Synthesis of oligomethylene-strapped chlorophyll derivatives and optical properties of their stereoisomers in a solution

Methyl pheophorbide-a/a′ derivatives covalently linked with oligomethylene chains at the 3-CH₂OCO– and 13²-COO– moieties in a molecule were prepared by modifying chlorophyll-a through intramolecular ring-closing metathesis of vinyl groups. At least, a C10-length between the 3³- and 13⁴-positions was necessary for the cyclization and connection of a C12-strap was the most suitable to achieve the highest closure yield. The oligomethylene chain in 13² R-epimers derived from methyl pheophorbide-a covered the α-face of the chlorin π-plane and the strap in the corresponding 13² S-epimers protected the β-face. Synthetic 13² R-epimer with a dodecamethylene chain gave a flat chlorin π-plane, while the decamethylene chain in the 13² R-epimer distorted the π-system due to its shorter linkage. The distortion by strapping in the 13² R-epimer induced a slight blue-shift of Qy peak in dichloromethane. CD spectra of the 13² R-epimers were similarly dependent on the chain length, i.e., the distortion of π-plane. Visible absorption and CD spectra of all the strapped 13² S-epimers were almost identical and only slightly different from those of the unstrapped. The strapping in the 13² S-epimers shifted the Qy peak bathochromically.     

Variation in bulk tissue, fatty acid and monosaccharide δ13C values between autotrophic and heterotrophic plant organs

The flowers of 23 species of grass and herb plants were collected from a mesotrophic grassland to assess natural variability in bulk, monosaccharide and fatty acid δ¹³C values from one plant community and were compared with previous analyses of leaves from the same species. The total mean bulk δ¹³C value of flower tissues was −28.1‰, and there was no significant difference between the mean δ¹³C_flower values for grass (−27.8‰) and herb (−28.2‰) species. On average bulk δ¹³C_flower values were 1.1‰ higher than bulk δ¹³C_leaf values, however, the δ¹³C_flower and δ¹³C_leaf values of grasses did not differ between organs suggesting that carbon isotope discrimination is different in grass and herb species. The abundance of different monosaccharides abundance varied between plant types, i.e. xylose concentrations in the grass flowers were as high as 40%, compared with up to 15% in the herb species, but the general relationship δ¹³C_arabinose > δ¹³C_xylose > δ¹³C_glucose > δ¹³C_galactose which had been observed in leaves was similar in flowers (total mean δ¹³C values = −25.9‰, −27.2‰, −28.8‰ and −28.1‰, respectively). However, the average 5.4‰ depletion in the δ¹³C values of the C_16:0, C_18:2 and C_18:3 fatty acids in flowers compared to bulk tissue was significantly greater than observed for leaves. The trend C_16:0 < C_18:2 < C_18:3 previously observed in leaves was also observed in grass flowers (δ¹³C_C16:0 = −33.8‰; δ¹³C_C18:2 = −33.1‰; δ¹³C_C18:3 = −34.2‰) but not herb flowers (δ¹³C_C16:0 = −34.1‰; δ¹³C_C18:2 = −32.4‰; δ¹³C_C18:3 = −34.5‰). We conclude: (i) that the biological processes influencing carbon isotope discrimination in grass flowers are different from herbs flowers; and, (ii) that a range of post-photosynthetic fractionation effects caused the observed differences between flower and leaf δ¹³C values, especially the significant ¹³C-depletion in flower fatty acid δ¹³C values.     

Seasonal variations in bulk tissue, fatty acid and monosaccharide δC values of leaves from mesotrophic grassland plant communities under different grazing managements

