Stable free radicals in insect cuticles: electron spin resonance spectroscopy reveals differences between melanization and sclerotization

Insect cuticles (exuviae; cast skins) were examined for the first time by ESR spectroscopy for the presence of stable free radicals, as found in melanins. All cuticles, except those from a locust albino strain, irrespective of the presence of melanin, provided single-line signals of varied g-values and linewidths. The ESR signals of melanins, isolated or in cuticles, were characterized by g-values <2.004 and small linewidths in the range of 4-6G, while sclerotized cuticles, lacking melanin, showed g-values >2.004 and broad linewidths of 5-11 G. The melanin spectra were comparable to those reported for eumelanins with indol-based monomers. Minor signals ascribed to pheomelanins were found in several probes. The 'sclerotin' spectra were broader and displayed unresolved hyperfine structure in some cases. As for melanins, the location and environment of the radicals in cuticles giving rise to the two types of ESR spectra could not be assigned. Changes in the radical environment due to insecticide or solvent treatment can be detected by ESR spectroscopy.     

Analysis of chitin structure by nuclear magnetic resonance spectroscopy and chitinolytic enzyme digestion

Solid-state 13C-NMR analysis of chitin prepared from cuticle of the tobacco hornworm, Manduca sexta (L.), and of crab yielded spectra that demonstrate a high degree of chemical homogeneity (greater than 95%) for the preparations. The chemical shifts of the well-resolved carbon signals from both samples matched closely those of the monomeric unit 2-acetamido-2-deoxy-D-glucopyranoside (GlcNAc). Chromatographic analysis of products from the digestion of chitin by the binary chitinase system (endo splitting chitinase and exo splitting beta-N-acetylglucosaminidase) isolated from M. sexta molting fluid showed that the major product from both chitin preparations is GlcNAc. Also detected was a minor product (product U) that had a chromatographic retention time on the carbohydrate analysis column intermediate between those of chitin penta- and hexasaccharides. Gel filtration chromatography of U indicated that U had an apparent molecular weight intermediate between that of GlcNAc and of N,N'-diacetylchitobiose. Cation-exchange chromatography of U after acid hydrolysis revealed the presence of glucosamine only. Derivatization with trinitrobenzenesulfonate showed the presence of a free amino group in U. Solution proton and carbon NMR spectroscopy were used to identify U as a N-monoacetylchitobiose [O-beta-D-2-amino-2-deoxyglucopyranosyl- (1----4)-2-acetamido-2-deoxy-beta-D-glucopyranose] with the residue at the nonreducing end deacetylated. These studies showed that chitin prepared from alkali- and heat-treated insect or crab cuticle contains trace levels of deacetylated residues that are released as a dead-end product, N-monoacetylchitobiose, after digestion by the binary enzyme system.     

The stabilisation of insect cuticles

The possible means by which insect cuticle is stabilised are examined: evidence is presented that secondary interactions between the components are very important in stabilisation. Cuticular proteins are shown to be able to adopt stable secondary structures. It is concluded that cuticle may well be stabilised by changes in the conformation of the proteins, changes in the secondary bonding between the proteins and the inclusion of a polymer filler. Crosslinking of the cuticular components may be of little consequence: the stabilisation would be a result of cuticular dehydration.     

Mechanical hysteresis of insect cuticles

Solid cuticles exhibit cyclic-hardening whereas intersegmental membranes undergo stress-softening on tensile mechanical hysteresis. These differences are related to the arrangement of chitin microfibrils with respect to direction of the applied load and to the nature and extent of matrix stabilization. It is suggested that cyclic-hardening provides a fine control for intra-instar larval growth in some caterpillars. In addition, it may also provide skeletal regidity on ecdysis before completion of bulk tanning of the presumptive exocuticle in more typical examples of solid cuticle.     

