Spectroscopic studies of lipids and biological membranes: carbon-13 and proton magic-angle sample-spinning nuclear magnetic resonance study of glycolipid-water systems.

We have obtained 1H and 13C magic-angle sample-spinning (MAS) nuclear magnetic resonance (NMR) spectra of three glycosyldiacylglycerol-water (1:1, weight ratio) mesophases, at 11.7 T, as a function of temperature, in order to probe lipid headgroup, backbone, and acyl chain dynamics by using natural-abundance NMR probes. The systems investigated were monogalactosyldiacyldiglyceride [MGDG; primarily 1,2-di[(9Z,12Z,15Z)octadec-9,12,15-trienoyl++ +]-3-beta-D-galactopyranosyl- sn-glycerol]; digalactosyldiacyldiglyceride [DGDG; primarily 1,2-di[(9Z,12Z,15Z)octadec-9,12,15-trienoyl++ +]-3- (alpha-D-galactopyranosyl-1-6-beta-D-glactopyranosyl)-sn-glycerol] ; and sulfoquinovosyldiacyldiglyceride [SQDG; primarily 1-[(9Z,12Z,15Z)octadec-9,12,15-trienoyl]-2 -hexadecanoyl-3-(6-deoxyl-6- sulfono-alpha-D-glucopyranosyl)-sn-glycerol]. At approximately 22 degrees C, all three lipid-water systems give well-resoled 13C and 1H MAS NMR spectra, characteristic of fluid, liquid-crystalline mesophases. 13C spin-lattice relaxation times of the headgroup and glycerol backbone carbons of all three materials give, within experimental error, the same NT1 values (approximately 400 ms), implying similar high-frequency motions, independent of headgroup size and charge. Upon cooling, pronounced line broadenings are observed, due to an increase in slow motional behavior. For each lipid, the onset of line broadening is seen with the glycosyl headgroup, glycerol backbone, and the first two or three carbons of the acyl chains. By approximately -20 degrees, all headgroup carbon resonances are broadened beyond detection. Both galactose moieties in DGDG "freeze out" together, implying a rigid-body motion of the disaccharide unit. Upon further cooling, the bulk polymethylene chain resonances in all three systems (in both 13C and 1H MAS) broaden greatly, followed by the olefinic and allylic carbon resonances.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elevated levels of Ca(II) modulate the activity and inhibition of serine proteases: implication in the mechanism of apoptosis

Elevated levels of intracellular Ca(II) are a prominent feature of apoptosis, a natural form of cell death involved in many physiological and pathological processes. Serine proteases play crucial roles in apoptosis and have been implicated in the genomic DNA degradation and the massive protein degradation that occur during apoptosis. In this study, the effects of the elevated level of Ca(II) on the activity and inhibition of serine proteases were examined by spectrophotometric methods. The effects of the elevated levels of Ca(II), Mg(II), K(I), and Na(I) on the activity and inactivation of three representative members of serine proteases were determined. The level of serine protease activity in CEM-C7-14 leukemic cells was also evaluated in the presence and absence of dexamethasone-induced apoptosis, and also in the presence of A23187, a Ca(II)-ionophore. Among the four metal-ions studied, only Ca(II) was found to significantly enhance the activity of mammalian serine proteases. Ca(II) was also found to significantly protect the enzymes from inhibition, while the other three metal-ions showed no significant effect on the inactivation of the enzymes. Compared to the control sample, the enzymic activity was found to be higher during apoptosis, and in the presence of the Ca(II)-ionophore. Results of this study indicate that Ca(II) can significantly enhance the catalytic efficiency of serine proteases during apoptosis.     

The timing of eruption of the primary dentition in Nigerian children

The objectives of this study were to clarify the eruption time and sequence for primary teeth in Nigerian children. It also investigated the effect of sex and socioeconomic status on the timing and sequence of eruption. A random sample of 1,657 children from ages of 3-40 months were examined--921 (55.6%) males and 736 (44.4%) females. The age of eruption of the teeth was estimated using probit regression. The results show that there was no effect of sex, socioeconomic status or breastfeeding status on the timing of eruption and pattern of teeth eruption in Nigerian children. Left and right teeth had similar eruption times. Eruption times of the lateral incisor, canine, and molars were similar for upper and lower teeth. Interpopulation studies however showed that though the sequence of eruption of primary teeth in Nigerian population is similar to that of their peers in other compared populations, there are observable sex differences in the timing of tooth eruption.     