Leaves of 26 grass, herb, shrub and tree species were collected from mesotrophic grasslands to assess natural variability in bulk, fatty acid and monosaccharide δ¹³C values under different grazing management (cattle- or deer-grazed) on three sample dates (May, July and October) such that interspecific and spatiotemporal variations in whole leaf tissues and compound-specific δ¹³C values could be determined. The total mean leaf bulk δ¹³C value for plants was −28.9‰ with a range of values spanning 7.5‰. Significant interspecific variation between bulk leaf δ¹³C values was only determined in October (P = <0.001) when δ¹³C values of the leaf tissues from both sites was on average 1.5‰ depleted compared to during July and May. Samples from May were significantly different between fields (P = 0.03) indicating an effect from deer- or cattle-grazing in young leaves. The average individual monosaccharide δ¹³C value was 0.8‰ higher compared with whole leaf tissues. Monosaccharides were the most abundant components of leaf biomass, i.e. arabinose, xylose, mannose, galactose and glucose, and therefore, fluctuations in their individual δ¹³C values had a major influence on bulk δ¹³C values. An average depletion of ca. 1‰ in the bulk δ¹³C values of leaves from the deer-grazed field compared to the cattle-grazed field could be explained by a general depletion of 1.1‰ in glucose δ¹³C values, as glucose constituted >50% total leaf monosaccharides. In October, δ¹³C values of all monosaccharides varied between species, with significant variation in δ¹³C values of mannose and glucose in July, and mannose in May. This provided an explanation for the noted variability in the tissue bulk δ¹³C values observed in October 1999. The fatty acids C_16:0, C_18:2 and C_18:3 were highly abundant in all plant species. Fatty acid δ¹³C values were lower than those of bulk leaf tissues; average values of −37.4‰ (C_16:0), −37.0‰ (C_18:2) and −36.5‰ (C_18:3) were determined. There was significant interspecific variation in the δ¹³C values of all individual fatty acids during October and July, but only for C_18:2 in May (P = <0.05). This indicated that seasonal trends observed in the δ¹³C values of individual fatty acids were inherited from the isotopic composition of primary photosynthate. However, although wide diversity in δ¹³C values of grassland plants ascribed to grazing management, interspecific and spatiotemporal influences was revealed, significant trends (P = <0.0001) for fatty acid and monosaccharide δ¹³C values: δ¹³C_16:0 < δ¹³C_18:2 < δ¹³C_18:3 and δ¹³C_arabinose > δ¹³C_xylose > δ¹³C_glucose > δ¹³C_galactose, respectively, previously described, appear consistent across a wide range of species at different times of the year in fields under different grazing regimes.     

δ13C as a marker to study digesta passage kinetics in ruminants: a combined in vivo and in vitro study

The aim of the current study was to explore the use of the tracer ¹³C as an internal marker to assess feed fraction-specific digesta passage kinetics through the digestive tract of dairy cows. Knowledge on feed-specific fractional passage rates is essential to improve estimations on the extent of rumen degradation and microbial protein efficiency; however, this information is largely lacking. An in vivo and in vitro experiment was conducted with grass silages (Lolium perenne L.) that were enriched with ¹³C by growing the grass under elevated ¹³CO₂ conditions. In a crossover design, two dairy cows received pulse doses of two ¹³C-enriched grass silages and chromium-mordanted neutral detergent fibre (Cr-NDF) into the rumen. The two ¹³C-enriched grass silages used differed in digestibility and were grown under identical field conditions as the bulk silages fed to the animals. Faecal excretion patterns of ¹³C-enriched dry matter (¹³C-DM), neutral detergent fibre (¹³C-NDF) and Cr-NDF were established, and a nonlinear multicompartmental model was used to determine their rumen passage kinetics. In addition, the ¹³C-enriched silages were incubated in rumen liquid in an in vitro batch culture system at different time intervals to determine the effect of fermentation on ¹³C-enrichment in the residue. The in vitro study showed that the ¹³C : ¹²C ratios in DM and NDF residues remained stable from 24 h of incubation onwards. In addition, in vitro fractional degradation rates for ¹²C in the DM and NDF did not differ from those of ¹³C, indicating that fermentative degradation does not affect the ¹³C : ¹²C ratio in the DM nor in the NDF fraction of the residue. Model fits to the faecal excretion curves showed a significant difference in fractional rumen passage rates between Cr-NDF, ¹³C-DM and ¹³C-NDF (P ⩽ 0.025). Silage type had no clear effect on rumen passage kinetics (P ⩾ 0.081). Moreover, it showed that peak enrichments for ¹³C-DM and ¹³C-NDF in faeces were reached at 30.7 and 41.7 h post dosing, respectively. This is well after the time (24 h) when the ¹³C : ¹²C ratios of the in vitro unfermented residues have reached stable enrichment level. Fractional rate constants for particle passage from the rumen are estimated from the descending slope of faecal excretion curves. The present study shows that the decline in ¹³C : ¹²C ratio after peak enrichment is not affected by fermentative degradation and therefore can be used to assess feed component-specific fractional passage rates.     

