Simple preparation of 1,2-dipalmitoyl-sn-glycero-3-phosphoric acid and deuterated choline derivatives

Analytically pure 1,2-dipalmitoyl-sn-glycero-3-phosphoric acid was prepared in gram amounts, using a simplified version of a previous procedure. The main step, enzymatic cleavage of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with freshly extracted phospholipase D, was performed in the presence of chloroform and the crude phosphatidic acid was purified by silica gel column chromatography. The interest of the method was illustrated by the synthesis of two dipalmitoyl phosphatidylcholines selectively deuterated on the polar headgroup.     

On the subtransitions of deuterated derivatives of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

By means of Fourier transform infrared spectroscopy the subtransition temperature of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the derivatives in which either or both of the acyl chains were deuterated were determined. It was found that, while the temperatures of the gel to liquid-crystal and the pre-transitions decrease with increasing deuteration, the temperature of the subtransition is independent of the degree of deuteration.     

The phase transition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine as seen by Fourier transform infrared difference spectroscopy

The potential of Fourier transform infrared difference spectroscopy for biochemical applications is demonstrated by the gel to liquid crystal phase transition of the title compound. While the changes occurring in the vibrational pattern of the hydrophobic palmitoyl chains are easily monitored, this technique also discriminates between no change in the choline moiety and a small yet significant change in the carbonyl moiety, both located in the hydrophylic head group.     

A low-temperature structural phase transition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers in the gel phase

A new thermotropic phase transition, at ?30°C and atmospheric pressure, was found to occur in the gel phase of aqueous DPPC dispersions. The Raman spectral changes at this phase transition are similar to those observed in the gel phase of DMPC dispersions at ?60°C. The thermotropic phase transition at ?30°C is equivalent to the barotropic GII to GIII phase transition observed in DPPC at 1.7 kbar and 30°C. It is shown that the rate of the large angle interchain reorientational fluctuations decreases gradually with decreasing temperature, and that the orientationally disordered acyl chain structure of the GII phase is extended into the GIII phase of DPPC. The interchain interaction, arising from the damping of the reorientational fluctuations, increases with decreasing temperature in the GII gel phase as well as in the GIII gel phase.     

The phase transition behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model membrane influenced by 2,4-dichlorophenol--an FT-Raman spectroscopy study

The effect of 2,4-dichlorophenol (DCP) on the structures and phase transitions of fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes was studied using FT-Raman spectroscopy. Whereas the Raman frequency shifts of the most frequently investigated bands of C-C and C-H stretching regions only indicate the main phase transition (P(beta')-L(alpha)) of the pure DPPC/water system, the Raman shift of C-H scissoring vibration at 1440 cm(-1) was found to be able to reveal the pretransition (L(beta')-P(beta')) as well. Analyzing the spectral parameters of the trans band at 1128 cm(-1), which does not overlap with DCP vibrational modes, a continuous decrease of trans conformations was found with increasing DCP concentration at 26 degrees C accompanying the phase transitions L(beta')-P(beta') and P(beta')-L(alpha). The intensity ratio of the symmetrical and asymmetrical methylene stretching bands (at 2850 cm(-1) and 2880 cm(-1)), defined as the disorder parameter by Levin [Levin, I.W., 1985. Two types of hydrocarbon chain interdigitation in sphingomielin bilayers. Biochemistry 24, 6282-6286], indicated that in the interdigitated phase (L(I)) the order is markedly high and comparable with that of L(beta). Both the phase transition P(beta')-L(alpha) in the DCP/DPPC molar ratio range of 10/100-50/100 and the phase transition L(I)-L(alpha) led to a significant increase of disordered chains and the presence of DCP molecules induced a more disordered chain region than that observed in the L(alpha) phase of DPPC. Nevertheless, it was found that the L(alpha) phase with DCP contains approximately the same amount of trans conformers than that without DCP.     

Crystal structure of conserved domains 1 and 2 of the human DEAD-box helicase DDX3X in complex with the mononucleotide AMP

DExD-box helicases are involved in all aspects of cellular RNA metabolism. Conserved domains 1 and 2 contain nine signature motifs that are responsible for nucleotide binding, RNA binding and ATP hydrolysis. The human DEAD-box helicase DDX3X has been associated with several different cellular processes, such as cell-growth control, mRNA transport and translation, and is suggested to be essential for the export of unspliced/partially spliced HIV mRNAs from the nucleus to the cytoplasm. Here, the crystal structure of conserved domains 1 and 2 of DDX3X, including a DDX3-specific insertion that is not generally found in human DExD-box helicases, is presented. The N-terminal domain 1 and the C-terminal domain 2 both display RecA-like folds comprising a central beta-sheet flanked by alpha-helices. Interestingly, the DDX3X-specific insertion forms a helical element that extends a highly positively charged sequence in a loop, thus increasing the RNA-binding surface of the protein. Surprisingly, although DDX3X was crystallized in the presence of a large excess of ADP or the slowly hydrolyzable ATP analogue ATPgammaS the contaminant AMP was seen in the structure. A fluorescent-based stability assay showed that the thermal stability of DDX3X was increased by the mononucleotide AMP but not by ADP or ATPgammaS, suggesting that DDX3X is stabilized by AMP and elucidating why AMP was found in the nucleotide-binding pocket.     

