From metabolic to metabolomic NMR spectroscopy of apoptotic cells

Over the past 10–15 years, nuclear magnetic resonance (NMR) spectroscopy has been employed to study metabolic events accompanying programmed cell death (apoptosis). The early studies were characterized by experiments focusing on specific metabolic parameters obtained by analyzing a limited number of biochemical compounds, e.g. selected metabolic species involved in the Krebs cycle, in energy metabolism, in phospholipid synthesis and degradation, or in mobile-lipid accumulation. However, during the past few years metabolic NMR spectroscopy has begun to refocus towards more comprehensive analyses of tissue metabolites detectable in NMR spectra. This review describes some requirements needed for the development of an integrated, metabolomic concept for NMR spectroscopy investigations of apoptotic cells, and presents recent studies approaching this goal. Metabolomic NMR spectroscopy allows one not only to distinguish between cells that are sensitive to apoptosis induction and resistant cells, but also, in conjunction with measurements of complementary biological parameters, to follow the temporal evolution of the apoptotic process and to analyze mechanisms of apoptosis resistance.     

Chirality of organophosphorus pesticides: Analysis and toxicity

Although the importance of chirality in organophosphorus compounds (OPs) is well recognized in relation to their biological effects, as with most chiral pesticides, OPs are generally marketed, used and released to the environment as racemates (i.e., equimolar mixtures of enantiomers). In addition, research on enantioselective environmental fate and effects of chiral OPs is still limited, particularly in the evaluation of enantioselectivity in their environmental degradation. A large number of OPs are chiral compounds, and yet enantioselectivity in their environmental fate and effects is rarely addressed. This paper highlights the current state of knowledge on the environmental occurrence and behavior of chiral OP pesticides. Developments in enantioselective analytical techniques, specifically gas chromatography (GC), high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), as applied in the evaluation of enantiomer-specific fate and effects of chiral OPs, are also discussed.     

NMR structural biology of sulfated glycans

Sulfated fucans, sulfated galactans, and glycosaminoglycans are extensively studied worldwide in terms of both structure and biomedical functions. Liquid-state nuclear magnetic resonance (NMR) spectroscopy is the most employed analytical technique in structural analysis of these sulfated glycans. This is due to the fact that NMR-based analyses enable a series of achievements such as (i) accurate structure characterization/determination; (ii) measurements of parameters regarding molecular motion (dynamics); (iii) assessment of the 3D structures (usually assisted by computational techniques of Molecular Modeling and/or Molecular Dynamics) of the composing monosaccharides (ring conformers) and the overall conformational states of the glycan chains either free in solution or bound to proteins; and (iv) analysis of the resultant intermolecular complexes with functional proteins through either the protein or the carbohydrate perspective. In this review, after a general introduction about the principal NMR parameters utilized for achieving this set of structural information, discussion is given on NMR-based studies of some representative sulfated fucans, sulfated galactans, and glycosaminoglycans. Due to the growing number of studies concerning both structure and function of sulfated glycans and the widely use of NMR spectroscopy in such studies, a review paper discussing (i) the most experiments employed for analysis, (ii) procedures used in data interpretation, and (iii) the general aspects of the sulfated glycans, is timely in the literature.     

Dynamic membrane interactions of antibacterial and antifungal biomolecules,and amyloid peptides,revealed by solid-state NMR spectroscopy

A variety of biomolecules acting on the cell membrane folds into a biologically active structure in the membrane environment. It is, therefore, important to determine the structures and dynamics of such biomolecules in a membrane environment. While several biophysical techniques are used to obtain low-resolution information, solid-state NMR spectroscopy is one of the most powerful means for determining the structure and dynamics of membrane bound biomolecules such as antibacterial biomolecules and amyloidogenic proteins; unlike X-ray crystallography and solution NMR spectroscopy, applications of solid-state NMR spectroscopy are not limited by non-crystalline, non-soluble nature or molecular size of membrane-associated biomolecules. This review article focuses on the applications of solid-state NMR techniques to study a few selected antibacterial and amyloid peptides. Solid-state NMR studies revealing the membrane inserted bent α-helical structure associated with the hemolytic activity of bee venom melittin and the chemical shift oscillation analysis used to determine the transmembrane structure (with α-helix and 3₁₀-helix in the N- and C-termini, respectively) of antibiotic peptide alamethicin are discussed in detail. Oligomerization of an amyloidogenic islet amyloid polypeptide (IAPP, or also known as amylin) resulting from its aggregation in a membrane environment, molecular interactions of the antifungal natural product amphotericin B with ergosterol in lipid bilayers, and the mechanism of lipid raft formation by sphingomyelin studied using solid state NMR methods are also discussed in this review article. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato.     

