Intracellular pH in Dictyostelium discoideum: a 31P nuclear magnetic resonance study

We have used phosphorus-31 nuclear magnetic resonance to determine intracellular pH in the cellular slime mold Dictyostelium discoideum. We devised an air-lift circulator to maintain the dense cell suspensions in a well-oxygenated and well-stirred state while causing minimal perturbation to the sample flowing through the detector coils. Cells continued to develop normally in this set-up. Spectra acquired under these conditions typically show two peaks in the inorganic phosphate region corresponding to pH values of 7.16 +/- 0.03 and 6.48 +/- 0.02. These peaks are believed to represent the mitochondrial and cytosolic compartments respectively, based on a comparison of these values with published data and the collapse of the two compartments upon addition of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone. Dictyostelium cells show a remarkable degree of intracellular pH homeostasis. Both mitochondrial and cytosolic pH remained unchanged as extracellular pH was varied from 4.3 to 8.1. There was also no apparent change in the pH of either compartment after up to 13.5 hours' development in suspension.     

Ascorbic acid prolongs the viability and stability of isolated perfused lungs: A mechanistic study using 31P and hyperpolarized 13C nuclear magnetic resonance

Ex vivo lung perfusion (EVLP) has recently shown promise as a means of more accurately gauging the health of lung grafts and improving graft performance post-transplant. However, reperfusion of ischemic lung promotes the depletion of high-energy compounds and a progressive loss of normal mitochondrial function, and it remains unclear how and to what extent the EVLP approach contributes to this metabolic decline. Although ascorbate has been used to mitigate the effects of ischemia–reperfusion injury, the nature of its effects during EVLP are also not clear. To address these uncertainties, this study monitored the energy status of lungs during EVLP and after the administration of ascorbate using ³¹P and hyperpolarized ¹³C NMR (nuclear magnetic resonance). Our experiments demonstrated that the oxidative phosphorylation capacity and pyruvate dehydrogenase flux of lungs decline during ex vivo perfusion. The addition of ascorbate to the perfusate prolonged lung viability by 80% and increased the hyperpolarized ¹³C bicarbonate signal by a factor of 2.7. The effect of ascorbate is apparently due not to its antioxidant quality but rather to its ability to energize cellular respiration given that it increased the lung’s energy charge significantly, whereas other antioxidants (glutathione and α-lipoic acid) did not alter energy metabolism. During ascorbate administration, inhibition of mitochondrial complex I with rotenone depressed energy charge and shifted the metabolic state of the lung toward glycolysis; reenergizing the electron transport chain with TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) recovered metabolic activity. This indicates that ascorbate slows the decline of the ex vivo perfused lung’s mitochondrial activity through an independent interaction with the electron transport chain complexes.     

The interaction of pseudohalides with the phospholipid head-group: a nuclear magnetic resonance study.

Liposomes have been studied by means of high-field magnetic resonance techniques. The choline N+(CH3)3 group showed two proton resonances for phosphatidylcholine whereas the addition of a charged species to the phospholipid resulted in a single N+(CH3)3 resonance. Upon the addition of either of two linear pseudohalide anions, the two resonances for phosphatidylcholine were further split whereas for the mixture of lipids containing a charged species, the single head-group resonance was now split. The presence of a negative charge on the liposome does not prevent the anion-liposome interaction observed for neutral liposomes. Incorporation of cholesterol into the negatively charged liposomes results in a clear initial splitting of the head-group proton signal in a manner very similar to that for neutral liposomes; this head-group signal is then further split upon anion addition. The small splitting involved suggests a weak pseudohalide-liposome interaction whose magnitude depends on the position of the anion in the lyotropic series. The phosphorous NMR signal from the head-group is unaffected by the pseudohalide interaction whereas the carbon signals from the N+(CH3)3 groups are affected, indicating that the initial anion interaction is localized to the region of the choline groups of the liposome. After the initial exposure of the liposome to the anion, however, the splitting decreases with time, indicating that the anions have entered the liposome and interact with both inside and outside head-groups.     

Acyl chain orientational order in large unilamellar vesicles: comparison with multilamellar liposomes: a 2H and 31P nuclear magnetic resonance study.

