Genetic basis of the MbrC "ploidy" phenotype in Escherichia coli

We measured the spin-lattice relaxation times (T1) of water protons and intermolecular cross-relaxation times (T(IS)) from irradiated protein protons (f2-irradiation at 1.95 or -4.00 ppm) of rabbit normal and monoiodoacetate-induced degenerated knee articular cartilages to observed water protons. The mean values of T1 (T1) for control and degenerated rabbit knee cartilages were 1.87+/-0.15 (mean+/-SD, n=29) and 1.82+/-0.13 s (n=34), respectively. The mean values of water content (W(H2O)) for control and degenerated rabbit knee cartilages were 82.9+/-2.09 (n=26) and 83.1+/-2.57% (n=28), respectively. These values were not statistically different from each other. However, the mean values of T(IS) (T(IS)) for normal knee articular cartilage were significantly different from those for degenerated cartilage: (normal), T(IS) (f2=1.95 ppm)=2.46+/-0.62 s (n=28), T(IS) (f2=-4.00 ppm)=4.25+/-1.26 s (n=26); (degenerated), T(IS) (f2=1.95 ppm)=1.99+/-0.76s (n=34), T(IS) (f2=-4.00 ppm)=3.33+/-0.76 s (n=31). Obtained results may be attributed to the reported switchover from type II to type I collagen syntheses in osteoarthritic cartilage, resulting in broad collagen fibers. This specificity of cross-relaxation effect may prove useful in the noninvasive and pathophysiological evaluation of cartilage tissues in vivo.     

Structure of gene 5 protein-oligodeoxynucleotide complexes as determined by 1H, 19F, and 31P nuclear magnetic resonance

1H nuclear magnetic resonance (NMR) spectra at 270 MHz of gene 5 protein from bacteriophage fd and its complexes with tetra- and octadeoxynucleotides show that approximately 12 of the 37 aromatic protons of the protein undergo upfield shifts upon nucleotide binding. In the complex with d(pT)8, the upfield shifts of the aromatic protons average approximately 0.3 ppm, while in the d(pA)8 complex the same resonances (assigned to tyrosyl protons) shift upfield approximately 0.8 ppm. These are interpreted as ring current shifts induced by stacking of the phenyl rings of three of the five tyrosyl residues with the bases of the nucleotides. 19FNMR of m-fluorotyrosyl gene 5 protein shows five separate resonances: two downfield from m-fluorotyrosine corresponding to "buried" tyrosyls and three near m-fluorotyrosine corresponding to "surface" tyrosyls. The latter (assigned to Tyr-26, -41, and -56, shown by chemical modification to be exposed to solvent) move upfield on nucleotide binding. The downfield 19F resonances are unaffected. Thus the aromatic protons shifted upfield on nucleotide binding appear to be those of Tyr-26, -41, and -56. In contrast to tetra-, octanucleotide binding to gene 5 protein induces large changes in the 1H resonances of the -CH3 groups of the Val, Leu, and Ile side chains. These may reflect conformational changes induced by protein-protein interactions between two monomers bound to the octanucleotide. 1H resonances of the epsilon-CH2 groups of the lysyl residues in the protein and the complexes with nucleotides are narrow with long T2 values, suggesting considerable rotational motion. Thus epilson-NH3+-phosphate interactions, if they occur, are on the surface of the complex and allow the epsilon-CH2 groups to retain considerable rotational freedom. 31P NMR of the bound nucleotides shows large decreases in T1 for the 3'-5' diesters, but little chemical shift suggesting no unusual distortion of the nucleotide backbone on binding to gene 5 protein. A three-dimensional model of a gene 5 protein-octanucleotide complex has been built based on predictions of the secondary structure from the amino acid sequence (87 AA) and tertiary folding dictated by known chemical and NMR features of the complex.     