Effects of body size and environment on diet-tissue δC fractionation in fishes

δ¹³C data are often used in trophodynamic research where diet-tissue fractionation (Δδ¹³C) is assumed to be 0-1‰ per trophic level and unaffected by the size of animals or their environment. Variation in Δδ¹³C will influence conclusions about food sources, energy pathways and trophic level. To assess the effects of body size, age and environmental conditions on Δδ¹³C, European sea bass (Dicentrarchus labrax) were reared on constant diets of dab (Limanda limanda) or (Ammodytes marinus) for 2years under natural environmental regimes. Bass were sampled approximately monthly to determine Δδ¹³C for muscle, heart and liver tissue and were 1.66‰, − 0.18‰, − 1.77‰ (sandeel diet) and 1.34‰, − 1.18‰, − 1.75‰ (dab diet) respectively. Arithmetic lipid correction increased Δδ¹³C to > 2‰ for muscle and liver. Δδ¹³C was dependent on body mass and experimental duration (age) and generally declined with weight or time even after correction for lipid content. For liver, increasing temperature increased Δδ¹³C. The Δδ¹³C estimates from this study were compared with all available published Δδ¹³C estimates for fish. Bass muscle Δδ¹³C was similar to previous estimates for fish white muscle Δδ¹³C (1.56 ± 1.10‰) and whole body Δδ¹³C (1.52 ± 1.13‰). Fractionations derived in this study, combined with those from the literature, support the use of diet-tissue fractionation values of between 1‰-2‰ for δ¹³C, rather than the commonly used 0‰ − 1‰. For muscle Δδ¹³C, 1.5‰ is appropriate.     

N-[1-13C]acetylimidazole as a reagent for tyrosyl modification in protein NMR studies: acetylation of cytochrome c

The reaction of N-[1-¹³C] acetylimidazole with cytochrome c and guanidinated cytochrome c was evaluated as a means of introducing NMR-detectable groups as conformation-dependent probes. Resonances from both N-[1-¹³C]acetyl lysyl and O-[1-¹³C]acetyl tyrosyl groups were observed when ferricytochrome c was acetylated. However, only O-[1-¹³C]acetyl tyrosyl resonances were seen with acetylated guanidinated ferricytochrome c. Chemical shifts of the four O-[1-¹³C]acetyl tyrosyl groups were conformation dependent and ranged from 172 to 176 ppm. A convenient method for the preparation of N-[1-¹³C]acetylimidazole is described.     

Mixotrophic metabolism in Burkholderia kururiensis subsp. thiooxydans subsp. nov., a facultative chemolithoautotrophic thiosulfate oxidizing bacterium isolated from rhizosphere soil and proposal for classification of the type strain of Burkholderia kururiensis as Burkholderia kururiensis subsp. kururiensis subsp. nov.

A thiosulfate-oxidizing facultative chemolithoautotrophic Burkholderia sp. strain ATSB13^T was previously isolated from rhizosphere soil of tobacco plant. Strain ATSB13^T was aerobic, Gram-staining-negative, rod shaped and motile by means of sub-terminal flagellum. Strain ATSB13^T exhibited mixotrophic growth in a medium containing thiosulfate plus acetate. A phylogenetic study based on 16S rRNA gene sequence analysis indicated that strain ATSB13^T was most closely related to Burkholderia kururiensis KP23^T (98.7%), Burkholderia tuberum STM678^T (96.5%) and Burkholderia phymatum STM815^T (96.4%). Chemotaxonomic data [G+C 64.0 mol%, major fatty acids, C_18:1 ω7c (28.22%), C_16:1 ω7c/15 iso 2OH (15.15%), and C_16:0 (14.91%) and Q-8 as predominant respiratory ubiquinone] supported the affiliation of the strain ATSB13^T within the genus Burkholderia. Though the strain ATSB13^T shared high 16S rRNA gene sequence similarity with the type strain of B. kururiensis but considerably distant from the latter in terms of several phenotypic and chemotaxonomic characteristics. DNA–DNA hybridization between strain ATSB13^T and B. kururiensis KP23^T was 100%, and hence, it is inferred that strain ATSB13^T is a member of B. kururiensis. On the basis of data obtained from this study, we propose that B. kururiensis be subdivided into B. kururiensis subsp. kururiensis subsp. nov. (type strain KP23^T = JCM 10599^T = DSM 13646^T) and B. kururiensis subsp. thiooxydans subsp. nov. (type strain ATSB13^T = KACC 12758^T).     