Large unilamellar vesicles (LUVs) composed of 1-[2H31]palmitoyl-2-oleoyl phosphatidylcholine (POPC-d31), with diameters of approximately 117 +/- 31 and 180 +/- 44 nm, were prepared by extrusion through polycarbonate filters with pore sizes of 0.1 and 0.2 microns, respectively. The 2H nuclear magnetic resonance (NMR) spectra obtained at 21 degrees C contain two components: a broad component (approximately 17 kHz linewidth) corresponding to the methylene groups and a narrower component originating from the methyl groups. Spectra with increasing powder pattern characteristics were obtained by reducing the rate of phospholipid reorientations by addition of glycerol (to increase the solvent viscosity) and by lowering the temperature. Full powder spectra, characteristic of liquid-crystalline bilayers, were obtained for both LUV samples at 0 degrees C in the presence of 50 wt% glycerol. Individual quadrupolar splittings were not resolved in these spectra, due to broader linewidths in the LUVs, which have significantly shorter values for spin-spin relaxation time T2 measured from the decay of the quadrupolar echo (90 microseconds) than the multilmellar vesicles (MLVs; 540 microseconds). Smoothed order parameter profiles (OPPs) were obtained for these samples by integration of the dePaked spectra. The OPPs were very similar to the OPP of POPC-d31 MLVs in 50 wt% glycerol at the same temperature, indicating that orientational order in MLVs and LUVs with a diameter of > or = 100 nm is essentially the same. The presence of 80 wt% glycerol was found to have a disordering effect on the vesicles.     

Aluminum-mediated metabolic changes in rat serum and urine: a proton nuclear magnetic resonance study

The toxic effects of Al(3+) have been studied in 90-days AlCl(3) orally treated male albino rats (n = 7) using (1)H NMR spectroscopy-based metabolic profile of rat serum and urine, serum enzyme tests, behavioral impairment, and histopathology of kidney and liver. Metabolic profile of 90-days Al(3+)-treated rat sera showed significantly elevated levels of alanine, glutamine, beta-hydroxy-butyrate, and acetoacetate and significantly decreased level of acetone when compared with that of control rats. However, metabolic profile of 90-days Al(3+)-treated rat urine showed significantly decreased levels of citrate, creatinine, allantoin, trans-aconitate, and succinate and significantly increased level of acetate when compared to control rats. The overall perturbations observed in the metabolic profile of serum and urine demonstrate the impairment in the tricarboxylic acid cycle, liver and kidney metabolism, which was further reinstated by clinical chemistry and histopathological observations. Moreover, "in vivo" behavioral impairment has also been observed as the indication of aluminum neurotoxicity.     

Alcohol interactions with lipids: a carbon-13 nuclear magnetic resonance study using butanol labeled at C-1

The interactions of carbon-13 enriched butanol with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied using C-13 nuclear magnetic resonance. It was found that above the gel to liquid crystal phase transition the resonance from the butanol could be resolved into two signals with similar chemical shifts but different T1 values and line widths. In contrast, only one narrow resonance was observed for ethanol, which has considerably less solubility in the lipids than butanol. Thermodynamic analyses of the effects of butanol on the phase transition temperature predict much greater solubility or butanol when the lipid is above the phase transition temperature than when it is below. It was concluded that the two butanol resonances represent two slowly exchanging populations, the free butanol in the aqueous phase and butanol dissolved in the liquid crystalline region of the lipid. No bound butanol was detected below the gel to liquid crystal phase transition. Relaxation studies were performed on the resonance of the bound butanol in DPPC and DMPC, including measurements of T1, line width, and nuclear Overhauser enhancement. Theoretical analysis of the relaxation parameters indicates that the lipid-bound alcohol has very high mobility within the fluid lipid bilayer. The data are consistent with butanol being present at the aqueous boundary or head group region of the lipid.     

Conformational variations in d(TGACGTCA) and its reverse sequence d(ACTGCAGT): a joint circular dichroism and nuclear magnetic resonance study

Circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques have been used to characterize the structural properties of the two self-complementary DNA octamers d(TGACGTCA) (I) and d(ACTGCAGT) (II). These display as distinctive features reverse sequences and central steps CpG and GpC, respectively. CD experiments lead to B-form DNA spectra characterized by larger magnitude signals in the case of octamer (I). NMR COSY spectra indicate that in the two octamers all the residues are predominantly south, S, (2'-endo) sugar conformation. NMR NOESY spectra show most of the glycosidic angles confined in the range predicted for B-form DNA although important heterogeneity is noticed along the chains, more pronounced in the case of octamer (I). Both the increase of north, N, (3'-endo) sugar conformation and P (pseudorotation phase angle) deviation from its standard B-form DNA value (162 degrees) express local sequence dependent structure distortions, remarkably visible in CpG step of octamer (I) and agreeing with NOESY cross-peaks intensities. Results interpreted according to Calladine's rules indicate higher cross-chain strains in octamer (I) than in octamer (II). All together, we find evidence to support for octamer (I) in solution local structures with A-DNA properties likely dictated by the central CpG step. Such structures could be involved in the DNA recognition by proteins and anticancerous drugs.     