Quantitative analysis of apoptotic cell death using proton nuclear magnetic resonance spectroscopy

Quantification of apoptotic cell death in vivo has become an important area of investigation in patients with acute lymphoblastic leukemia (ALL). We have devised a noninvasive analytical method to estimate the percentage of apoptotic lymphoblasts in doxorubicin-treated Jurkat T-cell ALL cultures, using proton nuclear magnetic resonance spectroscopy (1H NMR). We have found that the ratio of the methylene (CH2) resonance (at 1.3 ppm) to the methyl (CH3) resonance (at 0.9 ppm) signal intensity, as observed by 1H NMR, is directly proportional to the percentage of apoptotic lymphoblasts in vitro. The correlation between the CH2/CH3 signal intensity ratio and the percentage of apoptotic lymphoblasts was optimal 24 to 28 hours after doxorubicin treatment (r2 = .947, N = 27 samples). There was also a direct temporal relationship between an increase in the CH2/CH3 signal intensity ratio and the onset of apoptosis as detected by nuclear morphologic analysis, fluorescein-annexin V flow cytometry, and DNA gel electrophoresis. Thin-layer chromatography confirmed that a dynamic and/or compositional change of the plasma membrane, rather than increases in lipase activity or fatty acid production, appears to account for the increase in the CH2/CH3 signal intensity ratio during apoptosis. 1H NMR may have clinical utility for the early noninvasive assessment of chemotherapeutic efficacy in patients with ALL.     

sst2 somatostatin receptor expression reverses tumorigenicity of human pancreatic cancer cells

Among the five cloned somatostatin receptor subtypes (sst1 to sst5), sst2 mediates the antiproliferative effect of somatostatin analogues in vitro. Somatostatin analogues have been shown to inhibit cell growth in vitro and in vivo in pancreatic cancer models that expressed sst2. We recently demonstrated the loss of sst2 gene expression in human pancreatic adenocarcinomas and most of the derived pancreatic cancer cell lines. In the present study, we corrected the sst2 defect in human pancreatic cancer BxPC-3 and Capan-1 cells by stable transfection with human sst2 cDNA. In the absence of exogenous ligand, both BxPC-3 and Capan-1 cells expressing sst2 showed a significant reduction in cell growth. This inhibitory effect was blocked by treatment with antiserum to somatostatin. sst2-expressing cells produced somatostatin-like immunoreactivity that mainly corresponded to somatostatin 14, indicating the induction of a negative autocrine loop. In other respects, sst2 expression in Capan-1 cells induced a significant reduction of clonogenicity in soft agar. Moreover, a significantly reduced (Capan-1 cells) or suppressed (BxPC-3 cells) tumor growth in athymic nude mice was observed. The reversal of tumorigenicity induced by the restoration of sst2 expression suggests that the loss of sst2 contributes to the malignancy of human pancreatic cancers.     

Successful islet autotransplantation in humans: functional insulin secretory reserve as an estimate of surviving islet cell mass

Bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, can inhibit the growth of a variety of cultured cells in a dose-dependent manner, but its mechanism is unclear. The aim of this study was to examine whether bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis. The effect of bafilomycin A1 on tumour growth in vitro and in vivo was examined using an MTT assay and an in vivo tumour model. The presence or absence of apoptosis was determined by morphology and DNA analysis of tumour cells. The concentration of bafilomycin A1 for 50 per cent inhibition of cell viability during 72 h by the MTT assay was 5 nm. In DNA analysis, a ladder of fragmented DNA was detected in Capan-1 cells treated with bafilomycin A1 at concentrations greater than 10 nm for 24 h. Nude mice bearing a xenografted Capan-1 cell line tumour received 4 weeks of bafilomycin A1 (1.0 mg/kg per day). This treatment significantly inhibited tumour growth compared with controls after 21 days (P < 0.05). Histopathological examination of tumour cells in the treated group demonstrated signs of apoptosis with chromatin condensation and cell shrinkage. These observations suggest that bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis.     

