Search

A new synthetic scheme towards silica-supported organotrichlorotin derivatives has been developed. It involves the synthesis of (11-triethoxysilyl)undecyltricyclohexyltin, followed by sol-gel processing and, subsequently in the formation of the resulting hybrid silica, by electrophilic substitution of the tricyclohexyltin function by the target grafted trichlorotin using tin tetrachloride. HR-MAS (119)Sn and CP-MAS (29)Si NMR combined with N2-sorption and TEM measurements evidenced the formation of a mesoporous organic-inorganic hybrid silica including a functionally pure supported-organotrichlorotin species. This silica-grafted organotrichlorotin displays a satisfactory catalytic activity in the transesterification of ethyl acetate by 1-octanol. The catalyst could be recycled four times without significant loss of activity. Furthermore, tin leaching below 10 ppm evidences the benefits of the proposed strategy to limit tin contamination of the final products.

The self-aggregation and supramolecular micellar structure of two surfactants in aqueous solution, the anionic surfactant SDP2S (sodium dodecyl dioxyethylene-2 sulfate) and the nonionic surfactant Triton X-100 (octylphenol-polyoxyethylene ether with 9.5 ethoxy groups), were investigated by NMR spectroscopy. The critical micellar concentration (CMC), the size, and shape of the aggregates were determined by diffusion ordered NMR spectroscopy (DOSY), while 2D NOESY NMR spectra were used to study the mutual spatial arrangement of surfactant molecules in the aggregated state. A nonlinear increase of the micellar hydrodynamic radius, indicating possible sphere-to-rod shape transition, was found for SDP2S at higher surfactant concentrations. Triton X-100 micelles were found to be almost spherical at low surfactant concentrations, but formation of ellipsoid shaped particles and/or micellar aggregation was observed at higher concentrations. The NOESY data show that at low concentration Triton X-100 forms a two-layer spherical structure in the micelles, with partially overlapping internal and external layers of Triton X-100 molecules and no distinct hydrophilic-hydrophobic boundary.

Diffusion-ordered NMR spectroscopy (DOSY NMR) is presented as a tool for the determination of the diffusion coefficients of organic ligands in suspensions of titanium dioxide nanoparticles. The nanoparticles were prepared by a sol-gel process by hydrolysis and condensation reactions of titanium tetra-n-butoxide in the presence of pentane-2,4-dione (acacH: acetylacetone), as well as para-toluenesulfonic acid (pTsA) and n-butanol (nBuOH). NMR spectroscopic studies were performed in various deuterated solvents, on both dispersed xerosols and diluted sols. The bipolar-pulsed field gradient longitudinal eddy-current delay (LED) pulse sequence was used for data acquisition. The data were processed by inverse Laplace transformation (ILT), by using a maximum entropy algorithm, to afford 2D DOSY spectra. Different diffusion regimes for organic ligands in the bound and unbound states were successfully discriminated, more particularly in [D3]acetonitrile, thus allowing assessment of their interactions with the nanoparticles.

Dialkyltin dichloride grafted to a cross-linked polystyrene, with the formula [P-H]((1-t))[P-(CH2)nSnBuCl2]t (P=[CH2CH(pC6H4)], t=the degree of functionalization, and n=6 or 11), is investigated as a recyclable catalyst in the ring-opening polymerization (ROP) of epsilon-caprolactone (CL). It is demonstrated that high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy is an invaluable tool to characterize completely the supported catalyst. The 2D 1H-13C HSQC HR-MAS spectrum, in particular, allowed extensive assignment of the 1H and 13C resonances, as well as accurate measurement of the (n)J((1)H-(117/119)Sn) and (n)J((13)C-(117/119)Sn) coupling constants. 1H and 119Sn HR-MAS NMR spectroscopy is presented as a monitoring tool for catalytic processes based on organotin compounds, particularly for the investigation of the extent to which polymerization residues are observable in situ in the material pores and for the assessment of the chemical integrity and recycling conditions of the grafted catalyst. From polymerization experiments with CL, initiated by n-propanol and with [P-H]((1-t))[P-(CH2)nSnBuCl2]t of various compositions as the supported catalyst, it appears that a partial 'burst' of the polystyrene support occurs when the length of the alkyl spacer is limited to n=6, as a result of polymer chains growing within the pores of the support. However, extension of the length of the aliphatic polymethylene spacer from 6 to 11 carbon atoms preserves the support integrity and allows the production of catalyst-deprived poly(epsilon-caprolactone) (PCL) oligomers. A preliminary attempt to recycle the heterogeneous catalyst has shown that very good reproducibility can be obtained, in terms of both catalyst activity and molecular-weight parameters of the as-recovered PCL polyester chains.