Bleomycin-A2 complexes with poly(dA--dT): a proton nuclear magnetic resonance study of the nonexchangeable hydrogens

The mRNA coding for uteroglobin, a progesterone-induced uterine protein, has been partially purified from 4-day pregnant rabbit uterus. Double-stranded DNA synthesized from the partially purified mRNA preparation was inserted into the Pst I site of pBR 322. Bacterial transformants containing uteroglobin DNA sequences were identified by their ability to enrich for uteroglobin mRNA on hybridization with total uterine poly A-RNA. The identity of one recombinant was confirmed unambiguously by matching its nucleotide sequence with the amino acid sequence of the uteroglobin polypeptide.     

Cerebral creatine kinase deficiency influences metabolite levels and morphology in the mouse brain: a quantitative in vivo 1H and 31P magnetic resonance study

Creatine kinase (CK)-catalysed ATP-phosphocreatine (PCr) exchange is considered to play a key role in energy homeostasis of the brain. This study assessed the metabolic and anatomical consequences of partial or complete depletion of this system in transgenic mice without cytosolic B-CK (B-CK-/-), mitochondrial ubiquitous CK (UbCKmit-/-), or both isoenzymes (CK -/-), using non-invasive quantitative magnetic resonance (MR) imaging and spectroscopy. MR imaging revealed an increase in ventricle size in a subset of B-CK-/- mice, but not in animals with UbCKmit or compound CK mutations. Mice lacking single CK isoenzymes had normal levels of high-energy metabolites and tissue pH. In the brains of CK double knockouts pH and ATP and Pi levels were also normal, even though PCr had become completely undetectable. Moreover, a 20-30% decrease was observed in the level of total creatine and a similar increase in the level of neuronal N-acetyl-aspartate compounds. Although CKs themselves are not evenly distributed throughout the CNS, these alterations were uniform and concordant across different brain regions. Changes in myo-inositol and glutamate peaks did appear to be mutation type and brain area specific. Our results challenge current models for the biological significance of the PCr-CK energy system and suggest a multifaceted role for creatine in the brain.     

Application of 31P nuclear magnetic resonance technology in the study of phosphorus fractions and their translocation and transformation in sediments: a review

Phosphorus (P) release from sediments is one of the most important causes of lake eutrophication, while the activity of P is determined by P chemical form. Due to its advantages in improving our knowledge about the P fractions in environmental samples, the 31P nuclear magnetic resonance (31P NMR) technology has received extensive attention. This paper summarized the current studies on the characterization, translocation, and transformation of P fractions in sediments by using this technology, and described the technical principles, classification, analytical procedures, and specific application fields of this technology. At present, the researches of sediment P by using 31P NMR technology were focused on the characterization of different P forms, the effects of microbes on the P translocation and transformation, and the quantitative analysis of different P fractions. The studies on the P-extracting agents and extraction methods were the hot topics as well. The potential issues and research trends about the application of 31P NMR technology in environmental samples were also discussed.     

Dyke demolition led to a sharp decline in waterbird diversity due to habitat quality reduction: A case study of Dongting Lake,China

Dongting Lake, an important wintering habitat for migratory waterbirds in the East Asian–Australasian Flyway, has suffered serious degradation in recent decades. To restore habitats for biodiversity conservation and flood control, 459 dykes were demolished and 14 were preserved in 2017. However, the direct impact of dyke demolition on wintering waterbirds was not comprehensively assessed. In this study, based on annual waterbird census and habitat data (2013/14–2020/21), we compared the differences in habitat areas and species composition of waterbirds in the dyke‐demolished and preserved areas, and explored whether habitat changes caused by the dyke demolition were responsible for the changes in the number of species and percentages of waterbird individuals. The results indicate that the areas of water (including shallow water) and mudflat habitats significantly decreased, but the vegetation area significantly increased in the dyke‐demolished areas. The species numbers and percentages of waterbird individuals at the community and foraging guilds levels, and the percentages of nine species, were higher in the dyke‐preserved areas than those in the dyke‐demolished areas. Changes in the numbers of species and percentages of individuals of fish eaters, insectivores, and omnivores positively correlated with drastic changes in the percentages of water habitats (including shallow water) after dyke demolition. Effective measures should be carried out to restore hydrological regimes, providing waterbirds sufficient suitable habitats with different water depths. These findings improve our understanding of the influence of dyke demolition on waterbirds and provide insights for wetland management and waterbird conservation.     

