Modulation of islet G-proteins, α-glucosidehydrolase inhibition and insulin release stimulated by various secretagogues

Guanine nucleotide-binding proteins (G-proteins) are known to act as important modulators of insulin release from the islets of Langerhans. We have recently found that the deoxynojirimycin-derivative emiglitate, a recognized inhibitor of intestinal -glucosidehydrolase activity, is a powerful inhibitor of glucose-induced insulin release. With the use of isolated mouse islets the present investigation was performed in a primary attempt to elucidate whether this inhibitory mechanism in some way was linked to the -cell G-protein system. Treatment of freshly isolated islets with pertussis toxin (PTX), which is known to inactivate the G_i-proteins, abolished the inhibitory effect of the ₂-adrenoceptor agonist clonidine on insulin release stimulated by the phosphodiesterase inhibitor IBMX in the presence of the protein kinase C activator TPA and even changed it into an increase. Emiglitate did not display any inhibitory action on insulin release induced by these secretagogues. Similarly, clonidine-induced inhibition of glucose stimulated insulin release was reversed by PTX. However, PTX did not influence the suppressive action of emiglitate on glucose-induced insulin secretion. In contrast, the adenylate cyclase activator forskolin totally abolished the inhibitory effect of emiglitate, but not that of the glucose analogue mannoheptulose, on glucose-induced insulin release. Moreover, the stimulatory effect of forskolin and cholera toxin (CTX) (activator of G_s-proteins) on the secretion of insulin was markedly enhanced in the presence of emiglitate. In conclusion, our results suggest that the inhibitory effect of emiglitate on glucose-induced insulin release is not directly related to the G_j-proteins, but most likely exerted solely through the selective suppression of lysosomal -glucosidehydrolase activity, a step in between the proximal and the distal G_i-proteins, in glucose-induced stimulus-secretion mechanisms. Our data also suggests that the inhibitory action of emiglitate on glucose stimulated insulin release can be compensated for by an increased sensitivity of the cyclic AMP-protein kinase A pathway. Hence, emiglitate might indirectly elicit an increased activity of the G_s-proteins to facilitate the secretory process.     

Time dependent effect of indomethacin on the stimulation of protein synthesis in isolated rabbit muscle by insulin

Insulin (100 U/ml) stimulated protein synthesis and PGF₂ release in isolated rabbit muscle, but had little effect on the rate of protein degradation. The effect of insulin persisted for at least 5 h after removal of the hormone. Indomethacin, added at the start of the incubation, inhibited the stimulatory effect of insulin on protein synthesis and PGF₂ release, but did not block the binding of iodinated insulin. When added 2 h after insulin, indomethacin did not inhibit the stimulation of protein synthesis but completely inhibited the increase in PGF₂ release. The results suggest that the stimulation of protein synthesis by insulin is mediated by metabolites of membrane phospholipids but that these changes are involved during the phase of response that immediately follows the binding of insulin to its receptor.     

The effect of low oxygen uptake rate on the fatty acid profile of the oleaginous yeast Apiotrichum curvatum

Summary The effect of limiting the available oxygen on the fatty acid profile of Apiotrichum curvatum ATCC 20509 during growth on sulphuric acid casein whey was studied. At oxygen uptake rates (OUR) lower than 7 mmol O₂/l per hour, applied during the oil accumulating phase of the fermentation, a decrease in total unsaturated fatty acids was observed. It was possible to decrease the unsaturated fatty acids (oleate from 55% to 41% and linoleate from 9% to 3%) by limiting the OUR of the culture to <3 mmol O₂/l per hour. However at this low OUR, a lower oil coefficient (a measure of the efficiency of lactose substrate conversion to oil) was recorded. Furthermore the fermentation time was increased. An OUR of 5 mmol O₂/l per hour appeared to be the limit below which adverse effects on oil yields and increased fermentation times occurred. At this OUR, the accumulated oil contained 45% oleate and 5% linoleate. These effects were demonstrated in a 20-l air-lift fermentor and confirmed in a scaled down 500-l industrial type bubble column fermentor. Offprint requests to: R. J. Davies     

