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Biomedical subjects

K A Koehler

Publications and source records attributed to K A Koehler.

At least 19 recordsLinked to original sources
PubMed1997

Misinterpretation of body sensations in panic disorder.

Cognitive accounts of panic predict that panic disorder patients will be particularly prone to misinterpret autonomic sensations. Several studies have produced results consistent with this prediction, but each is open to alternative interpretation. To clarify matters, 2 studies administered the Body Sensations Interpretation Questionnaire (BSIQ) to panic patients and controls. Panic patients were more likely to interpret ambiguous autonomic sensations as signs of immediately impending physical or mental disaster and were more likely than other anxiety disorder patients and nonpatients to believe these interpretations. In a 3rd study, a brief version of the BSIQ was shown to have satisfactory test-retest reliability, to change with treatment, and to discriminate treatments that varied in their effects on panic.

Adult↗
PubMed1995

The pH-dependence of the equilibrium between the fast- and slow-folding forms of bovine prothrombin fragment 1: a role for pH-induced self-association?

Examination of the pH- and ionic strength (mu)-dependence of the equilibrium between fast- and slow-folding forms of bovine prothrombin fragment 1 reveals a sharp dependence of Keq ([% fast-folding form]/[% slow-folding form]) and % fluorescence quenching (%Q) on pH at low mu, and the absence of a pH-dependence of Keq at high mu (0.1 M NaCl) and much reduced pH-dependence of %Q, suggesting that the ionization process is coupled to other processes, such as self-association. The observed low mu pH effect on Keq amounts to a 10% increase in the % of the fast-folding form of prothrombin at low pH. We hypothesize the existence of a pH-dependent self-association of bovine prothrombin fragment 1. This process is associated with a conformation change involving an ionizing group with pKa in the neighborhood of 6.5 and a change in Keq from 0.27 to 0.45.

Animals↗
PubMed1995

Intramolecular domain-domain interactions and intermolecular self-association in bovine prothrombin. A potentiometric and laser light-scattering study.

The interaction of bovine prothrombin with Ca2+ and Mg2+ ions was investigated by following H+ release as a function of metal ion concentration at pH 6 and pH 7.4 at high and low ionic strength. Prothrombin Ca2+ and Mg2+ binding is characterized by high- and low-affinity sites. M2+ binding at these sites is associated with intramolecular conformational changes and also with intermolecular self-association. The pH dependence of H+ release by M2+ is bell shaped and consistent with controlling pKa values of 4.8 and 6.5. At pH 6 and low ionic strength, both Ca2+ and Mg2+ titrations following H+ release clearly show independent low- and high-affinity binding sites. Laser light scattering reveals that at pH 7.4 and low ionic strength, and at pH 6.0 and high ionic strength, the prothrombin molecular weight is between 73 and 98 kD. At pH 7.4 and high ionic strength, prothrombin is monomeric in the absence of metal ions, but appears to dimerize in the presence of M2+. At pH 6.0 and low ionic strength prothrombin exists as a dimer in the absence of metal ions and is tetrameric in the presence of Ca2+ and remains dimeric in the presence of Mg2+. These results and those for metal ion-dependent H+ release indicate that H+ release occurs concomitantly with association processes involving prothrombin.

Animals↗
PubMed1995

Photofrin photosensitization. Correlation of Photofrin:erythrocyte binding processes with photolysis.

Unilamellar suspensions of dimyristoylphosphatidylcholine (DMPC) can be utilized to remove Photofrin from the erythrocyte. This enables correlation of the Photofrin membrane-binding processes with Photofrin-sensitized photolysis. The observed rates of erythrocyte biding as well as the observed rates of removal of PHotofrin from the erythrocyte membrane suggest the existence of two Photofrin species that differ in their rates of exchange between the erythrocyte and buffer phases. Selective depletion and readdition of these Photofrin species to the erythrocyte membrane permits evaluation of their separate and joint photolytic efficiencies. These rapidly and slowly exchanging membrane-bound Photofrin species are separately much less efficient photosensitizers than the two species together. The two Photofrin species exhibit essentially identical fluorescence emission spectra in the presence of DMPC. Nevertheless, models consistent with the results involve partitioning by chemically distinct Photofrin components or partitioning of chemically similar Photofrin components into distinct membrane environments, or a combination of these.

Dihematoporphyrin Ether↗
PubMed1992

Correlation between photodynamic efficacy of differing porphyrins and membrane partitioning behavior.

