Osmeterial secretions of papilionid larvae in the genera Luehdorfia,Graphium and Atrophaneura (lepidoptera)

The osmeterial secretions of Luehdorfia larvae (two species and one subspecies) are chemically characterized as monoterpene hydrocarbons consisting mainly of α-pinene, β-myrcene (major component), limonene and terpinolene, whereas those of Atrophaneura larvae (one species) are marked by sesquiterpenic compounds (the principal component being δ-cadinene). In contrast, the osmetrical secretions of Graphium larvae (two species) predominantly consist of a mixture of aliphatic acids and esters (iso-butyric acid, 2-methylbutyric acid and their methyl and ethyl esters). In all the species examined of the three genera, the secretions of the last and the penultimate larval instars were of similar composition, which is unlike the results reported previously for larvae of Papilio protenor.     

13C-NMR, 1H-NMR and gas-chromatography mass-spectrometry studies of the biosynthesis of 13C-enriched L-lysine by Brevibacterium flavum

The basic metabolic pathways of lysine biosynthesis in Brevibacterium flavum, a strain which excretes excessive amounts of L-lysine, have been followed by using two 13C-labeled precursors. 13C- and 1H-NMR spectroscopies in conjunction with gas chromatography mass spectrometry (GC-MS) have revealed the various metabolic pathways leading to L-[13C]lysine. Discrete metabolic pathways give rise to distinct labeling patterns. L-Lysine resulting from [1-13C]glucose fermentation is relatively specifically labeled: L-[3,5-13C]lysine is the main product. Experimental and theoretical approaches based on the 13C-enrichment values of intracellular glutamate, a major intermediate metabolite, allowed us to assess the relative contribution of the major metabolic pathways forming lysine. The labeling pattern of glutamate reflects the isotope distribution in 2-oxoglutarate. When [2-13C]acetate is used as the sole carbon source in the culture, the energy-producing steps of the Krebs cycle are essential. The higher activity of the Krebs cycle, when endogenous carbohydrates are exhausted from the culture, is indicated by the increased 13C enrichment in C-1 of lysine and reveal a high content of isotopomers of four, five and six 13C atoms in the lysine molecule, pointing out that the four-carbon intermediates of the cycle are being derived from the glyoxylate shunt pathway. Such a phenomenon does not occur in glucose fermentation. GC-MS analyses of 13C enrichments and isotopomer distributions in metabolites and end products are in good agreement with the predicted contribution of each metabolic pathway. This new methodological approach of combined NMR and GC-MS has been demonstrated to be applicable to various other metabolic studies.     

Patterns of Amazonian area relationships based on raw distributions of papilionid butterflies (Lepidoptera: Papilioninae)

Diversifications within a biota are due to several factors. Although some of these are untestable with current analytical methods, hierarchical congruence obtained with different cladistic methods and based on independent taxa are undoubtedly important. In the recent past, most hypotheses of historical biogeography (e.g. refugial, riverine, disturbance, vicariance) were tested on the Amazonian biota, selecting a number of diverse organisms such as plants, anurans, lizards, butterflies, birds and monkeys. In this study we used parsimony analysis of endemicity to infer historical relationships among 16 interfluvial areas in the Amazonian lowlands based on raw distributions of 114 Papilioninae (Lepidoptera). The analysis yielded two most parsimonious trees of area relationships. One tree was characterized by two main clusters of areas which showed a separation of Guyanan + south-east Amazonian interfluvial areas from western Amazonian interfluvial areas. The second tree showed the Guyanan interfluvial areas basal to a cluster which included all the other interfluvial areas. This latter cluster was subdivided into two main groups of areas separating the south-east Amazonian and the western Amazonian interfluvial areas. This result is discussed in the light of previous hypotheses obtained with the same method using some vertebrate taxa in the Amazonian lowlands. Likewise, comparisons with other hypotheses on lineages of birds, mammals and butterflies obtained applying cladistic biogeographical methods are made. The two alternative vicariant patterns presented for papilionid butterflies are strictly congruent with those for birds.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 345–357.     

