Interaction between lipid bilayers and a new class of antineoplastic agents derived from arylchloroethylurea: a 2H solid-state NMR study

We have investigated the interaction between a new class of antineoplastic agents derived from arylchloroethylurea (CEU) and model membrane of dimyristoylphosphatidylcholine by deuterium nuclear magnetic resonance spectroscopy. The results indicate that the drug incorporates in the bilayer and causes an increase of the lipid acyl chain order, this effect being greater close to the interfacial region of the lipid bilayer. The increase in ordering is dependent on the nature (degree of ramification, length of the alkyl chain, and presence of a sulfur atom) as well as on the position of the R substituent and is correlated with the cytotoxicity of the drugs. More specifically, the more cytotoxic drugs, such as 4-sec-butyl CEU, are those having a bulky ramified substituent and those for which the ordering effect on the lipid bilayer is the smallest. On the other hand, the ordering effect is greater and seen all along the lipid acyl chains for the long-chain CEUs, such as n-hexadecyl CEU, which have been shown to have very weak cytotoxic activity. Finally, the results obtained as a function of the drug concentration indicate that the ordering effect is seen for lipid to drug molar ratios as low as 20:1.     

Membrane fusion induced by 11-mer anionic and cationic peptides: a structure-function study

We recently demonstrated that an amphipathic net-negatively charged peptide consisting of 11 amino acids (WAE 11) strongly promotes fusion of large unilamellar liposomes (LUV) when anchored to a liposomal membrane [Pecheur, E. I., Hoekstra, D., Sainte-Marie, J., Maurin, L., Bienvenue, A., and Philippot, J. R. (1997) Biochemistry 36, 3773-3781]. To elucidate a potential relationship between peptide structure and its fusogenic properties and to test the hypothesis that specific structural motifs are a prerequisite for WAE-induced fusion, three 11-mer WAE-peptide analogues (WAK, WAEPro, and WAS) were synthesized and investigated for their structure and fusion activity. Structural analysis of the synthetic peptides by infrared attenuated total reflection spectroscopy reveals a distinct propensity of each peptide toward a helical structure after their anchorage to a liposomal surface, emphasizing the importance of anchorage on conveying a secondary structure, thereby conferring fusogenicity to these peptides. However, whereas WAE and WAK peptides displayed an essentially nonleaky fusion process, WAS- and WAEPro-induced fusion was accompanied by substantial leakage. It appears that peptide helicity as such is not a sufficient condition to convey optimal fusion properties to these 11-mer peptides. Studies of changes in the intrinsic Trp fluorescence and iodide quenching experiments were carried out and revealed the absence of migration of the Trp residue of WAS and WAEPro to a hydrophobic environment, upon their interaction with the target membranes. These results do not support the penetration of both peptides as their mode of membrane interaction and destabilization but rather suggest their folding along the vesicle surface, posing them as surface-seeking helixes. This is in striking contrast to the behavior observed for WAE and WAK, for which at least partial penetration of the Trp residue was demonstrated. These results indicate that subtle differences in the primary sequence of a fusogenic peptide could induce dramatic changes in the way the peptide interacts with a bilayer, culminating in equally drastic changes in their functional properties. The data also reveal a certain degree of sequence specificity in WAE-induced fusion.     

Effect of sugars on headgroup mobility in freeze-dried dipalmitoylphosphatidylcholine bilayers: solid-state 31P NMR and FTIR studies

The effect of the carbohydrates trehalose, glucose, and hydroxyethyl starch (HES) on the motional properties of the phosphate headgroup of freeze-dried dipalmitoylphosphatidylcholine (DPPC) liposomes was studied by means of 31P NMR, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). The results show that trehalose, which is a strong glass former (Tg = 115 degreesC), elevates the onset of the lipid headgroup rotations and preserves some rotational mobility of the phosphate headgroups after cooling from the liquid-crystalline state. Glucose (Tg = 30 degreesC), a very effective depressant of the phase transition temperature of freeze-dried DPPC, markedly elevates the initiation of the temperature of headgroup rotations. On the other hand, the monosaccharide does not preserve the headgroup disordering when cooled from the liquid-crystalline state. These effects are consistent with formation of hydrogen bonds between the OH groups of the sugar and the polar headgroups of DPPC. They show, however, that hydrogen bonding is not sufficient for preservation of the dynamic properties of freeze-dried DPPC. HES, although a very good glass former (Tg > 110 degreesC), does not depress the phase transition temperature and affects only slightly the rotational properties of freeze-dried DPPC. This lack of effect of HES is associated with the absence of direct interactions with the lipid phosphates, as evidenced by the FTIR results. These data show that vitrification of the additive is not sufficient to affect the dynamic properties of dried DPPC.     

