Stable isotope and radiocarbon compositions of methane emitted from tropical rice paddies and swamps in Southern Thailand

Stable isotopes (¹³C, D) and radiocarbon weremeasured in methane bubbles emitted from rice paddies and swamps in southernThailand. Methane emitted from the Thai rice paddies was enriched in¹³C (mean ¹³C; –51.5 ±7.1 and–56.5 ± 4.6 for mineral soil and peat soil paddies,respectively)relative to the reported mean value of methane from temperate rice paddies(– 63 ± 5). Large seasonal variation was observed in¹³C(32) in the rice paddies, whereas variationinD was much more smaller (20), indicating that variation in¹³C is due mainly to changes in methane production pathways.Values of ¹³C were lower in swamps (–66.1 ±5.1)than in rice paddies. The calculated contribution of acetate fermentation from¹³C value was greater in rice paddies (mineral soils:62–81%, peat soils: 57–73%) than in swamps (27–42%). Din methane from Thai rice paddies (–324± 7 (n=46)) isrelativelyhigher than those from 14 stations in Japanese rice paddies ranging from–362 ± 5 (Mito: n=2) to –322 ± 8(Okinawa: n=3), due tohigher D in floodwaters. ¹⁴C content in methane produced fromThai rice paddies (127±1 pMC) show higher ¹⁴Cactivity compared with previous work in paddy fields and those from Thai swamps(110±2 pMC).     

γδ T-cell receptor repertoire in human peripheral blood and thymus

T-cell clones expressing the T-cell receptor (Tcr) were generated from peripheral blood lymphocytes (PBLs) and from a thymus sample. In the panel of ten thymus-derived clones, four Tcr phenotypes [as defined by the reaction of monoclonal antibodies (mAbs) directed against known V and V regions] were identified. All the clones lacked expression of the V3 V region, while seven clones were V1+ . V1 was found in combination with V9 or with undefined VVregions. In addition, two other Tcr phenotypes were identified on these clones: V9⁺ V1^– V3^– and V9^– V1^– V3^– One of the clones expressed CD4 and another was CD8positive. The remaining clones were CD4^– CD8^–. In the panel of 76 PBL-derived, Tcr-bearing clones, five Tcr phenotypes could be identified. In contrast to the thymus-derived clones, 30% of the clones were V3⁺ whereas V1 was expressed by a minority of the clones only. One clone was CD4-positive and approximately 30% of the clones were CD8-positive. Four of the five mAb-defined Tcr phenotypes could be identified on both thymus and PBL-derived T-cell clones. However, biochemical analysis of the Tcrs demonstrates differences in the usage of Ct- and C2-encoded y chains by T cells derived from the thymus and PBLs. The results therefore indicate that, at the clonal level, similarities and differences exist between the Tcr repertoires expressed in the thymus and by PBLs. Furthermore, they indicate that combinatorial Tcr heterogeneity is larger than has so far been described. The receptor diversity, combined with the potential of Tcr+ cells to express CD4 or CD8, indicates that these cells are a heterogeneous population that might mediate a number of immune functions.     

Autotrophic carbon sources for heterotrophic bacterioplankton in a floodplain lake of central Amazon

The relative contribution of autotrophic carbon sources (aquatic macrophytes, flooded forest, phytoplankton) for heterotrophic bacterioplankton was evaluated in a floodplain lake of the Central Amazon. Stable carbon isotopes (¹³C) were used as tracers. Values of ¹³C of different autotrophic sources were compared to those of dissolved organic carbon (DOC) and those of bacterially produced CO₂.The percentage of carbon derived from C₄ macrophytes for bacterially produced CO₂ was the highest, on average 89%. The average ¹³C value of CO₂ from bacterial respiration was –18.5 ± 3.3. Considering a fractionation of CO₂ of 3 by bacterial respiration, ¹³C value was –15.5, near C₄ macrophyte ¹³C value (–13.1).The average value of total DOC ¹³C was –26.8 ± 2.4. The percentage of C₄ macrophytes carbon for total DOC was on average 17%. Considering that bacteria consume mainly carbon from macrophytes, the dominance of C₃ plants for total DOC probably reflects a faster consumption of the former source, rather than a major contribution of the latter source.Heterotrophic bacterioplankton in the floodplain may be an important link in the aquatic food web, transferring the carbon from C₄ macrophytes to the consumers.     