The ability of a photosensitizer to partition into membrane is determined by its structure and physical properties. Partitioning behavior can be quantitated as the partition coefficient (Kp) for a particular drug. This property may be an important determinant of cytocidal efficacy in photodynamic therapy. The Kp of five photoactive drugs--13,17-ditetraammonium protoporphyrin (PH1008), photofrin II (PII), hematoporphyrin (Hp), benzoporphyrin derivative monoacid (BPD-MA), coproporphyrin (Cp), and uroporphyrin (Up)--was determined using a simple liposome system composed of sonicated egg phosphatidylcholine single bilayer vesicles. The cytocidal efficacy of each drug was compared by determining the concentration of drug resulting in 50% maximal lysis (C50) obtained by measuring the hemoglobin absorbance at 414 nm released from lysed human red blood cells. The percentage lysis at 1 microM final drug concentration was also determined. An argon-dye laser was used to administer light of 630-nm wavelength for a total exposure of 5 J/cm2. Porphyrins with a greater tendency to partition into phosphocholine bilayer membranes demonstrated a greater lytic efficacy in the rbc system utilized. The comparison of physical properties with lytic ability may be useful in understanding the mechanism by which PDT exerts its effects and in predicting the clinical efficacy of different drugs.

Cell Membrane↗
PubMed1992

A new porphyrin photosensitizer (PH1008) in model membranes, normal cells, and bladder cancer cells.

Newer photosensitizers continue to be sought for photodynamic therapy of bladder cancer particularly since local rather than systemic application is desired. Recent studies have indicated that a cationic dye, PH1008, a 13,17-N,N,N-dimethylethylethanolamine ester of protoporphyrin, sensitizes the photolysis of red blood cells. The study described in this report was designed to investigate the plasma membrane partitioning of PH1008 model lipid system and to compare partitioning of PH1008 in normal transitional cells and bladder cancer cells in vitro. Partition coefficient (Kp) values characterizing the distribution of PH1008 between aqueous buffer and normal and malignant transitional cells were 3.4 +/- 0.7 x 10(4) (CRL-7881) and 9.5 +/- 1.4 x 10(4) (HTB-9), resulting in a 20% difference in membrane photosensitizer concentration at a particular photosensitizer concentration. Significantly higher (2-5 fold) differences are observed between tumor and surrounding normal tissue for systemically delivered photofrin II. Cell-bound drug was 30-fold (CRL-7881) and 80-fold (HTB-9) more fluorescent when compared to aqueous buffer. The combined effects of partitioning and bound fluorescence suggest that a 3.2-fold increase in fluorescence of transformed vs. normal bladder urothelium exists. This difference in fluorescence suggests that PH1008 might be more useful as a diagnostic tool than as a phototherapeutic agent.

Humans↗
PubMed1991

Cooperativity in the calcium ion-induced quenching of the intrinsic fluorescence of a series of normal and GLA-deficient bovine prothrombin fragment 1 molecules.

Ca2+ titrations of the intrinsic fluorescence of a series of gamma-carboxyglutamic acid (GLA)-deficient bovine prothrombin fragments 1 yield response Hill plot parameters useful for characterization of the metal ion-binding process. 11-, 10-, and 9-GLA fragments 1 exhibit Tm (the (Ca2+)total concentration at which ln (B/F) = 0 in the response Hill plot) values between 0.2 and 0.3 mM. A 22-fold increase in Tm to 5.4 mM is observed for 8-GLA fragment 1. Tm decreases to 3.8 mM for the 7- and 6-GLA proteins. The value of h, about 2.8 +/- 0.2 for 11-, 10-, and 9-GLA fragments 1, abruptly decreases to 1.2-1.3 for 8-, 7-, and 6-GLA fragments 1. The observed degree of quenching induced by saturating levels of calcium ions is affected by both changes in the intrinsic fluorescence of the metal ion-free proteins and in the maximum possible degree of quenching in the presence of calcium. The kinetic characteristics of the calcium ion-induced quenching of the intrinsic fluorescence of 6-GLA fragment 1 are identical to those observed in 10-GLA fragment 1, suggesting that the fluorescence quenching observed in the 6- and 10-GLA fragments 1, while different in magnitude, involves similar processes. Observation of an abrupt change in the relative electrophoretic mobilities of 11- to 9-GLA fragments 1 compared to 8- to 6-GLA fragments 1, in the absence or presence of Ca2+, suggests the existence of a major protein conformation change which occurs concomitantly with the noted changes in Tm and h response Hill plot parameters. Molecular mechanics calculations suggest a structural hypothesis unifying these observations. Central to this model is the presumption of the existence of hydrogen bond-mediated interactions between metal ion-binding sites.