The biosynthesis of the antioxidant caffeic acid derivative subulatin by the liverwort Jungermannia subulata cultured in vitro

The in vitro cultured liverwort Jungermannia subulata produces the unique molecule subulatin. In this study, we examined the incorporation of [1-¹³C] and [1,2-¹³C₂] glucose, [2-¹³C] arabinose, [2-¹³C] caffeic acid, and [1-¹³C] phenylalanine into subulatin. The trilobatinoic acid C unit of subulatin incorporated ¹³C atoms from [1-¹³C] and [1,2-¹³C₂] glucose and from [2-¹³C] arabinose but not from any other of the other precursors. Based on these results and labeling patterns, the trilobatinoic acid C unit of subulatin appears to be biosynthesized from arabinose-5-phosphate and phosphoenolpyruvate.     

Fractionation of stable isotopes in the Arctic marine copepod Calanus glacialis: Effects on the isotopic composition of marine particulate organic matter

Fractionation of δ¹³C and δ¹⁵N between food, consumer, and faecal pellets was studied in the Arctic marine copepod Calanus glacialis Jaschnov, fed with isotopically distinct algal monocultures. Temporal variations in δ¹³C and δ¹⁵N of copepods that were fed ice algae and phytoplankton followed those of a control group consisting of starved animals. There were no significant trends in the δ¹³C and δ¹⁵N values of copepods that were starved for 42 days, suggesting that the isotopic composition of non-lipid body tissues is unaffected by the metabolic processes during prolonged periods of starvation. The stable isotopic composition of starved copepods therefore seems to reflect food consumed during the previous period of feeding and growth. Faecal pellets produced by feeding copepods were depleted in ¹³C and ¹⁵N by 6.3-11.2‰ and 0.7-9.1‰, respectively, relative to the food ingested. These results indicate that faecal pellet production is an important pathway for the trophic fractionation of δ¹³C, whereas other fractionation pathways, such as excretion of ammonia, may be relatively more important for δ¹⁵N. The strong depletion of ¹³C in faecal pellets compared to the food suggests that grazing by herbivorous copepods on primary production adds to the variability of δ¹³C in marine particulate organic matter.     

GC-MS and 13C-NMR studies on the biosynthesis of terpenoid defensive secretions by the larvae of papilionid butterflies (Luehdorfia and Papilio)

The biosynthetic pathway of some terpenic hydrocarbons present in the larval osmeterial secretions of Luehdorfia (homogeneous type) and Papilio (heterogeneous type) species was examined by in vivo experiments, using ¹³C-labelled acetic acid which was topically applied to the everted osmeteria. GC-MS investigation demonstrated that ¹³C was incorporated into mono- and/or sesquiterpene hydrocarbons with the enrichment factor of approx. 0.5% (L. puziloi), 1.0% (P. protenor) and 2.9% (P. helenus) by treatment with 1,2-¹³C-enriched acetic acid, thereby substantiating de novo biosynthesis of terpenes from acetate precursors by these larvae. The incorporation pattern of [2-¹³C]- or [1,2-¹³C]acetic acid into the carbon framework of β-myrcene (L. puziloi) and (E)-β-farnesene (P. helenus) as revealed by ¹³C-NMR spectroscopy definitely elucidated the biosyntheses of terpenic compounds in both species by the familiar terpenoid synthetic system with the isoprenoid skeletal units that is widely known in plants. Partial correction of previous assignment of ¹³C-NMR spectra of β-myrcene and (E)-β-farnesene is also made.     

Chiral-phase HPLC separation of (divinyl-)protochlorophyllide-a enantiomers as key precursors in chlorophyll biosynthesis from their 132-stereoisomeric prime forms