Sequential folding of transfer RNA: A nuclear magnetic resonance study of successively longer tRNA fragments with a common 5′ end

Most folding studies on proteins and nucleic acids have been addressed to the transition between the folded and unfolded states of an intact molecule, where an entire residue sequence is present during the folding event. However, since these polymers are synthesized sequentially from one terminus to the other in vivo, their folding pathways may be influenced greatly by the sequential appearance of the residues as a function of time.The three-dimensional structure of yeast tRNA^Phe in the crystalline state is correlated with 360 MHz proton nuclear magnetic resonances from three fragments plus an intact molecule of the tRNA that share a common 5′ end and are in a solution condition similar to that of the crystal structure. This has allowed identification of folded structures present in the fragments and presumably present in the growing tRNA molecule as it is being synthesized from the 5′ end. The experiments show that only the correct stems are formed in the fragments; no additional or competing helical region is produced. This suggests that in the biosynthesis of this tRNA, correct folding of helical stems occurs before the entire molecule is formed. Further, some of the tertiary interactions (hydrogen bonds) found in the crystal structure are also probably present before the synthesis is completed. These findings are generalized to consider the precursor of the tRNA as well as other tRNAs.     

Mixed micelle formation between gramicidin-S and a nonionic detergent: a nuclear magnetic resonance model study of peptide/detergent aggregation

The interaction of the cyclic decapeptide antibiotic gramicidin-S (GrS) with the nonionic detergent octaethylene glycol mono-n-dodecyl ether was studied by NMR spectroscopy. Detergent binding led to a slightly altered average conformation in the d-Phe side chains of the peptide. The changing diamagnetic shielding of nearby protons resulted in chemical shift variations, the largest effect being observed for the d-Phe C _α proton. The continuous upfield shift of this proton resonance, indicating rapid exchange of the peptide between detergent-associated and unassociated states, was employed for an evaluation of the detergent/peptide aggregation equilibria. The nonlinear binding plot thus obtained was attributed to essentially different aggregational states, depending on the detergent/peptide ratio. The almost linear dependence of the spin-lattice relaxation rate and of the hydrogen-deuterium exchange rate on the fraction of detergent-associated GrS could be reconciled with a simple model, comprising binding of detergent monomers and cooperative binding of micelles at low and high detergent/peptide molar ratios, respectively. Thus, GrS provides a useful model for a study of backbone dynamics and water penetration in detergent- and membrane-bound peptides and proteins. The results will also be discussed with reference to the interaction of GrS with biological membranes. Received: 22 June 1998 / Revised version: 5 October 1998 / Accepted: 9 October 1998     

Cerebral metabolism in male patients with schizophrenia who have seriously and dangerously violently offended: a 31P magnetic resonance spectroscopy study

There is biochemical evidence to suggest that membrane phospholipid metabolism may be impaired in some patients with schizophrenia. The aim of this study was to test the hypothesis that patients with schizophrenia who have violently offended while psychotic suffer from changes in cerebral phospholipid metabolism. Cerebral 31-phosphorus magnetic resonance spectroscopy was carried out in 15 male patients with schizophrenia who had violently offended (homicide, attempted murder, or wounding with intent to cause grievous bodily harm) while psychotic and in a control group of 13 age-matched healthy male control subjects. Spectra were obtained from 70x70x70mm(3) voxels in the brain using an image-selected in vivo spectroscopy pulse sequence. betaNTP was lower (P < 0.04) and gammaNTP was higher (P < 0.04) in the patient group compared with the normal control group. Our results are suggestive of increased cerebral energy metabolism taking place in the forensic patients.     

Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500-MHz proton nuclear magnetic resonance study

The equilibrium binding of influenza virus hemagglutinin to derivatives of its cell-surface ligand, sialic acid, was measured by nuclear magnetic resonance (NMR) spectroscopy. Binding was quantified by observing perturbations of sialic acid resonances in the presence of protein. The major perturbation observed was a chemical shift of the N-acetyl methyl resonance, presumably due to the proximity of the methyl group to tryptophan 153. X-31 hemagglutinin binds to the methyl alpha-glycoside of sialic acid with a dissociation constant of 2.8 mM and does not bind to the methyl beta-glycoside. Replacing the 4-hydroxyl group of sialic acid with an acetyl group has little effect, while replacing the 7-hydroxyl group with an acetyl prevents binding. Experiments with sialylated oligosaccharides confirm literature reports that mutations at amino acid 226 change the specificity of hemagglutinin for alpha(2,6) and alpha(2,3) glycosidic linkages. The NMR line broadening of sialyloligosaccharides suggests that sialic acid is the only component that contacts the protein. Saccharides containing two sialic acid residues appear to have two separate binding modes. Hemagglutinin that has undergone a low pH induced conformational change retains the ability to bind sialic acid.     

Determination of choline in erythrocytes using high-resolution proton nuclear magnetic resonance spectroscopy: Comparison with a choline oxidase method

Detailed operating conditions are reported for the determination of choline in human erythrocytes using proton nuclear magnetic resonance spectroscopy in conjunction with the inversion-recovery spin-echo pulse sequence. The results of the NMR method were in excellent agreement with those obtained using an enzymatic (choline oxidase) assay; however, they were approximately three times higher than those reported using gas chromatography/mass spectrometry techniques. The differences may be partly due to the method of preparing or sampling cells since there is a distribution of choline in cells of different ages. However, choline levels were not affected by the methods used in the present study for storing or preparing cells.     

大堤型湖滨带生态系统健康状态驱动因子——以太湖为例

湖滨带是湖泊生态系统的重要组成部分,对维持湖泊生态系统健康和改善水环境功能具有积极作用。由于防洪需要,我国许多湖泊在湖滨带中修建了防洪大堤,防洪大堤对湖滨带生态系统的影响还缺乏研究,识别引起大堤型湖滨带生态健康退化的驱动因子是开展湖滨带生态修复的必要前提。针对太湖湖滨带的特点,在定性筛选了太湖主体营养状态、入湖河流污染负荷通量、岸带类型、风浪强度4个主要影响因子的基础上,分别采用"多元线性逐步回归法"和"偏相关系数法"进行驱动因子识别,2种方法确定的驱动因子个数均为3个,且3者的驱动力大小排序也相同,即太湖主体营养状态>岸带类型>入湖河流污染负荷通量。3个驱动因子对太湖湖滨带生态系统健康的影响均为负效应,也即太湖主体的营养状态综合指数越高、入湖河流污染负荷通量越重、岸带状况越差,湖滨带生态系统也就越不健康。另外,从统计方法的角度解释了"风浪强度"没有入选为驱动因子的原因;澄清了现阶段环境学、生态学领域对"驱动因子"识别方法的误解及不足之处。研究结果为大堤型湖滨带的生态修复方案提供了理论支持。     

Step-wise thermal denaturation of cobrotoxin, a snake venom neurotoxin fromNaja naja atra: A proton nuclear magnetic resonance study

Temperature dependence of proton nuclear magnetic resonance spectra has been followed for cobrotoxin, a postsynaptic neurotoxin fromNaja naja atra venom. Several aromatic amino-acid residues, including the functionally essential Trp-29 located at the tip of the central loop of the molecule, have been found to undergo a thermal structural transition above the global thermal denaturation temperature. It is suggested that a local structure around these residues behaves somehow independently of the rest of the molecule, and that such structural organization may be favorable for a conformational change of a neurotoxin molecule on binding to acetylcholine receptor.     

Brain cell membrane motion-restricted phospholipids: a cerebral 31-phosphorus magnetic resonance spectroscopy study of patients with schizophrenia

This study directly assessed, for the first time, whether there was a change in brain cell motion-restricted membrane phospholipids in vivo in patients with schizophrenia with mild to moderate negative symptoms, by quantification of the underlying broad resonance signal of cerebral 31-phosphorus magnetic resonance scans. Cerebral 31-phosphorus magnetic resonance spectroscopy was carried out in 16 schizophrenia patients and 16 age- and gender-matched normal controls. Spectra were obtained from 70x70x70 mm3 voxels using an image-selected in vivo spectroscopy pulse sequence. There was no significant difference in the broad resonances between the two groups, with the mean (S.E.) percentage signal being 59.4 (5.6) for the patients and 53.5 (5.9) for the controls. The phosphomonoesters and phosphodiesters narrow signals also did not differ significantly, their ratio being 0.26 (0.01) in the patients and 0.25 (0.01) in the controls. These results appear to be at variance with the changes expected under the membrane phospholipid hypothesis of schizophrenia.