Contractile dysfunction caused by normothermic ischaemia and KCL arrest in the isolated pig heart: a 31P NMR study

The aims of this study were to assess (1) whether contractile dysfunction caused by ischaemia under hyperkalaemic conditions ("cardioplegic ischaemia") is associated with impaired energy production or abnormalities in regulation of contractility and (2) whether hyperkalaemia itself contributes to contractile dysfunction. We used 31P and 23Na NMR spectroscopy in conjunction with measurements of mechanical function and oxygen consumption in Langendorff perfused pig hearts to evaluate the mechanism of contractile failure caused by (1) total global cardioplegic (17 mM [K+]) ischaemia (36 degrees C, 50 min KCl arrest, 45 min ischaemia, 20 min reflow with high KCl) and (2) KCl arrest alone (115 min) without flow cessation. KCl arrest plus ischaemia and subsequent reperfusion (Group I) resulted in decreases in ATP (mean +/- S.D.; 61 +/- 13% of initial, n = 5; P < 0.01) and pressure-rate product (PRP) (31 +/- 9%, n = 17; P = 0.0001) while phosphocreatine (PCr), Pi, total creatine (Cr) and intracellular Na+ levels were unaffected. KCl arrest itself (Group II, n = 6) did not affect PCr, ATP or total Cr levels but decreased the PRP to 59 +/- 12% (P < 0.001). Oxygen consumption rates (Vo2) were reduced in both groups to similar levels (67 +/- 18, P < 0.01 and 77 +/- 13%, P < 0.02, respectively). The efficiency of energy conversion to mechanical work (PRP/delta VO2) decreased to 51 +/- 15 (P < 0.001) and 67 +/- 13% (P < 0.012) of initial levels, respectively. To assess metabolic and contractile reserves of post-ischaemic (n = 7) and KCl-treated (n = 3) hearts, the effects of isoproterenol (Iso) and increased Ca2+ were compared with those in normal beating hearts (Group III, n = 3). In all groups treatment with Iso (0.1 micron) greatly increased PRP (to 526 +/- 116, 203 +/- 16 and 198 +/- 8% of the level prior to stimulation (baseline), P < 0.01, respectively) and Vo2 (162 +/- 9, 153 +/-16 and 128 +/-10% of baseline, P < 0.05, Respectively). Increasing [Ca2+] from 1 to 1.66 mM produced less stimulation than Iso: PRP increased to 195 +/- 23, 156 +/- 13 and 163 +/- 22% (P < 0.05) and Vo2 increased to 138 +/- 22 (P < 0.05), 115 +/- 4 and 120 +/- 10% of baseline in Groups I, II and III, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

A high-resolution NMR study (1H, 13C, 31P) of the interaction of paramagnetic ions with phospholipids in aqueous dispersions

In the surgical treatment of hernias in children, many differences of opinion still exist, especially in the selection of the type of procedure to be used. The authors have applied the Ferguson Method in the repair of Inguinal hernias as a method of choice. Along with a high resection of Processus Vaginalis of the peritoneum, the anterior wall of Canalis Inguinalis is reinforced without transposing the Funiculus Spermaticus. To evaluate the success of this method, the authors made a late postoperative analysis. Examinations were made on 106 children of various ages who had unilateral inguinal hernia repairs one to two years prior. The unoperated contralateral inguinal canal served as a control by which eventual changes of testis could be evaluated. Clinical examinations involved determination of whether or not a recidive hernia exits, as well as the condition, size and consistency of the testis. Accordingly, poor postoperative results using the Ferguson method were less than one percent.     

Poly(L-lysine)-graft-dextran copolymer is a novel stabilizer of triplex DNA (I): stabilization of poly(dA).2poly(dT) triplex

Comb-type polylysine copolymer having grafted hydrophilic side chains was newly designed as a novel stabilizer of triplex DNAs. The comb-type copolymer elevated melting temperature of poly(dA).2poly(dT) triplex by 50 degrees C without affecting reversibility, melting and reassociation, of the triplex in buffer with physiological salt concentrations. The stabilizing effect of the copolymer was greater than spermine. Our results indicate that the molecular designing of polycation with comb-type structure is a successful strategy for creating an effective triplex stabilizer.     

