A 13C-methylation study of glycophorin A in intact erythrocytes by 13C-NMR spectroscopy

N-terminal $\text{N}{}^{\mathrm{\alpha }}\text{-[}{}^{13}\text{C]monomethylamino}$ derivatives for the N-terminal serine and leucine residues of glycophorins A^M and A^N, respectively, were obtained by reductively ¹³C-methylating homozygous human erythrocytes (MM, NN). The ¹³C-labeled glycophorins, A^M and A^N, were then isolated. A unique structural state was observed in solution reductively ¹³C-methylated glycophorin A^M that was not observed in glycophorin A^M derived from ¹³C-methylated erythrocytes. We attribute this state to the fact that some of the glycophorin A^M forms a head-to-head dimer when subjected to reductive ¹³C-methylation in aqueous solution. The ¹³C chemical shift data and pH titration data for the N-terminal [¹³C]dimethylamino and [¹³C]monomethylamino groups of glycophorin A^M and A^N derived from reductively ¹³C-methylated erythrocytes were in agreement with the chemical shift and titration data previously obtained for the N-terminal [¹³C]dimethylamino groups of solution reductively ¹³C-methylated glycophorins and related glycopeptides and peptides and N-terminal [¹³C]monomethylamino groups of related glycopeptides and peptides.     

pH titration studies of 13C reductively methylated N-terminal serine of glycophorin AM

The ¹³C resonances of N^α,N-[¹³C]dimethylserine of partially ¹³C reductively methylated glycophorin A^M were monitored as a function of pH at 45°C. For comparison, limited data are also presented for the pH dependence of the ¹³C resonances of N^α,N- [¹³C]dimethylserine of fully ¹³C reductively methylated deglycosylated glycophorin A^M. The ‘major’ component of N^α,N- [¹³C]dimethylserine of glycophorin A^M did not titrate, whereas the ‘minor’ component titrated with a pK_a of 7.80 (Hill coefficient of 0.95). Similar results are also indicated for the N^α,N- [¹³C]dimethylserine resonances of ¹³C reductively methylated deglycosylated glycophorin A^M.     

Simultaneous <Superscript>1</Superscript>H- or <Superscript>2</Superscript>H-, <Superscript>15</Superscript>N- and multiple-band-selective <Superscript>13</Superscript>C-decoupling during acquisition in <Superscript>13</Superscript>C-detected experiments with proteins and oligonucleotides

Significant resolution improvement in ¹³C,¹³C-TOCSY spectra of uniformly deuterated and ¹³C, ¹⁵N-labeled protein and ¹³C,¹⁵N-labeled RNA samples is achieved by introduction of multiple-band-selective ¹³C-homodecoupling applied simultaneously with ¹H- or ²H- and ¹⁵N-decoupling at all stages of multidimensional experiments including signal acquisition period. The application of single, double or triple band-selective ¹³C-decoupling in 2D-[¹³C,¹³C]-TOCSY experiments during acquisition strongly simplifies the homonuclear splitting pattern. The technical aspects of complex multiple-band homonuclear decoupling and hardware requirements are discussed. The use of this technique (i) facilitates the resonance assignment process as it reduces signal overlap in homonuclear ¹³C-spectra and (ii) possibly improves the signal-to-noise ratio through multiplet collapse. It can be applied in any ¹³C-detected experiment.     

Initial organic products of assimilation of [N]ammonium and [N]nitrate by tobacco cells cultured on different sources of nitrogen