Multinuclear magnetic resonance studies on serine protease transition state analogues

31P Nuclear Magnetic Resonance (NMR) studies were performed on mono- and diisopropylphosphoryl derivatives of alpha-chymotrypsin, trypsin, and subtilisin. Questions addressed included the pKa of the active center Asp...His...Ser triad in both species. While the pKa in the diisopropylphosphoryl derivatives is near 7.4 (found in this and other laboratories earlier) and reflects a nearly normal imidazolium titration curve, the apparent pKa in the monoisopropylphosphoryl enzymes (obtained by "aging" of the diisopropylphosphoryl derivatives and monitored by 31P NMR) is between 9.7 and 11.4 depending on the protease. This latter "titration" of the 31P NMR signal is reversible and presumably reflects the interaction of the imidazolium positive charge with the monoanionic phosphodiester. Of the two tetrahedral intermediates, the properties of the monoisopropylphosphoryl enzyme are probably more representative of the tetrahedral oxyanionic intermediate invoked during peptide hydrolysis. The same NMR technique was used to determine the action of PAM (pyridine-2-aldoxime methiodide, a known "antidote" for acetylcholinesterase inactivated by diisopropylfluorophosphate), on the inactivated enzymes. It was clear that the "antidote" could reverse the diisopropylphosphorylation but was ineffective on the monoisopropylphosphoryl ("aged") enzyme. 11B NMR studies were performed on phenylboronic (PBA) acid and 3,5-bis-trifluoromethylphenylboronic acid in the absence and presence of chymotrypsin and subtilisin. At 22 degrees C the former, but not the latter, compound was in fast exchange between the free and enzyme bound states. The relaxation parameters could be calculated for the bound PBA in chymotrypsin and the fluorinated analogue in subtilisin and clearly indicated that the boron nucleus was tetrahedral in the active centers, a good analogue for the tetrahedral oxyanionic intermediate.     

19F NMR studies of changes in membrane potential and intracellular volume during dexamethasone-induced apoptosis in human leukemic cell lines

The induction of apoptosis in leukemic cells by dexamethasone is well known, but the mechanism of this type of cell death and of dexamethasone resistance by some variants is still poorly understood. Apoptotic cell death is preceded by many changes in cellular properties, such as glucose metabolism, cell size, cell density, and others. In this study, ¹⁹F-NMR has been used to characterize changes in cell membrane potential and intracellular accessible volume during dexamethasone induced apoptosis. One dex-sensitive (CEM-C7) and three dex-resistant variants (CEM-C1, CEM-ICR27, and CEM-4R4) were examined. We have observed separate intracellular and extracellular resonances for trifluoroacetate and trifluoroacetamide added to suspended leukemic cells. From the equilibrium distribution of these fluoro-compounds between intra and extracellular spaces, the changes in membrane potential and intracellular accessible volume were calculated. The membrane potential for CEM-C7 cells was found to significantly decrease in the presence of dexamethasone (9-mV decrease within 18 h of dexamethasone treatment), while that of CEM-ICR27 was found in some samples to increase on dexamethasone incubation. The membrane potential for CEM-C1 decreased slightly, while that of CEM-4R4 was not appreciably affected by dexamethasone. The reduction of membrane potential seems to be an early step in the mechanism of dexamethasone induced apoptosis. Although the intracellular volume varied with cell type and dexamethasone incubation (for CEM-C7), the fractional intracellular volume (α = V_in/V_cell was found to be the same (0.82 ± 0.06) for all the cell lines in the presence and absence of dexamethasone. © 1993 Wiley-Liss, Inc.     

Metabolism of D-glucose in a wall-less mutant of Neurospora crassa examined by 13C and 31P nuclear magnetic resonances: effects of insulin

13C NMR and 31P NMR have been used to investigate the metabolism of glucose by a wall-less strain of Neurospora crassa (slime), grown in a supplemented nutritionally defined medium and harvested in the early stationary stage of growth. With D-[1-13C]- or D-[6-13C]glucose as substrates, the major metabolic products identified from 13C NMR spectra were [2-13C]ethanol, [3-13C]alanine, and C1- and C6-labeled trehalose. Several observations suggested the existence of a substantial hexose monophosphate (HMP) shunt: (i) a 70% greater yield of ethanol from C6- than from C1-labeled glucose; (ii) C1-labeled glucose yielded 19% C6-labeled trehalose, while C6-labeled glucose yielded only 4% C1-labeled trehalose; (iii) a substantial transfer of 13C from C2-labeled glucose to the C2-position of ethanol. 31P NMR spectra showed millimolar levels of intracellular inorganic phosphate (Pi), phosphodiesters, and diphosphates including sugar diphosphates and polyphosphate. Addition of glucose resulted in a decrease in cytoplasmic Pi and an increase in sugar monophosphates, which continued for at least 30 min. Phosphate resonances corresponding to metabolic intermediates of both the glycolytic and HMP pathways were identified in cell extracts. Addition of insulin (100 nM) with the glucose had the following effects relative to glucose alone: (i) a 24% increase (P less than 0.01) in the rate of ethanol production; (ii) a 38% increase (P less than 0.05) in the rate of alanine production; (iii) a 27% increase (P less than 0.05) in the rate of glucose disappearance. Insulin thus increases the rates of production of ethanol and alanine in these cells, in addition to increasing production of CO2 and glycogen, as previously shown.     

Monitoring Copper Bioaccumulation in Cocoa from Copper-Based Pesticide–Treated Cocoa Farms Using Fuzzy Similarity Method

Cocoa production has been hindered by pests, leading to application of pesticides. These pesticide applications have raised health concerns. This study is therefore aimed at developing accumulation classifications for copper (Cu) in cocoa from plantations where Cu-based pesticides have been applied using fuzzy similarity method (FSM). Cocoa pods, seeds, leaves, and soil samples were collected from five different plantations from three states in Nigeria. The plant samples were digested using standard methods, whereas the Cu in soil samples was extracted with 1 M ammonium acetate. The products were analyzed for Cu using flame atomic absorption spectrophotometer. The Cu concentrations obtained were used to model the accumulation degree using FSM. The Cu concentrations were higher in the leaves than the seeds and the pods. The persistency of Cu was displayed by high concentrations (11.5–375 mg/kg) and enrichment factor in CRIN sites where this pesticide application has been stopped for over a decade. The leaves from Ondo sites had the highest Cu concentration, which indicates impact of recent applications. The FSM membership function and algorithm indicated that the plant parts had low degrees of accumulation for fresh leaves, pods, and seeds, the valued part applied in beverages, but for the dry leaves, the accumulation degree is high. It may be concluded that the applied Cu-based pesticide had more impact and accumulation on the leaves than the commercial valued seeds, but the Cu concentration in the seeds is above the recommended value.     

Paediatric Acute Kidney Injury in a Tertiary Hospital in Nigeria: Prevalence,Causes and Mortality Rate

Background

The modest decline in child mortality in Africa raises the question whether the pattern of diseases associated with acute kidney injury (AKI) in children in Nigeria has changed.

Methods

A database of children, aged between one month and 16 years, with AKI (using modified pediatric RIFLE criteria) was reviewed. The cause of AKI was defined as the major underlying disease. The clinical and laboratory features of children with AKI who survived were compared to those who died.

Results

Of the 4 015 children admitted into Lagos University Teaching Hospital between July 2010 and July 2012, 70 episodes of AKI were recorded equalling 17.4 cases per 1000 children. The median age of the children with AKI was 4.8 (range 0.1–14.4) years and 68.6% were males. Acute kidney injury was present in 58 (82.9%) children at admission with 70% in ‘failure’ category. Primary kidney disease (38.6%), sepsis (25.7%) and malaria (11.4%) were the commonest causes. The primary kidney diseases were acute glomerulonephritis (11) and nephrotic syndrome (8). Nineteen (28.4%) children with AKI died. Need for dialysis [odds ratio: 10.04 (2.94–34.33)], white cell >15 000/mm³ [odds ratio: 5.72 (1.65–19.89)] and platelet <100 000/mm³ [odds ratio: 9.56 (2.63–34.77)] were associated with death.