DNA duplex dynamics: NMR relaxation studies of a decamer with uniformly 13C-labeled purine nucleotides

Dynamics in a DNA decamer duplex, d(CATTTGCATC). d(GATGCAAATG), were investigated via a detailed 13C NMR relaxation study. Every 2'-deoxyadenosine and 2'-deoxyguanidine was chemically enriched with 15% 13C and 98% 15N isotopes. Six nuclear relaxation parameters [R(13Cz), R(1Hz), R(2(1)Hz13Cz), R(13Cx), R(2(1)Hz13Cx) and steady-state 13C?1H? NOE] were measured at 600 MHz and three were measured at 500 MHz (1H frequency) for the CH spin systems of sugar 1', 3', and 4' as well as base 8 and 2 positions. A dependence of relaxation parameter values on chemical position was clearly observed; however, no sequence-specific variation was readily evident within our experimental error of approximately 5-10%, except for 3' and 5' termini. It was demonstrated that the random 15% 13C enrichment effectively suppressed both scalar and dipolar contributions of the neighboring carbons and protons on the relaxation parameters. To analyze dynamics via all observed relaxation parameters, full spectral density mapping (1992, J. W. Peng and G. Wagner, J. Magn. Reson. 98, 308) and the "model-free" approach (1982, Lipari and Szabo, J. Am. Chem. Soc. 104, 4546) were applied complementarily. A linear correlation between three spectral density values, J(omegaC), J(omegaH - omegaC), and J(omegaH + omegaC) was observed in plots containing all measured values, but not for the other spectral density terms including J(0). These linear correlations reflect the effect of overall motion and similar internal motions for each CH vector in the decamer. The correlations yielded two correlation times, 3-4 ns and 10-200 ps. One value, 3-4 ns, corresponds to the value of 3.3 ns obtained for the overall isotropic tumbling correlation time determined from analysis of 13C T1/T2 ratios. The possibility of overall anisotropic tumbling was examined, but statistical analysis showed no advantage over the assumption of simple isotropic tumbling. Lack of correlations entailing J(0) implies that a relatively slow chemical exchange contributes to yielding of effective Jeff(0) values. Based on spectral density mapping and the T1/T2 ratio analysis, three basic assumptions were initially employed (and subsequently justified) for the model-free calculation: isotropic overall tumbling, one internal motion, and the presence of chemical exchange terms. Except for terminal residues, the order parameter S2 and the corresponding fast internal motion correlation time were determined to be about 0.8 +/- 0.1 and 20 +/- 20 ps, respectively, for the various CH vectors. Only a few differences were observed between or within sugars and bases. The internal motion is very fast (ps-ns time scale) and its amplitude restricted; e.g., assuming a simple wobble-in-a-cone model, the internal motion is restricted to an angular amplitude of +/-22. 5 degrees for each of the 1', 3', 4', 2, and 8 positions in the purine nucleotides in the entire duplex.     

Three-dimensional solution structure and stability of phage 434 Cro protein

1H NMR resonances of the phage 434 Cro protein were assigned using standard 2D NMR methods, and its solution structure determined using 867 distance constraints in distance geometry (DIANA) calculations ultimately refined by restrained molecular dynamics (GROMOS). In the 20 best NMR structures, the average pairwise backbone and heavy atom RMSDs are 0.63 +/- 0.14 and 1.53 +/- 0.15 A, respectively, for the structurally well-defined residues 4-65. Residues 1-3 and 66-71 at the N- and C-termini are structurally disordered. The region 4-65 includes five alpha-helices and tight turns which define the hydrophobic core of the protein. The backbone and heavy atom RMSDs for residues 4-65 are 0.92 +/- 0.12 and 1.99 +/- 0.12 A, respectively, for the NMR versus the crystal structures, but there are significant differences in the side-chain conformations and solvent accessibilities for some core residues. Analytical ultracentrifugation experiments confirm that 434 Cro is monomeric even at the high NMR concentrations. 434 Cro folding under NMR solution conditions is two-state as indicated by coincident urea denaturation curves from circular dichroism and intrinsic fluorescence measurements. They yield values for 434 Cro stability which show good correspondence to the free energy for global unfolding determined by NMR hydrogen exchange measurements for the slowest exchanging amide protons.     