Glutamine is the first major organic product of assimilation of ¹³NH₄⁺ by tobacco (Nicotiana tabacum L. cv. Xanthi) cells cultured on nitrate, urea, or ammonium succinate as the sole source of nitrogen, and of ¹³NO₃⁻ by tobacco cells cultured on nitrate. The percentage of organic ¹³N in glutamate, and subsequently, alanine, increases with increasing periods of assimilation. ¹³NO₃⁻, used for the first time in a study of assimilation of nitrogen, was purified by new preparative techniques. During pulse-chase experiments, there is a decrease in the percentage of ¹³N in glutamine, and a concomitant increase in the percentage of ¹³N in glutamate and alanine. Methionine sulfoximine inhibits the incorporation of ¹³N from ¹³NH₄⁺ into glutamine more extensively than it inhibits the incorporation of ¹³N into glutamate, with cells grown on any of the three sources of nitrogen. Azaserine inhibits glutamate synthesis extensively when ¹³NH₄⁺ is fed to cells cultured on nitrate. These results indicate that the major route for assimilation of ¹³NH₄⁺ is the glutamine synthetase-glutamate synthase pathway, and that glutamate dehydrogenase also plays a role, but a minor one. Methionine sulfoximine inhibits the incorporation of ¹³N from ¹³NO₃⁻ into glutamate more strongly than it inhibits the incorporation of ¹³N into glutamine, suggesting that the assimilation of ¹³NH₄⁺ derived from ¹³NO₃⁻ may be mediated solely by the glutamine synthetase-glutamate synthase pathway.     

Spectral editing: selection of methyl groups in multidimensional solid-state magic-angle spinning NMR

A simple spectroscopic filtering technique is presented that may aid the assignment of ¹³C and ¹⁵N resonances of methyl-containing amino-acids in solid-state magic-angle spinning (MAS) NMR. A filtering block that selects methyl resonances is introduced in two-dimensional (2D) ¹³C-homonuclear and ¹⁵N–¹³C heteronuclear correlation experiments. The 2D ¹³C–¹³C correlation spectra are recorded with the methyl filter implemented prior to a ¹³C–¹³C mixing step. It is shown that these methyl-filtered ¹³C-homonuclear correlation spectra are instrumental in the assignment of C_δ resonances of leucines by suppression of C_γ–C_δ cross peaks. Further, a methyl filter is implemented prior to a ¹⁵N–¹³C transferred-echo double resonance (TEDOR) exchange scheme to obtain 2D ¹⁵N–¹³C heteronuclear correlation spectra. These experiments provide correlations between methyl groups and backbone amides. Some of the observed sequential ¹⁵N–¹³C correlations form the basis for initial sequence-specific assignments of backbone signals of the outer-membrane protein G.     

Continuous chlorophyll degradation accompanied by chlorophyllide and phytol reutilization for chlorophyll synthesis in Synechocystis sp. PCC 6803

Chlorophyll synthesis and degradation were analyzed in the cyanobacterium Synechocystis sp. PCC 6803 by incubating cells in the presence of ¹³C-labeled glucose or ¹⁵N-containing salts. Upon mass spectral analysis of chlorophyll isolated from cells grown in the presence of ¹³C-glucose for different time periods, four chlorophyll pools were detected that differed markedly in the amount of ¹³C incorporated into the porphyrin (Por) and phytol (Phy) moieties of the molecule. These four pools represent (i) unlabeled chlorophyll (¹²Por¹²Phy), (ii) ¹³C-labeled chlorophyll (¹³Por¹³Phy), and (iii, iv) chlorophyll, in which either the porphyrin or the phytol moiety was ¹³C-labeled, whereas the other constituent of the molecule remained unlabeled (¹³Por¹²Phy and ¹²Por¹³Phy). The kinetics of ¹²Por¹²Phy disappearance, presumably due to chlorophyll de-esterification, and of ¹³Por¹²Phy, ¹²Por¹³Phy, and ¹³Por¹³Phy accumulation due to chlorophyll synthesis provided evidence for continuous chlorophyll turnover in Synechocystis cells. The loss of ¹²Por¹²Phy was three-fold faster in a photosystem I-less strain than in a photosystem II-less strain and was accelerated in wild-type cells upon exposure to strong light. These data suggest that most chlorophyll appears to be de-esterified in Synechocystis upon dissociation and repair of damaged photosystem II. A substantial part of chlorophyllide and phytol released upon the de-esterification of chlorophyll can be recycled for the biosynthesis of new chlorophyll molecules contributing to the formation of ¹³Por¹²Phy and ¹²Por¹³Phy chlorophyll pools. The phytol kinase, Slr1652, plays a significant but not absolutely critical role in this recycling process.     

