Ghrelin to obestatin ratio in maternal serum in pregnancies complicated by intrauterine growth restriction.

Ghrelin, an endogenous for the growth hormone secretagogue receptor, has been shown to participate in fetal growth. Obestatin, encoded by the same gene as ghrelin, is described as a physiological opponent of ghrelin. This study was designed to determine the changes of ghrelin/obestatin ratio in maternal serum in pregnancies with intrauterine growth restriction (IUGR). The authors found that the ghrelin levels in maternal serum were significent lower in IUGR group than in control group (236.34 ± 14.58 pg/ml vs. 321.49 ± 18.19 pg/ml, p = 0.003). However, the difference of obestatin levels in maternal serum in IUGR group than in control group was not significent (276.25, ±20.54 pg/ml vs. 256.34 ± 21.21 pg/ml, p = 0.308). The ratio of ghrelin to obestatin in maternal serum were significent lower in UGR group than in control group (1.05 ± 0.09 vs. 0.82 ± 0.08, p = 0.03). Meanwhile, the maternal serum growth hormone (GH) concentration in IUGR group was lower than that in control group (1.08 ± 0.08 pg/ml vs. 1.41 ± 0.09 pg/ml, p = 0.009), and the maternal serum pla- cental growth hormone (PGH) concentration in IUGR group was lower than that in control group (2.21 ± 1.24 pg/ml vs. 2.92 ± 0.27 pg/ml,p = 0.031). The ratio of ghrelin to obestatin in maternal serum were positively correlation with GH and PGH concentrations in IUGR group (r = 0.876, p = 0.52; r = 0.764, p = 0.64). The findings of this study suggest that the ratio of ghrelin to obestatin in maternal serum were low, and were positively correlated with GH and PGH concentration in IUGR group, which can been considered as evidencees of ghrelin/obestatin balance disorder role in pathogenesis of IUGR.

[Method and significance of specimens standardized pathological treatment in pancreaticoduodenectomy for pancreatic head cancer].

Pancreatic ductal adenocarcinoma is a highly aggressive disease with a grim prognosis. Surgical resection offers the best chance for long-term survival. Negative-margin resection still remains the goal, the influence of margin status on outcomes in pancreatic head carcinoma remains controversial, as conflicting data have been plagued by a lack of standardization in R0 resection and margin definitions, pathologic analysis, and reporting. In contrast to common belief, a high rate of R1 resections in pancreatic cancer is not a marker of low-quality surgery but rather of high-quality pathology. The international pathological consensus of pancreatic head carcinoma is still needed to fully understand the prognostic value of margin status in order to optimize treatment strategy for this disease.

Inhibitor of signal transducer and activator of transcription 3 (STAT3) suppresses ovarian cancer growth, migration and invasion and enhances the effect of cisplatin in vitro.

The aim of the present study was to investigate the anti-ovarian cancer effect of the inhibitor of signal transducer and activator of transcription 3 (STAT3), WP1066. Western blot was used to detect the phosphorylation of STAT3 in ovarian cancer cell line SKOV3 and cisplatin-resistant ovarian cancer cell line SKOV3/DDP. MTT and colony-forming assays were performed to evaluate the viability and growth of ovarian cancer cells. The apoptosis of ovarian cancer cells was determined by flow cytometry. The wound healing assay and Transwell assay were performed to examine the migration and invasion of ovarian cancer cells. WP1066 significantly inhibited the phosphorylation of STAT3 in SKOV3 and SKOV3/DDP cells. WP1066 treatment inhibited the proliferation and clonogenicity of both SKOV3 and SKOV3/DDP cells. After WP1066 treatment for 24 h, the apoptosis rates of SKOV3 and SKOV3/DDP cells were significantly increased compared with the control cells. After treatment with WP1066, the reduction of the wound gaps was significantly less in both SKOV3 and SKOV3/DDP cells. WP1066 also significantly inhibited the invasion capacity of SKOV3 and SKOV3/DDP cells compared with the control group. Treatment with WP1066 combined with cisplatin significantly increased proliferation inhibition and apoptosis in SKOV3 and SKOV3/ DDP cells compared with treatment with cisplatin alone. A synergistic action between WP1066 and cisplatin on the proliferation and apoptosis of ovarian cancer cells was determined. In conclusion, inhibition of STAT3 may suppress the proliferation, migration and invasion, induce apoptosis and enhance the chemosensitivity of ovarian cancer cells, indicating that STAT3 is a new therapeutic target of ovarian cancer.