1-Carboxyglutamic Acid↗
PubMed1988

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.

Humans↗
PubMed1988

Studies on Ca(II) binding to gamma-carboxyglutamic acid. Use of thermal decarboxylation to probe metal ion/gamma-carboxyglutamic acid interactions.

The thermal decarboxylation of N-benzyloxycarbonyl-L-gamma-carboxyglutamic acid alpha-methyl ester [Z)-L-Gla-OMe) has been studied. In the presence of increasing amounts of calcium or magnesium ions, lyophilized powders of (Z)-L-Gla-OMe exhibit a corresponding increase in thermal stability. Both magnesium and calcium form relatively tight, thermally stable complexes with (Z)-L-Gla-OMe at high metal ion concentrations. Differences between Ca(II) and Mg(II) binding are noted at low metal ion concentrations, where (Z)-L-Gla-OMe is in excess. Under these conditions, complex formation with Mg(II) apparently favors a 2:1 Gla-magnesium ion complex in which both Gla residues are unstable to thermal decarboxylation. Calcium ion complexes, however, are found to favor a 3:1 Gla-calcium ion complex in which 1 of the 3 Gla residues is thermally stable.

1-Carboxyglutamic Acid↗
PubMed1987

Mg(II) binding by bovine prothrombin fragment 1 via equilibrium dialysis and the relative roles of Mg(II) and Ca(II) in blood coagulation.

The first direct equilibrium dialysis titration of the blood coagulation protein bovine prothrombin fragment 1 with Mg(II) is presented. Fragment 1 has fewer thermodynamic binding sites for Mg(II) than Ca(II), less overall binding affinity, and significantly less cooperativity. Several nonlinear curve fitting models were tested for describing the binding of fragment 1 with Mg(II), Ca(II), and mixed metal binding data. The Mg(II) data is represented by essentially five equivalent, noninteracting sites; for Ca(II), a model with three tight, cooperative sites and four "loose", equal affinity, noninteracting sites provides the best model. Based on the reported equilibrium dialysis data and in conjunction with other experimental data, a model for the binding of Ca(II) and Mg(II) to bovine prothrombin fragment 1 is proposed. The key difference between the binding of these divalent ions is that Ca(II) apparently causes a specific conformational change reflected by the cooperativity observed in the Ca(II) titration. The binding of Ca(II) ions to the three tight, cooperative sites establishes a conformation that is essential for phospholipid X Ca(II) X protein binding. The filling of the loose sites with Ca(II) ions leads to charge reduction and subsequent phospholipid X Ca(II) X protein complex interaction. Binding of Mg(II) to bovine prothrombin fragment 1 does not yield a complex with the necessary phospholipid-binding conformation. However, Mg(II) is apparently capable of stabilizing the Ca(II) conformation as is observed in the mixed metal ion binding data and the synergism in thrombin formation.

Animals↗
PubMed1987

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.

Acetates↗
PubMed1986

Chemical modification of bovine prothrombin fragment 1 in the presence of Tb3+ ions.

The formaldehyde-morpholine method for the conversion of gamma-carboxyglutamyl (Gla) residues to gamma-methyleneglutamyl (gamma-MGlu) residues has been applied to the modification of bovine prothrombin fragment 1. In the absence of Tb3+ ions or at Tb3+ ion concentrations of 2 Km app and 25 Km app the action of 10,000-fold molar excess of formaldehyde and morpholine, pH 5.0, converts the 10 Gla residues of the protein into 10 gamma-MGlu residues. Modification of the protein using the same conditions but increasing the Tb3+ concentration to 100 Km app provided a homogeneous protein containing 3 gamma-MGlu and 7 Gla residues, bovine 3 gamma-MGlu-fragment 1. The modified protein binds the same number of Ca2+ ions (6-7) as bovine fragment 1. However, the positive cooperatively associated with Ca2+ binding is abolished and the overall affinity for Ca2+ ions is reduced. Fluorescence titrations of 3 gamma-MGlu-fragment 1 using either Ca2+ or Mg2+ ions indicate that the modified protein retains a fluorescence quenching behavior similar to that of the native protein. The modified protein does not bind to phosphatidylserine/phosphatidylcholine vesicles in the presence of Ca2+ ions. Thus the metal ion-induced fluorescence transition exhibited by the bovine protein appears to be a necessary but not sufficient condition for phospholipid binding.