Conclusion

Acute kidney injury is common in children admitted to hospitals. The common causes remain primary kidney diseases, sepsis and malaria but the contribution of sepsis is rising while malaria and gastroenteritis are declining. Acute kidney injury-related mortality remains high.     

Carbon-13 nuclear magnetic resonance spectroscopy of lipids: differential line broadening due to cross-correlation effects as a probe of membrane structure

We have obtained proton-coupled carbon-13 nuclear magnetic resonance (NMR) spectra of a variety of lipid-water and lipid-drug-water systems, at 11.7 T, as a function of temperature, using the "magic-angle" sample-spinning (MAS) NMR technique. The resulting spectra show a wide range of line shapes, due to interferences between dipole-dipole and dipole-chemical shielding anisotropy interactions. The differential line-broadening effects observed are particularly large for aromatic and olefinic (sp2) carbon atom sites. Coupled spectra of the tricyclic antidepressants desipramine and imipramine, in 1,2-dimyristoyl-sn-glycero-3-phosphocholine-water mesophases, show well-resolved doublets having different line shapes for each of the four aromatic methine groups, due to selective averaging of the four C-H dipolar interactions due to rapid motion about the director (or drug C2) axis. 2H NMR spectra of [2,4,6,8-2H4]desipramine (and imipramine) in the same 1,2-dimyristoyl-sn-glycero-3-phosphocholine-water mesophase exhibit quadrupole splittings of approximately 0-2 and approximately 20 kHz, indicating an approximate magic-angle orientation of the C2-2H(1H) and C8-2H(1H) vectors with respect to an axis of motional averaging, in accord with the 13C NMR results. Selective deuteration of imipramine confirms these ideas. Spectra of digalactosyl diglyceride [primarily 1,2-di[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl ]-3- (alpha-D-galactopyranosyl-1-6-beta-D-galactopyranosyl)-sn-glycerol]-H2O (in the L alpha phase) show a large differential line broadening for C9 but a reduced effect for C10, consistent with the results of 2H NMR of specifically 2H-labeled phospholipids [Seelig, J., & Waespe-Saracevic, N. (1978) Biochemistry 17, 3310-3315].(ABSTRACT TRUNCATED AT 250 WORDS)     

31P NMR characterization of cellular metabolism during dexamethasone induced apoptosis in human leukemic cell lines

³¹P NMR has been used to study the effects of dexamethasone on phosphorus metabolism in one dexamethasone (dex)-sensitive (CEM-C7) and three different dex-resistant (CEM-C1, CEM-4R4, and CEM-ICR27) human leukemic cell lines. The use of these cell lines, containing widely varying amounts of glucocorticoid receptors, made it possible to evaluate the receptor-mediated contributions to the modes of action of dexamethasone in these cells. To evaluate the effects of dexamethasone without any significant contribution from experimental conditions, all the experiments were done with parallel controls. Results obtained showed: (1) significantly different levels of phosphorylethanolamine (PE) and phosphorylcholine (PC) among cell lines, suggesting significant differences in phospholipid metabolism; (2) the dexamethasone induced reduction of phosphomonoester (PE + PC), ATP, and metabolic rates probably through glucocorticoid receptor mediated mechanisms; (3) the dexamethasone induced stimulation of cellular metabolism in a process which seems to be independent of glucocorticoid receptors; and (4) the dexamethasone induced alkaline shift of intracellular pH in all the cell lines except ICR27. The reduction in PME levels seems to be an earlier step in dexamethasone-induced apoptosis than the reduction in ATP. The degree of alkaline shift was found to correlate with the number of glucocorticoid receptors present. The possible involvement of phospholipid metabolites as second messengers in dexamethasone-induced apoptosis is discussed. © 1994 Wiley-Liss, Inc.