Quantitative determination of catecholic degradation products from insect sclerotized cuticles

Acid hydrolysates of cuticle from various insect species were quantitatively analyzed for five catecholic amino acid adducts. Four of the adducts are ketocatechols; in three of them the amino acid moiety, either lysine, glycine or beta-alanine, is connected via its amino group to the alpha-carbon atom of 3,4-dihydroxyacetophenone, in the fourth a tyrosine residue is connected to the same position via its phenolic group. The fifth adduct contains histidine linked via its imidazole-ring to the beta-position of the dopamine sidechain. The three ketocatecholic adducts containing alpha-amino acids were obtained in significant yields from adult cuticles of the locust Schistocerca gregaria, the cockroaches Blaberus craniifer and Periplaneta americana, and the beetles Pachynoda sinuata and Tenebrio molitor, but only in trace amounts from larval and pupal cuticles of T. molitor, pupal cuticles of the moths Manduca sexta and Hyalophora cecropia, and puparia of the blowfly Calliphora vicina. The beta-alanine-containing ketocatechol was not obtained from cuticle of locusts and T. molitor larvae and pupae, but it was present in the hydrolysates of the other cuticles. The beta-histidine-dopamine adduct was obtained from all the cuticles, the highest yield was obtained from adult P. sinuata and the lowest yield was from adult S. gregaria. The beta-histidine-dopamine adduct is derived from the product formed by reaction of p-quinone methides of N-acetyldopamine (NADA) or N-beta-alanyldopamine (NBAD) with histidine residues in the cuticular proteins. The ketocatecholic adducts are assumed to be degradation products of crosslinks formed when oxidized dehydro-NADA reacts with the cuticular proteins. The insect species investigated appear to use both pathways for sclerotization, but to widely differing extents; the dehydro-NADA pathway dominates in cuticles which are exposed to strong deforming forces, such as those of adult locusts and cockroaches, and the p-quinone methide pathway dominates in cuticle of lepidopteran pupae and blowfly puparia, which are not exposed to strong mechanical forces but have to be effectively protected against microbial and fungal attacks.     

Protein-structural heterogeneity in a non-allosteric monomeric insect hemoglobin monitored by proton magnetic resonance spectroscopy

Proton NMR has revealed two modes of structural heterogeneity in the monomeric hemoglobin I of Chironomus thummi thummi, CTT I; rotational disorder caused by a 180 degree rotation of the heme about the alpha, gamma-meso axis (primary heterogeneity), which varies for each preparation or reconstitution of this hemoglobin, and a 'silent' amino acid replacement [Thr/Ala exchange in position 98(FG4)] in the vicinity of the heme group, which is invariant under all experimental conditions. The heme rotational disorder (primary heterogeneity) can be removed by reconstitution of CTT I with the symmetrical protoheme III. The secondary splitting is not affected; the ratio of intensities of the two types of resonance remains constant. The 8-methyl and 3-methyl and one of the alpha-vinyl proton resonances for the major heme rotational component and the 5-methyl and 1-methyl and one of the alpha-vinyl proton resonances for the minor heme rotational component have been identified and assigned by reconstitution with deuterium-labeled heme. Decoupling experiments have been employed to assign vinyl beta protons in cis and trans position to the respective vinyl alpha protons. Hyperfine shifts for the heme protons exhibited no pH influence above pH 6, in accord with the lack of the alkaline Bohr effect. Below pH 6, pH effects are most strongly reflected by the 8-methyl and 5-methyl proton resonances possibly reflecting titration of the propionate groups.     

An introduction to biological nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful analytical techniques available to biology. This review is an introduction to the potential of this method and is aimed at readers who have little or no experience in acquiring or analyzing NMR spectra. We focus on spectroscopic applications of the magnetic resonance effect, rather than imaging ones, and explain how various aspects of the NMR phenomenon make it a versatile tool with which to address a number of biological problems. Using detailed examples, we discuss the use of ¹H NMR spectroscopy in mixture analysis and metabolomics, the use of ¹³C NMR spectroscopy in tracking isotopomers and determining the flux through metabolic pathways (‘fluxomics’) and the use of ³¹P NMR spectroscopy in monitoring ATP generation and intracellular pH homeotasis in vivo. Further examples demonstrate how NMR spectroscopy can be used to probe the physical environment of a cell by measuring diffusion and the tumbling rates of individual metabolites and how it can determine macromolecular structures by measuring the bonds and distances which separate individual atoms. We finish by outlining some of the key challenges which remain in NMR spectroscopy and we highlight how recent advances—such as increased magnet field strengths, cryogenic cooling, microprobes and hyperpolarisation—are opening new avenues for today's biological NMR spectroscopists.     