Synthesis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with labeled acyl groups

A facile procedure for the small scale chemical synthesis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPL) is reported. Under optimal conditions, 36% of the sn-glycero-3-phosphocholine was converted to DPL. This procedure is suitable for small scale preparation of DPL with very high specific radioactivity from labeled palmitic acid. Furthermore, the labeled DPL obtained will serve as precursor for the chemical or enzymatic synthesis of other labeled phospholipids.     

Microsomal activation of thioacetamide-S-oxide to a metabolite(s) that covalently binds to calf thymus DNA and other polynucleotides

In the presence of NADPH liver microsomes isolated from phenobarbital-pretreated rats catalyze the conversion of [³H]thioacetamide-S-oxide to a reactive intermediate(s) which covalently binds to calf thymus DNA, calf liver RNA, polyguanylic acid (poly(G)) and polyadenylic acid (poly(A)). The highest level of binding of radioactivity was obtained with poly(G), followed by poly(A), RNA and DNA. The incorporation of radioactivity into DNA was linear for 30 min and there was a requirement for NADPH for time-dependent covalent binding to occur. Performing the microsomal incubations in an atmosphere of 80% CO/20% O₂ or adding partially purified anti cytochrome P-450 immune serum to the microsomal incubations inhibited the total metabolism of thioacetamide-S-oxide and had a small, but insignificant, inhibitory effect on binding of radioactivity to calf thymus DNA. Using a reconstituted monooxygenase system containing cytochrome P-450 purified from phenobarbital-treated rats we were unable to detect any metabolism of thioacetamide-S-oxide. Only background levels of radioactivity were incorporated into calf thymus DNA when microsomes isolated from phenobarbital-treated rats were incubated with [³H]thioacetamide in the presence of NADPH. These results suggest that thioacetamide-S-oxide is an obligatory intermediate in the metabolic activation of thioacetamide to a reactive metabolite(s) which binds to calf thumus DNA.     

Identification of N-methylprotoporphyrin IX in livers of untreated mice and mice treated with 3, 5-diethoxycarbonyl- 1, 4-dihydrocollidine: source of the methyl group

Administration of the porphyrogenic agent, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to mice, leads to the accumulation of N-methylprotoporphyrin IX in liver. This porphyrin is a potent inhibitor of ferrochelatase activity and accounts for the porphyria produced after DDC administration. The N-methylprotoporphyrin IX extracted from DDC-treated mice is primarily of one isomeric form, as shown by nuclear magnetic resonance spectroscopy. The methyl group of N-methylprotoporphyrin IX isolated from DDC-treated mice is derived mostly from the 4-methyl group of DDC. The transfer of this methyl group and its subsequent covalent attachment to protoporphyrin IX may be mediated by a form of hepatic microsomal cytochrome P-450. N-Methylprotoporphyrin IX is also found in livers of untreated mice at levels that are low but significant.     

Fungal oxidation of 3-methylcholanthrene: Formation of proximate carcinogenic metabolites of 3-methylcholanthrene

The filamentous fungus, Cunninghamella elegans, was found to metabolize the potent carcinogen, 3-methylcholanthrene (3-MC) to 1-hydroxy-3-MC, 2-hydroxy-3-MC, 1-keto-3-MC, 2-keto-3-MC and trans-9,10-dihydrodiols of 1-hydroxy-3-MC. In addition several unidentified derivatives of 3-MC were found. The metabolites formed were separated by high pressure liquid chromatography (HPLC) and identified by comparison of retention times, absorbance, fluorescence and mass spectra with those of synthetic standards. Incubation of (±)-1-hydroxy-3-MC and (±)-2-hydroxy-3-MC with cells of C. elegans indicated that 1-hydroxy-3-MC is metabolized to form diasteromerically related trans-9,10-dihydrodiols of 1-hydroxy-3-MC. Experiments with 3-[¹⁴C]MC showed that over a 48-h period, 8.7% of the hydrocarbon was oxidized to organic solvent-soluble metabolic products. Most of the metabolites were polar products, some of which co-chromatographed with trans-9,10-dihydrodiols of 1-hydroxy-3-MC. The results show that C. elegans has the ability to oxidize 3-MC to metabolites that have been implicated as proximate carcinogenic forms of 3-MC in higher organisms.     