Speciation problems in the Papilio machaon group of butterflies (Lepidoptera: Papilionidae)

Abstract. The taxonomic and genetic relationships between P.machaon rathjensi Warnecke from the west side of southern Arabia and P.machaon muetingi Seyer from the east are discussed, together with suggestions of their relationship to P.machaon saharae Oberthiir from the northern Hejaz, Sinai and desert North Africa. It is concluded that though P.m.rathjensi and P.m.saharae are subspecifically distinct, they are very closely related to each other, and both should be regarded as specifically distinct from the rest of the machaon complex.
The remainder of the machaon complex in its turn presents a somewhat similar problem. Thus P.machaon gorganus Fruhstorfer and P.hospiton Géné are normally regarded as good species, but Fl caterpillars have been found in the wild and the cross can be readily produced in captivity with no upset in the sex ratio. Furthermore, recently we have obtained sparse F2 offspring between the two species.
We therefore suggest that the entire P.machaon complex is in a labile state as regards speciation throughout its range and the relationships between the various forms are not easily expressed through the traditional species and subspecies concepts. Our tentative conclusions are that the status of P.machaon and P.hospiton as distinct species probably remains valid. The saharaelrathjensi complex might be considered distinct from P.machaon using the biological species concept, while their genetics indicate a more conservative approach. However, more important than the exact status of the taxa in question is the fact that the P.machaon complex illustrates evoluton in action, the end result of which cannot be predicted.     

Conformation and backbone dynamics of bacteriorhodopsin revealed by (13)C-NMR

It is demonstrated here how the secondary structure and dynamics of transmembrane helices, as well as surface residues, such as interhelical loops and N- or C-terminus of bacteriorhodopsin (bR) in purple membrane, can be determined at ambient temperature based on very simple (13)C-NMR measurements, together with a brief experimental background. In contrast to the static picture of bR, currently available from X-ray diffraction or cryo-electron microscopy, the structure consists of dynamically heterogeneous domains which undergo various types of local fluctuations with a frequency range of 10(2)--10 (8) Hz. The significance of this picture is discussed in relation to the biological function of this protein.     

Independent evolution of sexual dimorphism and female‐limited mimicry in swallowtail butterflies (Papilio dardanus and Papilio phorcas)

Several species of swallowtail butterflies (genus Papilio) are Batesian mimics that express multiple mimetic female forms, while the males are monomorphic and nonmimetic. The evolution of such sex‐limited mimicry may involve sexual dimorphism arising first and mimicry subsequently. Such a stepwise scenario through a nonmimetic, sexually dimorphic stage has been proposed for two closely related sexually dimorphic species: Papilio phorcas, a nonmimetic species with two female forms, and Papilio dardanus, a female‐limited polymorphic mimetic species. Their close relationship indicates that female‐limited polymorphism could be a shared derived character of the two species. Here, we present a phylogenomic analysis of the dardanus group using 3964 nuclear loci and whole mitochondrial genomes, showing that they are not sister species and thus that the sexually dimorphic state has arisen independently in the two species. Nonhomology of the female polymorphism in both species is supported by population genetic analysis of engrailed, the presumed mimicry switch locus in P. dardanus. McDonald–Kreitman tests performed on SNPs in engrailed showed the signature of balancing selection in a polymorphic population of P. dardanus, but not in monomorphic populations, nor in the nonmimetic P. phorcas. Hence, the wing polymorphism does not balance polymorphisms in engrailed in P. phorcas. Equally, unlike in P. dardanus, none of the SNPs in P. phorcas engrailed were associated with either female morph. We conclude that sexual dimorphism due to female polymorphism evolved independently in both species from monomorphic, nonmimetic states. While sexual selection may drive male–female dimorphism in nonmimetic species, in mimetic Papilios, natural selection for protection from predators in females is an alternative route to sexual dimorphism.     

13C-NMR studies of acetate and methanol metabolism by methylotrophic Pseudomonas strains

The metabolism of [2-13C]acetate by Pseudomonas M27(Icl-) and Pseudomonas MA(Icl+) was studied in vivo using 13C-NMR spectroscopy. The flux of 13C-label into bicarbonate, glutamate and citrate was observed in both organisms. In addition 13C-labelled alpha, alpha-trehalose was synthesized as a major metabolite by Pseudomonas M27 but not by Pseudomonas MA. The presence of this disaccharide in cell extracts of Pseudomonas AM1(Icl-) grown with [13C]methanol was also observed. The data from analysis of the trehalose multiplet signal observed in the spectra of Pseudomonas M27 cell extracts were consistent with the absence of the glyoxylate cycle in this methylotroph.     

In vivo 13C-NMR studies on the metabolism of the lugworm Arenicola marina

13C-NMR natural-abundance spectra of specimens of Arenicola marina obtained, showed seasonal changes in the concentration of some metabolites, with the osmolite alanine as well as triacylglyceride storage compounds present at high concentrations. Glycogen was sometimes only barely detectable due to the low natural abundance level of 13C. Glycogenic metabolism of the lugworm A. marina was studied in vivo by 13C-NMR spectroscopy using 13C-labelled glucose. During recovery from a hypoxic period [1-13C]glucose was incorporated into glycogen. [1-13C]Glucose was injected 5 h after the end of hypoxia to guarantee sufficient and reliable 13C labelling of glycogen. An earlier injection of [1-13C]glucose led to considerably diminished incorporation of 13C-labelled glucosyl units into glycogen, probably due to the consumption of the available glucose as fuel for ATP production. No scrambling of 13C into the C6 position of glycogen was observed, indicating a lack of gluconeogenic activity. 13C was also incorporated into the C3 positions of alanine and alanopine. To assign correctly this last 13C-NMR resonance, the compound was synthesized biochemically. No labelling of glycogen was observed when [3-13C]alanine was injected into the coelomic cavity with similar incubation conditions being used. The 13C of [1-13C]glucose, incorporated into glycogen, showed a very low turnover rate in normoxic lugworms as shown by two 13C(1H)-NMR spectra, one obtained 48 h after the other. On the other hand, in hypoxia lugworms the signal due to 13C-labelled glycogen decreased very rapidly proving a high turnover rate. The disappearance of 13C from glycogen during the first 24 h of hypoxia indicates that the last glycosyl units to be synthesized are the first to be utilized. Lugworms were quite sensitive to the 1H-decoupling field used for obtaining the 13C(1H)-NMR spectra, especially at 11.7 T. Using bi-level composite-pulse decoupling and long relaxation delays, no tissue damage or stress-dependent phosphagen mobilization, as judged by 31P-NMR spectroscopy, was observed.     

Structural and dynamical characterization of piroxicam by 1H- and 13C-NMR relaxation studies

Carbon spin-lattice relaxation rates of an anti-inflammatory drug, piroxicam, have been measured. These results have been used in determining the reorientational rates of the proton carbon vectors. An analysis of internal motions within the pyridinyl moiety of piroxicam was carried out. Selective proton-carbon nuclear Overhauser effect (NOE) measurements were made in order to determine the solution structure of piroxicam. The effect of indirect NOE arising from exchangeable protons has been analyzed and considered.     

The ultrastructure of the osmeterium and the nature of its secretion in Papilio larvae (lepidoptera)

Two main cell types constitute the defensive osmeterium gland of Papilio larvae. Ellipsoid gland cells have an extensively infolded basal plasma membrane, abundant ribosomes and whorls of smooth endoplasmic reticulum. The apical plasma membrane bears long microvilli extending into a mass of granular material containing electron-lucid cavities. Tangential slits occur in the epicuticle. Tubular arm cells contain heterogeneous, electron-dense inclusions, extensively-branched nuclei and large mitochondria sometimes distended with electron-dense material. The apical plasma membrane bears short microvilli. The inner, dense epicuticle forms a complex ramifying system. The two-phase defensive fluid consists mainly of water, 2-methyl propionic acid, and 2-methyl butyric acid.     

Characterization of oligosaccharides from the chondroitin sulfates. (1)H-NMR and (13)C-NMR studies of reduced disaccharides and tetrasaccharides.

Chondroitin sulfates were fragmented using the enzymes chondroitin sulfate ABC endolyase and chondroitin ACII lyase; both disaccharide and tetrasaccharide fragments were isolated after reduction to the corresponding 2-deoxy-2-N-acetylamino-D-galactitol (GalNAc-ol) form. These have the structures: Delta UA(beta 1--3)GalNAc4S-ol, Delta UA(beta 1--3)GalNAc6S-ol, Delta UA2S(beta 1--3)GalNAc6S-ol, Delta UA(beta 1--3)GalNAc4S(beta 1--4)L-IdoA(alpha 1--3)GalNAc4S-ol, Delta UA(beta 1--3)GalNAc4S(beta 1--4)GlcA(beta 1--3)GalNAc4S-ol, Delta UA(beta 1--3)GalNAc6S(beta 1--4)GlcA(beta 1--3)GalNAc4S-ol, Delta UA(beta 1--3)GalNAc6S(beta 1--4)GlcA(beta 1--3)GalNAc6S-ol, Delta UA2S(beta 1--3)GalNAc6S(beta 1--4)GlcA(beta 1--3)GalNAc4S-ol and Delta UA2S(beta 1--3)GalNAc6S(beta 1--4)GlcA(beta 1--3)GalNAc6S-ol, where Delta UA represents a 4,5-unsaturated hexuronic acid (4-deoxy-alpha-Lthreo-hex-4-enepyranosyluronic acid) and 6S/4S/2S represent O-ester sulfate groups at C6/C4/C2 sites. Complete (1)H-NMR and (13)C-NMR data are derived for these species, which may help to alleviate some of the significant difficulties resulting from signal complexity that are currently hindering the characterization and assignment of major and minor structural components within chondroitin sulfate and dermatan sulfate polymers.     