Molecular characterization of β-thalassemia in Czechoslovakia

Summary We have identified different -thalassemia mutations in 93 members of 34 families of Czech or Slovakian descent using gene amplification, hybridization with specific ³²P-labeled oligonucleotide probes, sequencing of amplified DNA, and gene mapping. The GA mutation at IVS-I-1 was found in 18 families; other Mediterranean mutations were IVS-II-1 (GA), IVS-II-745 (CG), IVS-I-110 (GA), and codon 39 (CT); these were present in 9 additional families. The GT mutation at codon 121, known to cause Heinzbody -thalassemia, was present in 3 families, and the frameshift at codons 82/83 (-G), first described in the Azerbaijanian population, in 2 families. A newly discovered allele was a frameshift at codons 38/39 (-C). One -thalassemia allele was incompletely characterized. We observed in 2 families a TC mutation at position +96 UTR (untranslated region) relative to the termination codon; this mutation likely is a rare polymorphism, -Thalassemia was rare; only one person carried the -^3.7 heterozygosity, and one other had a yet to be identified -thalassemia-1, while seven had the ^{anti 3.7} triplication.     

Specialized and generalized dispersal systems: where does ‘the paradigm’ stand?

I explore the specialization versus generalization paradigm in frugivory and seed dispersal. This view predicts that some tropical trees produce nutritious fruits adapted for use by a small coterie of specialized frugivores that provide reliable seed dissemination. Other tree species are expected to offer superabundant fruits of lower nutritional reward, relying instead on common opportunistic frugivores that are individually less reliable, but collectively disperse seeds effectively. Though widely referenced, many aspects of the paradigm are untested with tropical trees and avian frugivores, primarily because plant ecologists rarely determine whether specialist or generalist foragers are responsible for different patterns of seed distribution, while students of foraging behavior rarely determine the effects seed dispersal by different animals for survival of seeds or seedlings of specialist or generalist trees.Ecological paradigms provide alternative hypotheses, without evolutionary arguments. Keystone species have ecological effects disproportionate to their abundance; it is important for management considerations to know whether fruiting trees or frugivores serve as keystone mutualists in tropical forests. Alternatively, the extent to which vertebrate seed dispersers influence density-dependent seed, seedling, sapling, or adult mortality may have important consequences for spatial dispersion and population dynamics of tree species in tropical forests.     

Purification and characterization of aspartate aminotransferase from developing embryos of the camel tick Hyalomma dromedarii

Aspartate transaminase (AST) activity in the camel tick Hyalomma dromedarii was followed throughout embryogenesis. During purification of AST to homogeneity, ion exchange chromatography lead to four separate forms (termed I, II, III and IV). AST II with the highest specific activity was pure after chromatography on Sephacryl S-300. The molecular mass of AST II was 52KDa for the native enzyme, composed of one subunit of 50KDa. AST II had a Km value of 0.67mM for -ketoglutarate and 15.1mM for aspartate. AST II had a pH optimum of 7.5 with heat stability up to 50°C for 15min. The enzyme was activated by MnCl₂, and inhibited by CaCl₂, MgCl₂, NiCl₂, and ZnCl₂.     

Genetics of fertility restoration in hybrid rice

Summary The cross combination involving 14 male-sterile lines in rice, when crossed with different maintainers, showed fertility restoration in certain combinations. When F₂ segregating populations were classified based on spikelet fertility, fertility restoration was shown to be governed by 31, 9331, and 1231, due to allelic differences. This indicated that the cytosterility of the same group showed monogenic fertility restoration, whereas crossing plants belonging to different cystosterile groups showed a digenic pattern of segregation.     

