Karl A. Koehler

Tissue and plasma carcinoembryonic antigen in early breast cancer. A prognostic factor.

A prospective study of plasma and tissue carcinoembryonic antigen (P‐CEA and T‐CEA) levels in 63 patients with early (Stage I and II) breast cancer was undertaken to determine if the presence of CEA in tissue and/or plasma at the time of primary surgery can be used as a prognostic factor. Thirty‐two Stage I and 31 Stage II patients were evaluable with a median follow‐up time of 26 months: 29/63 were T‐CEA and/or P‐CEA positive while 34/63 were T‐CEA and P‐CEA negative; 9/63 were both T‐CEA and P‐CEA positive; 13/63 were P‐CEA positive alone, while 25/63 were T‐CEA positive alone; 5/29 T‐CEA and/or P‐CEA positive showed disease progression with a mean DFI of 11.8 months, compared with 0/34 T‐CEA and P‐CEA negative patients (P < 0.02); 2/9 T‐CEA and P‐CEA positive compared with 0/34 negative patients progressed (P < 0.01). There was a significant difference (P < 0.05) between P‐CEA positive (3/13) patients with recurrence and P‐CEA negative (2/50). When T‐CEA positive patients (4/25) were compared with T‐CEA negative (1/38), the difference approaches significance. When the recurrences were analyzed with respect to CEA, estrogen receptor (ER) and nodal status, only in the CEA+ versus CEA– group was there a significant difference. The early data show that patients with positive T‐CEA and/or P‐CEA have a higher recurrence rate with probable poor prognosis. Prognosis correlates better with CEA status than with ER or nodal status.

A lithium-7 NMR investigation of the lithium ion interaction with phosphatidylcholine-phosphatidylglycerol membranes. Observation of calcium and magnesium ion competition

The observation of Hauser and Shipley (I) that lithium ion-dimyristoylphosphatidyl serine interactions lead to separation of a Li:DMPS phase consisting of approximately 1: 1 PS:Li mole ratio possessing an elevated transition temperature, suggests that membrane interactions may significantly immobilize the lithium ion. Calcium and magnesium ions also cause phase separations in PS-containing membranes. Competition between the divalent cations and lithium ions for interaction sites on the membrane surface occur and are an important feature of the mechanism of lithium action. Phase separations induced by metal ions may have consequences for the interaction of extrinsic membrane proteins with the membrane surface as well as for the modulation of the activities of intrinsic membrane proteins. In the case of lithium action, alterations in membrane-associated processes may have direct consequences for cellular intracellular interactions, and therefore may effect neural function. We have employed lithium-7 NMR spectroscopy to examine the Li-membrane interactions and competition of calcium and magnesium ions with lithium in this interaction. Multilayer membranes of defined composition were employed. 7Li is a quadrupolar ion with spin 3. While the resonance of lithium ion in solution is a sharp singlet with linewidth at half height of 9 Hz (50 mm LiCl at 8.45 T), significant immobilization of this ion at the membrane surface may lead to observation of the quadrupolar triplet. Such an observation might lead to details of ion binding. In such a case, the immobilized lithium triplet central line is coincident with the position of the singlet resonance of uncomplexed lithium. Such quadrupolar splittings have not previously been reported for lithium ions in systems of biological importance. Dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine and Trizma were obtained from Sigma Chemical Company. The buffer was 20 mA4 Trizma, pH 7.5, and lithium chloride was Baker analytical grade. 7Li NMR spectra were obtained

Interleukin-2 self-association

The self-association of human recombinant interleukin-2 (IL-2) from E. coli was explored. Self-association, with an apparent Kd of 0.6 micromolar, has pronounced effects on (1) the surface exposure of Trp-121, deduced from quenching studies employing potassium iodide and acrylamide, (2) the apparent quantum yield of Trp-121, the fluorescence of Trp-121 in IL-2 aggregates is 4-fold lower than in IL-2 monomers, and (3) IL-2-mediated phospholipid vesicle fusion/aggregation.

Relative affinity of Ca(II) and Mg(II) ions for human and bovine prothrombin and fragment 1

Equilibrium dialysis results are presented for Ca(II) and Mg(II) ion binding to human and bovine prothrombin and fragment 1. Ca(II) ions bind cooperatively, Mg(II) does not.

Comparison of the membrane-related effects of cytarabine and other agents on model membranes

UNLABELLEDnThe membrane-associated effects of a series of chemotherapeutic and other drugs were examined via differential scanning calorimetry and by their modulation of the action of porcine phospholipase A2 (PLA2) on bilayer substrates. The drugs examined included: cytarabine, amino-glycoside antibiotics, adriamycin, dibucaine, butacaine, and VP-16. The bilayers employed were phase-separated ternary lipid mixtures containing dimyristoylphosphatidylcholine: palmitoyllysolecithin: and either hexadecanoic acid (fatty acid ternary mixture) or hexadecanol (alcohol ternary mixture). Effects of the more hydrophilic drugs (cytarabine and aminoglycoside antibiotics) on the calorimetric profiles of the negatively charged (fatty acid-containing) and the neutral (hexadecanol-containing) ternary lipid mixtures indicate that the interaction of these drugs with biomembranes is likely to be dominated by electrostatic interactions. All of the drugs investigated, including the more hydrophobic adriamycin, dibucaine, butacaine, and VP-16, affected the phase equilibrium in the membrane and exhibited apparent noncompetitive inhibition of the action of PLA2 on bilayers composed of ternary lipid substrates. In addition, cytarabine inhibited fusion of fatty acid-containing ternary mixtures.nnnCONCLUSIONSnThese drug:membrane interactions leading to a shift in the phase equilibria were apparently regiospecific. Hydrophilic drug:membrane interactions included an important electrostatic component. The effects of all of the drugs employed in this study on the action of PLA2 on a bilayer substrate (fatty acid-containing ternary lipid mixture) are hypothesized to be a result of the drug-mediated shift in phase equilibria away from the optimally active phase distribution. As a result, PLA2 binds with normal affinity to the membrane, but its membrane substrate is not catalytically turned over. It is evident that these drugs can directly affect cellular homeostasis in a manner that can show a dependence on the nature of the membrane surface.