Chemical proteomics from a nuclear magnetic resonance spectroscopy perspective

Proteomics is the study of the protein complement of a genome and employs a number of newly emerging tools. One such tool is chemical proteomics, which is a branch of proteomics devoted to the exploration of protein function using both in vitro and in vivo chemical probes. Chemical proteomics aims to define protein function and mechanism at the level of directly observed protein-ligand interactions, whereas chemical genomics aims to define the biological role of a protein using chemical knockouts and observing phenotypic changes. Chemical proteomics is therefore traditional mechanistic biochemistry performed in a systems-based manner, using either activity- or affinity-based probes that target proteins related by chemical reactivities or by binding site shape/properties, respectively. Systems are groups of proteins related by metabolic pathway, regulatory pathway or binding to the same ligand. Studies can be based on two main types of proteome samples: pooled proteins (1 mixture of N proteins) or isolated proteins in a given system and studied in parallel (N single protein samples). Although the field of chemical proteomics originated with the use of covalent labeling strategies such as isotope-coded affinity tagging, it is expanding to include chemical probes that bind proteins noncovalently, and to include more methods for observing protein-ligand interactions. This review presents an emerging role for nuclear magnetic resonance spectroscopy in chemical proteomics, both in vitro and in vivo. Applications include: functional proteomics using cofactor fingerprinting to assign proteins to gene families; gene family-based structural characterizations of protein-ligand complexes; gene family-focused design of drug leads; and chemical proteomic probes using nuclear magnetic resonance SOLVE and studies of protein-ligand interactions in vivo.     

Chemical proteomics from a nuclear magnetic resonance spectroscopy perspective

Proteomics is the study of the protein complement of a genome and employs a number of newly emerging tools. One such tool is chemical proteomics, which is a branch of proteomics devoted to the exploration of protein function using both in vitro and in vivo chemical probes. Chemical proteomics aims to define protein function and mechanism at the level of directly observed protein–ligand interactions, whereas chemical genomics aims to define the biological role of a protein using chemical knockouts and observing phenotypic changes. Chemical proteomics is therefore traditional mechanistic biochemistry performed in a systems-based manner, using either activity- or affinity-based probes that target proteins related by chemical reactivities or by binding site shape/properties, respectively. Systems are groups of proteins related by metabolic pathway, regulatory pathway or binding to the same ligand. Studies can be based on two main types of proteome samples: pooled proteins (1 mixture of N proteins) or isolated proteins in a given system and studied in parallel (N single protein samples). Although the field of chemical proteomics originated with the use of covalent labeling strategies such as isotope-coded affinity tagging, it is expanding to include chemical probes that bind proteins noncovalently, and to include more methods for observing protein–ligand interactions. This review presents an emerging role for nuclear magnetic resonance spectroscopy in chemical proteomics, both in vitro and in vivo. Applications include: functional proteomics using cofactor fingerprinting to assign proteins to gene families; gene family-based structural characterizations of protein–ligand complexes; gene family-focused design of drug leads; and chemical proteomic probes using nuclear magnetic resonance SOLVE and studies of protein–ligand interactions in vivo.     

Application of nuclear magnetic resonance spectroscopy to proteins.

