Studies on the interaction of anesthetic steroids with phosphatidylcholine using 2H and 13C solid state NMR

The effects of the anesthetic steroid alphaxalone and its inactive analog delta 16-alphaxalone on model phospholipid membranes were studied using 13C and 2H solid-state nuclear magnetic resonance spectroscopy. Aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) with specific 13C and 2H labels as endogenous probes at the carbonyl and the C-7 methylene groups, respectively, of the sn-2 chain were used to study the conformational and dynamical properties of the bilayer as a function of temperature. There were no significant changes between the 13C and 2H spectra of the DPPC preparation containing the inactive steroid and that of DPPC with no drug. However, the physiologically active steroid produces significant spectral 2H and 13C changes. These changes include a reduction of the main phase transition temperature and a broadening of that transition. Alphaxalone also increases the relative number of gauche conformers in the liquid-crystalline phase of DPPC and increases the rate of axial diffusion in both the gel and liquid-crystalline phase. The thermotropic properties of the above preparations, as monitored by differential scanning calorimetry, were congruent with the spectroscopic data.     

Heterokaryosis inPolyporus ostreiformis for improved yield of exo-1,4-α-d-glucosidase

An amylolytic strain ofPolyporus ostreiformis was subjected to multistep mutagenic treatment and a heterokaryotic mutant was finally selected which produced exo-1,4-α-d-glucosidase in a yield of 2.54 U/mL against the parental yield of 0.42 U/mL. The crude enzyme extract of the mutant was used to hydrolyze soluble starch, maltose and amylopectin and chromatography of the hydrolyzates revealed exo-1,4-α-D-glucosidase activity predominant in the enzyme system.     

Modification by diacylglycerol of the structure and interaction of various phospholipid bilayer membranes

The effects of incorporating diacylglycerol (DG) derived from egg phosphatidylcholine (PC) into PC, egg phosphatidylethanolamine (PE), and bovine phosphatidylserine (PS) have been measured. In excess solution DG induces a multilamellar-to-hexagonal (L-H) structural transition in PE and PC that is temperature dependent. At 37 degrees C it begins at about 3 and 30 mol%, respectively. In PC at lower DG concentrations a modified lamellar phase is formed; at about 70 mol% DG a single primitive cubic phase forms. An L-H transition induced by 20-30 mol% DG in PS is dependent on ionic strength and degree of lipid hydration, with the appearance of crystalline acyl chains at the higher DG levels. Calcium precipitates of DG/PS (1/1) mixtures have melted chains. Structural parameters were derived for the lamellar phases at subtransition levels of DG in PE and PC. The area per polar group is increased, but by contrast with cholesterol, the polar group spreading is not accompanied by an increase in bilayer thickness. DG does not affect the equilibrium separation of PC or PE bilayers. Measured interbilayer forces as they vary with bilayer separation show that DG at 20 mol% does not effect closer apposition of PC bilayers at any separation. Spreading the polar groups may effect the binding of protein kinase C or the activation of phospholipases; the nonlamellar phases may be linked to the biochemical production of DG in cellular processes involving membrane fusion.     

Membrane channel forming polypeptides. Molecular conformation and mitochondrial uncoupling activity of antiamoebin, an alpha-aminoisobutyric acid containing peptide

The conformations of the 16-residue fungal peptide antiamoebin I (Ac-Phe-Aib-Aib-Aib-D-Iva-Gly-Leu-Aib-Aib-Hyp-Gln-D-Iva-Hyp-Aib-Pro-P hol) have been investigated in dimethyl sulfoxide solution by one- and two-dimensional NMR techniques. A substantial number of resonances in the 270-MHz 1H NMR spectrum have been assigned. Intramolecularly hydrogen-bonded (solvent inaccessible) NH groups have been identified by determining solvent and temperature dependence of NH chemical shifts and rates of hydrogen-deuterium exchange. Ten backbone NH groups are inaccessible to solvent, while three NH groups assigned to the Phe(1), Aib(2), and Aib(8) residues are exposed to solvent. Interresidue nuclear Overhauser effects are consistent with psi values of approximately 120 +/- 30 degrees for Phe(1) and Leu(7). The NMR results, together with the stereochemical constraints imposed by the presence of alpha-aminoisobutyryl, isovalyl, prolyl, and 4-hydroxyprolyl residues, favor a highly ordered structure. Two backbone conformations consistent with the data are considered. Antiamoebin is shown to be an effective uncoupler of oxidative phosphorylation in rat liver mitochondria, providing evidence for its membrane-modifying activity.     

Chromosomal damage induced in human lymphocytes by low doses of D-T neutrons

Unstable chromosome aberrations induced by in vitro irradiation with zero plus seven low doses of 14.8 MeV D-T neutrons in the range 3.55-244 mGy have been analysed in human peripheral blood lymphocytes. In order to obtain the required large numbers of scored cells for such low doses, fourteen laboratories participated in the experiment. The dose responses for dicentrics, excess acentrics and total aberrations, fitted well to the Y = alpha D model. The alpha coefficient of yield for dicentrics, 1.60 +/- 0.07 X 10(-2) Gy-1, compares well with the values obtained in previous studies with D-T neutrons at somewhat higher doses. Results from a previous collaborative study using 250 kVp X-rays over a comparable dose range indicated the possible existence of a threshold below 50 mGy. In the present study there is no clear evidence for neutrons for such a threshold. However, the data were insufficient to permit the rejection of a possible threshold below approximately 10 mGy.     

Higher incidence of spontaneous sister-chromatid exchanges (SCEs) and X-ray-induced chromosome aberrations in peripheral blood lymphocytes during pregnancy

In vitro cultures of peripheral blood lymphocytes from human and muntjac (barking deer) females who were at an advanced stage of pregnancy (32-37 weeks pregnant women and 20-24 weeks pregnant muntjacs) showed an enhanced frequency of SCEs and X-ray-induced chromosome aberrations when compared with those of nonpregnant females. Lymphocyte cultures of nonpregnant females to which sex hormones progesterone, oestrogen and human chorionic gonadotropin (HCG) were added together exogenously also showed higher frequency of SCEs. The plausible reason(s) for such high incidence of SCEs during pregnancy is discussed.     

Stomatal Movement and Sucrose Uptake by Guard Cell Protoplasts of Commelina benghalensis L.

Sucrose concentration in guard cells of epidermal strips ofCommelina benghalensis increased with stomatal opening. Sucroseuptake patterns were investigated using guard cell protoplastsof C. benghalensis. Sucrose (0.5 mM) uptake into these protoplastswas sensitive to pH, with an optimum at pH 6. Uptake of sucroseinto guard cell protoplasts was inhibited by 2,4-dinitrophenol(DNP), diethylstilbestrol (DES) and (ptrifluoromethoxy)carbonylcyanide phenylhydrozone (FCCP), while DCMU and o-phenanthrolinehad no effect on the uptake of sucrose. Fusicoccin (FC) stimulatedsucrose influx. The influence of pH and the effect of the metabolicinhibitors on the sucrose uptake into the guard cell protoplastsare consistent with an energy dependent membrane-function. (Received July 7, 1986; Accepted September 26, 1986)     

Purification and characterization of human seminal plasma acid phosphatase

A 81-fold purification of human seminal plasma acid phosphatase was obtained by a three-step procedure, involving ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Homogeneity of the preparation during purification steps was tested by polyacrylamide gel electrophoresis and only one major band was obtained after the final step. The pH optimum for the activity of the purified enzyme was 5.6 and thermal stability was obtained even up to 40 degrees C. PNPP was the most specific synthetic substrate. The Km of purified seminal acid phosphatase towards PNPP was 1.5 X 10(-3) M. Among the metal ions tested, Hg+2 showed an I50 value of 4.2 X 10(-7) M. Studies with PCMB, PMSF and EDTA did not show any inhibition, whereas NaF and L(+)tartrate, at 1 mM concentration, inhibited the enzyme by 95% and 85%, respectively.     

Amino acid replacements in yeast iso-1-cytochrome c. Comparison with the phylogenetic series and the tertiary structure of related cytochromes c

The structural and folding requirements of eukaryotic cytochromes c have been investigated by determining the appropriate DNA sequences of a collection of 46 independent cyc 1 missense mutations obtained in the yeast Saccharomyces cerevisiae and by deducing the corresponding amino acid replacements that abolish function of iso-1-cytochrome c. A total of 33 different replacements at 19 amino acid positions were uncovered in this and previous studies. Because all of these nonfunctional iso-1-cytochromes c are produced at far below the normal level and because a representative number are labile in vitro, most of the replacements appear to be affecting stability of the protein or heme attachment. By considering the tertiary structure of related cytochromes c, the loss of function of most of the mutant iso-1-cytochromes c could be attributed to either replacements of critical residues that directly interact with the heme group or to replacements that disrupt the proper folding of the protein. The replacements of residues interacting with the heme group include those required for covalent attachment (Cys-19 and Cys-22), ligand formation (His-23 and Met-85), and formation of the immediate heme environment (Leu-37, Tyr-53, Trp-64, and Leu-73). Proper folding of the protein is prevented by replacements of glycine residues at sites that cannot accommodate side chains (Gly-11 and Gly-34); by replacements of residues with proline, which limit the torsion angle (Leu-14 and His-38); and by replacements apparently unable to direct the local folding of the backbone into the proper conformation (Pro-35, Tyr-72, Asn-75, Pro-76, Lys-84, Leu-99, and Leu-103). Even though most of the missense mutations occurred at sites corresponding to evolutionarily invariant or conserved residues, a consideration of the replacements in functional revertants indicates that the requirement for residues evolutionarily preserved is less stringent than commonly assumed.     

Neutrophil kinetics in O2-exposed rabbits

Hyperoxic injury results in an influx of polymorphonuclear leukocytes (PMN) into the lung. To better understand the role of the PMN in this injury, kinetic studies were used to assess the survival of PMNs in the circulation. The rate of deposition of PMNs in the lungs of rabbits exposed to hyperoxia was also examined. The half-lives (T1/2) of [3H]thymidine-labeled PMNs in the circulation in rabbits exposed to air or to 95% O2 for less than or equal to 48 h varied between 3.9 and 4.5 h. After 72 h of hyperoxic exposure, T1/2 fell to 2.2 h, the marginal and circulating PMN pool increased and 3H deposition in the lung increased 10-fold. Autoradiographs confirmed that [3H]thymidine was initially nuclear- and cellular-associated but, with time, [3H]thymidine dispersed throughout the lung, suggesting PMN disintegration. These PMN events seem to occur in the later phases of O2 toxicity, and because PMNs are an additional source of oxyradicals, they may further amplify oxidant injury.