Discovery of subtype selective muscarinic receptor antagonists as alternatives to atropine using in silico pharmacophore modeling and virtual screening methods

Muscarinic acetylcholine receptors (mAChRs) have five known subtypes which are widely distributed in both the peripheral and central nervous system for regulation of a variety of cholinergic functions. Atropine is a well known muscarinic subtype non-specific antagonist that competitively inhibits acetylcholine (ACh) at postganglionic muscarinic sites. Atropine is used to treat organophosphate (OP) poisoning and resulting seizures in the warfighter because it competitively inhibits acetylcholine (ACh) at the muscarinic cholinergic receptors. ACh accumulates due to OP inhibition of acetylcholinesterase (AChE), the enzyme that hydrolyzes ACh. However, atropine produces several unwanted side-effects including dilated pupils, blurred vision, light sensitivity, and dry mouth. To overcome these side-effects, our goal was to find an alternative to atropine that emphasizes M1 (seizure prevention) antagonism but has minimum M2 (cardiac) and M3 (e.g., eye) antagonism so that an effective less toxic medical countermeasure may be developed to protect the warfighter against OP and other chemical warfare agents (CWAs). We adopted an in silico pharmacophore modeling strategy to develop features that are characteristics of known M1 subtype-selective compounds and used the model to identify several antagonists by screening an in-house (WRAIR-CIS) compound database. The generated model for the M1 selectivity was found to contain two hydrogen bond acceptors, one aliphatic hydrophobic, and one ring aromatic feature distributed in a 3D space. From an initial identification of about five hundred compounds, 173 compounds were selected through principal component and cluster analyses and in silico ADME/Toxicity evaluations. Next, these selected compounds were evaluated in a subtype-selective in vitro radioligand binding assay. Twenty eight of the compounds showed antimuscarinic activity. Nine compounds showed specificity for M1 receptors and low specificity for M3 receptors. The pK_i values of the compounds range from 4.5 to 8.5 nM in comparison to a value of 8.7 nM for atropine. 2-(diethylamino)ethyl 2,2-diphenylpropanoate (ZW62841) was found have the best desired selectivity. None of the newly found compounds were previously reported to exhibit antimuscarinic specificity. Both theoretical and experimental results are presented.     

Interaction of platinum compounds with short oligodeoxynucleotides containing guanine and cytosine

The products resulting from reaction of cis-Pt(NH3)2Cl2 with d(CpCpGpG), d(GpCpG), d(pCpGpCpG), d(pGpCpGpC) and d(CpGpCpG) and from reaction of [Pt(dien)Cl]Cl with d(CpCpGpG) and d(GpCpG) have been characterized with the aid of proton NMR spectroscopy, circular dichroic spectroscopy and Pt analysis. The binding sites of the Pt compounds were determined by pH-dependent NMR spectroscopy. Binding of the two Pt compounds invariably occurs at the guanine N7 atoms. In all compounds containing [cis-Pt(NH3)2]2+ chelates are formed by coordination of platinum to two guanines of the same oligonucleotide. The resulting intrastrand-cross-linked oligonucleotides contain either d(GpG) . cisPt units, or d(GpCpG) . cisPt units. In the latter case the middle cytosine is not coordinated to platinum. As a result the conformational changes originating from these two chelates are different from each other. In the case of [Pt(dien)Cl]Cl as a starting product, two types of oligonucleotide adducts are formed, i.e. those with one Pt atom/molecule and those with two Pt atoms/molecule. The NMR spectra of the adducts containing only one Pt(dien)2+ show that only one adduct is formed, although two guanine bases are present. This indicates a preference for one of the N7 atoms in the molecule.     

Anaerobic degradation of non-substituted aromatic hydrocarbons

Aromatic hydrocarbons are among the most prevalent organic pollutants in the environment. Their removal from contaminated systems is of great concern because of the high toxicity effect on living organisms including humans. Aerobic degradation of aromatic hydrocarbons has been intensively studied and is well understood. However, many aromatics end up in habitats devoid of molecular oxygen. Nevertheless, anaerobic degradation using alternative electron acceptors is much less investigated. Here, we review the recent literature and very early progress in the elucidation of anaerobic degradation of non-substituted monocyclic (i.e. benzene) and polycyclic aromatic hydrocarbons (PAH such as naphthalene and phenanthrene). A focus will be on benzene and naphthalene as model compounds. This review concerns the microbes involved, the biochemistry of the initial activation and subsequent enzyme reactions involved in the pathway.     