Ethyl pyruvate administration suppresses growth and invasion of gallbladder cancer cells via downregulation of HMGB1-RAGE axis.

High mobility group box B1 (HMGB1)-receptor for advanced glycation end products (RAGE) axis has been previously known to be involved in carcinogenesis and development of multiple malignancies. Some studies have confirmed that Ethyl pyruvate (EP), a potent inhibitor of HMGB1, exerts the therapeutic effects on metastatic live tumor from gastric cancer. However, the effects and possible molecular mechanisms of EP on gallbladder cancer (GBC) need to be further explored. In the present study, human GBC cell lines (GBC-SD and SGC-996) were treated with different concentrations of EP. Then, the expression levels of HMGB1, RAGE and some transcription factors were identified by Real-time PCR and Western blot assays. Cell proliferative activities indicated by MTT assay, invasive potential by Transwell assay and cell apoptosis and cycle distribution were performed for functional analysis of GBC cell lines in vitro. As a result, EP decreased the expression of HMGB11, RAGE, PCNA and matrix metallopeptidase-9 (MMP-9), while it increased the expression of p53. Moreover, EP administration decreased GBC cell proliferation, inhibited the invasive potential, and induced apoptosis and cycle arrest in S phase in GBC cells. In conclusion, EP administration inhibits growth and invasion of gallbladder cancer cells possibly via down-regulation of the HMGB1-RAGE axis, suggesting that EP may play a critical role in the treatment of cancer in conjunction with other therapeutic agents.

Three-dimensional cavity nanoantennas with resonant-enhanced surface plasmons as dynamic color-tuning reflectors.

As plasmonic antennas for surface-plasmon-assisted control of optical fields at specific frequencies, metallic nanostructures have recently emerged as crucial optical components for fascinating plasmonic color engineering. Particularly, plasmonic resonant nanocavities can concentrate lightwave energy to strongly enhance light-matter interactions, making them ideal candidates as optical elements for fine-tuning color displays. Inspired by the color mixing effect found on butterfly wings, a new type of plasmonic, multiresonant, narrow-band (the minimum is about 45 nm), high-reflectance (the maximum is about 95%), and dynamic color-tuning reflector is developed. This is achieved from periodic patterns of plasmonic resonant nanocavities in free-standing capped-pillar nanostructure arrays. Such cavity-coupling structures exhibit multiple narrow-band selective and continuously tunable reflections via plasmon standing-wave resonances. Consequently, they can produce a variety of dark-field vibrant reflective colors with good quality, strong color signal and fine tonal variation at the optical diffraction limit. This proposed multicolor scheme provides an elegant strategy for realizing personalized and customized applications in ultracompact photonic data storage and steganography, colorimetric sensing, 3D holograms and other plasmon-assisted photonic devices.

Mechanism of hemolysis of red blood cell mediated by ethanol.

The effects of ethanol on hemolysis of human red blood cells (RBCs) were studied at 21 +/- 1 degrees C in the saline buffer (138 mM NaCl, 6.1 mM Na2HPO4, 1.4 mM NaH2PO4, 5 mM glucose and pH 7.4). The hemolysis process for ethanol-treated RBCs was preceded by the leakage of the small cation K+ from the cells indicating the colloid-osmotic nature of lysis. Since the extent of membrane lesion increased with an increasing ethanol concentration, osmotic protection experiments by using solutes varying in size were carried out to estimate the diameter of the pore. Quantitative analysis of the data by considering the effect of molecular seiving of the protectants with different sizes indicated that ethanol induced formation of membrane pores with a diameter of approximately 13 A. There was no detectable release of membrane fragments as assayed by the acetylcholinesterase activity, but the membrane structures were significantly perturbed, presumably at the membrane cytoskeletal protein, as evidenced by the altered rheological properties of RBC in the presence of ethanol. It is suggested that the creation of membrane pores might involve in the deranged cytoskeletal network of ethanol-treated RBC.