Imaging H2(17)O distribution in a phantom and measurement of metabolically produced H2(17)O in live mice by proton NMR

Previously we suggested a method for indirect detection of H2(17)O via a combination of 1H spin-echo sequence and 17O decoupling. The method is based on the shortening of the water proton T2 due to the residual spin-spin coupling with the 17O nucleus, and on our finding that this shortening is abolished by applying an radio frequency field at the 17O resonance frequency. In the present work we demonstrate how the method can be used for imaging low concentrations of H2(17)O, and report for the first time an application of this method to in vivo detection of H2(17)O in live newborn mice, where the H2(17)O was introduced either directly i.p. or by inhalation of 17O2 where it was formed by oxidative metabolic activity.     

Dissociation between lactate accumulation and acidosis in middle cerebral artery-occluded rats assessed by 31P and 1H NMR metabolic images under a 2-T magnetic field

The relationships among tissue edema, lactate accumulation, and intracellular pH in middle cerebral artery (MCA)-occluded rats were investigated with multiecho 1H magnetic resonance imaging and spatially resolved metabolic images constructed by 1H and 31P nuclear magnetic resonance (NMR) chemical shift imaging (CSI). For the effective and sensitive detection of NMR signals from the brain, outer volume suppression (OVS), reduced k-space sampling and proton irradiation were incorporated into the CSI sequences. The consecutive three measurements of calculated T2 image, lactate image, and pH image which were required for 3.75 h were repeated for four cycles of 1-16 h after MCA occlusion. Tissue edema and lactate accumulation in the infarcted region were gradually and consistently increased during the 15-h observation period. In contrast, severe acidosis was already detected on the first pH image (2-4.7 h after MCA occlusion); thereafter, the degree of acidosis became milder and showed no further progression. The dissociation between the time courses of the lactate accumulation and pH decrease was clearly demonstrated by the NMR metabolic images. Acid-base balance in cerebral infarction might be affected not only by lactate production but also by complicated interactions with tissue edema and some other factors.     

Comparison between the dynamics of lipid/gramicidin A systems in the lamellar and hexagonal phases: a solid-state 13C NMR study

Adolescence is a developmental period marked by multiple challenges and demands which create a heightened vulnerability to the development of emotional disorders. Primary care physicians are in an ideal position to intervene in the early stages and prevent the tragic consequences which can occur with an untreated mental health disorder. This article reviews the assessment and treatment of adolescent mental health in the primary care medical setting. Knowledge of these disorders and their manifestations in the primary care environment will enable clinicians to provide higher quality medical care and will reduce the potential for continual life disruptions into the adult years.     

Structural characterization of an N-acetyl-2-aminofluorene (AAF) modified DNA oligomer by NMR, energy minimization, and molecular dynamics

An N-acetyl-2-aminofluorene (AAF) modified deoxyoligonucleotide duplex, d(C1-C2-A3-C4-[AAF-G5]-C6-A7-C8-C9).d(G10-G11-T12-G13-C14-++ +G15-T16-G17-G18), was studied by one- and two-dimensional NMR spectroscopy. Eight of the nine complementary nucleotides form Watson-Crick base pairs, as shown by NOEs between the guanine imino proton and cytosine amino protons for G.C base pairs or by an NOE between the thymine imino proton and adenine H2 proton for A.T base pairs. The AAF-G5 and C14 bases show no evidence of complementary hydrogen bond formation to each other. The AAF-G5 base adopts a syn conformation, as indicated by NOEs between the G5 imino proton and the A3-H3' and A3-H2'/H2" protons and by NOEs between the fluorene-H1 proton of AAF and the G5-H1' or C6-H1' proton. The NOEs from the C4-H6 proton to C4 sugar protons are weak, and thus the glycosidic torsion angle in this nucleotide is not well defined by these NMR data. The remaining bases are in the anti conformation, as depicted by the relative magnitude of the H8/H6 to H2' NOEs when compared to the H8/H6 to H1' NOEs. The three base pairs on each end of the duplex exhibit NOEs characteristic of right-handed B-form DNA. Distance restraints obtained from NOESY data recorded at 32 degrees C using a 100-ms mixing time were used in conformational searches by molecular mechanics energy minimization studies. The final, unrestrained, minimum-energy conformation was then used as input for an unrestrained molecular dynamics simulation. Chemical exchange cross peaks are observed, and thus the AAF-9-mer exists in more than a single conformation on the NMR time scale. The NMR data, however, indicate the presence of a predominant conformation (> or = 70%). The structure of the predominant conformation of the AAF-9-mer shows stacking of the fluorene moiety on an adjacent base pair, exhibiting features of the base-displacement [Grunberger, D., Nelson, J. H., et al. (1970) Proc. Natl. Acad. Sci. U.S.A. 66, 488-494] and insertion-denaturation models [Fuchs, R.P.P., & Daune, M. (1971) FEBS Lett. 14, 206-208], while the distal ring of the fluorene moiety protrudes into the minor groove.     