The application of 19F nuclear magnetic resonance to investigate microbial biotransformations of organofluorine compounds

Fluorinated organic compounds, although rare in nature, are significant environmental contaminants owing to the numerous applications for which this class of compounds is employed. It is important that biodegradation of these compounds can be readily assessed in order to provide information on their fate in the environment. Fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy has emerged as a very useful technique to readily determine the catabolism of fluorinated aromatic compounds by microorganisms, either in whole cell or cell-free systems. The principal advantage of this technique is that fluorinated compounds can be observed directly in the culture supernatant or enzyme assay, without purification or derivatization. In this review an account of the application of 19F NMR in the study of microbial metabolism of organofluorine compounds is presented.     

Endocrine disrupting compounds: effect of octylphenol on reproduction over three generations

With the growing concern that environmental chemicals might impair human and animal fertility, it is important to investigate the possible influence of these substances on sexual differentiation and genital development of mammals. Many of these substances are suspected to interfere with endocrine processes, and exposure during critical periods of prenatal development might affect reproductive performance over several generations. Alkylphenols and their metabolites are lipophilic substances exerting apparent estrogenic action in in vitro and in vivo testing systems. With the widespread industrial use of alkylphenols, these are disseminated in the environment with sewage sludge, and domestic animals and humans are likely to be exposed via the food chain. Using the pig as an in vivo model, we studied the effect of intrauterine exposure to tertiary octylphenol (OP) on essential reproductive parameters over 3 generations. Sows were treated daily from D 23 to 85 of pregnancy with either 0, 10 or 1000 micrograms OP/kg body weight. Treatment with OP extended pregnancy length and induced basal cell proliferation in the cervical epithelium of the parental generation. In F1 offspring of sows treated with the low dosage of OP, onset of puberty was accelerated. Furthermore, when F1 gilts and F1 boars originating from sows treated with high dosages of OP were bred, the litter size was reduced. The results of the present study are compared with previous reports on estrogenicity of OP, and the usefulness of in vivo animal or embryo models for the evaluation of possible consequences of human exposure to endocrine disrupting compounds is discussed. Furthermore, possible consequences of exposure to endocrine disrupting compounds for the embryo transfer industry are addressed.     

Detoxification of G- and V-series nerve agents by the phosphotriesterase OpdA

Abstract

Intoxication by organophosphorous (OP) insecticides and nerve agents is often lethal and currently available therapeutics are often ineffective. A range of catalytic and stoichiometric OP scavengers have been investigated for use as potential treatments for OP poisoning. Recent studies have shown that one enzyme, OpdA, an enzyme involved in organophosphorous degradation, was an effective treatment for OP insecticide poisoning in animal models. Here we have tested OpdA for its ability to detoxify G- and V-type nerve agents in vitro. Although OpdA was found to have high catalytic activities for G-series toxins (soman and cyclosarin), it was substantially less active with V-type nerve agents. The activity towards V-series agents was close to the theoretical maximum for this enzyme (i.e. the rate determined by the chemistry of the leaving group); it seems unlikely that enzyme engineering or directed evolution could be used to improve upon this activity without a significant change in its reaction mechanism.     

The 5-hydrazino-3-methylisothiazole-4-carboxylic acid,its new 5-substituted derivatives and their antiproliferative activity

Currently, the basic method of treatment of colon cancer is surgery. The range of anticancer drugs used in the treatment of colorectal cancer is small and is based mainly on systemic combination chemotherapy. As a result of the designed syntheses, we received new isothiazole derivatives with anticancer activity. The synthesized 5-hydrazino-3-methylisothiazole-4-carboxylic acid has never been obtained before. It is also a substrate for the synthesis of its innovative derivatives, i.e. compounds that are Schiff bases. The identification of the structure of new compounds was carried out using mass spectrometry (MS), proton nuclear magnetic resonance spectroscopy (¹H NMR), carbon nuclear magnetic resonance spectroscopy (¹³C NMR) and infrared spectroscopy (IR). Potential antitumor activity was confirmed in antiproliferative MTT and SRB tests. The selected, most biologically active substances were characterized by high selectivity towards leukemia and colon cancer cell lines. They caused high inhibition of proliferation of human biphenotypic B cell myelomonocytic leukemia MV4-11 (13 compounds), human colon adenocarcinoma cell lines sensitive LoVo (8 compounds) and resistant to doxorubicin LoVo/DX (12 compounds). However, in the conducted studies, their activity against breast adenocarcinoma MCF-7 and normal non-tumorigenic epithelial cell line derived from mammary gland MCF-10A was substantially lower. The result of this work is claimed Polish patent application.     

NMR studies of drug interaction with membranes and membrane-associated proteins

This review focuses on the recent developments in the study of drug interactions with biological membranes and membrane-associated proteins using nuclear magnetic resonance (NMR) spectroscopy and other spectroscopic techniques. Emphasis is placed on a class of low-affinity neurological agents as exemplified by volatile general anesthetics and structurally related compounds. The technical aspects are reviewed of how to prepare membrane-mimetic systems and of NMR approaches that are either in current use or opening new prospects. A brief literature survey covers studies ranging from drug distribution in simplified lipid matrix to specific drug interaction with neuronal receptors reconstituted in complicated synthetic membrane systems.     

Recent excitements in protein NMR: Large proteins and biologically relevant dynamics

The advent of Transverse Relaxation Optimized SpectroscopY (TROSY) and perdeuteration allowed biomolecular NMR spectroscopists to overcome the size limitation barrier (~20 kDa) in de novo structure determination of proteins. The utility of these techniques was immediately demonstrated on large proteins and protein complexes (e.g. GroEL-GroES, ClpP protease, Hsp90-p53, 20S proteasome, etc.). Further, recent methodological developments such as Residual Dipolar Couplings and Paramagnetic Relaxation Enhancement allowed accurate measurement of long-range structural restraints. Additionally, Carr-Purcell-Meiboom-Gill (CPMG), rotating frame relaxation experiments (R₁ρ) and saturation transfer experiments (CEST and DEST) created never-before accessibility to the μs–ms timescale dynamic parameters that led to the deeper understanding of biological processes. Meanwhile, the excitement in the field continued with a series of developments in the fast data acquisition methods allowing rapid structural studies on less stable proteins. This review aims to discuss important developments in the field of biomolecular NMR spectroscopy in the recent past, i.e., in the post TROSY era. These developments not only gave access to the structural studies of large protein assemblies, but also revolutionized tools in the arsenal of today’s biomolecular NMR and point to a bright future of biomolecular NMR spectroscopy.     

Studies on the Chemical Constituents of Annona squamosa(5)

From the seeds of Annona squamosa L.,three compounds were obtained,in which compounds i and 2 are known compounds,i.e. bullatacin and annonin I,respectively.Compounds 3 is a new non -adjacent bis -tetrahydrofuranyl annonaceous acetogenin,named squamostatin C.Based on the analysis of IR MS,1H NMR and 13 C NMR spectra,the structure of compound 3 was elucidated as (3).     

The structural and topological analysis of membrane-associated polypeptides by oriented solid-state NMR spectroscopy: established concepts and novel developments

Solid-state NMR spectroscopy is a powerful technique for the investigation of membrane-associated peptides and proteins as well as their interactions with lipids, and a variety of conceptually different approaches have been developed for their study. The technique is unique in allowing for the high-resolution investigation of liquid disordered lipid bilayers representing well the characteristics of natural membranes. Whereas magic angle solid-state NMR spectroscopy follows approaches that are related to those developed for solution NMR spectroscopy the use of static uniaxially oriented samples results in angular constraints which also provide information for the detailed analysis of polypeptide structures. This review introduces this latter concept theoretically and provides a number of examples. Furthermore, ongoing developments combining solid-state NMR spectroscopy with information from solution NMR spectroscopy and molecular modelling as well as exploratory studies using dynamic nuclear polarization solid-state NMR will be presented.     

Refinement of structural leads for centrally acting oxime reactivators of phosphylated cholinesterases

We present a systematic structural optimization of uncharged but ionizable N-substituted 2-hydroxyiminoacetamido alkylamine reactivators of phosphylated human acetylcholinesterase (hAChE) intended to catalyze the hydrolysis of organophosphate (OP)-inhibited hAChE in the CNS. Starting with the initial lead oxime RS41A identified in our earlier study and extending to the azepine analog RS194B, reactivation rates for OP-hAChE conjugates formed by sarin, cyclosarin, VX, paraoxon, and tabun are enhanced severalfold in vitro. To analyze the mechanism of intrinsic reactivation of the OP-AChE conjugate and penetration of the blood-brain barrier, the pH dependence of the oxime and amine ionizing groups of the compounds and their nucleophilic potential were examined by UV-visible spectroscopy, (1)H NMR, and oximolysis rates for acetylthiocholine and phosphoester hydrolysis. Oximolysis rates were compared in solution and on AChE conjugates and analyzed in terms of the ionization states for reactivation of the OP-conjugated AChE. In addition, toxicity and pharmacokinetic studies in mice show significantly improved CNS penetration and retention for RS194B when compared with RS41A. The enhanced intrinsic reactivity against the OP-AChE target combined with favorable pharmacokinetic properties resulted in great improvement of antidotal properties of RS194B compared with RS41A and the standard peripherally active oxime, 2-pyridinealdoxime methiodide. Improvement was particularly noticeable when pretreatment of mice with RS194B before OP exposure was combined with RS194B reactivation therapy after the OP insult.     

