoplasmin Skin Test on the Serological Diagnosis of Histoplasmosis

Numerous reports have indicated that a single histoplasmin skin test may stimulate humoral antibodies to Histoplasma capsulatum antigens in histoplasmin-hypersensitive individuals. Although these investigations concur that antibody elevations are evoked, they vary in the reported degree of incidence and response induced, and they cast doubt on the interpretation of serological tests in the diagnosis of histoplasmosis. Histoplasmin-hypersensitive subjects (114) were bled prior to administration of the skin test, 2 days later, at the time this test was read, and 15 and 30 days after testing. No significant antibody titers were observed at 2 days. At 15- and 30-day intervals, only 17 (15%) of the subjects demonstrated circulating antibodies. All 17 showed agar gel bands; 5 demonstrated no complement-fixation (CF) titers, 10 produced CF antibodies ranging from 1:8 to 1:16, and 2 demonstrated titers of 1:32. The data suggest that skin testing does not interfere significantly with antibody levels in sera drawn approximately 2 days after administration of antigen. However, since titers as high as 1:32 were obtained at later intervals, such reactions should be evaluated cautiously and only after consideration of clinical findings.