可溶性聚醚醚酮改性环氧树脂的研究

采用热熔法制备了一系列可溶性聚醚醚酮(s-PEEK)改性环氧树脂(EP),并与普通聚醚醚酮(PEEK)改性环氧体系进行比较,探讨了聚醚醚酮类型、用量对改性树脂固化体系的凝胶时间、冲击强度、弯曲性能和断裂形貌的影响,并对含s-PEEK树脂体系的玻璃化转变温度(Tg)和热稳定性进行了分析.结果表明,s-PEEK和PEEK可在提高环氧体系冲击性能的同时,提高材料的弯曲性能、玻璃化温度和热稳定性;当m(s-PEEK):m(E-51)和m(PEEK):m(E-51)均为5:100时,冲击强度达到42.6和46.6 kJ/m2,分别比未改性的环氧体系提高69.1%和85.6%;m(s-PEEK):m(E-51)=25:100时,Tg=179.1℃,比未改性环氧树脂提高20℃左右;且含s-PEEK的体系是均相体系,含s-PEEK的固化物是颗粒增强体系.     

Expression of truncated utrophin improves pH recovery in exercising muscles of dystrophic mdx mice: a 31P NMR study

Since the introduction of benzodiacepines in the medical practice their use has been generalized to numerous clinical situations. One of them is schizophrenia. In this article we analize the main settings for its use and the possible mechanisms of action, trying to draw some recomendations applicable to the psychiatric practice.     

Synthesis of a self-gelatinizable grafting copolymer of poly(vinyl alcohol) for construction of an amperometric peroxidase electrode

PURPOSE: To describe the clinical presentation and course of Whipple disease in an adult. METHODS: A 34-year-old man with phthisis bulbi in the right eye secondary to uveitis-induced neovascular glaucoma presented with severe acute posterior uveitis in the left eye. He underwent esophagogastroduodenoscopy and jejunal biopsy for evaluation of anemia. The posterior uveitis was treated with a subtenon injection of triamcinolone. RESULT: The diagnosis of Whipple disease was confirmed by polymerase chain reaction analysis of the jejunal biopsy that demonstrated Tropheryma whippelii rDNA. CONCLUSION: Although Whipple disease is typically evident with malabsorption, it can also present as uveitis without prominent gastrointestinal symptoms.     

The substrate binding site of human liver cytochrome P450 2C9: an NMR study

Purified recombinant human liver cytochrome P450 2C9 was produced, from expression of the corresponding cDNA in yeast, in quantities large enough for UV-visible and 1H NMR experiments. Its interaction with several substrates (tienilic acid and two derivatives, lauric acid and diclofenac) and with a specific inhibitor, sulfaphenazole, was studied by UV-visible and 1H NMR spectroscopy. At 27 degrees C, all those substrates led to an almost complete conversion of CYP 2C9 to high-spin (S = 5/2) CYP 2C9-substrate complexes characterized by a Soret peak at 390 nm; their KD values varied between 1 and 42 microM. On the contrary, sulfaphenazole led to a low-spin (S = 1/2) CYP 2C9 complex upon binding of its NH2 group to CYP 2C9 iron. Interactions of the five substrates with the enzyme were studied by paramagnetic relaxation effects of CYP 2C9-iron(III) on the 1H NMR spectrum of each substrate. Distances between the heme iron atom and substrate protons were calculated from the NMR data, and the orientation of the substrate relative to iron was determined from those distances. Finally, a model for substrate positioning in the CYP 2C9 active site was constructed by molecular modeling studies under the constraint of the iron-proton distances. It points out two structural characteristics for a compound to be selectively recognized by CYP 2C9: (i) the presence of an anionic site able to establish an ionic bond with a putative cationic residue of the protein and (ii) the presence of an hydrophobic zone between the substrate hydroxylation site and the anionic site. Sulfaphenazole was easily included in that model; its very high affinity for CYP 2C9 is due to a third structural feature, the presence of its NH2 function which binds to CYP 2C9 iron.     