Protochlorophyllide(PChlide)-a and its 8-vinylated analog, divinyl(DV)-PChlide-a, are common and essential intermediates in the biosynthesis of all naturally occurring chlorophyll (Chl) pigments. These porphyrinoid-type pigments have a single optically active (asymmetric) carbon atom at the 13²-position, so their stereoisomers are (13²R)- and (13²S)-enantiomers. The former and latter are called (DV-)PChlide-a and (DV-)PChlide-a′, respectively. In this study, chiral-phase HPLC separation of enantiomeric (DV-)PChlides-a/a′ was demonstrated. The (13²R)-enantiomeric PChlide-a was eluted more slowly than the corresponding (13²S)-enantiomeric PChlide-a′ under the present HPLC conditions. On the other hand, the elution order of (13²R)-DV-PChlide-a and (13²S)-DV-PChlide-a′ was reverse to that of PChlides-a/a′. After the separation of each enantiomer by the chiral-phase HPLC, the stereoisomeric configuration at the 13²-position was characterized by means of circular dichroism spectroscopy. The present chiral-phase HPLC method enables us to evaluate optical purities of (DV-)PChlide-a species. For example, PChlide-a and/or DV-PChlide-a extracted from the spent medium and harvested cells of cultured purple photosynthetic bacterial mutants, the former of which has been often used as the source of (DV-)PChlide-a substrates for enzymatic reactions, were revealed to be mostly racemized, giving enantiomeric mixtures of (DV-)PChlides-a/a′.     

Passage kinetics of concentrates in dairy cows measured with carbon stable isotopes

Fractional passage rates form a fundamental element within modern feed evaluation systems for ruminants, but knowledge on feed-specific fractional passage is largely lacking. Commonly applied tracer techniques based on externally applied markers, such as chromium-mordanted neutral detergent fibre (Cr-NDF), have been criticised for behaving differently to feed particles. This study describes the use of the carbon stable isotope ratio (¹³C : ¹²C) as an internal digesta marker to quantify the fractional passage rate of concentrates through the digestive tract of dairy cows. In a crossover study, five dairy cows were fed low (24.6%) and high (52.6%) levels of concentrates (dry matter (DM) basis) and received a pulse-dosed Cr-NDF and ¹³C isotopes. The latter was administered orally by exchanging part of the dietary concentrates of low ¹³C natural abundance with a pulse dose of maize bran-based concentrates of high ¹³C natural abundance. Fractional passage rates from the rumen (K₁) and from the large intestine (K₂) were determined from faecal marker concentrations of Cr-NDF and of ¹³C in the DM (¹³C-DM), NDF (¹³C-NDF) and neutral detergent soluble (¹³C-NDS). No differences in K₁ estimates were found for the two concentrate levels fed but significant differences between markers (P<0.001) were observed. Faecal Cr-NDF excretions gave lower K₁ estimates (0.037–0.039/h) than ¹³C-DM (0.054–0.056/h) and ¹³C-NDF (0.061–0.063/h). The ¹³C-NDS was calculated by the difference of ¹³C in the DM and NDF, and K₁ values (0.039–0.043/h) were comparable to Cr-NDF. Total mean retention time was considerably higher for Cr-NDF (40.9–42.0 h) as compared to ¹³C-DM and ¹³C-NDF (32.0–33.5 h; P<0.001). The accuracy of the curve fits for Cr-NDF and ¹³C-DM and ¹³C-NDF was overall good (mean prediction error of 9.9–13.9%). Fractional passage rate of Cr-NDF was comparable to studies where this marker was assumed to represent the fractional passage of roughages. However, K₁ estimates based on the ¹³C : ¹²C ratio varied considerably from studies based on external markers. Our results suggest that the use of ¹³C isotopes as digesta passage markers can provide feed component-specific K₁ estimates for concentrates and provides new insight into passage kinetics of NDF from technologically treated compound feed.     

The effect of feeding on CO2 production and energy expenditure in ponies measured by indirect calorimetry and the 13C-bicarbonate technique

Energy expenditure (EE) can be estimated based on respiratory gas exchange measurements, traditionally done in respiration chambers by indirect calorimetry (IC). However, the ¹³C-bicarbonate technique (¹³C-BT) might be an alternative minimal invasive method for estimation of CO₂ production and EE in the field. In this study, four Shetland ponies were used to explore the effect of feeding on CO₂ production and EE measured simultaneously by IC and ¹³C-BT. The ponies were individually housed in respiration chambers and received either a single oral or intravenous (IV) bolus dose of ¹³C-labelled sodium bicarbonate (NaH¹³CO₃). The ponies were fed haylage 3 h before (T₋₃), simultaneously with (T₀) or 3 h after (T₊₃) administration of ¹³C-bicarbonate. The CO₂ produced and O₂ consumed by the ponies were measured for 6 h with both administration routes of ¹³C-bicarbonate at the three different feeding times. Feeding time affected the CO₂ production (P<0.001) and O₂ consumption (P<0.001), but not the respiratory quotient (RQ) measured by IC. The recovery factor (RF) of ¹³C in breath CO₂ was affected by feeding time (P<0.01) and three different RF were used in the calculation of CO₂ production measured by ¹³C-BT. An average RQ was used for the calculations of EE. There was no difference between IC and ¹³C-BT for estimation of CO₂ production. An effect of feeding time (P<0.001) on the estimated EE was found, with higher EE when feed was offered (T₀ and T₊₃) compared with when no feed was available (T₋₃) during measurements. In conclusion, this study showed that feeding time affects the RF and measurements of CO₂ production and EE. This should be considered when the ¹³C-BT is used in the field. IV administration of ¹³C-bicarbonate is recommended in future studies with horses to avoid complex ¹³C enrichment-time curves with maxima and shoulders as observed in several experiments with oral administration of ¹³C-bicarbonate.     

Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis

Coenzyme Q (Q or ubiquinone) is a redox-active polyisoprenylated benzoquinone lipid essential for electron and proton transport in the mitochondrial respiratory chain. The aromatic ring 4-hydroxybenzoic acid (4HB) is commonly depicted as the sole aromatic ring precursor in Q biosynthesis despite the recent finding that para-aminobenzoic acid (pABA) also serves as a ring precursor in Saccharomyces cerevisiae Q biosynthesis. In this study, we employed aromatic ¹³C₆-ring-labeled compounds including ¹³C₆-4HB, ¹³C₆-pABA, ¹³C₆-resveratrol, and ¹³C₆-coumarate to investigate the role of these small molecules as aromatic ring precursors in Q biosynthesis in Escherichia coli, S. cerevisiae, and human and mouse cells. In contrast to S. cerevisiae, neither E. coli nor the mammalian cells tested were able to form ¹³C₆-Q when cultured in the presence of ¹³C₆-pABA. However, E. coli cells treated with ¹³C₆-pABA generated ¹³C₆-ring-labeled forms of 3-octaprenyl-4-aminobenzoic acid, 2-octaprenyl-aniline, and 3-octaprenyl-2-aminophenol, suggesting UbiA, UbiD, UbiX, and UbiI are capable of using pABA or pABA-derived intermediates as substrates. E. coli, S. cerevisiae, and human and mouse cells cultured in the presence of ¹³C₆-resveratrol or ¹³C₆-coumarate were able to synthesize ¹³C₆-Q. Future evaluation of the physiological and pharmacological responses to dietary polyphenols should consider their metabolism to Q.     

The dendroclimatic value of oak stable isotopes

Tree-ring stable oxygen and carbon isotope ratios (δ¹⁸O and δ¹³C) are an important archive for climate reconstructions. However, it remains unclear whether the polyvinyl acetate emulsion, often used for the preservation and fixation of wood samples, influences δ¹⁸O and δ¹³C signals. Further uncertainties are associated with the possible effects of geographical origin and cambial age of historical samples. Here, we present annually-resolved and absolutely-dated δ¹⁸O and δ¹³C measurements of 21 living oaks (Quercus robur and Q. petraea) from the Czech Republic. We find that the δ¹⁸O and δ¹³C signals in the extracted alpha-cellulose are not affected by polyvinyl acetate treatment. Covering the entire 20^th century and reaching until 2018 CE, our dataset reveals spatial and temporal coherency within and between the individual δ¹⁸O and δ¹³C chronologies of different oak species, sample locations, and tree ages. Highly significant (p < 0.01) Pearson’s correlation coefficients of the site-specific δ¹³C and δ¹⁸O chronologies range from 0.48–0.77 and 0.36–0.56, respectively. The isotopic inter-series correlations of Q. robur and Q. petraea from the same site are 0.75 and 0.43 for the mean δ¹³C and δ¹⁸O values, respectively. Significant (p < 0.01) correlations of 0.49 and 0.84 are found for δ¹³C and δ¹⁸O, respectively, when all measurements from all sampling locations and tree ages are included. Our study shows that non-pooled oak δ¹⁸O and δ¹³C measurements from both species, different locations, and diverse tree ages can be combined into robust isotopic chronologies for climate reconstructions.     