Interaction between the Pentose Phosphate Pathway and Gluconeogenesis from Glycerol in the Liver

After exposure to [U-¹³C₃]glycerol, the liver produces primarily [1,2,3-¹³C₃]- and [4,5,6-¹³C₃]glucose in equal proportions through gluconeogenesis from the level of trioses. Other ¹³C-labeling patterns occur as a consequence of alternative pathways for glucose production. The pentose phosphate pathway (PPP), metabolism in the citric acid cycle, incomplete equilibration by triose phosphate isomerase, or the transaldolase reaction all interact to produce complex ¹³C-labeling patterns in exported glucose. Here, we investigated ¹³C labeling in plasma glucose in rats given [U-¹³C₃]glycerol under various nutritional conditions. Blood was drawn at multiple time points to extract glucose for NMR analysis. Because the transaldolase reaction and incomplete equilibrium by triose phosphate isomerase cannot break a ¹³C-¹³C bond within the trioses contributing to glucose, the appearance of [1,2-¹³C₂]-, [2,3-¹³C₂]-, [5,6-¹³C₂]-, and [4,5-¹³C₂]glucose provides direct evidence for metabolism of glycerol in the citric acid cycle or the PPP but not an influence of either triose phosphate isomerase or the transaldolase reaction. In all animals, [1,2-¹³C₂]glucose/[2,3-¹³C₂]glucose was significantly greater than [5,6-¹³C₂]glucose/[4,5-¹³C₂]glucose, a relationship that can only arise from gluconeogenesis followed by passage of substrates through the PPP. In summary, the hepatic PPP in vivo can be detected by ¹³C distribution in blood glucose after [U-¹³C₃]glycerol administration.     

Utilization of 13C-13C Coupling in Structural and Biosynthetic Studies Double Labeling Method

A new method which utilizes ¹³C-¹³C coupling for structural and biosynthetic studies on acetate-derived metabolites is described. The ¹³C-NMR spectra of dihydrolatumcidins separately labeled with ¹³CH₃¹³C0₂Na and with a 1: 1 mixture of ¹³CH₃CO₂-Na and CH313C02-Na gave enough information to establish its structure.     

(13)C-labeled indolequinone-DTPA-Gd conjugate for NMR probing cytochrome:P450 reductase-mediated one-electron reduction

We designed and synthesized a new class of ¹³C-labeled NMR probe, ¹³C-IQ-Gd, to monitor one-electron reductions by cytochrome:P450 (CYP450) reductase under hypoxic conditions. ¹³C-IQ-Gd consisted of a Gd³⁺-diethylene triamine pentaacetic acid (DTPA) complex unit and an indolequinone (¹³C-IQ) unit bearing a ¹³C-labeled methoxy group. The ¹³C NMR signal of ¹³C-IQ-Gd was suppressed because of the intramolecular paramagnetic effect of Gd³⁺, whereas enzymatic reduction mediated by CYP450 reductase under hypoxic conditions yielded an intensed ¹³C NMR signal due to enzymatic activation of the IQ unit followed by release of the DTPA-Gd unit from ¹³C-IQ-Gd. This ¹³C NMR spectral change allowed the monitoring of CYP450 reducatase-mediated one-electron reduction.     

Synthesis of a 13C-Labeled Tracer for Stream DOC: Labeling Tulip Poplar Carbon with 13CO2

Ecosystem tracer-level additions would benefit from a stable isotope-labeled source of complex organic molecules. We tested a method to label tree C with ¹³C and create a stable isotope tracer for stream dissolved organic carbon (DOC) using tulip poplar (Liriodendron tulipifera L.) seedlings. In 2000, seedlings were grown with 0.82 moles of ¹³CO₂ to assess the distribution and level of ¹³C enrichment in the tree tissues. In 2001, seedlings were grown with 25 times more ¹³CO₂ to generate tissues with a ¹³C signal strong enough for a ¹³C-DOC stream tracer addition. ¹³C enrichment in the trees varied in each year and by tissue age and type. Tissues formed during labeling (new) were more enriched in ¹³C than tissues established prior to the ¹³CO₂ injection (old). Stems were most enriched in ¹³C in both new and old tissues. A higher percentage of ¹³CO₂ was incorporated into seedlings in 2000 (59% ±1) than 2001 (43% ±0). Percent ¹³C incorporation among tree tissue types paralleled biomass distributions. Although tree C and ¹³C were equally soluble in both years, a greater percentage of tree C went into solution in 2001 (30%) than 2000 (20%). The water-soluble tree C accounted for approximately 12% of the injected ¹³CO₂ and had both humic and polysaccharide components. Results from a whole-stream ¹³C-DOC tracer addition demonstrated that tree C could be sufficiently labeled with ¹³CO₂ to create a stream DOC isotope tracer with some polymeric constituents.     