Trehalose synthesis from maltose by a thermostable trehalose synthase from Thermus caldophilus

Purified trehalose synthase from Thermus caldophilus GK24 produced 18–86% trehalose from 10 mM–1 M maltose. The enzyme also catalyzed the conversion of ,-trehalose into maltose but did not act on other disaccharides. The yield of trehalose from maltose by this enzyme increased 30% more at 40°C than at 80°C and was independent of the substrate concentration. The maximum yield of ,-trehalose from 10 mM maltose reached 86% at 40°C. In addition, ,-trehalose was also formed from maltose or ,-trehalose at 3.5% yield at 80°C. © Rapid Science Ltd. 1998     

Neutral changes during divergent evolution of hemoglobins

Summary A comparison of the mRNAs for rabbit and human-hemoglobins shows that synonymous changes in codons have accumulated three times as rapidly as nucleotide replacements that produced changes in amino acids. This agrees with predictions based on the so-called neutral theory. In addition, seven codon changes that appear to be single-base changes (according to maximum parsimony) are actually two-base changes. This indicates that the construction of primordial sequences is of limited significance when based on inferences that assume minimum base changes for amino acid replacements.     

Characterization of groups of the zygomycete genusRhizopus

Rhizopus is a zygomycetous genus. Several species of this taxon may infect humans and lower animals. Seventeen isolates ofRhizopus species in three distinct morphological groups were studied: the stolonifer group (sporangiophores greater than 1 mm in height, sporangial diameters of 100–275 µm, branched rhizoids); the arrhizus group (sporangiophores greater than 1 mm in height, branched rhizoids, sporangial diameters of 100–240 µm); and the microsporus group (sporangiophores less than 0.8 mm in height, sporangial diameters less than 100 µm, simple rhizoids). Maximal growth temperatures were characteristic: the stolonifer group grew at 30°C, the arrhizus group grew at 36°C, and the microsporus group grew at 45°C. The DNA mol% G + C base composition of all isolates ranged from 34.9 to 40.2% Species within the three groups were grouped by DNA differences. The arrhizus group was most distinctive with a value of 34.9–36.3%; the stolonifer and microsporus groups had G + C values of 37.0–39.3% and 37.8–40.2%, respectively. Our research clarifies and defines the G-C values of the three important groups ofRhizopus species.     

Circadian changes in synaptic ribbons and spherules in pinealocytes of the Syrian hamster (Mesocricetus auratus)

Summary In the present study, synaptic ribbons were studied morphologically and quantitatively in hamster pineal gland. The number of ribbons and spherules of hamster pinealocytes was counted over a 24-h period. The 24-h variations in the quantity of synaptic ribbons were found to parallel fluctuations in pineal melatonin concentrations. No significant circadian changes were observed for synaptic spherules, indicating different roles for these two structures.     

Zur Wirkung des Lichtes auf die circadiane Periodik des Stoffwechsels und der Aktivität beim Buchfinken (Fringilla coelebs L.)