Comparisons of lipid dynamics and packing in fully interdigitated monoarachidoylphosphatidylcholine and non-interdigitated dipalmitoylphosphatidylcholine bilayers: cross polarization/magic angle spinning 13C-NMR studies.

13C-NMR spectra have been obtained at 50.3 MHz for monoarachidoylphosphatidylcholine (MAPC) and dipalmitoylphosphatidylcholine (DPPC) dispersions from 25 degrees C to 55 degrees C and for DPPC polycrystals at 25 degrees C using the cross polarization/magic angle spinning technique. Differential scanning calorimetric studies on DPPC and MAPC dispersions show comparable lipid phase transitions with transition temperatures at 41 degrees C and 45 degrees C, respectively, and thus enable the comparison of thermal, structural and dynamic differences between these two systems at corresponding temperatures. Conformational-dependent 13C chemical shift studies on DPPC dispersions demonstrate not only the coexistence of the tilted gel (L beta') and liquid-crystalline (L alpha) phases in the rippled gel (P beta') phase, but also the presence of an intermediate third microscopic phase as evidenced by three resolvable peaks for omega - 1 methylene carbon signals at the temperature interval between Tp and Tm. By comparing chemical shifts of MAPC in the hydrocarbon chain region with those of DPPC at similar reduced temperatures, it can be concluded that the packings are perturbed markedly in the middle segment of the fatty acyl chain during the lamellar to micellar transition. However, terminal methylene and methyl groups of interdigitated MAPC lamellae were found to be more ordered than those of non-interdigitated DPPC bilayers in the gel state. Interestingly, the terminal methyl groups of MAPC in the micelles remain to be relatively ordered; in fact, they are more ordered than the corresponding acyl chain end of DPPC in the liquid-crystalline state. Analysis of data obtained from rotating frame proton spin-lattice relaxation measurements shows a highly mobile phosphocholine headgroup, a rigid carbonyl group and an ordered hydrocarbon chain for lamellar MAPC in the interdigitated state. Furthermore, results suggest that free rotations of the glycerol C2-C3 bond within MAPC molecules may occur in the interdigitated bilayer, whereas intramolecular exchange between two conformations of the glycerol backbone in DPPC become possible at temperatures close to the pretransition temperature. Without isotope enrichment, we conclude that high-resolution solid-state 13C-NMR is indeed a useful technique which can be employed to study the packing and dynamics of phospholipids.

Biophysical correlates of lysophosphatidylcholine- and ethanol-mediated shape transformation and hemolysis of human erythrocytes. Membrane viscoelasticity and NMR measurement.

The effects of monopalmitoylphosphatidylcholine (MPPC or lysophosphatidylcholine) and a series of short-chain primary alcohols (ethanol, 1-butanol and 1-hexanol) on cell shape, hemolysis, viscoelastic properties and membrane lipid packing of human red blood cells (RBCs) were studied. For MPPC, the effective membrane concentration to induce the formation of stage 3 echinocytes (8 x 10(6) molecules per cell) was one order of magnitude lower than that needed to induce 50% hemolysis (7 x 10(7) molecules per cell). In contrast, short-chain alcohols induced both shape changes and hemolysis within close concentration range (2.5 x 10(8) to 3.5 x 10(8) molecules per cell). Viscoelastic properties of the RBCs were studied by micropipette aspiration and correlated with shape change. Ethanol-treated RBCs showed a decrease in membrane elastic modulus and an increase in membrane viscosity in the recovery phase at the early stage of shape change. MPPC-treated cells showed the same type of viscoelastic changes, but these were not observed until the formation of stage 2 echinocytes. High-resolution solid-state 13C nuclear magnetic resonance technique was applied to study membrane lipid packing in the ghost membrane by following the chemical shift of hydrocarbon chains. Both MPPC and ethanol caused the 13C-NMR chemical shift to move upfield, indicating that membrane lipids were expanded due to the intercalation of these exogenous molecules. Using data obtained from model compounds, we convert values of chemical shift into a lipid packing parameter, i.e., number of gauche bonds for fatty acyl hydrocarbon chains. Approximately 10(8) interacting molecules per cell are required to induce a detectable change of lipid packing by both MPPC and ethanol. The results indicate that homolysis occurs at a smaller surface area for MPPC- than ethanol-treated RBCs. Our findings suggest that progressive changes in the molecular packing in the membrane lead eventually to hemolysis, but the mode responsible for shape transformation varies with these amphipaths.