The active sites of enzymes can be studied in great detail using nuclear magnetic resonance spectroscopy. The determination of pKa values of active site histidine residues in bovine pancreatic ribonuclease and the characterization of the binding of peptide hormones to carrier proteins are two such examples. The study of the active site of staphylococcal nuclease is another example and is presented in detail in this paper. The structure of 3'5'-thymidine diphosphate bound in the active site of staphylococcal nuclease has been studied by measuring the relaxation rate enhancement of substrate analog nuclei by a paramagnetic metal ion. The lanthanide ion, Gd(III), was substituted for Ca(II) in the formation of the ternary complex of nuclease: Gd(III) : 3'5'-thymidine diphosphate. Measurements were made of the transverse relaxation rates of protons and the longitudinal and transverse relaxation rates of the phosphorus nuclei of bound nucleotide. Internuclear distances between the metal ion and atoms of the 3'5'-thymidine diphosphate nucleotide were determined from these data by using the Solomon-Bloembergen equation. In general, these distances corresponded closely to those determined by previous X-ray crystallography of the thymidine diphosphate complex. These internuclear distances were also used with a computer program and graphics display to solve for metal : nucleotide geometries which were consistent with the experimental data. A geometry similar to the structure of the metal : nucleotide complex bound to nuclease determined by X-ray analysis was one of the solutions to this computer modeling process. For staphylococcal nuclease the NMR and X-ray methods yield compatible high resolution information about the structure of the active site.     

Nucleotide binding to tubulin-investigations by nuclear magnetic resonance spectroscopy

In an attempt to distinguish between the interaction of GTP and ATP with tubulin dimer, high-resolution ¹H- and ³¹P-NMR experiments have been carried out on the nucleotides in the presence of tubulin. The location of the ATP binding sites on the protein in relation to the GTP sites is still not clear. Using NMR spectroscopy, we have tried to address this question. Evidence for the existence of a site labelled as X-site and another site (labelled as L-site for both the nucleotides on tubulin has been obtained. It is suggested that this X-site is possibly the putative E-site. In order to gain further insight into the nature of these sites, the Mg(II at the N-site has been replaced by Mn(II and the paramagnetic effect of Mn(II on the linewidth of the proton resonances of tubulin-bound ATP and GTP has been studied. The results show that the L-site nucleotide is closer to the N-site metal ion compared to the X-site nucleotide. On the basis of these results, it is suggested that the L-site of ATP is distinct from the L-site of GTP while the X-site of both the nucleotides seems to be same. By using the paramagnetic effect of the metal ion, Mn(II), at the N-site on the relaxation rates of tubulin-bound ATP at L-site, distances of the protons of the base, sugar and phosphorous nuclei of the phosphorous moiety of ATP, from the N-site metal ion have been mapped. The base protons are 2 0.7–1 nm distant from the N-site metal ion, while the protons of the sugar are 2 0.8-1 nm from this metal ion site. On the other hand, the phosphorous nuclei of the phosphate groups are somewhat nearer (2 0.4–0.5 nm from the N-site metal ion.     

Nuclear magnetic resonance Fourier transform spectroscopy

The development and current status of Fourier transform spectroscopy is described.Nobel Lecture given on December 9, 1991 by Professor R. Ernst and published in Les Prix Nobel 1991, printed in Sweden by Norstedts Tryckeri, Stockholm, Sweden, 1992, republished here with the permission of the Nobel Foundation, the copyright holder.     

The application of nuclear magnetic resonance spectroscopy to parasite metabolism

Nuclear magnetic resonance (NMR) has become a valuable tool for the study of metabolism in a wide variety of biological systems. Its inherent advantages are that it is non-destructive and non-invasive. Observations can be carried out not only on extracts and media but also on whole cells and whole tissues under varying conditions and over varying times. The information gained gives considerable insight into cellular metabolism. There has been, to date, relatively little literature on the application o f NMR to the biochemistry of parasites, presumably reflecting the paucity of interfaces between parasitologists and NMR practitioners as well as the inherent difficulties in obtaining sufficient parasite material for NMR experiments. These difficulties are being overcome and William O'Sullivan, Michael Edwards and Raymond Norton believe that NMR has a great deal to offer those interested in parasite metabolism. In particular, it has the capacity to turn up the unexpected, on important factor as so many parasites appear to have developed their own variations on orthodox metabolic pathways.     