Octoxynol presence in bear-bile

The presence of octoxynol from dried bear-bile was examined. Octoxynol was coextracted when glycolipids by Folch-Suzuki partition method. Octoxynol formed mixed-micelles with glycosphingolipids. The glycolipids were purified by DEAE-Sephadex A-25 column chromatography. The fractions containing mixed micelles were obtained from linear gradient solvent of 0.05M-0.5M ammonium acetate in methanol. HPLC ( Bondapak-NH(2) - linked to a Bondapak-C(18) column) chromatogram showed five peaks. Two possible structures for the fourth peak fraction were proposed as (CH(3))(3)C-CH(2)-C(CH(3))(2)-C(6)H(4)-OR and (CH(3))(3)C-C(CH(3))(2)-CH(2)-C(6)H(4)-OR by NMR spectroscopy. The structure was further confirmed by electrospray tandem mass spectrometry (ESI MS/MS). The spectrum showed a protonated molecule at m/z 559 and three different series of ions with mass difference of 44 were detected in the MS/MS spectrum. Therefore, the structure of the fourth peak fraction from HPLC was confirmed as octoxynol, (CH(3))(3)C-CH(2)-C(CH(3))(2)-C(6)H(4)-(OCH(2)-CH(2))n-OH, based on mass spectrometry and NMR spectroscopy.     

Effect of ischemic preconditioning and PKC activation on acidification during ischemia in rat heart

Ischemic preconditioning (PC) has been shown to attenuate intracellular acidification during a subsequent period of ischemia, to minimize stunning, and to decrease infarct size, PKC activation has been suggested to be involved in this phenomenon. The present study is designed to test whether PKC activation could mimic and PKC inhibition could block the PC effects on intracellular acidification during ischemia and on stunning during reflow in Langendorff perfused rat hearts. Prior to 20 min of sustained global normothermic ischemia, groups of hearts were treated with the PKC activators 4 beta-phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoyl-srt-glycerol (DOG), a group of hearts was treated with the PKC inhibitor chelerythrine (CH), a group was treated with DOG plus CH, a group was preconditioned with four cycles of 5 min of ischemia and 5 min of reflow, and a group was treated with CH during PC. Recovery of left ventricular developed pressure (% of initial, pretreatment, preischemic LVDP), measured after 20 min of reflow, was improved in hearts treated with DOG, but not PMA (80 +/- 3% (DOG), 55 +/- 3% (PMA) v 51 +/- 3% (control), P < 0.05 between DOG and control), although both caused a similar degree of PKC translocation (measured by fractionation followed by an assay of PKC activity using incorporation of 32P into histone). The improved recovery of LVDP in the PC group and in the DOG group was blocked by chelerythrine. Measurement of pH (by 31P NMR) showed that DOG reduced acidification at 15-20 min of ischemia, although the effect was not as great as PC, while PMA did not reduce acidification. The effect of DOG on pHi was attenuated by CH; however, the PC-induced attenuation of the fall in pHi, was not affected by CH. High energy phosphates (measured by 31P NMR) were not significantly different between any of the groups during ischemia or reflow. This study confirms that the protective effect of ischemic preconditioning on stunning in rat heart can be eliminated by inhibition of PKC, but suggests that the effect of PC on the fall in pHi during sustained ischemia is not mediated by PKC.     

Accuracy of histone H3 messenger RNA in situ hybridization for the assessment of cell proliferation in human tissues

Histone H3 mRNA in situ hybridization was compared to a reference method, iododeoxyuridine (IdUrd) immunohistochemistry of tissues labeled in vivo, as a means for assessing the proportion of S-phase cells (labeling index, LI) in oral tumor and normal mucosa. Paraffin sections from 16 patients with oral squamous cell carcinoma were studied. Patients received an IdUrd infusion before the biopsy was taken. Tissue sections were coded before counting the percentages of S-phase cells. A high correlation was found between the results obtained by the two techniques. The average histone H3 and IdUrd LIs of the tumors were 28.5 +/- 2.4% and 29.2 +/- 2.7%, respectively (P = 0.85), with a Spearman correlation coefficient r = 0.95 (P < 0. 0001). The histone H3 LI of the basal layer of normal mucosa was 3.1 +/- 0.8%, whereas the IdUrd LI was 2.7 +/- 0.9% (P = 0.74), with r = 0.78 (P = 0.004). In the suprabasal layers, these parameters were 21. 3 +/- 2.3% and 23.9 +/- 3.2%, respectively (P = 0.56), with r = 0.93 (P < 0.0001). In sections stained for both histone H3 and IdUrd, most cells were double labeled, with very few cells containing only one of the labels. In some specimens, large areas of H3-stained cells did not contain IdUrd-labeled cells, suggesting that during the IdUrd infusion, the precursor did not reach these areas. Two specimens were histone H3 negative. They were also negative when hybridized with beta-actin probe, indicating degradation of mRNAs in these samples. The results of this study demonstrate that the histone H3 mRNA in situ hybridization performed in human formalin-fixed, paraffin-embedded tissues provides the same data as does labeling the tumors in vivo with halogenated pyrimidine.     

Stochastic model for a wavelike exothermal reaction in condensed heterogeneous systems

The labile protons of two 32-base-pair, four-arm models of immobile Holliday junctions have been studied by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. Overlap of resonances in the imino-imino region of two-dimensional nuclear Overhauser enhancement (NOE) spectra necessitates the use of a multi-pathway approach for obtaining sequence-specific assignments wherein all possible NOE connectivities to the labile protons are utilized, including those from the 2H of adenine, 5CH3 of thymine, and 5H of cytosine. Resonance assignments are obtained for all slowly exchanging imino and cytosine amino protons. Base-pairing up to and including the junction point is found in all four arms of both Holliday junctions. Several cross-arm NOE connectivities are identified and can be used to infer the geometry of the helical stacking domains. The two Holliday junctions studied, which differ only by the exchange of two base pairs at the branch point, appear to have opposite arm stacking geometries. These assignments form an important part of the critical background for detailed NMR analysis of Holliday junction structure and dynamics.     

Mortality and cancer incidence following radiotherapy for seminoma of the testis

The model peptides glycylglycyltyrosylalanine (Gly-Gly-Tyr-Ala), glycylglycylthreonylalanine (Gly-Gly-Thr-Ala) and glycylglycylserylalanine (Gly-Gly-Ser-Ala) were phosphorylated at the hydroxyl groups of their tyrosyl, threonyl and seryl residues, respectively, and characterized by 31P and 1H NMR spectroscopy. The pKa-value of the phosphoryl group in the tyrosine-containing peptide determined from the pH dependence of chemical shifts is 5.9, the 31P chemical shifts at low pH (4.0) and high pH (8.0) are -3.8 and 0.2 ppm, respectively. Phosphorylation also leads to significant shifts of the 1H NMR resonances of the tyrosine residue; the amide resonance is shifted -0.02 ppm, the H alpha resonance 0.06 ppm, the H beta resonances 0.10 and -0.04 ppm, the H delta resonances 0.02 ppm and the H epsilon resonances 0.26 ppm. The pKa-value of the phosphoryl group in the threonine peptide determined from the pH dependence of chemical shifts is 6.1; the 31P chemical shifts at low pH (4.0) and high pH (8.0) are -0.1 and 4.8 ppm, respectively. The corresponding values for the serine peptide are 6.1 (pKa), 0.6 ppm and 4.9 ppm. Phosphorylation also leads to significant shifts of the 1H NMR resonances of the threonine and serine residues. In the threonine residue the amide resonance is shifted 0.25 ppm, the H alpha-resonance -0.43 ppm, the H beta-resonance 0.03 ppm and the H gamma-resonance 0.09 ppm. In the serine residue the amide resonance is shifted 0.21 ppm, the H alpha-resonance -0.17 ppm, and the H beta-resonances 0.17 ppm.     

Conformational changes in yeast pyruvate kinase studied by 205Tl+ NMR

The interaction of the monovalent cation with yeast pyruvate kinase (yPK) has been investigated by 205Tl+ NMR. TlNO3 activates yPK to 80-90% activity compared to KCl with an apparent Ka of 1.00 +/- 0.03 mM in the presence of 4 mM Mn(NO3)2 as the activating divalent cation. At higher concentrations of Tl+, enzyme inhibition is observed with an apparent KI of 180 +/- 10 mM. The extent of inhibition is dependent on the nature and concentration of the divalent cation. The effect of Mn2+ on the 1/T1 and 1/T2 values of 205Tl+ in the presence of yPK was determined at 173.02 MHz (300 MHz, 1H) and 346.03 MHz (600 MHz, 1H). The temperature dependence of the relaxation rates indicates that fast exchange conditions prevail for 205Tl+ longitudinal relaxation rates. The correlation time, tauc, for the Mn2+-205Tl+ interaction was estimated by a frequency dependence of 1/T1m for several enzyme complexes, and an average value of tauc was determined to be 0.91 ns. The distance between Tl+ and Mn2+ at the active site of yPK was calculated from the paramagnetic contribution of Mn2+ to the longitudinal (1/T1m) relaxation rates of Tl+ bound to yPK. For the apo yPK complex, the Tl+ to Mn2+ distance is 6.7 +/- 0.2 A. Upon addition of phosphoenolpyruvate (PEP) to form the yPK-Tl-Mn-PEP complex, the inter-cation distance decreases to 6.1 +/- 0.3 A. The addition of the allosteric activator fructose 1,6-bisphosphate (FBP) to form the yPK-Tl+-Mn2+-PEP-FBP complex gives an intermetal distance of 6.2 +/- 0.2 A. In the yPK-Tl-Mn-FBP complex, a Tl+-Mn2+ distance of 6.0 +/- 0.1 A is observed, indicating that FBP causes a conformational change at the active site in the absence of PEP. Analogous 205Tl NMR experiments with competitive inhibitors of PEP (oxalate, BrPEP) indicate that these ligands do not induce the same conformational changes as do the physiological substrates and activators. Similar experiments with the nonallosteric rabbit muscle PK were also performed and analyzed.     

Inhibitory effect of the non-steroidal anti-inflammatory drugs, indomethacin and piroxicam on 2-acetylaminofluorene-induced hepatocarcinogenesis in male ACI/N rats

The modifying effects of the non-steroidal anti-inflammatory drugs, indomethacin (IMC) and piroxicam (PC) on hepatocarcinogenesis induced by 2-acetylaminofluorene (AAF) were investigated in male ACI/N rats. Rats were divided into 6 groups: group 1 was fed a diet containing 200 ppm AAF for 16 weeks, starting at 6 weeks of age; group 2 was fed an AAF together with 130 ppm PC-containing diet; group 3 received an AAF diet and IMC (10 ppm) in their drinking water; group 4 was fed a PC diet alone; group 5 was given IMC alone; and group 6 served as controls. The PC diet, or the drinking water containing IMC, was given to the rats starting at 5 weeks of age until 1 week after the carcinogen exposure. At termination of the experiment (week 36), the incidences of iron-excluding altered liver cell foci (24.2 +/- 5.2/cm2) and liver cell tumors (1/10, 10%), and the tumor multiplicity (0.10/rat) in rats of group 2 were significantly smaller than those of group 1 (foci incidence, 42.6 +/- 6.7/cm2; tumor incidence, 10/10, 100%; and multiplicity, 4.00/rat) (P < 0.05). Similarly, the incidence of iron-excluding hepatocellular foci (27.4 < 1.2/cm2) and liver cell tumors (1/10, 10%) and the tumor multiplicity (0.10/rat) in rats of group 3 were significantly lower than those of group 1 (P < 0.05). There were no liver cell lesions (foci and neoplasms) in rats of groups 4, 5 and 6. Thus, PC and IMC inhibited the hepatocarcinogenesis induced by AAF when administered concurrently with the carcinogen and the results may indicate possible involvement of altered arachidonic metabolism in the initiation phase of AAF-induced liver carcinogenesis.     

Secretory immunoglobulins in colonic neoplasms

1. The phosphonium analogues of choline, phosphorylcholine, CDPcholine and phosphatidylcholine were synthesized chemically and characterized by 1H-NMR and 31P-NMR; in 1,2-distearoyl-DL-glycero-3-phosphorylphosphocholine, the 31P-NMR chemical shift of phosphonium relative to phosphate was--28.2 ppm. 2. A comparison was made of the rates of reaction of choline kinase, cholinephosphate cytidyltransferase, cholinephosphotransferase and phospholipase C on natural and phosphonium substrates. Enzyme reaction rates were similar for all but the cytidyltransferase, which exhibited a 3-fold preference for the normal substrate. 3. Weanling rats were maintained for 6 weeks on a diet in which choline was fully replaced by phospho[1,2-14C2]choline mixed with a trace of [Me-3H] choline. Incorporation of phosphocholine into liver lipids was detectable by 31P-NMR even in crude tissue homogenates. Choline-based phospholipids of liver, kidney, lung and brain were extracted, and phosphocholine incorporation calculated from 31P-NMR peak area ratios. The phosphatidylcholine analogues were separated by preparative thin-layer chromatography. Incorporation of phosphocholine ranged from 33% in lung phosphatidylcholine to 6% in kidney sphingomyelin. Variations in 14C/3H ratio between feed and phospholipid extracts indicated preferences for exogenous choline over phosphocholine varying from 1.3: 1 in brain to 3.2: 1 in liver. The results indicated that phosphocholine is a potentially useful 31P-NMR probe for the study of membrane lipids.     

Characterization of the interaction between bovine pancreatic trypsin inhibitor and thiocyanate by NMR

The interaction between Bovine Pancreatic Trypsin Inhibitor and thiocyanate was studied using NMR spectroscopy following several experimental approaches. The chemical shift variations of the BPTI protons in the absence and in the presence of increasing thiocyanate concentrations (up to 0.2 M) were significant (> 0.05 ppm) for 30 protein protons belonging to 20 residues. The largest deviation, 0.2 ppm, was observed for the amide backbone proton of Arg42 in the absence of thiocyanate and in the presence of 40 molar equivalents of thiocyanate. The influence of the presence of thiocyanate on the electrostatic potential surrounding the protein was demonstrated by NOESY spectra selective at the water frequency: the presence of SCN- favours acid catalysed exchange and disfavours base catalysis. However, a specific effect of thiocyanate was pointed out since the comparison of the chemical shifts in the presence of 40 molar equivalents of KSCN and KCl, respectively, showed much more as well as larger deviations compared to measurements in the absence of salt. A dissociation constant, KD, for a 1/1 complex between BPTI and thiocyanate was calculated from chemical shifts measurements: KD = 89 +/- 8 mM. A second value, KD = 99 +/- 10 mM, was extracted from SC15N relaxation time measurements.     