Preliminary crystallographic analysis of cardiotoxin V with major fusion activity from Taiwan cobra (Naja naja atra) venom.

Crystals of a cardiotoxin from Taiwan cobra venom have been obtained by the vapor diffusion method using methyl pentanediol as precipitant. The crystals belong to the hexagonal space group P6(1)22 (or P6(5)22), with cell dimensions a = b = 47.5 A, c = 111.3 A, alpha = beta = 90 degrees and gamma = 120 degrees and diffract to a resolution of 2.2 A. There is one molecule per asymmetric unit and the solvent content is estimated to be 53%.

C-CAM1, a candidate tumor suppressor gene, is abnormally expressed in primary lung cancers.

Previous studies have shown that the expression of the cell-cell adhesion molecule (C-CAM1), located at chromosome 19, is down-regulated in several types of human cancers, including prostate and breast cancers. Two major isoforms of C-CAM1, the long or L-form C-CAM1 and the short or S-form C-CAM1, are derived from the C-CAM1 gene through alternative splicing. Tumor cells transfected with L-form C-CAM1, which contains a cytoplasmic domain, display significantly lower growth rates and less tumorigenicity in both in vitro and in vivo models compared with untransfected tumor cells, suggesting that L-form C-CAM1 may be a tumor suppressor. The transfection of the cytoplasmic domain of L-form C-CAM1 could also cause suppression of tumor growth, further supporting the role of L-form C-CAM1 in tumorigenesis. In contrast to reports of most of the tumor types tested, Ohwada et al. (Am. J. Respir. Cell Mol. Biol., 11: 214-220, 1994) reported that C-CAM1 was not down-regulated or even up-regulated in lung cancer. Because the cytoplasmic domain of L-form C-CAM1 is critical for the tumor suppressor function of C-CAM1, we hypothesized that switching of the isoform rather than down- regulation of C-CAM1 gene expression occurs during lung tumorigenesis. To test this hypothesis, we analyzed pairs of tumor tissue and corresponding normal-appearing lung tissue from 51 patients with non-small cell lung cancer (NSCLC) and 43 cell lines to determine expression profiles of L-form C-CAM1 and S-form C-CAM1 using reverse transcription-PCR. We found that L-form C-CAM1 was the predominant form (75%; 38 of 51) in normal-appearing lung tissue, whereas most (84%; 43 of 51) of the primary NSCLC tissue samples expressed predominantly S-form C-CAM1 (P < 0.0001). Similarly, 19 (79%) of the 24 NSCLC cell lines and 17 (85%) of the 20 small cell lung cancer cell lines expressed predominantly S-form C-CAM1. The frequent alteration of the C-CAM1 expression pattern suggests that C-CAM1 has an important role in lung tumorigenesis.

Cobra cardiotoxin and phospholipase A2 as GAG-binding toxins: on the path from structure to cardiotoxicity and inflammation.