Analysis of the essential oil from the roots of Eupatorium cannabinum subsp. corsicum (L.) by GC, GC-MS and 13C-NMR

Eupatorium cannabinum subsp. corsicum (L.) is an endemic subspecies from the island of Corsica. The essential oil from the roots of this aromatic plant has been studied by GC, GC-MS and by 13C-NMR. In contrast to the essential oil from the aerial parts, which is dominated by hydrocarbon compounds (76.9%) and particularly by sesquiterpene components (43.3%), the essential oil from the roots was characterized by a high content of oxygenated compounds (61.0%), particularly oxygenated monoterpenes (54.0%). In the root oil, 106 components were identified representing 96.1% of the total amount. This oil was dominated by the monoterpenes esters (33%), the major components of which were neryl isobutyrate (17.6%), thymyl methyl oxide (15.1%), delta-2-carene (14.5%) and beta-pinene (5.7%). Aromatic esters, nerol derivatives (esters and diesters) and a benzofuran were investigated by GC-MS using different ionization modes including electron impact ionization, and positive- and negative-chemical ionization. These components have not previously been reported in the essential oil of aerial parts of E. cannabinum from Corsica island.     

13C-NMR studies of the membrane structure of enveloped virions (vesicular stomatitis virus).

The mobility of the lipids in the bilayer of the envelope of vesicular stomatitis virus has been probed over its complete space by the biosynthetic incorporation of [N-13CH3]- choline as a probe for the polar head groups and [3-13C]- and [11-13C] oleic acid and [16-13C]- palmitic acid for the hydrophobic region of the bilayer. These precursors were effectively incorporated as established by the concomitant administration of the same precursors in radioactive form. Spin lattice relaxation time measurements (T1) of the 13C enriched segments in complete virus envelope allowed estimation of their mobility. The mobility of the polar head groups is restricted, probably due to ionic interactions with neighbouring acidic phospholipids (phosphatidylserine) and/or acidic side chains of the glycoprotein (G-protein). The rigidity of the hydrophobic part of the bilayer is due to the high cholesterol content and interaction with the immersing polypeptide chains of the G- and possibly M-protein. The rigidity is limited to a depth of about 15 A ranging from the inner and outer surface, whereas the inner core of the bilayer is fluid. Tryptic cleavage of the hydrophilic part of the G-protein allows the lipophilic immersing polypeptide fragment to enter further the bilayer which then reduces the fluidity of the hydrocarbon chains in the core region by lipid-protein interactions.     

Dynamics of ethanol translocation in Saccharomyces cerevisiae as detected by (13)C-NMR

(13)C-NMR has yielded to the dynamics study of ethanol as carbon and energy source in the metabolic oscillation of Saccharomyces cerevisiae. Three ethanol fractions such as media, cytoplasm and mitochondria were observed and characterised by different longitudinal relaxation times and chemical shifts.     

The hydration response of poly (L-lysine) dynamics measured by 13C-NMR spectroscopy.

13C-nmr measurements are reported for samples of poly (L-lysine) both static and spinning at the magic angle in the beta-sheet form as a function of water content. The addition of water decreases the side-chain line widths considerably. Measurements of the cross-polarization time constants indicate that hydration by either H2O or D2O increases the time constant. Measurements of spin-lattice relaxation times in the laboratory frame and the rotating frame indicate that hydration does not change the dynamics of the backbone carbon atoms in the beta-sheet structure appreciably, but the side-chain atoms experience considerable increase in local mobility with increasing hydration. Deuteration of the exchangeable protons or the water has only small effects on the carbon relaxation times, indicating that relaxation is driven by intramolecular dipole-dipole interactions.     

Composition of the leaf, stem bark and root bark oils of Isolona cooperi investigated by GC (retention index), GC-MS and 13C-NMR spectroscopy

The leaf, stem bark and root bark oils of Isolona cooperi Hutchinson & Dalziel from the Ivory Coast have been analysed by GC (retention index), GC-MS and 13C-NMR spectroscopy. Two types of essential oil were produced by the plant. The leaf and stem bark oils were monoterpene-rich, containing principally (Z)-beta-ocimene and gamma-terpinene and three lactones, 5-[(E and Z)-hexylidene]-5H-furan-2-ones and massoia lactone, were present in appreciable amounts. Conversely, the root bark oil was dominated by 5-isopentenylindole and (E)-beta-caryophyllene. The strategy for the analysis of each oil was adapted according to the nature of the components.     

13C-NMR and transient kinetic studies on lactate dehydrogenase [Cys(13CN)165]. Direct measurement of a rate-limiting rearrangement in protein structure

Chemical modification of cysteine-165 in pig heart lactate dehydrogenase to produce lactate dehydrogenase [Cys(13CN)165] introduces an covalently bound, enriched 13C probe at a position adjacent to the active cen. The signal from the thiocyanate probe is clearly visible at 47 ppm relative to dioxane. On formation of binary complexes with NAD+ and NADH, no signal change is detected. Formation of the ternary complexes E-NADH-oxamate and E-NAD+-oxalate results in an upfield shift of the signal of 1.2 ppm. These results interpreted as demonstrating that binding of the substrate analogue induces a conformational change a position adjacent to the active centre. Exchange experiments in which the enzyme is poised in dynamic equilibrium between binary and ternary complexes show that the rate at which the probe senses a change environment is the same as the kinetically observed unimolecular event which limits the enzyme-catalyst reduction of pyruvate. The two processes show the same dependence on temperature, solvent composition and pH. These results indicate that the rate-limiting isomerisation corresponds to a rearrangement of the protein in the region of cysteine-165.     

13C-NMR studies of the effects of the carcinogen acetylaminofluorene on the conformation of dinucleoside monophosphate

13C-NMR spectra are obtained in aqueous solution of dinucleoside monophosphates (ApG and GpA) and of their adducts formed by the addition of the carcinogen acetylaminofluorene (AAF) to the C8 position of the guanine. The base and sugar carbons of all dimers and adducts are assigned. The task of assigning base and carbohydrate resonances was accomplished using a series of reference compounds. Significant changes in many of the carbon resonances of the adducts are observed suggesting three general conformational changes, namely: (1) chemical shift changes are noted in base carbon atom resonances as a function of temperature and adduct formation which are indicative of stacking effects; (2) large upfield shifts of the furanose C2' resonance of the guanosine-adduct indicate a shift to higher populations of the syn conformation. Other shifts of carbohydrate resonances are indicative of a change in conformation of the carbohydrate itself. (3) Large temperature effects on linewidth of several fluorine and furanose resonances indicate interconversion of various conformers in the dimer adduct.     

In vivo 31P- and 13C-NMR studies of ATP synthesis and methane formation by Methanosarcina barkeri

Carbon and phosphorus metabolism of cell suspensions of Methanosarcina barkeri strain MS (DSM 800), grown on methanol, were probed in vivo by NMR. The experimental conditions, which involved thick cell suspensions, did not significantly affect the efficiency of the rate of methanol uptake by cells. Following exposure to methanol an acidification of both the intracellular and the extracellular spaces was observed and a gradient of 0.5 pH units across the cytoplasmic membrane was determined from the 31P-NMR data. High levels of intracellular ATP up to 4 mM were detected. The ADP concentration determined in a suspension of starved cells was only 2 mM, suggesting that a significant amount of ADP may be immobilized and is thus not detectable by NMR. In the presence of the protonophore, 3,3',4',5-tetrachlorosalicylanilide, the proton gradient was dissipated and the synthesis of ATP stopped. The inhibitor of the ATP synthase, N,N'-dicyclohexylcarbodiimide, was rather inefficient in inhibiting ATP synthesis. High concentrations of N,N'-dicyclohexylcarbodiimide (corresponding to 300 nmol/mg protein-1) were required to decrease the ATP content by approximately 60%, and, under these conditions, formation of acetyl phosphate was detected. However, the methanol consumption rate was not affected.