Zusammenfassung Bei 8 Buchfinken (4, 4) wurden Sauerstoffverbrauch und Hüpfaktivitätsowohl unter Bedingungen eines künstlichen Licht-Dunkel-Wechsels (LD 1212 Std; ohne Dämmerung) als auch im Dauerlicht (LL) bei konstanter Umgebungstemperatur (18–20° C) kontinuierlich gemessen. Im 1. Teil des Versuchs wurde die Beleuchtungsstärke während der Dunkelzeit (D) zwischen 0,01 und 1,0 Lux bei gleichbleibender Beleuchtungsstärke während der Lichtzeit (10 Lux) geändert. Im 2. Teil des Versuchs wurden die Vögel im Dauerlicht von 1 und 10 Lux untersucht. Vier Vögel wurden zusätzlich bei 100 Lux gemessen.Das mittlere Niveau des Sauerstoffverbrauchs (pro Std) und die mittlere Aktivitätsmenge (pro Stunde) während einer Periode sind bei der Mehrzahl der untersuchten Vögel linear und positiv miteinander korreliert (Abb. 2 und 3; Tabelle 1).Änderungen des mittleren Aktivitätsumsatzes (Differenz von mittlerem Niveau des Gesamtumsatzes und des Ruheumsatzes) sind ebenfalls positiv mit Änderungen der Aktivitätsmenge korreliert. Diese Aussage beruht auf der Voraussetzung, daß sich die circadiane Periodik des Gesamtstoffwechsels aus mindestens zwei Periodizitäten zusammensetzt: der Periodik des Grundstoffwechsels (vegetative Periodik) und der Periodik von Ruhe und Aktivität (Aktivitätsperiodik).Bei Änderungen des Zeitgebers (Erniedrigung der Zeitgeberamplitude und gleichzeitige Erhöhung der mittleren Beleuchtungsstärke) werden folgende Parameter signifikant verändert: die positive Phasenwinkel-Differenz zwischen Stoffwechselperiodik und Zeitgeber wird vergrößert, die Dauer der Aktivität (Aktivitätsumsatz ₃ ) wird verlängert, die Schwingungsbreite der Stoffwechselperiodik wird erniedrigt (Abb. 4; Tabelle 2).Unter Dauerlicht-Bedingungen werden durch eine Erhöhung der Beleuchtungsstärke von 1 auf 10 Lux folgende circadianen Parameter erhöht: die Frequenz, das mittlere Niveau der Stoffwechselperiodik (Gleichwert), der Ruheumsatz, das -Verhältnis (Verhältnis Aktivitätszeit/Ruhezeit) und die Aktivitätsmenge pro Zeiteinheit. Die Schwingungsbreite der Stoffwechselperiodik bleibt unverändert (Abb. 5). Außerdem besteht eine regelhafte Beziehung zwischen dem Formfaktor der Stoffwechselschwingung und der Beleuchtungsstärke, beziehungsweise der Spontanfrequenz. Erhöhung der Beleuchtungsstärke von 10 auf 100 Lux hat unterschiedliche Wirkungen auf die circadianen Parameter der 4 gemessenen Vögel.Die unter Dauerlicht-Bedingungen gewonnenen Ergebnisse folgen der modifizierten circadianen Regel, die eine positive Korrelation zwischen Frequenz und Gleichwert der autonomen circadianen Schwingung fordert (Abb. 7).Die unter Zeitgeber-Bedingungen (Licht-Dunkel-Wechsel) gewonnenen Ergebnisse werden auf der Grundlage einer vorwiegenden Beeinflussung des circadianen Schwingers durch die Amplitude der Zeitgeber-Periodik (Zeitgeberstärke) gedeutet. Änderungen der Beleuchtungsstärke während der Dunkelzeit (im Bereich der gebotenen Lichtintensitäten) haben keinen regelhaften Einfluß auf die Stoffwechselparameter der Periodik.

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On the effect of light upon the crcadian rhythms of metabolism and activity in the chaffinch (Fringilla coelebs L.)

Summary Oxygen consumption and hopping activity of 8 chaffinches (4 males, 4 females) were measured simultaneously (I) in an artificial 1212 hr light-dark-cycle (LD; without twilight) and (II) in continuous illumination, both at constant temperature (18–20°C). During the first part of the experiment illumination during darktime (D) was varied in both directions between 0.01 and 1.0 lux. Light intensity in L (lighttime) remained constant (10 lux). In the second part of the experiment the birds were exposed to a continuous illumination of 1 and 10 lux. Four birds were also measured at 100 lux.For the majority of the birds, the mean level of oxygen consumption per hour and the average amount of activity (contacts per hour) during one circadian period are positively and linearly correlated to each other (Fig. 2 and 3; Table 1).Changes in the level of the activity metabolism — i.e. the difference between mean level of total metabolism and resting metabolism — are also positively correlated to changes in the amount of activity. This statement is based on the assumption that there exist at least 2 rhythms as basic components of the circadian rhythm of total metabolism: the rhythm of basal metabolic rate (BMR) or vegetative rhythm and the rhythm of activity and rest (activity rhythm).By variation of the Zeitgeber — simultaneous decrease in amplitude and increase in mean level of light intensity — the following parameters are significantly changed: the phase-angle difference between the circadian rhythm of metabolism and the Zeitgeber is increased (positive phase-angle differences are changed in positive direction), the activity time is lengthened, and the amplitude of the rhythm (range of oscillation) is diminished (Fig. 4; Table 2).With an increase in light intensity from 1 to 10 lux (continuous illumination), the following parameters are increased: the frequency of the rhythm, the mean level of metabolism per period, the resting metabolic rate, the -ratio (i.e. ratio activity time/rest time), and the total amount of activity per unit time. The range of oscillation remains unchanged (Fig. 5). There is also a regular change of the form-factor characterizing the shape of the metabolic oscillation, which depends upon light intensity as well as upon frequency (Fig. 8). Increasing light intensity from 10 to 100 lux affected differently the circadian parameters of the four birds tested.The results obtained in continuous illumination follow the modified circadian rule which postulates the positive correlation between frequency and mean level of the autonomous circadian oscillation (Fig. 7).The results obtained under the influence of a Zeitgeber (light-dark-cycle) are interpreted on the basis of the primary influence of the Zeitgeber amplitude (strength of the Zeitgeber) upon the circadian oscillation. The given changes in light intensity during darktime have no significant effect on the metabolic parameters of the rhythm.