Temperature effects on the structure of poly(dA).poly(dT): an X-ray fiber diffraction study

X-ray fiber diffraction of poly(dA).poly(dT) subjected to variation in the relative humidity, has allowed us to demonstrate the effects of temperature on the conformation of the polynucleotide. When the temperature of the poly(dA).poly(dT) is greater than 30 degrees C and the relative humidity near 80%, a new diffraction pattern is obtained. We observe a transition between the classical alpha B' form of poly(dA).poly(dT) and a double helical structure, B*, which remains stable at a temperature up to 70 degrees C. This new conformation of poly(dA).poly(dT) is a right-handed double helix with 11.4 nucleotide pairs per turn and a pitch of 36.7 A.     

Determination of DMPC hydration in the L(alpha) and L(beta'') phases by 2H solid state NMR of D2O

OBJECTIVE: To compare the effect of vaginal misoprostol with that of placebo when used prior to dilation and aspiration in women with a missed abortion. METHOD: Eighty-four pregnant women with a missed abortion were randomized to receive either vaginal misoprostol (200 micrograms) or placebo the day before the planned dilatation and aspiration under inhalation anesthesia. RESULT: Thirty-five women (83.33%) in the misoprostol group and 6 women (17.14%) in the placebo group aborted spontaneously prior to the scheduled dilatation and aspiration, P < 0.0001. The mean insertion to spontaneous expulsion time was 11.63 +/- 6.14 h in the misoprostol group compared to 11.95 +/- 5.43 h in placebo. In the misoprostol group two women required intramuscular pethidine for analgesia. In the placebo group there were two cases of blood loss in excess of 500 ml and one woman with a uterine perforation. CONCLUSION: Vaginal administration of misoprostol to women with a missed abortion produced spontaneous expulsion of the pregnancy and reduced the need for surgical treatment.     

Detection and quantitation of phosphorus metabolites in crude tissue extracts by 1H and 31P NMR: use of gradient assisted 1H-31P HMQC experiments, with selective pulses, for the assignment of less abundant metabolites

The lungs and blood of Yusho patients and healthy Japanese people have already been contaminated with methylsulphonyl polychlorinated biphenyls (MSF-PCBs) at relatively high concentration. Therefore, we should give due attention to their biological and toxicological effects to man. In this study, in order to mainly evaluate non-S-dependent genotoxicity of five MSF-PCB congeners, namely, 3-MSF-4, 5, 3', 4'-tetrachlorobiphenyl (TCB), 3-MSF-4, 5, 2', 3'-TCB, 3-MSF-2, 5, 2', 4', 5'-pentachlorobiphenyl (PenCB), 4-MSF-2, 5, 2', 3', 4'-PenCB and 4-MSF-2, 5, 2', 3', 5', 6'-hexachlorobiphenyl (HCB). We have examined their effects on the induction of micronucleated cells, which has been frequently used to estimate the dose of ionizing radiation and truly radiomimetic, non-S-dependent, clastogens, in cultured human lymphocytes in the absence or presence of 2, 3, 4, 7, 8-pentachlorodibenzofuran (PenCDF), 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) or 3, 4, 5, 3', 4'-pentachlorobiphenyl (Co-PenCB). The following results were obtained. 1) 4 x 10(-5) M7, 8-benzoflavone (ANF) significantly enhanced the frequency of micronucleated cells and all of the five MSF-PCB congeners failed to induce the formation of micronucleated cells at doses of 5.2 to 9.6 ppm, which were about 35,000 times higher than the concentrations in the lungs and adipose tissue of healthy Japanese people.(ABSTRACT TRUNCATED AT 250 WORDS)