31P nuclear magnetic resonance spectroscopy of lipopolysaccharides from Pseudomonas aeruginosa

Intact lipopolysaccharide antigens isolated from seven different immunotypes of Pseudomonas aeruginosa have been examined by 31P-NMR spectroscopy. These macromolecular complexes contain phosphorus covalently attached to the carbohydrate residues present in the lipid A moiety and the 'core' oligosaccharide region. The spectral signals for various ortho- and pyrophosphoric esters were observed. All phosphate groups appeared to be monoesterified. Certain shifts characteristic for phosphate diester groups, observed in lipopolysaccharide complexes from other Gram-negative bacteria, were absent. Furthermore, no evidence was found to indicate that phosphate groups are involved in the covalent linkage of individual lipopolysaccharide complexes to form dimers or trimers.     

Applications of nuclear magnetic resonance in parasitology

The basis of the nuclear magnetic resonance (NMR) phenomenon is described in a classical framework with emphasis on magnetic nuclei of 1/2 spin, including 1H, 13C, and 31P. Biological applications of NMR spectroscopy and magnetic resonance imaging (MRI) are outlined briefly. NMR spectroscopic studies on parasitic protozoa, cestodes, nematodes, trematodes, and hymenopterous insect parasites are reviewed. NMR and MRI investigations on the pathophysiology of the host are also discussed, and the potential future of NMR applications in parasitology outlined.     

Phosphorus-31 nuclear magnetic resonance of insect hemolymph sera

Sera from larval and pupal stages of the tobacco hornworn, Manduca sexta, have been investigated using phosphorus-31 pulsed Fourier transform nuclear magnetic resonance. Spectra of larval and pupal sera containing 5 mm EDTA were characterized by four major peaks and one or more minor resonances. A phosphorus-31 spectrum of dialyzed larval serum showed several weak signals which indicated the presence of some higher-molecular-weight phosphorylated compounds as well. None of those signals, however, corresponded to any of the ones seen with undialyzed sera. Three of the four prominent peaks and one minor peak in the whole larval serum had the same chemical shifts as those in the pupal samples. The pupal sera, in addition, displayed an extra peak well upfield from those of the larval stage. All of the low-molecular-weight resonances detectable in the hemolymphs have been identified and included four compounds not previously reported; trehalose-6-phosphate, phosphoarginine, phosphatidylcholine, and phosphatidylethanol-amine. The phosphometabolites found at millimolar or higher concentrations in larval hemolymph were α-glycerolphosphate, phosphorylcholine, phosphorylethanolamine, inorganic phosphate, trehalose-6-phosphate, phosphatidylcholine, and phosphatidylethanolamine. All of the above compounds were found in pupal sera as well except for the addition of phosphoarginine and the deletion of phosphorylethanolamine. The levels of the phosphometabolites in common between the two stages of development, however, were quite different as were their stabilities after extraction. While the intensities of the larval phosphates remained virtually constant in the presence of EDTA at pH 7.8, those of the pupal sera changed rapidly. This was especially true for arginine phosphate which disappeared quickly.     

P nuclear magnetic resonance spectroscopy of lipopolysaccharides from pseudomonas aeruginosa

Intact lipopolysaccharide antigens isolated from seven different immunotypes of Pseudomonas aeruginosa have been examined by ³¹P-NMR spectroscopy. These macromolecular complexes contain phosphorus covalently attached to the carbohydrate residues present in the lipid A moiety and the ‘core’ oligosaccharide region. The spectral signals for various ortho- and pyro-phosphoric esters were observed. All phosphate groups appeared to be mono-esterified. Certain shifts characteristic for phosphate diester groups, observed in lipopolysaccharide complexes from other Gram-negative bacteria, were absent. Furthermore, no evidence was found to indicate that phosphate groups are involved in the covalent linkage of individual lipopolysaccharide complexes to form dimers or trimers.     