     

Restriction fragment analysis of the secalin loci of rye

Analyses of wheat/rye addition lines by Southern blotting confirmed the presence of sequences related to theSec 1, Sec 2, andSec 3 loci on chromosomes 1R and 2R. Comparison of the 1R and 2R addition lines allowed the identification of -secalin genes atSec 1 andSec 2, respectively, while -secalin and -secalin genes atSec 1 were discriminated by comparative hybridization with three probes: -secalin, total -secalin, and 3 -secalin. The high molecular weight (HMW) secalin genes atSec 3 were identified using a homologous HMW subunit probe from wheat. Gene copy numbers were estimated as about 40–60 for -secalins, 5–10 for -secalins, and 2 for HMW secalins. Comparison of individual plants of cv. Gazelle showed a high degree of polymorphism, particularly for sequences related to -secalins and HMW secalins.     

Measurement of the protein backbone dihedral angle φ based on quantification of remote CSA/DD interference in inter-residue 13C′(i − 1)-13Cα(i) multiple-quantum coherences

A novel triple-resonance NMR method is presented for the measurement of the protein backbone dihedral angle based on differential multiple-quantum relaxation induced by relaxation interference between ¹H(i)-¹³C(i) dipolar and ¹³C(i–1) (carbonyl) chemical shift anisotropy mechanisms. The method employs a simultaneous transfer of ¹⁵N magnetization to the inter- and intra-residue ¹³C carbons as well as the directly attached carbonyl carbon ¹³C. Results obtained on ¹³C,¹⁵N-labeled ubiquitin demonstrate the potential of the method.     

Characterization of a new Bacillus stearothermophilus isolate: a highly thermostable α-amylase-producing strain

A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known -amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable -amylase. The half-time of inactivation of this -amylase was 5.1 h at 80°C and 2.4 h at 90°C. The temperature optimum for activity of the -amylase was 70°C; the pH optimum for activity was relatively low, in the range 5.5–6.0. -Amylase synthesis was regulated by induction and repression mechanisms. An inverse relationship was found between growth rate and -amylase production. Low starch concentrations and low growth temperatures were favourable for enzyme production by the organism. At the optimal temperature for growth, 65°C, the -amylase was a growth-associated enzyme. The optimal temperature for -amylase production, however, was 40°C, with -amylase increasing from 3.9 units (U)/ml to 143 U/ml when lowering the growth temperature from 65°C to 40°C. Maximal -amylase production in a batch fermentor run at 65°C was 102 U/ml, which was 26-fold higher than in erlenmeyer flasks at 65°C. The dissolved O₂ concentration was found to be a critical factor in production of the -amylase.     