Bioactive constituents from the leaves of Clinacanthus nutans Lindau

Three chlorophyll derivatives (phaeophytins) were isolated from the chloroform extract of Clinacanthus nutans Lindau leaves by means of chromatographic techniques and bioactivity-guided fractionation to give three pure compounds. Structure elucidation of the isolated compounds was carried out on the basis of spectral analyses. Three of these were known compounds with structures related to chlorophyll a and chlorophyll b namely 13²-hydroxy-(13²-R)-phaeophytin b, 13²-hydroxy-(13²-S)-phaeophytin a and 13²-hydroxy-(13²-R)-phaeophytin a. These compounds, which have not previously been reported in this plant, were shown to have anti-herpes simplex activity. They exhibited anti-HSV-1F activity at subtoxic concentrations. Their inhibitory activity affected the virus before viral entry to the host cells. This effect might be virucidal or interference with viral adsorption or penetration.     

13C structuring shifts for the analysis of model β-hairpins and β-sheets in proteins: diagnostic shifts appear only at the cross-strand H-bonded residues

The present studies have shown that ¹³C=O, ¹³C^α and ¹³C^β of H-bonded strand residues in β-hairpins provide additional probes for quantitating the extent of folding in β-hairpins and other β-sheet models. Large differences in the structuring shifts (CSDs) of these ¹³C sites in H-bonded versus non-H-bonded sites are observed: the differences between H-bonded and non-H-bonded sites are greater than 1.2 ppm for all three ¹³C probes. This prompts us to suggest that efforts to determine the extent of hairpin folding from ¹³C shifts should be based exclusively on the observation at the cross-strand H-bonded sites. Furthermore, the statistics suggest the ¹³C′ and ¹³C^β CSDs will provide the best differentiation with 100 %-folded CSD values approaching ?2.6 and +3 ppm, respectively, for the H-bonded sites. These conclusions can be extended to edge-strands of protein β-sheets. Our survey of reported ¹³C shifts in β-proteins indicates that some of the currently employed random coil values need to be adjusted, particularly for ionization-induced effects.     

An acyclic diterpene from the brown alga Bifurcaria bifurcata

Structure elucidation and total assignment of the ¹³C NMR spectrum of 12-(S)-hydroxygeranylgeraniol, a new acyclic diterpene from the grown alga Bifurcaria bifurcata, was accomplished through the use of ¹HNMR, ¹³C NMR and 2D NMR spectroscope including 2D long range ¹H-¹³C chemical shift correlations.     

An NMR study of the loss of carbon-20 in the biosynthesis of gibberellin A3 by Gibberella fujikuroi

Resuspension cultures of Gibberella fujikuroi, strain GF-1a, were shown to metabolise potassium [3′-¹³C] mevalonate to ¹³C-enriched C₁₉-gibberellins, plus ¹³CO₂ (derived from the loss of carbon-20). The formation of [¹³C]-gibberellins could be observed in vivo using ¹³C NMR; however that of ¹³CO₂ could not. In contrast, removal of the mycelium and concentration of the filtrate at pH 12 enabled the ¹³CO₂ produced to be observed using ¹³C NMR. During incubations of H¹⁴CO₂Na with this fungus, complete conversion to other radioactive products was observed, and the significance of these results in the light of previous work is discussed.     

Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR

Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved ¹³C and ¹⁵N signals of [1-¹³C]Tyr-, [¹⁵N]Pro- and [1-¹³C]Val-, [¹⁵N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1-¹³C]Val-, [¹⁵N]Pro signals to the specific residues in bR. The previous assignments of [1-¹³C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent ¹⁵N chemical shifts of Pro bonded to Val in the presence of ¹³C-¹⁵N correlation, although no assignment of peak is feasible for ¹³C nuclei not bonded to Pro. ¹³C or ¹⁵N spin-lattice relaxation times (T₁), spin-spin relaxation times under the condition of CP-MAS (T₂), and cross relaxation times (T_CH and T_NH) for ¹³C and ¹⁵N nuclei and carbon or nitrogen-resolved, ¹H spin-lattice relaxation times in the rotating flame (¹H T_1ρ) for the assigned signals were measured in [1-¹³C]Val-, [¹⁵N]Pro-bR. It turned out that V69-P70 in the BC loop in the extracellular side has a rigid β-sheet in spite of longer loop and possesses large amplitude motions as revealed from ¹³C and ¹⁵N conformation-dependent chemical shifts and T₁, T₂, ¹H T_1ρ and cross relaxation times. In addition, breakage of the β-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.