31P NMR chemical shifts of phosphate covalently bound to proteins

31P nuclear magnetic resonance (NMR) spectroscopy for characterizing the nature of covalently bound phosphate in proteins is relatively unexploited by the biochemist. 31P NMR chemical shifts of phosphate covalently bound to naturally occurring phosphoproteins, phosphorylated enzyme intermediates and chemically phosphorylated proteins have been compiled in this review. The chemical shifts (31P NMR) of selected reference compounds are reported to assist in the assignment of 31P resonances of phosphate covalently attached to proteins. 31P NMR chemical shifts of phosphate and phospho compounds non-covalently bound to selected proteins as well as the pH dependence of 31P NMR resonance have also been compiled.     

Prospects for in vivo NMR methods in xenobiotic research in plants

The application of non-invasive nuclear magnetic resonance (NMR) methods in xenobiotic research is reviewed in relation to: (i) the characterisation of the effects of xenobiotics on the metabolism of plants and plant cell suspensions; (ii) the direct detection of xenobiotics and their degradation products in vivo; and (iii) the spatial localisation of xenobiotics and their derivatives at the subcellular and tissue levels. Novel information has been generated by in vivo NMR studies of both agrochemicals and heavy metals, but a lack of generality in the methods makes it difficult to extrapolate from one successful application to the next. In vivo NMR spectroscopy is shown to be informative when a xenobiotic perturbs metabolic pathways that are accessible to the technique, and it is useful for probing the partitioning of paramagnetic metal ions between the cytoplasm and the vacuole. The successful application of 19F NMR to the analysis of plant tissue extracts also suggests that in vivo 19F NMR spectroscopy may have a role in biotransformation studies of fluorinated xenobiotics. In contrast NMR imaging techniques have been little used for xenobiotic research in plants, and while the method has been shown to be capable of monitoring the uptake and translocation of paramagnetic ions in plants, the potential use of high resolution 1H and 19F NMR imaging for mapping agrochemicals in tissues is still in its infancy.     

Choosing membrane mimetics for NMR structural studies of transmembrane proteins

The native environment of membrane proteins is complex and scientists have felt the need to simplify it to reduce the number of varying parameters. However, experimental problems can also arise from oversimplification which contributes to why membrane proteins are under-represented in the protein structure databank and why they were difficult to study by nuclear magnetic resonance (NMR) spectroscopy. Technological progress now allows dealing with more complex models and, in the context of NMR studies, an incredibly large number of membrane mimetics options are available. This review provides a guide to the selection of the appropriate model membrane system for membrane protein study by NMR, depending on the protein and on the type of information that is looked for. Beside bilayers (of various shapes, sizes and lamellarity), bicelles (aligned or isotropic) and detergent micelles, this review will also describe the most recent membrane mimetics such as amphipols, nanodiscs and reverse micelles. Solution and solid-state NMR will be covered as well as more exotic techniques such as DNP and MAOSS.     

C8- and C9-Alkylphenols and Ethoxylates: II. Assessment of Environmental Persistence and Bioaccumulation Potential

C8- and C9-alkylphenols and their ethoxylates (APE) are widely used commercial products mainly used in industrial applications, in the formulation of crop protection chemicals, and in industrial and household cleaners. Recent regulatory focus on these compounds has included an assessment of their potential to meet criteria for persistent, bioaccumulative, and toxic compounds (PBT). To fully evaluate either the relative persistence or bioaccumulation potential of any APE, degradation intermediates and metabolic by-products of these compounds should also be considered. To facilitate the evaluation of the ultimate fate of APE in the environment, a review of the degradation pathways and identification of degradation intermediates was performed (part I of a two-part series). In part II of this series, the relative persistence of APE as indicated by degradation half-lives was examined based on a review of abiotic and biological degradation data. To assess the bioaccumulation potential of APE, the relevant literature was also reviewed. The available data for C8- and C9-APE show that the commercial products and their degradation intermediates do not meet any national or international criteria for identifying these compounds as PBT substances.