An ab initio mo study of calcium and magnesium complexes with malonate and formate

Abstract Quantum mechanical SCF calculations were performed on magnesium and calcium complexes of malonate and malonate/formate coordination. The calcium malonate structure is in substantial agreement with recent X-ray results on α-ethylmalonate. The magnesium complexes are generally more tightly bound than calcium, although qualitatively, the calcium and magnesium structures are similar. Experimental binding results for divalent ions with malonates and EDTA are discussed in view of the current calculations.

DIHEMATOPORPHYRIN ETHER‐PHOSPHOLIPID INTERACTIONS. THE ROLES OF SURFACE CHARGE and LATERAL PHASE SEPARATIONS

Abstract Fluorescence spectroscopy was utilized to investigate the equilibrium interaction of dihem‐atoporphyrin ether(s) (DHE) with binary and ternary phospholipid mixtures of defined composition in order to define the roles of net negative surface charge and lateral phase separations in DHE‐membrane partitioning. Binary phospholipid mixtures employed were composed of dimyristoyl‐phosphatidylcholine (DMPC) mixed with increasing weight percentages of dimyristoylphosphatidylgly‐cerol (DMPG) providing controlled variation of net membrane surface charge. Two types of ternary phospholipid mixtures were utilized. Ternary acid mixtures contained various percentages of palmitoyl‐lysophosphatidyl choline (LPC) + palmitic acid (PA) dispersed in DMPC. Ternary alcohol mixtures contained various percentages of LPC + hexadecanol (OL) dispersed in DMPC. The ternary phospholipid mixtures are known to be phase separated. At total DHE concentrations of 0.33 μA/ and using 100% DMPC, the DHE partition coefficient (P) is 250 000. This partition coefficient is to some extent dependent on the DHE concentration. The observed partition coefficients show little dependence on surface charge in DMPC‐DMPG mixtures. However, P decreases markedly with increasing phase separation in the ternary lipid mixtures. The fluorescence of membrane‐bound DHE is dependent on the composition of the ternary mixtures in a manner suggesting micropartitioning of DHE into the phospholipid bulk phase as well as into the disordered regions between laterally phase separated phospholipid domains.

Dye binding probes of lipid-binding structures: An investigation of 2-p-Toluidinvinaphthylene-6-sulfonate binding to human and bovine prothrombin and fragment 1 in the presence and ansence of calcium and magnesium ions

TNS (2-p-toluidinylnaphthylene-6-sulfonate) binds to human and bovine prothrombin and Fragment 1 in the absence and presence of added Ca2+. The stoichiometry of TNS binding is 1:1 for human and bovine prothrombin and Fragment 1. The Ca2+-dependence of the fluorescence of TNS bound to bovine prothrombin Fragment 1 yields a modified Hill plot slope of 2.7, which is consistent with the slope obtained by monitoring the Ca2+ dependence of protein fluorescence quenching, CD changes and phospholipid binding. Mg2+ has have no effect on the fluorescence of TNS-prothrombin fluorescence. TNS binding to the amino-terminal region of prothrombin is the first relatively simple probe of the subtle and complex relationship which exists between protein structure and phospholipid binding.

Estimation of apparent quadrupolar coupling constants for complexes of magnesium ions with mono- and dicarboxylic acid ligands. applications to magnesium ion: protein interactions

25Mg+2 ion NMR studies of complexes of magnesium ions with acetate and malonate ligands have yielded apparent quadrupolar coupling constants, chi, of approximately 1.5 MHz. The aquo magnesium ion yields a smaller chi value of 0.12 MHz, consistent with its expected higher symmetry. chi values for magnesium ion: acetate and magnesium ion: malonate complexes are utilized to calculate observed linewidths for magnesium ion: bovine prothrombin fragment 1 and magnesium ion: human Factor XII interactions. These calculated values are compared with observed values and implications of the agreement are discussed.

UTILIZATION OF HEAVY‐ATOM EFFECT QUENCHING OF PYRENE FLUORESCENCE TO DETERMINE THE INTRAMEMBRANE DISTRIBUTION OF HALOTHANE

The interaction of halothane (CF3CHBrCl) with dipalmitoylphosphatidylcholine (DPPC) membranes containing varying amounts of dipalmitoylphosphatidylglycerol (DPPG) was examined via heavy atom effect quenching of pyrene fluorescence by halothane. The effect of halothane on pyrene fluorescence is consistent with a kinetic model based upon the assumption of the existence of two populations of pyrene in the membrane: one accessible to interactions with halothane; the second inaccessible to halothane on the time scale of the pyrene fluorescence excited state. Both populations of pyrene are affected by the presence of halothane in the membrane. The rate of halothane quenching of pyrene fluorescence is increased significantly for all DPPG/DPPC membranes compared to pure DPPC membranes indicating that any DPPG in the membrane facilitates interaction between halothane and pyrene even though the measured partition coefficients indicate that little change in total halothane concentration in the membrane as a whole occurs as a function of percent DPPG in DPPG/DPPC mixtures.