Glycosaminoglycans (GAGs) represent the sulfated carbohydrate moieties of proteoglycans which occur abundantly in tissues of the cardiovascular system. Many proteins bind specifically to GAGs and perform an important role in inflammation, cell proliferation, and blood coagulation processes. Recently, in vitro GAG-binding studies of cardiotoxins (CTXs) and basic phospholipase A(2) (PLA(2)) from cobra venom established the toxins as two new families of GAG-binding proteins. In particular, discontinuous basic residues in beta-sheet CTXs may form a cationic cradle suitable for heparin binding, as in the case of fibronectin module III-13. The binding specificity of beta-sheet proteins to different GAGs can be further enhanced by involving other cationic clusters near the flexible loop of the molecule. Since the three-dimensional structures of many CTXs and PLA(2) are available, these two toxins may serve as models for the elucidation of the molecular recognition of GAG-binding proteins and also as polypeptide templates for further improvement of the binding specificity suitable for future biomedical application. Research along the line of GAG-guided toxicity of cobra venom components may help us to understand the functional role of GAGs and the action mechanism of cobra venom components in the cardiovascular system.

Heparin binding to cobra basic phospholipase A2 depends on heparin chain length and amino acid specificity.

Heparin is shown to bind specifically to the carboxy-terminal region of toxic type I phospholipase A2 from Naja nigricollis (N-PLA2) by competition assay using synthetic polypeptides and heparin affinity chromatography. The binding strength is seen to depend on heparin chain length and the presence of N-sulfate groups of heparin. It is observed that both electrostatic and non-electrostatic interactions are involved in the specific binding of heparin to the carboxy-terminus. When heparin's size is at least a decasaccharide, about two molecules of N-PLA2 bind to one molecule of heparin, as evidenced by the chemical estimate of protein to carbohydrate ratio in such N-PLA2/heparin complexes. Based on such a stoichiometric measurement and computer modeling of the N-PLA2/heparin complex, it is suggested that the binding sites of the two N-PLA2 molecules on one heparin molecule lie on the opposite sides of the heparin chain.

Expression of glutathione S-transferase-cardiotoxin fusion protein in Escherichia coli.

We report here the construction of cardiotoxin V gene, from cobra snake venom (Naja naja atra), by chemically synthesized oligonucleotides and its expression as a glutathione S-transferase-cardiotoxin fusion protein in the inclusion bodies of Escherichia coli. The expression of cardiotoxin fusion protein in protein with a yield of about 35 mg/liter culture was confirmed by highly specific anti-peptide antibodies generated against the unique amino acid residues located at the tip of loop II of cardiotoxin V. Since the fusion protein can be easily treated by CNBr to free the toxin moiety, as revealed by immunoblotting of the cleaved protein, the results provide an avenue for future structural and functional studies of cardiotoxin molecules.

Effect of aging on phosphate metabolites of rat brain as revealed by the in vivo and in vitro 31P NMR measurements.

Changes of phosphate metabolism in brains of neonate, weaning and adult rats were compared using both in vivo and in vitro nuclear magnetic resonance spectra. Ratios of phosphocreatine/nucleoside triphosphate (PCr/NTP) were the same in neonatal brain in both in vivo and in vitro studies, but not in weaning and adult brains. This discrepancy may have resulted from extended cerebral hypoxia due to slowed freezing of the brain by the increased skull thickness and brain mass in the weaning and adult rats. Variations in in vitro extraction condition for this age-related study may lead to systematic errors in the adult rats. Nevertheless, the phosphomonoester/nucleoside triphosphate (PME/NTP) ratios in extracts of brain from neonatal rats were higher than those obtained in vivo. In addition, the glycerophosphorylethanolamine plus glycerophosphorylcholine/nucleoside triphosphate (GPE+GPC/NTP) ratios, which were not measurable in vivo, showed age-dependent increase in extracts of rat brain. Some of the phosphomonoester and phosphodiester molecules in rat brain may be undetectable in in vivo NMR analysis because of their interaction with cellular components. The total in vitro GPE and GPC concentration in brain from neonatal rat was estimated to be 0.34 mmole/g wet tissue.