Separation and chemical differentiation of alpha and beta subunits in yeast phosphofructokinase

The two types of subunits alpha and beta constitutive of yeast phosphofructokinase have been separated by ion-exchange chromatography under denaturating conditions. Amino acid analysis and peptide mapping were performed on the isolated subunits. The frequence of most of the amino acids significantly differs between the two types of polypeptide chains. Moreover, tryptic peptide maps of alpha and beta subunits are clearly not superimposable. These chemical differences seem sufficient to account for the distinct catalytic and regulatory functions of beta and alpha subunits in the yeast phosphofructokinase reaction.     

Infrared and raman spectroscopy of carbohydrates. 3. Raman spectra of the polymorphic forms of amylose

The Raman spectra of V_a-, Vh_h-, and B-amylose have been recorded, and are interpreted in terms of the proposed mechanism for conversion from the V- into the B-form. Lines occurring at 1263 and 946cm^-1 with V-amylose shift to 1254 and 936 cm^-1 on conversion into the B-form; at the same time intensity changes are observed for the lines at 2940 and 1334 cm^-1. These effects are consistent with the mechanism proposed for V→B conversion, involving an extension of the helix and changes in the intramolecular hydrogen-bonding. In addition, the spectra of amylose dissolved in aqueous salt solution and in methyl sulfoxide have been recorded. The results indicate that amylose does not adopt the V-conformation in methyl sulfoxide solution.     

Metabolism of N-nitrosomorpholine by the rat in vivo and by rat liver microsomes and its oxidation by the Fenton system

N-Nitrosomorpholine is converted into N-nitroso-2-hydroxymorpholine by rat liver microsomes and by the Fenton oxidation system. The hydroxy derivative was also synthesised by the oxidation of N-nitrosomorpholine with permanganate and characterized as the methoxime and the 2,4-dinitrophenylhydrazone. The Fenton system also afforded products believed to be N-nitroso-2-morpholone, and the 2-hydroperoxy- and 2-peroxy-derivatives ofN-nitrosomorpholine. The only urinary metabolite definitely identified was N-itrosodiethanolamine.

The significance of metabolic 2-hydroxylation in relation to the carcinogenic action of N-nitrosomorpholine is discussed.     

delta5 desaturation of fatty acids in L-M cells

L-M cells grown in a lipid-free medium containing ¹⁴C-labeled 9,12-linoleic acid incorporated most of this acid into glycerolipids as linoleic acid. Only a small amount (3%) was elongated to eicosadienoic acid. No Δ6 desaturation occurred. When the cells were incubated with ¹⁴C-labeled 8, 11, 14-eicosatrienoic acid, 22% of the activity was found in 5,8,11,14-eicosatetraenoic acid. Treatment of the cells for 24 hr with N-isopropylethanolamine, a choline analog, depressed this desaturation reaction to about 60% of control values. The identity of the tetraene product was established by two different chromatographic analyses of the fatty acid methyl esters. Location of the double bond at position C-5 was determined by ozonolysis and subsequent reduction of the ozonides to aldesters followed by gas-liquid chromatography. These results prove that L-M cells have a Δ5 desaturase and an elongation enzyme converting 18:2 to 20:2, but lack a Δ6 desaturase.     

Blood group i and I activities of "lacto-N-norhexaosylceramide" and its analogues: the structural requirements for i-specificities.

A pure straight chain ceramide hexasaccharide (“lacto-N-norhexaosylceramide” Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glc→Ceramide) showed strong i-activity determined by hemagglutination inhibition and by radioimmunoassay with five out of six anti-i antisera. Two repeating Galβ1→4GlcNAc residues and GlcNAcβ1→3Gal residues could be essential for the full expression of this activity; eleven closely related analogues including those derived by chemical modification had lower or no detectable activity. The same structure reacted also with some anti-I antisera. The strong i-activity and the moderate I-activity were both abolished by elimination of the terminal Gal or by removal of the N-acetyl groups of the two GlcNAc residues.     

Blood group A glycolipid (Ax) with globo-series structure which is specific for blood group A1 erythrocytes: one of the chemical bases for A1 and A2 distinction

A new blood group A-active glycolipid fraction, termed Ax, showing a chromatographic mobility between Aa and Ab was found in blood group A1 erythrocytes but not in A2 erythrocytes. Ax was identified by its conversion to "globo H" by alpha-N-acetylgalactosaminidase and by 1H-NMR spectroscopy as GalNAc alpha l----3[Fuc alpha l----2]Gal beta l----3GalNAc beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer. Globo-H (Fuc alpha l----2Gal beta l----3GalNac beta l----3Gal alpha l----4Gal beta l----4Glc beta l----lCer) was found in blood group A, and O but not in A1 erythrocytes. Thus, one of the A1-specific determinants must be an A determinant carried by globo-series structure.