Characterization of lipids from human brain tissues by multinuclear magnetic resonance spectroscopy

Multinuclear ((1)H, (13)C, and (31)P) magnetic resonance spectroscopy are applied to the biochemical characterization of the total lipid fraction of healthy and neoplastic human brain tissues. Lipid extracts from normal brains, glioblastomas, anaplastic oligodendrogliomas, oligodendrogliomas, and meningiomas are examined. Moreover, the unknown liquid content of a cyst adjacent to a meningioma is analyzed. Two biopsies from glioblastomas are directly studied by (1)H-NMR without any treatment (ex vivo NMR). The (1)H- and (13)C-NMR analysis allows full characterization of the lipid component of the cerebral tissues. In particular, the presence of cholesteryl esters and triglycerides in the extracts of high grade tumors is correlated to the vascular proliferation degree, which is different from normal brain tissue and low grade neoplasms. The (31)P spectra show that phosphatidylcholine is the prominent phospholipid and its relative amount, which is higher in gliomas, is correlated to the low grade of differentiation of tumor cells and an altered membrane turnover. The ex vivo (1)H-NMR data on the glioblastoma samples show the presence of mobile lipids that are correlated to cell necrotic phenomena. Our data allow a direct correlation between biochemical results obtained by NMR and the histopathological factors (vascular and cell proliferations, differentiation, and necrosis) that are prominent in determining brain tumor grading.     

Proton magnetic resonance spectroscopy in diagnostics of epilepsy

Present paper presents proton MRS investigation results. The investigation was carried out with Magnetom Vision device. Twenty-five patients in the age of 20-44 years suffering with generalization epileptic fits validated by EEG (no visible changes on MRT) were examined. In all cases independently on the localization of the changes, decreasing of NAA and increasing of Cho were recorded. At one side temporal lobe injury recorded by EEG at the damaged part decreasing of NAA/Cr and NAA/Cho + Cr ratios were registered. Patients with bilateral changes registered by EEG showed non-equal changes of metabolite concentration on both sides. Examination of patients suffering with distinct symptoms of temple epileptics has shown ipsilaterality decrease of NAA and Cr concentration. But on the injured side NAA/Cr ratio decrease was more distinct. In general, the laterality was recorded in 14 patients out of 22 with pathological changes registered by proton MRS and in 10 patients out of 14 the above mentioned changes corresponds to the side of the fit initiation. In the patients with bilaterality changes NAA/Cr ratio asymmetry was recorded in all cases, but the most distinctly in the medium part of the temple lobe. Comparison of data recorded in 8 patients suffering with one side fit complex has shown significant asymmetry of metabolites which was observed in ipsilaterality and contra laterality NAA ratio obtained in hippocampal areas. Difference in NAA ratio obtained between left and right sides are 19-25%. Left-right ratio of other metabolites corresponded to that ratio in the control group and was symmetrical.     

Molecular aspects of magnetic resonance imaging and spectroscopy

Magnetic resonance imaging (MRI) is a well known diagnostic tool in radiology that produces unsurpassed images of the human body, in particular of soft tissue. However, the medical community is often not aware that MRI is an important yet limited segment of magnetic resonance (MR) or nuclear magnetic resonance (NMR) as this method is called in basic science. The tremendous morphological information of MR images sometimes conceal the fact that MR signals in general contain much more information, especially on processes on the molecular level. NMR is successfully used in physics, chemistry, and biology to explore and characterize chemical reactions, molecular conformations, biochemical pathways, solid state material, and many other applications that elucidate invisible characteristics of matter and tissue. In medical applications, knowledge of the molecular background of MRI and in particular MR spectroscopy (MRS) is an inevitable basis to understand molecular phenomenon leading to macroscopic effects visible in diagnostic images or spectra. This review shall provide the necessary background to comprehend molecular aspects of magnetic resonance applications in medicine. An introduction into the physical basics aims at an understanding of some of the molecular mechanisms without extended mathematical treatment. The MR typical terminology is explained such that reading of original MR publications could be facilitated for non-MR experts. Applications in MRI and MRS are intended to illustrate the consequences of molecular effects on images and spectra.