TRAIL-R2 is not correlated with p53 status and is rarely mutated in non-small cell lung cancer.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors play an important role in regulating apoptosis. Recently, it was shown that the expression of TRAIL-R2, also known as KILLER, Trick or DR5, can be induced by either DNA damage or overexpression of a wild-type p53 transgene, suggesting a role for p53 in the death-signaling pathway. Furthermore, mutations in the death domain of TRAIL-R2 were reported in 10.6% of non-small cell lung cancer (NSCLC) patients in a Korean population, suggesting a role for TRAIL-R2 in lung tumorigenesis. MATERIALS AND METHODS: To determine the association between expression of TRAIL-R2 and p53 mutation status in lung cancers, we compared the two events in 20 small-cell lung cancer (SCLC) cell lines, 20 NSCLC cell lines, and 30 primary NSCLC tumors. We also sequenced the death domain of TRAIL-R2 in a total of 100 primary NSCLC. RESULTS: Lack of TRAIL-R2 expression was found in eight of 20 (40%) SCLC cell lines and in eleven of 20 (55%) NSCLC cell lines. Interestingly, in primary NSCLC, TRAIL-R2 was overexpressed in seven (23%) of the 30 tumors tested, and all primary tumors expressed TRAIL-R2. No association was found between the expression status of TRAIL-R2 and p53 mutation status in primary NSCLC tumors, SCLC cell lines or NSCLC cell lines. Further analysis of the death domain of TRAIL-R2 failed to identify any mutation in 100 primary NSCLC tumors. CONCLUSIONS: Our data indicate that the expression profile of TRAIL-R2 is significantly different in lung cancer cell lines and primary tumors, that the expression of TRAIL-R2 is independent from p53 mutation status and that mutations in the death domain of TRAIL-R2 play a minimal role in NSCLCs in white Americans.

Heparin reduces the alpha-helical content of cobra basic phospholipase A(2) and promotes its complex formation.

The interaction of phospholipase A(2) (PLA(2)) with glycosaminoglycans (GAGs) has recently attracted attention in view of its implication on inflammation and cell proliferation. By using Fourier Transformed Infrared (FTIR) spectroscopic measurements, we demonstrate here that binding of cobra basic phospholipase A(2) from Naja nigricollis (N-PLA(2)) to heparin may induce a significant conformational change observed in the amide I region of the enzyme's alpha-helical and beta-sheet structure. It is observed that notable conformational change of N-PLA(2) due to heparin binding occurs only when heparin's chain length is at least an octasaccharide as evidenced by circular dichroism and optical density measurements. This correlation may be an important factor in the aggregation of N-PLA(2) and N-PLA(2)-heparin complexes. Heparin induced change in conformation of PLA(2) is suggested to be a notable link in understanding the diversity in PLA(2) activity when rendered to the extracellular matrix of cell membranes that is full of GAG molecules.

Solvent effect on phosphatidylcholine headgroup dynamics as revealed by the energetics and dynamics of two gel-state bilayer headgroup structures at subzero temperatures.

The packing and dynamics of lipid bilayers at the phosphocholine headgroup region within the temperature range of -40 to -110 degrees C have been investigated by solid-state nuclear magnetic resonance (NMR) measurements of selectively deuterium-labeled H2O/dimyristoylphosphatidylcholine (DMPC) bilayers. Two coexisting signals with 2H NMR quadrupolar, splittings of 36.1 and 9.3 (or smaller) kHz were detected from the -CD3 of choline methyl group. These two signals have been assigned to two coexisting gel-state headgroup structures with fast rotational motion of -CD3 and -N(CD3)3 group, respectively, with a threefold symmetry. The largest quadrupolar splitting of the NMR signal detected from the -CD2 of C alpha and C beta methylene segment was found to be 115.2 kHz, which is 10% lower than its static value of 128.2 kHz. Thus, there are extensive motions of the entire choline group of gel-state phosphatidylcholine bilayers even at a subzero temperature of -110 degrees C. These results strongly support the previous suggestion (E. J. Dufourc, C. Mayer, J. Stohrer, G. Althoff, and G. Kothe, 1992, Biophys. J. 61:42-57) that 31P chemical shift tensor elements of DMPC determined under similar conditions are not the rigid static values. The free energy difference between the two gel-state headgroup structures was determined to be 26.3 +/- 0.9 kJ/mol for fully hydrated bilayers. Furthermore, two structures with similar free energy difference were also detected for "frozen" phosphorylcholine chloride solution in a control experiment, leading to the conclusion that the two structures may be governed solely by the energetics of fully hydrated phosphocholine headgroup. The intermolecular interactions among lipids, however, stabilize the static headgroup structure as evidenced by the apparently lower free energy difference between the two structures for partially hydrated lipid bilayers. Evidence is also presented to suggest that one of the headgroup structures with trimethylammonium group rotation, which is not compatible with the static headgroup structure in crystals, is due to the dielectric relaxation of the slowly reorienting inter bilayer water molecules near the physical edge of membrane surface. Finally, a molecular model of the hydration-induced conformational changes at the torsion angle a5 of the O-C-CN+ bond is proposed to explain the two detected coexisting headgroup structures. These results emphasize the important role of the trimethylammonium group in monitoring the structure and dynamics of the lipid headgroup.