A novel strategy for the assignment of side-chain resonances in completely deuterated large proteins using 13C spectroscopy

The assignment of the aliphatic ¹³C resonances of trimeric Bacillus Subtilis chorismate mutase, a protein with a molecular mass of 44 kDa, consisting of three 127-residue monomers is presented by use of two-dimensional (2D) ¹³C-start and ¹³C-observe NMR experiments. These experiments start with ¹³C excitation and end with ¹³C observation while relying on the long transverse relaxation times of ¹³C spins in uniformly deuterated and ¹³C,¹⁵N-labeled large proteins. Gains in sensitivity are achieved by the use of a paramagnetic relaxation enhancement agent to reduce ¹³C T ₁ relaxation times with little effect on ¹³C T ₂ relaxation times. Such 2D ¹³C-only NMR experiments circumvent problems associated with the application of conventional experiments for side-chain assignment to proteins of larger sizes, for instance, the absence or low concentration of the side-chain ¹H spins, the transfer of the side-chain spin polarization to the ¹H^N spins for signal acquisition, or the necessity of a quantitative reprotonation of the methyl moieties in the otherwise fully deuterated side-chains. We demonstrate that having obtained a nearly complete assignment of the side-chain aliphatic ¹³C resonances, the side-chain ¹H chemical shifts can be assigned in a semiautomatic fashion using 3D ¹⁵N-resolved and ¹³C-resolved NOESY experiments measured with a randomly partially protonated protein sample. We also discuss perspectives for structure determination of larger proteins by using novel strategies which are based on the ¹H,¹H NOEs in combination with multiple residual dipolar couplings between adjacent ¹³C spins determined with 2D ¹³C-only experiments.     

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.     

A C-detection NMR approach for large glycoproteins

NMR spectroscopy has great potential to provide us with information on structure and dynamics at atomic resolution of glycoproteins in solution. In larger glycoproteins, however, the detrimental fast ¹H transverse relaxation renders the conventional ¹H-detected NMR experiments difficult. ¹³C direct detection potentially offers a valuable alternative to ¹H detection to overcome the fast T₂ relaxation. Here, we applied ¹³C-detected NMR methods to observe the NMR signals of ¹³C-labeled glycans attached to the Fc fragment of immunoglobulin G with a molecular mass of 56 kDa. Spectral analysis revealed that a ¹³C-detected ¹³C-¹³C NOESY experiment is highly useful for spectral assignments of the glycans of large glycoproteins. This approach would be, in part, complementary to ¹³C-¹³C TOCSY and ¹H-detection experiments.     

C1 metabolism and the Calvin cycle function simultaneously and independently during HCHO metabolism and detoxification in Arabidopsis thaliana treated with HCHO solutions

Formaldehyde (HCHO) is suggested to be detoxified through one‐carbon (C1) metabolism or assimilated by the Calvin cycle in plants. To further understand the function of the Calvin cycle and C1 metabolism in HCHO metabolism in plants, HCHO elimination and metabolism by Arabidopsis thaliana in HCHO solutions was investigated in this study. Results verified that Arabidopsis could completely eliminate aqueous HCHO from the HCHO solutions. Carbon‐13 nuclear magnetic resonance (¹³C‐NMR) analysis showed that H¹³CHO absorbed by Arabidopsis was first oxidized to H¹³COOH. Subsequently, a clear increase in [U‐¹³C]Gluc peaks accompanied by a strong enhancement in peaks of [2‐¹³C]Ser and [3‐¹³C]Ser appeared in Arabidopsis. Pretreatment with cyclosporin A or L‐carnitine, which might inhibit the transport of ¹³C‐enriched compounds into chloroplasts and mitochondria, caused a remarkable decline in yields of both [U‐¹³C]Gluc and [3‐¹³C]Ser in H¹³CHO‐treated Arabidopsis. These results suggested that both the Calvin cycle and the C1 metabolism functioned simultaneously during HCHO detoxification. Moreover, both functioned more quickly under high H¹³CHO stress than low H¹³CHO stress. When a photorespiration mutant was treated in 6 mm H¹³CHO solution, formation of [U‐¹³C]Gluc and [2‐¹³C]Ser was completely inhibited, but generation of [3‐¹³C]Ser was not significantly affected. This evidence suggested that the Calvin cycle and C1 metabolism functioned independently in Arabidopsis during HCHO metabolism.     