Interfacial conformation of dipalmitoylglycerol and dipalmitoylphosphatidylcholine in phospholipid bilayers

Diacylglycerols are minor constituents of membrane lipids, yet are essential in the activation and membrane association of protein kinase C. Solid-state 13C NMR experiments have been used to characterize the orientation of the glycerol backbone of dipalmitoylglycerol (DPG) and dipalmitoylphosphatidylcholine (DPPC) in egg phosphatidylcholine (PC) bilayers. The 13C NMR spectra of both DPG and DPPC specifically 13C-labeled at the sn-2 chain carbonyl exhibit a single narrow resonance (approximately 2 ppm) in liquid-crystalline egg PC bilayers. In contrast, specific 13C-labeling of both the sn-1 and sn-2 chain carbonyls results in an additional broad component (24-32 ppm) with an axially symmetric line shape. These data reveal that DPG has a distinct motionally-averaged structure in PC bilayers that is similar to that of DPPC and is not significantly affected by the absence of the large polar PC headgroup. The NMR line shapes are roughly consistent with the results of previous FTIR and NMR studies that indicate the sn-1 chain extends from the C1 carbon of the glycerol backbone into the hydrophobic interior of the bilayer, while the sn-2 chain first extends parallel to the bilayer surface and incorporates a bend at the ester linkage in order to keep the sn-1 and sn-2 chains parallel. However, the data suggest that the time-averaged orientation of the glycerol backbone is tilted from the bilayer normal, in contrast to the nearly parallel orientation observed in the crystal structures of phosphatidylcholines and phosphatidylethanolamines or the perpendicular orientation observed in the crystal structures of diacylglycerols.     

Ischemic preconditioning: effects on pH, Na and Ca in newborn rabbit hearts during Ischemia/Reperfusion

In adult hearts, ischemic preconditioning (PC) has been shown to decrease ischemia-induced changes in intracellular pH (pHi) and [Ca] ([Ca]i) and decrease associated injury. These results are consistent with the interpretation that PC decreases the stimulus for Na uptake via Na/H exchange, thereby decreasing intracellular Na (Nai) accumulation, and thus decreasing the change in force driving Na/Ca exchange, which otherwise contributes to ischemia-induced increases in [Ca]i. Given documented age-related differences in myocardial responses to ischemia, we tested the hypothesis that in newborn hearts, PC will diminish intracellular [H], Nai, and [Ca]i during ischemia/reperfusion. NMR was used to measure pHi, Nai, [Ca]i, ATP, and PCr in isolated newborn (4-7 days) rabbit hearts Langendorff-perfused with Krebs-Henseleit solution equilibrated with 95% O2/5% CO2 at 36+/-1 degrees C. Control hearts were perfused 30 min before initiating 40 min global ischemia followed by 40 min reperfusion. PC hearts were treated the same except four 5-min intervals of ischemia each followed by 10 min of perfusion which preceded global ischemia. At end ischemia, pHi was higher in PC than control hearts (6.31+/-0.03 v 5.83+/-0.05; P<0.05). Similarly, PC diminished Nai-accumulation during ischemia and reperfusion (P<0.05). Control Nai rose from 16.2+/-2.6 to 108.8+/-10.3 (mEq/kg dry weight) and recovered to 55.2+/-10.1 and the corresponding values for PC hearts were 25.6+/-6.2, 70.0+/-7.9 and 21.9+/-5.2. PC also improved [Ca]i recovery during reperfusion (P<0.05). Control [Ca]i rose from 418+/-43 to 1100+/-78 (nm/l) and recovered to 773+/-63, whereas in PC hearts the values were 382+/-40, 852+/-136 and 371+/-45, respectively. In addition, PC decreased coronary resistance during reperfusion (P<0.05) as reflected by lower perfusion pressures under constant flow conditions (65.9+/-1.5 v 56. 1+/-4.1 mmHg at end of reperfusion). Finally, PC improved recovery of left-ventricular developed pressure (LVDP-43.8+/-12.0 v 17.2+/-3. 0% of control; P<0.05) and diminished CK release (607+/-245 v 2432+/-639 IU/g dry weight; P<0.05) during reperfusion. The results are consistent with the hypothesis.