Complexation affinity of laurate ligands (C(12)H(23)O(2)) grafted onto the surface of cerium(IV) oxide nanoparticles can be probed and quantified in situ, by pulsed field gradient (1)H NMR through the dependence of the diffusion coefficient on the size of a species.

The anomeric specificity of D-glucose metabolism and functional effects is currently assessed either over about 5 min at 37 degrees C or longer periods at much lower temperatures. In the present study, the half-life for the interconversion of D-glucose anomers at 30 degrees C was found to be increased (P<0.005) from 17.2+/-0.9 min in a mixture of H2O/D2O (9/1, v/v) to 58.6+/-1.9 min in pure D2O. It is proposed that advantage can be taken from this delayed interconversion in D2O in experiments dealing with the anomeric specificity of D-glucose metabolic fate and functional effects. Thus, even over 60-min incubation at 30 degrees C, the integrated mean abundance of alpha- and beta-D-glucose would remain close to 82 and 89%, respectively, of the corresponding initial value (100%).

This study aims at assessing the conversion of exogenous D-[1-13C]fructose, D-[2-13C]fructose or D-[6-13C]-fructose (10 mM) to 13C-enriched and either hydrogenated or deuterated D-glucose, L-lactate and L-alanine released by rat liver cells prepared from Goto-Kakizaki rats and incubated for 120 min in the presence of unlabelled D-glucose (also 10 mM) and D2O. The results of this study are relevant to the relative contribution of fructokinase and hexokinase isoenzyme to the phosphorylation of D-fructose, the capacity of D-glucose to confer to glucokinase positive cooperativity towards D-fructose, the circulation of D-fructose 6-phosphate in the pentose phosphate pathway, the regulation of the cytosolic NADD/NADH ratio, the respective fate of D-fructose-derived D-glyceraldehyde and dihydroxyacetone phosphate, the deuteration of fructose-derived glycolytic intermediates at the phosphoglucoisomerase, phosphomannoisomerase, enolase, pyruvate kinase and glutamate-alanine transaminase levels, and the unequal generation of L-[1-13C]lactate by cells exposed to D-[1-13C]fructose or D-[6-13C]fructose versus D-[2-13C]-fructose.

Pulsed field gradient (1)H NMR spectroscopy has been applied to investigate the association behavior of the Sn(12)-oxo cluster macrocation [(BuSn)(12)O(14)(OH)(6)](2+) with two different and smaller anions, p-toluenesulfonate (PTS(-)) and diphenylphosphinate (Ph(2)PO(2) (-)). By monitoring the translational diffusion coefficients of the various species involved, it is shown that the association depends on the anion involved and on the solvent used. Moreover, the possibility to individually monitor the diffusion characteristics of multiple anionic and cationic species in mixtures, by virtue of resolved (1)H resonances available from each species, allows us to evidence the occurrence of ion exchange in such systems. Thus when [(BuSn)(12)O(14)(OH)(6)](PTS)(2) is mixed with two equivalents of Ph(2)PO(2)NMe(4), PTS(-) is displaced by Ph(2)PO(2) (-), highlighting the greater affinity of the organotin macrocation for the diphenylphosphinate. This example clearly illustrates the potential of pulsed field gradient (1)H NMR spectroscopy in inorganic/organometallic chemistry, to follow preferential ion pairing in multi-ion systems at the level of each individually charged species.

The ring-opening polymerization (ROP) of epsilon-caprolactone (CL) was carried out in toluene at 100 degrees C with n-propanol (nPrOH) in the presence of Bu(2)SnCl(2). It comes out that the molar mass of the polyester chains can be predicted from the initial monomer-to-alcohol molar ratio in accordance with a controlled ROP mechanism involving an O-acyl cleavage of the monomer to selectively form (alpha-propyloxy)(omega-hydroxy)poly(epsilon-caprolactone) chains. In order to gain fundamental understanding of the mechanistic factors governing the polyester chain growth, advanced (1)H, (13)C, and (119)Sn NMR investigations were performed in situ in [D(8)]toluene, as well as with model solutions that contained Bu(2)SnCl(2) and binary mixtures of the components at various concentrations and temperatures. This has enabled us to propose a mechanism in which Bu(2)SnCl(2) behaves as a catalyst, while nPrOH is the actual initiator. It involves non-aggregated, six-coordinate Bu(2)SnCl(2) complexes in which ligands exchange fast on the (119)Sn NMR observational timescale, and the simultaneous interactions of CL and alcohol function in such a way that it favors insertion/propagation reactions over transesterification ones, up to high monomer conversion.

Isolated hepatocytes from fed rats were exposed for 120 min to D-glucose (10 mM) and either D-[1-13C]fructose, D-[2-13C]fructose or D-[6-13C]fructose (also 10 mM) in the presence of D2O. The identification and quantification of 13C-enriched D-fructose and its metabolites (D-glucose, L-lactate, L-alanine) in the incubation medium and the measurement of their deuterated isotopomers indicated, by comparison with a prior study conducted in the absence of exogenous D-glucose, that the major effects of the aldohexose were to increase the recovery of 13C-enriched D-fructose, decrease the production of 13C-enriched D-glucose, restrict the deuteration of the 13C-enriched isotopomers of D-glucose to those generated by cells exposed to D-[2-13C]fructose, and to accentuate the lesser deuteration of the C, (as compared to C5) of 13C-enriched D-glucose derived from D-[2-13C]fructose. The ratio between C2-deuterated and C2-hydrogenated L-lactate, as well as the relative amounts of the CH3-, CH2D-, CHD, and CD3- isotopomers of 13C-enriched L-lactate were not significantly different, however, in the absence or presence of exogenous D-glucose. These findings indicate that exogenous D-glucose suppressed the deuteration of the C1 of D-[I-13C]glucose generated by hepatocytes exposed to D-[1-13C]fructose or D-[6-13C]fructose, as otherwise attributable, in part at least, to gluconeogenesis from fructose-derived [3-13C]pyruvate, and apparently favoured the phosphorylation of D-fructose by hexokinase isoenzymes, probably through stimulation of D-fructose phosphorylation by glucokinase.

Isolated hepatocytes from fed rats were exposed for 120 min to D-[1-(13)C]fructose, D-[2-(13)C]fructose, or D-[6-(13)C]fructose in the presence of H(2)O or D(2)O. The identification and quantification of (13)C-enriched metabolites (D-glucose, L-lactate) in the incubation medium and the measurement of their deuterated isotopomers indicated that the ketohexose was phosphorylated predominantly at the intervention of fructokinase and that the majority of the D-glyceraldehyde molecules generated from d-fructose 1-phosphate were further metabolized, e.g., after phosphorylation to D-glyceraldehyde 3-phosphate. It is proposed that the present procedure may help to further characterize the regulation of D-fructose metabolism in both hepatocytes and other cell types.

Hepatocytes from fed rats were incubated for 120 min in the presence of alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM), both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM), alpha-D-glucose penta[2-13C]acetate (1.7 mM), or D-[1,2-13C]glucose (8.3 mM). The amounts of 13C-enriched L-lactate and D-glucose and those of acetate and beta-hydroxybutyrate recovered in the incubation medium were comparable under the first two experimental conditions. The vast majority of D-glucose isotopomers consisted of alpha- and beta-D[1,2-13C]glucose. The less abundant single-labeled isotopomers of D-glucose were equally labeled on each C atom. The output of 13C-labeled L-lactate, mainly L-[2-13C]lactate and L-[3-13C]lactate, was 1 order of magnitude lower than that found in hepatocytes exposed to 8.3 mM D-[1,2-13C]glucose, in which case the total production of the single-labeled species of D-glucose was also increased and that of the C3- or C4-labeled hexose was lower than that of the other 13C-labeled isotopomers. In cells exposed to alpha-D-glucose penta[2-13C]acetate, the large majority of 13C atoms was recovered as [2-13C]acetate and, to a much lesser extent, beta-hydroxybutyrate labeled in position 2 and/or 4. Nevertheless, L-[2-13C]lactate, L-[3-13C]lactate, and single-labeled D-glucose isotopomers were also produced in amounts higher or comparable to those found in cells exposed to alpha-D-[1,2-13C]glucose pentaacetate. However, a modest preferential labelling of the C6-C5-C4 moiety of D-glucose, relative to its C1-C2-C3 moiety, and a lesser isotopic enrichment of the C3 (or C4), relative to that of C1 (or C6) and C2 (or C5), were now observed. These findings indicate that, despite extensive hydrolysis of alpha-D-glucose pentaacetate (1.7 mM) in the hepatocytes, the catabolism of its D-glucose moiety is not more efficient than that of unesterified D-glucose, tested at the same molar concentration (1.7 mM) in the presence of the same molar concentration of unesterified acetate (8.5 mM), and much lower than that found at a physiological concentration of the hexose (8.3 mM). The present results also argue against any significant back-and-forth interconversion of D-glucose 6-phosphate and triose phosphates, under conditions in which sizeable amounts of D-glucose are formed de novo from 13C-enriched Krebs cycle intermediates generated from either D-[1,2-13C]glucose or [2-13C]acetate.

Tumoral pancreatic islet cells of the RINm5F line were incubated, in groups of 25x106 cells each, for 120 min at 37 degrees C in media (5. 0 ml) containing either alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM) or both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM). In both cases, the amounts of 13C-enriched metabolites (D-glucose, L-lactate and acetate) and non-enriched metabolites (acetate) recovered in the incubation medium after incubation were close to the initial amount of esterified or non-esterified D-[1, 2-13C]glucose and acetate, respectively. The 13C-enriched metabolites corresponded mainly to double-labelled D-[1, 2-13C]glucose, L-[2,3-13C]lactate and [1,2-13C]acetate. The output of L-[2,3-13C]lactate and [1,2-13C]acetate was about 3-4 times lower in the cells exposed to alpha-D-[1,2-13C]glucose pentaacetate than in those incubated with unesterified D-[1,2-13C]glucose. These findings indicate that, despite extensive hydrolysis of alpha-D-[1, 2-13C]glucose pentaacetate in the RINm5F cells, the hexose moiety of the ester is less efficiently metabolized than unesterified D-[1, 2-13C]glucose tested at the same molar concentration (1.7 mM) in the presence of 8.5 mM acetate. Thus, a higher utilization of the hexose moiety of alpha-D-glucose pentaacetate than that of unesterified D-glucose, as previously documented in isolated pancreatic islets, represents a far-from-universal situation.

Hepatocytes prepared from overnight-fasted rats were incubated for 120 minutes in the presence of 2.5 mmol/L [1,3-13C]glycerol-1,2,3-tris(methylsuccinate) or glycerol-1,2,3-tris(methyl[2,3-13C]succinate). The identification and quantification of 13C-enriched metabolites by a recently developed method for the deconvolution of nuclear magnetic resonance (NMR) spectra with multiplet structures and constraints documented a virtually complete recovery of [1,3-13C]glycerol-1,2,3-tris(methylsuccinate) in 13C-labeled glycerol, lactic acid, and glucose. In hepatocytes exposed to [1,3-13C]glycerol-1,2,3-tris(methylsuccinate), glucose was symmetrically labeled, with the vast majority of hexose molecules being enriched with 13C on both C1 and C3 and/or C6 and C4. The respective abundance of glucose isotopomers labeled either on both C3 and C4 or on only 1 of these 2 C atoms indicated that the triose phosphates generated from [1,3-13C]glycerol represented 44% +/- 1% of the total amount of triose phosphates incorporated into the hexose. In hepatocytes exposed to glycerol-1,2,3-tris(methyl[2,3-13C]succinate), the recovery of [2,3-13C]succinate, [2,3-13C]fumarate, and either double- or single-labeled malate, lactate, alanine, and glucose accounted for about half the initial 13C content of the ester. The majority of the glucose molecules were now labeled in both C, and C2 or C6 and C5, with a preferential labeling of C6-C5 relative to C1-C2, the paired C6/C1 and C5/C2 ratios averaging 1.33 +/-0.04. These findings show that glycerol-1,2,3-tris(methylsuccinate) is efficiently and extensively metabolized in hepatocytes. They reinforce the concept that the asymmetry of glucose 13C-labeling by triose phosphates generated from Krebs cycle intermediates is modulated by the availability of glycerol-derived triose phosphates. Lastly, the present study indicates that the latter triose esters, under the present experimental conditions which do not aim at duplicating the physiological in vivo situation, are largely directly channelled in the gluconeogenic pathway, with only a limited intrahepatic contribution of the "indirect" pathway involving their back-and-forth interconversion to and from pyruvate.

To obtain insight into the link between proline (Pro) accumulation and the increase in osmotolerance in higher plants, we investigated the biochemical basis for the NaCl tolerance of a Nicotiana plumbaginifolia mutant (RNa) that accumulates Pro. Pro biosynthesis and catabolism were investigated in both wild-type and mutant lines. (13)C-Nuclear magnetic resonance with [5-(13)C]glutamate (Glu) as the Pro precursor was used to provide insight into the mechanism of Pro accumulation via the Glu pathway. After 24 h under 200 mM NaCl stress in the presence of [5-(13)C]Glu, a significant enrichment in [5-(13)C]Pro was observed compared with non-stress conditions in both the wild type (P2) and the mutant (RNa). Moreover, under the same conditions, [5-(13)C]Pro was clearly synthesized in higher amounts in RNa than in P2. On the other hand, measurements of enzyme activities indicate that neither the biosynthesis via the ornithine pathway, nor the catabolism via the Pro oxidation pathway were affected in the RNa mutant. Finally, the regulatory effect exerted by Pro on its biosynthesis was evaluated. In P2 plantlets, exogenous Pro markedly reduced the conversion of [5-(13)C]Glu into [5-(13)C]Pro, whereas Pro feedback inhibition was not detected in the RNa plantlets. It is proposed that the origin of tolerance in the RNa mutant is due to a mutation leading to a substantial reduction of the feedback inhibition normally exerted in a wild-type (P2) plant by Pro at the level of the Delta-pyrroline-5-carboxylate synthetase enzyme.

The metabolism of [1,3-(13)C]glycerol-1,2,3-tris(methylsuccinate) and glycerol-1,2,3-tris(methyl[2,3-(13)C] succinate) was examined in hepatocytes prepared from hereditarily diabetic Goto-Kakizaki rats. Over 120 min incubation in the presence of one of the two (13)C-labelled esters (2.5 mM), the output of (13)C-enriched glucose averaged 57.1 +/- 18.5 and 54.1 +/- 22.7 nmol per 10(6) cells, when expressed as [1,3-(13)C]glycerol and [2,3-(13)C] succinate equivalent, respectively. In the case of [1,3-(13)C]glycerol-1,2,3-tris(methyl-succinate), the molecules of glucose were symmetrically labelled. In the case of glycerol-1,2,3-tris(methyl[2,3-(13)C] succinate), however, both the single-labelled and double-labelled isotopomers of glucose contained more (13)C atoms in their C(6)-C(5)-C(4) than C(1)-C(2)-C(3) moiety. These findings indicate that glycerol-1,2,3-tris(methylsuccinate), recently proposed as a novel insulinotropic tool for the treatment of non-insulin-dependent diabetes mellitus, is efficiently metabolized in hepatocytes from diabetic rats, the high rate of gluconeogenesis coinciding with channelling of D-glyceraldehyde-3-phosphate between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.

The metabolism of [2,3-13C]succinic acid dimethyl ester ([2,3-13C]-SAD) 10 mmol/L was examined in hepatocytes from overnight-fasted normal rats, 3-day starved rats, and overnight-fasted hereditarily diabetic Goto-Kakizaki (GK) rats. The amount of 13C-labeled succinate, fumarate, malate, lactate, alanine, and aspartate released by the hepatocytes was much higher in fasted normal rats than in starved or diabetic animals. Although the integrated areas of the 13C2 and 13C3 signals assigned to double-labeled malate, lactate, or alanine were not significantly different, the amount of single-labeled malate, lactate, alanine, and aspartate was higher in C3- versus C2-labeled isotopomers. The release of 13C-labeled glucose by the hepatocytes was lower in fasted versus starved or diabetic rats. Virtually all hexose molecules double-labeled in the C1-C2-C3 and/or C6-C5-C4 moieties corresponded to the [1,2-13C] and/or [5,6-13C] isotopomers. However, in the case of the single-labeled species, 13C-labeling of C1 (or C6) exceeded that of C2 (or C5). Both the single- and double-labeled molecules enriched with 13C in the C1-C2-C3 moiety were less abundant than those labeled in the C6-C5-C4 moiety, with such asymmetry being most marked in overnight-fasted normal rats, less pronounced in diabetic animals, and virtually absent in starved rats. These findings document that SAD is efficiently metabolized in hepatocytes, with its use as a gluconeogenic precursor being influenced by the nutritional and hormonal status of the animals. The present experiments also reinforce the view that asymmetrical labeling of glucose by 13C-labeled precursors is modulated by the relative contribution of exogenous and endogenous nutrients to the production of triose phosphates incorporated into the hexose.

Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-(13)C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-(13)C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.

The generation of 13C-labeled D-glucose isotopomers by rat hepatocytes incubated for 30 or 120 min in the presence of 10 mM [3-(13)C]pyruvate was assessed by 13C NMR. The amount of C1-labeled D-glucose exceeded that of C2-labeled hexose, which was itself higher than that of C3-labeled D-glucose. A comparable hierarchy was observed in the C6-C5-C4 moiety of the hexose. The latter moiety of D-glucose was more efficiently labeled, however, than the C3-C2-C1 moiety. This finding is similar to that both previously reported and again observed in the present study when hepatocytes were exposed to [2(-13)C]pyruvate. These converging observations thus support the concept of enzyme-to-enzyme channeling of D-glyceraldehyde 3-phosphate between glyceraldehyde-3-phosphate dehydrogenase and phospho-fructoaldolase.

In rat hepatocytes exposed to [2-13C]pyruvate, newly formed glucose was more efficiently labeled in the carbon C5 than C2, as well as in the carbon C6 than C1, suggesting enzyme-to-enzyme channeling of D-glyceraldehyde 3-phosphate between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase. Likewise the C1/C2 and C6/C5 ratios for 13C abundance in newly formed glucose, which largely exceeded the C3/C2 ratio of lactate or alanine and could reflect reversibility in the fumarase reaction, were compatible with the enzyme-to-enzyme tunneling of symmetrical Krebs cycle intermediates in the sequence of reactions catalyzed by succinyl-CoA synthetase, succinate dehydrogenase, and fumarase. This study further indicates that the major fraction of pyruvate is metabolized via pyruvate carboxylase rather than pyruvate dehydrogenase.

The generation of 13C-labelled lactate by colon carcinoma cells of the Caco-2 line incubated for 120 min in the presence of [2-13C]propionate (10 mM) was assessed by 13C NMR. About 10% of the total amount of 13C-labelled lactate was recovered in the cell pellet and displayed a [2-13C]lactate/[3-13C]lactate isotopomer ratio of 1.18 +/- 0.01. An even higher isotopomer ratio of 1.53 +/- 0.14 was observed in the case of 13C-labelled lactate released by the cells into the incubation medium. These findings indicate that, in the Caco-2 cells, metabolic intermediates of the Krebs cycle undergo enzyme-to-enzyme channelling in the sequence of reactions catalysed by succinyl-CoA synthetase, succinate dehydrogenase and fumarase.

Tumoural islet cells of the RINm5F line were incubated for 120 min in the presence of [2-13C]propionate (10 mmol/l), and the 13C enrichment of lactate released in the incubation medium was monitored by 13C nuclear magnetic resonance. The C3/C2 ratio of resonance areas was much lower than that found with naturally 13C-enriched lactate. This reveals that symmetric Krebs cycle intermediates undergo oriented transfer in the sequence of reactions catalysed by succinate thiokinase, succinate dehydrogenate and fumarase in the mitochondria of islet cells.

Two S-(2-aminoethyl)L-cysteine (AEC) resistant lines were isolated by screening mutagenized protoplasts from diploid N. sylvestris plants. Both lines accumulated free lysine at levels 10 to 20-fold higher than in controls. Lysine overproduction and AEC-resistance were also expressed in plants regenerated from the variant cultures. A feedback insensitive form of dihydrodipicolinate synthase (DHPS), the pathway specific control enzyme for lysine synthesis, was detected in callus cultures and leaf extracts from the resistant lines. Aspartate kinase (AK), the other key enzyme in the regulation of lysine biosynthesis, was unaltered in the mutants. Crosses with wild type plants indicated that the mutation conferring insensitivity to feedback in DHPS, with as result overproduction of lysine and resistance to AEC, was inherited as a single dominant nuclear gene.