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.     

Comparison of <Superscript>13</Superscript>CH<Subscript>3</Subscript>, <Superscript>13</Superscript>CH<Subscript>2</Subscript>D,and <Superscript>13</Superscript>CHD<Subscript>2</Subscript> methyl labeling strategies in proteins

A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins, using ¹³CH₃,¹³CH₂D, and ¹³CHD₂ methyl isotopomers, is presented. For each labeling scheme, ¹H–¹³C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue enzyme, malate synthase G, with ¹³CH₃,¹³CH₂D, and ¹³CHD₂ labeling in Ile δ1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence’s performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence has previously been identified as a transverse relaxation optimized experiment for ¹³CH₃-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (¹³CH₃ HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version of the ¹³CH₃ HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both ¹³C and ¹H spins. The HSQC and HMQC spectra of ¹³CH₂D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, ¹³CH₂D TROSY, that exploits cross-correlation effects to record ¹H–¹³C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of ¹³CHD₂-labeled methyl groups can be recorded with HSQC or HMQC; a new ¹³CHD₂ HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here. When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the ¹³CH₃ labeling strategy is the most beneficial from the perspective of sensitivity (gains ≥2.4 relative to either ¹³CH₂D or ¹³CHD₂ labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented here.     

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.     

Experiments and strategies for the assignment of fully13 C/15N-labelled polypeptides by solid state NMR

High-resolution heteronuclear NMR correlation experiments and strategies are proposed for the assignment of fully¹³ C/¹⁵N-labelled polypeptides in the solid state. By the combination of intra-residue and inter-residue¹³ C-¹⁵N correlation experiments with¹³ C-¹³C spin-diffusion studies, it becomes feasible to partially assign backbone and side-chain resonances in solid proteins. The performance of sequences using ¹⁵N instead of¹³ C detection is evaluated regarding sensitivity and resolution for a labelled dipeptide (L-Val-L-Phe). The techniques are used for a partial assignment of the ¹⁵N and ¹³C resonances in human ubiquitin.     

The 13C NMR spectra of some rosane diterpenoids

The ¹³C NMR signals of rosenonolactone have been assigned utilizing the ¹³C-¹³C couplings in material obtained biosynthetically from sodium [1,2-¹³C₂] acetate.     

Regional differences in the carbon isotopic compositions of teak from two monsoonal regimes of India

Inter and intra-annual carbon isotope compositions (δ¹³C) of several annual growth rings of teak trees from two monsoonal regimes from India were studied and compared with the corresponding oxygen isotopic (δ¹⁸O) variations. In teak from both the regimes, amplitudes of intra-annual δ¹³C were ∼2-3 times lower than that observed in δ¹⁸O. Seasonal cycle with lower δ¹³C values at the middle and higher at ring boundaries was observed for teak from central India, dominated by the southwest monsoon. Positive correlations of intra-annual δ¹³C values with the corresponding δ¹⁸O values of the same rings and with relative humidity (RH) of the concurrent period suggest a dominant role of RH in controlling δ¹³C values of teak from central India. Intra-annual δ¹³C variations of teak from southern India, receiving both the southwest and northeast monsoons, revealed an initial decreasing trend followed by an increasing trend before culminating in depleted ¹³C values at the end of the growing season. No correlation was observed between intra-annual δ¹³C and δ¹⁸O variations of teak trees from southern India. Regional differences in the climatology of δ¹³C of atmospheric CO₂ or the lengths of growing season could be likely reasons for differing intra-annual δ¹³C variations of teak from the two climatic regimes.