Synthesis of hexaploid (AABBCC) somatic hybrids: a bridging material for transfer of ‘tour’ cytoplasmic male sterility to different Brassica species

Most of the alloplasmic cytoplasmic male sterility (CMS) systems are known to be associated with a number of floral abnormalities that result from nuclear-cytoplasmic incompatibilities. One such system, tour, which is derived from Brassica tournefortii, induces additional floral abnormalities and causes chlorosis in Brassica spp. While the restorer for this CMS has been reported to be present in B. napus, in B. juncea, where the abnormalities are more pronounced, no restorer has yet been identified. Rectification of these floral abnormalities through mitochondrial recombinations and chloroplast replacement might result in the improvement of this CMS system. As organelle recombinations can possibly be achieved only by somatic cell hybridization, fusion experiments were carried out between hygromycin-resistant B. juncea AABB carrying tour cytoplasm and phosphinotricin-resistant, normal B. oleracea CC to generate AABBCC hexaploid somatic hybrids. The presence of selectable marker genes facilitated the selection of hybrids in large numbers. The resulting hybrids showed wide variation in floral morphology and organelle composition. Regenerants with normal, male-sterile flowers having recombinant tour-or oleracea-type mitochondria and oleracea-type chloroplasts were obtained. Hybrids with male-fertile flowers were also obtained that had recombined tour mitochondria. The AABBCC hexaploid hybrids synthesized in the present study were successfully utilized as a bridging material for transferring variability in the organelle genome simultaneously to all the digenomic Brassica species, and all of these hybrids are now being stabilized through repeated backcrosses to the allopolyploid crop brassicas.     

Theβ-subunit of human chorionic gonadotropin containsN-glycosidic trisialo tri- and tri′-antennary carbohydrate chains

TheN-linked carbohydrate chains of the-subunit of highly purified urinary human chorionic gonadotropin have been re-investigated. The oligosaccharides were released enzymatically by peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F, and fractionated by a combination of FPLC and HPLC. As a result of the application of improved fractionation methods, apart from the earlier reported carbohydrate chains, also small amounts of trisialo tri- and tri-antennary oligosaccharides were found. The primary structures of the latter carbohydrate chains have been determined by 500-MHz¹H-NMR spectroscopy to beAbbreviations hCG human chorionic gonadotropin - hCG- -subunit - hCG- -subunit - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52) - endo-F endo--N-acetylglucosaminidase-F (E.C. 3.2.1.96) - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - CBB coomassie brilliant blue R 250 - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

The taxonomic significance of the trunk limbs of the chydoridae (Cladocera)

Summary The differences in the structure of the trunk limbs allow to outline three sections of Chydoridae (see table I and fig. 1), coinciding with the sections distinguished according to the structure of the head pores.

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Chydoridae (Cladocera)

Chydoridae (. ), , .

     

Correlation of spinule dynamics and plasticity of the horizontal cell spectral response in cyprinid fish retina: quantitative analysis

Summary A negative feedback interaction between luminosity type horizonatal cells (HCs) and green-sensitive cones generates the long-wavelength-sensitive depolarizing response in biphasic chromaticity type HCs. This interaction is suppressed in the dark and is potentiated by light adaptation of the retina. HCs are morphologically plastic; during light adaptation, their dendritic terminals within cone pedicles extend, giving rise to spinules. This paper examines whether there is a quantitative correlation between the time course of light-dependent formation of the spinules and enhancement of the feedback interaction. The strength of the feedback interaction in isolated retinac of the roach was determined as the neutral wavelength at which reversal of spectral response polarity occurred in biphasic HCs. A good correlation was found between the neutral wavelength and the spinule/ribbon ratios of retinae. Biphasic HCs were intracellularly stained with horseradish peroxidase and the correlative ultrastructure of the contacted pedicles was examined. Neutral wavelength was found to be correlated with the spinule number, weighted according to the number of synaptic contacts mediating feed-forward transmission. The latter was estimated from the total number of labelled C_b/H2 HC processes (central and lateral) at synaptic triads. A model in which spinules mediate the negative feedback interaction of HCs in the retina of cyprinid fish is presented.     

TROSY experiment for refinement of backbone psi and phi by simultaneous measurements of cross-correlated relaxation rates and 3,4J(H alpha HN) coupling constants

The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential H-C/H^N-N dipole/dipole and H-C/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the ^3,4 J _{H HN} coupling constants measured in an E.COSY manner. The conformational restraints for the and angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants. Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle. The application of the [¹⁵N,¹³C]-DQ/ZQ-[¹⁵N,¹H]-TROSY-E.COSY experiment to the 16 kDa apo-form of the E. coli Heme Chaperon protein CcmE is described. Overall good agreement is achieved between and angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformers.