13C NMR based profiling unveils different α-ketoglutarate pools involved into glutamate and lysine synthesis in the milk yeast Kluyveromyces lactis.

BACKGROUND: The construction of efficient cell factories for the production of metabolites requires the rational improvement/engineering of the metabolism of microorganisms. The subject of this paper is directed towards the quantitative understanding of the respiratory/fermentative Kluyveromyces lactis yeast metabolism and its rag8 casein kinase mutant, taken as a model for all rag gene mutations. METHODS: (13)C NMR spectroscopy and [1,2-(13)C2]glucose were used as metabolic stable-isotope tracer to define the metabolic profiling of a K. lactis yeast and its derivative mutants. RESULTS: Rag8 showed a decrease of all (13)C glutamate fractional enrichments, except for [4-(13)C]glutamate that was higher than wild type ones. A decrease of TCA cycle flux in rag8 mutants and a contribution of a [4-(13)C]ketoglutarate pool not originating from mitochondria were suggested. (13)C lysine enrichments confirmed the presence of two compartmentalized α-ketoglutarate (α-KG) pools participating to glutamate and lysine synthesis. Moreover, an increased transaldolase, as compared to transketolase activity, was observed in the rag8 mutant by (13)C NMR isotopomer analysis of alanine. CONCLUSIONS: (13)C NMR-based isotopomer analysis showed the existence of different α-KG metabolic pools for glutamate and lysine biosynthesis. In the rag8 mutant, (13)C labeled pentose phosphate intermediates participated in the synthesis of this compartmentalized α-KG pool. GENERAL SIGNIFICANCE: A compartmentalization of the α-KG pools involved in lysine biosynthesis has been revealed for the first time in K. lactis. Given its great impact in metabolic engineering field, its existence should be validated/compared with other yeasts and/or fungal species.

Depletion of casein kinase I leads to a NAD(P)(+)/NAD(P)H balance-dependent metabolic adaptation as determined by NMR spectroscopy-metabolomic profile in Kluyveromyces lactis.

BACKGROUND: In the Crabtree-negative Kluyveromyces lactis yeast the rag8 mutant is one of nineteen complementation groups constituting the fermentative-deficient model equivalent to the Saccharomyces cerevisiae respiratory petite mutants. These mutants display pleiotropic defects in membrane fatty acids and/or cell walls, osmo-sensitivity and the inability to grow under strictly anaerobic conditions (Rag(-) phenotype). RAG8 is an essential gene coding for the casein kinase I, an evolutionary conserved activity involved in a wide range of cellular processes coordinating morphogenesis and glycolytic flux with glucose/oxygen sensing. METHODS: A metabolomic approach was performed by NMR spectroscopy to investigate how the broad physiological roles of Rag8, taken as a model for all rag mutants, coordinate cellular responses. RESULTS: Statistical analysis of metabolomic data showed a significant increase in the level of metabolites in reactions directly involved in the reoxidation of the NAD(P)H in rag8 mutant samples with respect to the wild type ones. We also observed an increased de novo synthesis of nicotinamide adenine dinucleotide. On the contrary, the production of metabolites in pathways leading to the reduction of the cofactors was reduced. CONCLUSIONS: The changes in metabolite levels in rag8 showed a metabolic adaptation that is determined by the intracellular NAD(P)(+)/NAD(P)H redox balance state. GENERAL SIGNIFICANCE: The inadequate glycolytic flux of the mutant leads to a reduced/asymmetric distribution of acetyl-CoA to the different cellular compartments with loss of the fatty acid dynamic respiratory/fermentative adaptive balance response.

Monitoring of metabolic profiling and water status of Hayward kiwifruits by nuclear magnetic resonance.

The metabolic profiling of kiwifruit (Actinidia deliciosa, Hayward cultivar) aqueous extracts and the water status of entire kiwifruits were monitored over the season (June-December) using nuclear magnetic resonance (NMR) methodologies. The metabolic profiling of aqueous kiwifruit extracts was investigated by means of high field NMR spectroscopy. A large number of water-soluble metabolites were assigned by means of 1D and 2D NMR experiments. The change in the metabolic profiles monitored over the season allowed the kiwifruit development to be investigated. Specific temporal trends of aminoacids, sugars, organic acids and other metabolites were observed. The water status of kiwifruits was monitored directly on the intact fruit measuring the T(2) spin-spin relaxation time by means of a portable unilateral NMR instrument, fully non-invasive. Again, clear trends of the relaxation time were observed during the monitoring period. The results show that the monitoring of the metabolic profiling and the monitoring of the water status are two complementary means suitable to have a complete view of the investigated fruit.

Gut microbiome-derived metabolites characterize a peculiar obese urinary metabotype.

Obesity is a complex multifactorial disease involving genetic and environmental factors and influencing several different metabolic pathways. In this regard, metabonomics, that is the study of complex metabolite profiles in biological samples, may provide a systems approach to understand the global metabolic regulation of the organism in relation to this peculiar pathology. In this pilot study, we have applied a nuclear magnetic resonance (NMR)-based metabolomic approach on urinary samples of morbidly obese subjects. Urine samples of 15 morbidly obese insulin-resistant (body mass index>40; homeostasis assessment model of insulin resistance>3) male patients and 10 age-matched controls were collected, frozen and analyzed by high-resolution (1)H-NMR spectroscopy combined with partial least squares-discriminant analysis. Furthermore, two obese patients who underwent bariatric surgery (biliopancreatic diversion and gastric bypass, respectively) were monitored during the first 3 months after surgery and their urinary metabolic profiles were characterized. NMR-based metabolomic analysis allowed us to identify an obesity-associated metabolic phenotype (metabotype) that differs from that of lean controls. Gut flora-derived metabolites such as hippuric acid, trigonelline, 2-hydroxyisobutyrate and xanthine contributed most to the classification model and were responsible for the discrimination. These preliminary results confirmed that in humans the gut microflora metabolism is strongly linked to the obesity phenotype. Moreover, the typical obese metabotype is lost after weight loss induced by bariatric surgery.

An NMR spectroscopy study of bendaline-albumin interactions.

The complete assignment of the 1H and 13C NMR spectra of bendaline (BNDL) was performed by mono-dimensional and homo- and hetero-correlated two-dimensional NMR experiments. The interaction between bendaline and albumin was also studied by the analysis of the motional parameters spin-lattice relaxation times, allowing the motional state of the BNDL free and bound with albumin to be defined. In absence of albumin the indazolacetic and benzylic moieties are characterized by roughly the same mobility and by positive sigma (cross-relaxation rates) values. In the presence of the macromolecule, the indazolacetic and benzylic moieties and the lysine change their motional behaviour to different extents, as indicated by correlation times. Data obtained in absence and in presence of the protein show that the molecular moiety of the bendaline most involved in the binding with albumin is the fragment H-4 H-5. The binding constant was evaluated at 2.4x10(3)M(-1).

Investigation of lipase-catalysed hydrolysis of naproxen methyl ester: use of NMR spectroscopy methods to study substrate-enzyme interaction.

(+/-)-2-(6-Methoxy-2-naphthyl)propionic acid methyl ester (methyl ester of Naproxen), the precursor of therapeutically important nonsteroidal anti-inflammatory drugs (NSAIDs) was enantioselectively hydrolysed using as biocatalyst Candida rugosa lipase. In research aimed at studying the structure-activity relationship (SAR), NMR spectroscopy methods were employed to identify which Naproxen molecular moiety was essential to the substrate-enzyme interaction. The experimental results, in agreement with previous computer modelling studies and reported kinetic data, gave new information on the enzyme-substrate complex formation in solution.

Molecular characterization and action of usnic acid: a drug that inhibits proliferation of mouse polyomavirus in vitro and whose main target is RNA transcription.

Usnic acid is a normal component of lichen cells. This natural compound has shown different biological and physiological activities that might have a great relevance in pharmacology and clinics. For instance, usnic acid is known for its antibacterial and antiparasitic action. Also, the drug has a potential interest in cancer therapy because of its antimitotic and antiproliferative action. The molecular structure of usnic acid has been validated and further explored in this investigation. Many biological properties of this drug are known; however its potential antiviral action has not yet been evaluated. In this paper, we demonstrate that usnic acid is a potent inhibitor of the proliferation of mouse polyomavirus. Its action is not exerted at the level of virion entry into the host cell. Moreover, the abolition of viral DNA replication is an indirect consequence of the drastic inhibition of RNA transcription.

Notch signalling acts in postmitotic avian myogenic cells to control MyoD activation.

During Drosophila myogenesis, Notch signalling acts at multiple steps of the muscle differentiation process. In vertebrates, Notch activation has been shown to block MyoD activation and muscle differentiation in vitro, suggesting that this pathway may act to maintain the cells in an undifferentiated proliferative state. In this paper, we address the role of Notch signalling in vivo during chick myogenesis. We first demonstrate that the Notch1 receptor is expressed in postmitotic cells of the myotome and that the Notch ligands Delta1 and Serrate2 are detected in subsets of differentiating myogenic cells and are thus in position to signal to Notch1 during myogenic differentiation. We also reinvestigate the expression of MyoD and Myf5 during avian myogenesis, and observe that Myf5 is expressed earlier than MyoD, consistent with previous results in the mouse. We then show that forced expression of the Notch ligand, Delta1, during early myogenesis, using a retroviral system, has no effect on the expression of the early myogenic markers Pax3 and Myf5, but causes strong down-regulation of MyoD in infected somites. Although Delta1 overexpression results in the complete lack of differentiated muscles, detailed examination of the infected embryos shows that initial formation of a myotome is not prevented, indicating that exit from the cell cycle has not been blocked. These results suggest that Notch signalling acts in postmitotic myogenic cells to control a critical step of muscle differentiation.

Delta 1-activated notch inhibits muscle differentiation without affecting Myf5 and Pax3 expression in chick limb myogenesis.

The myogenic basic helix-loop-helix (bHLH) transcription factors, Myf5, MyoD, myogenin and MRF4, are unique in their ability to direct a program of specific gene transcription leading to skeletal muscle phenotype. The observation that Myf5 and MyoD can force myogenic conversion in non-muscle cells in vitro does not imply that they are equivalent. In this paper, we show that Myf5 transcripts are detected before those of MyoD during chick limb development. The Myf5 expression domain resembles that of Pax3 and is larger than that of MyoD. Moreover, Myf5 and Pax3 expression is correlated with myoblast proliferation, while MyoD is detected in post-mitotic myoblasts. These data indicate that Myf5 and MyoD are involved in different steps during chick limb bud myogenesis, Myf5 acting upstream of MyoD. The progression of myoblasts through the differentiation steps must be carefully controlled to ensure myogenesis at the right place and time during wing development. Because Notch signalling is known to prevent differentiation in different systems and species, we sought to determine whether these molecules regulate the steps occurring during chick limb myogenesis. Notch1 transcripts are associated with immature myoblasts, while cells expressing the ligands Delta1 and Serrate2 are more advanced in myogenesis. Misexpression of Delta1 using a replication-competent retrovirus activates the Notch pathway. After activation of this pathway, myoblasts still express Myf5 and Pax3 but have downregulated MyoD, resulting in inhibition of terminal muscle differentiation. We conclude that activation of Notch signalling during chick limb myogenesis prevents Myf5-expressing myoblasts from progressing to the MyoD-expressing stage.

Paraxis is expressed in myoblasts during their migration and proliferation in the chick limb bud.

The Paraxis gene encodes a basic helix-loop-helix (bHLH) transcription factor expressed in paraxial mesoderm and whose mutant displays an inability to form epithelial somites. We analyzed the later expression pattern of Paraxis transcripts in the chick limb. Paraxis transcripts are located in the Pax3-expressing myoblasts migrating from the somites and persist in proliferating myoblasts in the limb bud. Moreover, the QH1 antibody, which recognizes quail endothelial cells, reacts with a subset of the Paraxis/Pax3 expressing cells in the quail limb. Paraxis is then down-regulated during muscle differentiation.

1H NMR relaxation investigation of inhibitors interacting with Torpedo californica acetylcholinesterase.

Two naphthyridines interacting with Torpedo californica acetylcholinesterase (AChE) were investigated. (1)H NMR spectra were recorded and nonselective, selective, and double-selective spin-lattice relaxation rates were measured. The enhancement of selective relaxation rates could be titrated by different ligand concentrations at constant AChE (yielding 0.22 and 1.53 mM for the dissociation constants) and was providing evidence of a diverse mode of interaction. The double-selective relaxation rates were used to evaluate the motional correlation times of bound ligands at 34.9 and 36.5 ns at 300 K. Selective relaxation rates of bound inhibitors could be interpreted also in terms of dipole-dipole interactions with protons in the enzyme active site.

Delineation of conformational and structural features of the amikacin-Cu(II) complex in water solution by 13C-NMR spectroscopy.

The copper (II) complex of amikacin in water solution at pH 5.5 was investigated by 13C-NMR. The temperature dependence of spin-lattice relaxation rates was measured and fast exchange conditions were shown to apply. The motional correlation time of the complex was approximated by the pseudo-isotropic rotational correlation time of free amikacin in water solution (tau c = 0.17 ns at 300 K). Formation of a pseudo-tetrahedral 1:1 complex was demonstrated by relaxation rates analysis and also by UV-Vis spectrophotometry. Two amino nitrogens of amikacin, together with the amide nitrogen and the hydroxyl in the hydroxyl-aminopropyl carbonyl side chain, were assigned as the copper-binding sites and a model of the complex was built by using copper-carbon distances obtained by NMR analysis as input parameters.

Chemical analysis of water of the Anticolana Valley: isolation of humic compounds.

The purpose of this study was to identify and characterize humic compounds in Anticolana Valley (Fiuggi) water. The capacity of this class of compounds to mobilize metals from solid phases could have an important role in calculi solubilization. Humic compounds were isolated, purified and characterised by FTIR, 1H-NMR and 13C-NMR spectroscopy, thermogravimetry and elemental analysis. Only fulvic acids were found. They are mainly composed of aliphatic chains, made of six -CH2O- groups and contain a number of carboxylic groups, responsible for their metal complexing capacity.

1H NMR studies on the interaction of beta-carboline derivatives with human serum albumin.

1H NMR studies were performed on two beta-carboline derivatives interacting with human serum albumin. The spin-lattice relaxation rates of the two derivatives, having side chains of different length and polarity, were used to demonstrate a diverse motional behavior in solution together with slightly different relaxation pathways. Single- and double-selective excitation made it possible to evaluate dynamics in the free and protein-bound states. Occurrence of a relatively long hydrophilic chain interacting with the proton-acceptor nitrogen of the beta-carboline moiety was shown to yield lower association constants, slower dissociation rates, and diverse interacting modes with the indole hydrophobic site of the protein.

Conformational study of [Met5]enkephalin-Arg-Phe in the presence of phosphatidylserine vesicles.

The interaction of [Met5]enkephalin-Arg.Phe with phosphatidylserine (PtdSer) was studied by circular dichroism (CD), two-dimensional nuclear magnetic resonance spectroscopy, hybrid distance geometry simulated annealing (DG-SA) and molecular dynamics (MD) calculations. The very low solubility of [Met5]enkephalin-Arg-Phe and the instability of the solution containing PtdSer vesicles at low pH values did not allow us to observe the amide proton resonances in the usual two-dimensional NMR work. NOESY cross-peaks of protons of side chains from two-dimensional NMR were converted into distances which were used as restraints for modelling with DG-SA and MD. Our results indicate that, in aqueous solutions at pH 7.68 [Met5]enkephalin-Arg-Phe exists in the absence of PtdSer as a random distribution of conformers, whereas in the presence of PtdSer it adopts conformations containing a common orientation of the bonds of C alpha 2, C alpha 3, C alpha 4, and C alpha 5, although different orientations of the peptide planes are consistent with the results. Two of the reported conformers from MD simulations are characterized by the presence of a 2<--4 gamma and inverse gamma turns centered on Gly3. A gradual decline of order was observed when moving from the central moiety of the peptide to both the N-terminus and C-terminus. Finally, the DG-SA and MD calculations resulted in a structure such that the orientation of the Phe4 and Met5 side chains favours hydrophobic interactions with the apolar portion of the PtdSer vesicle to form a hydrophobic cluster. These data support the hypothesis of a role of lipids to modify the conformation of [Met5]enkephalin-Arg-Phe to permit the interactions with the receptor site.

Enzymatic catalysis by lipase from Candida cylindracea: enantiomeric activity evaluation by 1H and 13C NMR.

Lipolytic enzymes represent an important class of biocatalysts and are widely used in the resolution of racemic mixtures. The activity of lipase from Candida cylindracea at different temperatures has been studied by NMR determination of the enantiomeric excess in the enantioselective hydrolysis of 2-arylpropionic acid esters of pharmacological interest. At a purpose, a system based on Europium (III) chiral shift reagent has been settled and utilized.

1H and 23Na NMR relaxation times study of pectin solutions and gels.

1H and 23Na longitudinal and transverse relaxation times have been measured to examine the concentration dependence of the dynamic behaviour of pectin solutions and gels. T1 and T2 relaxation times were measured in HDE pectin solutions and gels prepared with different cosolutes. A lowering of T1 and T2 values was observed in relation to a better efficiency of the intermolecular forces between polymer molecules and/or the formation of more extended junction zones. The T1/T2 ratio was also exploited indicating that even bulk water experiences anisotropic motions due to the gel formation. A correspondence of NMR results with previous reported data of rheological measurements of pectin gels prepared with the same cosolutes was found.

A new tripeptide, Pol 509, influences biochemical events associated with antigen presentation efficiency of PPD-specific EBV-B cells.

A synthetic tripeptide (pGLU-LEU-TRP-OCH3) Pol 509, derived from snake venom, was studied directly by analyzing the interactions with synthetic lipid bilayers using NMR spectroscopy. Functional studies were also performed by measuring the effects: i), on early biochemical events (adenyl cyclase and phospholipase C activation products), intermediate (surface Ag expression) and late (DNA synthesis) parameters following B-cell activation elicited by PPD-linkage to specific membrane Ig; and ii), on the presentation of PPD to Ag-specific T-cell lines. Comparative experiments using PMA and IFN-gamma were also performed. We found that all parameters studied were affected by Pol 509 treatment. In fact, while PPD linkage to mlg reversed the balance between cAMP and IP3 existing in unstimulated EBV-B cells, Pol 509 reduced the PPD-induced accumulation of cAMP to control values and induced a further decrease of IP3 level. Pol 509-mediated decrease of these second messenger levels was accompanied by a slight increase of HLA-DR molecule expression and DNA synthesis inhibition. Furthermore, Pol 509 enhanced the efficiency of PPD presentation to T-cell lines. Taken together, these observations suggest that Pol 509, which enhances Ag presentation by modifying second messenger levels, may be considered as a new immunomodulatory drug with immunopotentiating activity.

23Na NMR evaluation of human erythrocytes Na+/K+/CL-cotransport. A study in elderly hypertensives.

The behaviour of sodium transport systems across the cell membrane has been poorly investigated in elderly hypertension. Sodium efflux driven by Na+/K+/Cl-cotransport activity was therefore investigated (using a novel NMR-spectroscopy method) in 5 elderly hypertensive males (mean age 78 +/- 5 years) and 5 normotensive controls (mean age 79 +/- 3 years). In order to exclude any change in cotransport activity secondary to metabolic abnormalities, both patients and controls were non-obese and had normal glucose and lipid metabolisms. The Na+/K+/Cl-cotransport evaluation was performed after three months of pharmacological wash-out, under a diet containing 120 mEq of Na+/day. The resulting data showed that Na+ efflux due to outward Na+/K+/Cl-cotransport was higher in hypertensive group than in the normotensive one (0.50 +/- 0.10 mmol Na+/l cells/hr. vs 0.33 +/- 0.03 mmol Na+/l cells/hr., respectively, p < 0.05). Intracellular Na+ content was similar in both groups. At variance with previous data from the literature, our findings indicate that the Na+/K+/Cl-cotransport activity is elevated in elderly hypertensives.

Improved resolution of 31P nuclear magnetic resonance spectra of phospholipids from brain.

A method is described wherein the resolution of 31P nuclear magnetic resonance spectra of the lipophilic fraction from a Bligh-Dyer extract of rat brain can be enhanced. The lipids are dispersed as micelles in aqueous solution with sodium deoxycholate, and spectral resolution is further optimized by adjusting the pH and by removing ions from the solution. The application of the method to the study of aging in rat brain serves as an example.