Molecular order and hydration property of amine group in phosphatidylethanolamine and its N-methyl derivatives at subzero temperatures.

The molecular order and hydration properties of the amine group in phosphatidylethanolamine and its N-methyl derivatives were studied by 2H-NMR at subzero temperatures. Three coexisting signals with 2H-NMR quadrupolar splittings of 146, 106, and 28.8 KHz were detected from the fully hydrated phosphatidylethanolamine/D2O at the lowest studied temperature of -120 degrees C by using short recycle time in the applied NMR pulse sequence. These signals have been assigned to originate from frozen D2O in the interbilayer space and the deuterated amine group, i.e., -ND, with and without threefold symmetric motions. Comparative 2H-NMR studies of phosphatidylethanolamine/D2O with different degrees of methylation over a temperature range between -40 and -120 degrees C lead to the following conclusions. First, the bond angle of -D attached to the nitrogen atom of the amine group may be determined by the 2H-NMR quadrupolar splittings, i.e., 106 and 28.8 KHz, of the two coexisting signals of the deuterated amine group and found to be 112.9 for the gel-state phosphatidylethanolamine. Second, assuming the applicability of the empirical equation for the hydrogen bond distance of N+D--O with deuteron quadrupole coupling constants and using the intermolecular hydrogen bond distance of the amine group determined in single crystals of phosphatidylethanolamine bilayers, the largest measured quadrupolar splitting (delta nu Q) of N-D in this study, i.e., 106 KHz, is close to the static value. This interpretation is also consistent with the fact that the delta nu Q value determined remains constant in the temperature range between -70 and -120 degrees C. Third, the molecular order parameter of the amine group, as calculated from the ratio of the libration-averaged and static delta nu Q value for the lipid with different degrees of methylation, suggests that the perturbation of the headgroup interaction is most significant for the final methylation step. Finally, measurement of the spectral intensity of isotropic unfrozen D2O signals in D2O/phospholipid dispersions at temperatures below the homogeneous nucleation temperature of ice formation for D2O, i.e., below -34 degrees C, suggests that the first methylation step perturbs the neighboring water most significantly. Assuming that the molecular order of the amine group and the amount of unfrozen water detected under the present experimental condition can be taken as a measure of the hydrogen-bonding ability and the extent of perturbation caused by the methyl group, respectively, the gradual methylation of the amine group perturbs the interactions of the N-methylated headgroups in a nonlinear fashion. The results provide a molecular explanation for the phase behavior of phospholipids with different degrees of methylation.

A new method of superoxide free radical determination in ischemia and reperfusion injury of rat liver.

The present study was designed to utilize the chemiluminescence (CL) method for O2- analysis in ischemia and reperfusion of the whole rat liver. The results indicated that the O2- level was lower after 30 and 60 min ischemia. After 40 min reperfusion, the level of O2- increased obviously, reaching about 8.3-fold (30 min ischemia) and 9.0-fold (60 min ischemia) (P < 0.001, P < 0.01) respectively. The results also show that Chinese Traditional Medicines ligustrazine and salvia compound can scavenge O2- effectively.