Amino Acids↗
PubMed1986

The effect of divalent metal ions on the electrophoretic mobility of bovine prothrombin and bovine prothrombin fragment 1.

Examination of metal ion-dependent effects on the electrophoretic mobility of bovine prothrombin and fragment 1 provides a useful and sensitive method for investigation of conformational processes in these proteins. Utilization of this method reveals a conformational change in bovine prothrombin and fragment 1 which occurs at low metal ion concentrations. Equilibrium dialysis studies indicate that the metal ion-induced shape change occurs concomitant with binding of a single calcium ion/molecule of prothrombin or fragment 1. Mixed metal electrophoretic mobility studies with Mg2+ and Ca2+ have demonstrated the "synergistic" effect for fragment 1 observed by others. Mixed metal equilibrium dialysis has provided experimental support for this observation and allows us to conclude that two tight Ca2+ sites are not affected by low Mg2+ concentrations and that the third Ca2+ site is also a tight site for Mg2+. Thus, at low Mg2+ concentrations and upon the addition of Ca2+, there are effectively three tight sites; consequently more Ca2+ will bind to the protein at lower total Ca2+ ion concentrations.

Animals↗
PubMed1986

Synthesis of a gamma-carboxyglutamic acid containing heptapeptide corresponding to bovine prothrombin residues 17-23.

The vitamin K-dependent proteins involved in blood coagulation processes each contain a small cystine loop with the sequence -Gla-Cys-X-Gla-Gla-X-Cys-. A method for the synthesis of the heptapeptide derivative representing the 17-23 sequence of bovine prothrombin, Z-Gla-Cys-Leu-Gla-Gla-Pro-Cys-OBzl, has been developed. The 25Mg2+ n.m.r. spectrum of the heptapeptide derivative has been obtained and is compared to the n.m.r. spectra obtained from the interaction of 25Mg2+ with Z-Gla-OMe and Z-Gla-Gla-OMe.

1-Carboxyglutamic Acid↗
PubMed1986

Conformation of the 18-23 loop region of bovine prothrombin in the absence and presence of a model Ca2+ ion. An energy minimization study.

The conformation of the cyclic peptide Ac-Cys-Leu-Gla-Gla-Pro-Cys-NHMe, representing the 18-23 disulfide loop of bovine prothrombin, was studied by energy minimization with the ECEPP (Empirical Conformational Energy Program for Peptides) algorithm. Parameters for charge and geometry for the gamma-carboxyglutamic acid (Gla) residue were obtained for inclusion in the ECEPP data set. Construction of the 18-23 cyclic peptide, for which no crystal structure is available, was carried out by using a scheme that took advantage of the constraints imposed by the requirement of disulfide ring closure and utilized known low-energy structures of single residues and dipeptides. Both cis and trans isomers about the Gla 21-Pro 22 peptide bond were considered. The lowest-energy conformation found for the isolated 18-23 cyclic peptide with arbitrary reduction of the charge on the Gla residues (to simulate hydration roughly) is a trans form, differing in energy by 11 kcal-mol-1 from the lowest-energy cis form. However, when the energy calculation includes one model Ca2+ ion, X2+, introduced at a fixed distance of 2.3 A from a single oxygen atom of either of the side-chain carboxyl groups of Gla with the C delta-O-X2+ bond angle fixed at one of three values, the lowest-energy cis conformation is about 1 kcal-mol-1 lower in energy than the lowest-energy trans conformation; i.e. the two structures have similar energies. In these structures, four oxygen atoms, two from each Gla side-chain, approach the model Ca2+ ion closely, in a manner similar to that seen in crystals of calcium alpha-ethylmalonate (Zell, A., Einspahr, H. & Bugg, C.E. (1985) Biochemistry 24, 533-537). It appears that the binding of Ca2+ to the 18-23 cyclic peptide may alter the equilibrium between cis and trans structures such that the fraction of cis isomers is greater in the presence of Ca2+.

Animals↗
PubMed1985

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

UNLABELLED: The 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. CONCLUSIONS: These 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.

Anti-Bacterial Agents↗