Mapping of brain activity in mesencephalic and diencephalic structures of toads during presentation of visual key stimuli: a computer assisted analysis of (14C)2DG autoradiographs

Summary The (¹⁴C)2DG autoradiographic technique has been employed to quantitatively map glucose utilization in the mesencephalon, the diencephalon and the cerebellum, of toads in response to configurational moving visual stimuli: (i) a 0.4 cm × 2.8 cm worm-like stripe (W) which elicited prey catching responses, (ii) a 8.4 cm × 8.4 cm square (S) that released predator avoidance responses, and (iii) a 2.8 cm × 0.4 cm antiworm-like stripe (A) which elicited no motor activity.For various brain nuclei different relationships were obtained: The optic tectum showed statistical significant higher 2DG uptake during worm-stimulation (¯X _W) than during antiworm stimulation (¯X _A), i.e.¯X _W>¯X _A. The latter visual pattern led to a 2DG utilization that was statistically significant stronger than during stimulation with a square (¯X _S), i.e.¯X _A>¯X _S. Thus, in comparison between right and left hemisphere as well as between brains the following ratios were obtained:Optic tectum:¯X _W>¯X _A>¯X _S; nucleus isthmi:¯X _W>¯X _A-¯X _s; posterodorsal lateral thalamic nucleus:¯X _S>¯X _A>¯X _W; posteroventral lateral thalamic nucleus:¯X _S>¯X _A¯X _W; posterior thalamic nucleus:¯X _W>¯X _A¯X _S; anteripr division of the lateral thalamic nucleus:¯X _W>¯X _A¯X _S; anterior thalamic nucleus:¯X _A>¯X _S>¯X _W; nucleus of Bellonci and dorsal division of the ventrolateral thalamic nucleus:¯X _W¯X _A¯X _S; cerebellum:¯X _S¯X _W>¯X _A.Abbreviations A anterior thalamic nucleus - Cb cerebellum - Hyp hypothalamus - Ist nucleus isthmi - cl. Ist contralateral Ist - La lateral thalamic nucleus, anterior division - Lpd lateral thalamic nucleus, posterodorsal division - Lpv lateral thalamic nucleus, posteroventral division - MP medial pallium - NB/VLd nucleus of Bellonci and ventrolateral thalamic nucleus, dorsal division - P posterior thalamic nucleus - PO preoptic area - Sna snapping evoking area=ventrolateral tectum - Str striatum - Tec tectum opticum     

Partial purification and characterization of a folatebinding protein from human choroid plexus

A folate-binding protein (binder) from human choroid plexus was solubilized with Triton X-100 and partially purified in three steps: (1) affinity chromatography, (2) Sephadex G-200 column chromatography, and (3) polyacrylamide gel electrophoresis. When the partially purified binder was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the binding activity was located in the region of the gel with a molecular weight between 45,000 and 60,000. The specific activity of the binder after the three purification steps was 1.2 g folic acid/mg protein, a 316-fold purification. Binding activity of the partially purified binder decreased below pH 6.0 and above pH 8.0 was unaffected by treatment with ribonuclease or deoxyribonuclease, but was abolished with trypsin, chymotrypsin, or protease (Streptomyces griesus). The binding of folic acid to the human binder was inhibited by folate > H₄-folate > methyl-H₄-folate dihydrofolate pteroic acid methotrexate aminopterin.     

Incorporation of 14CO2 into C4 acids by leaves of C3-C4 intermediate and C3 species of Moricandia and Panicum at the CO2 compensation concentration

Comparative ¹⁴CO₂ pulse-¹²CO₂ chase studies performed at CO₂ compensation ()-versus air-concentrations of CO₂ demonstrated a four-to eightfold increase in assimilation of ¹⁴CO₂ into the C₄ acids malate and aspartate by leaves of the C₃-C₄ intermediate species Panicum milioides Nees ex Trin., P. decipiens Nees ex Trin., Moricandia arvensis (L.) DC., and M. spinosa Pomel at . Specifically, the distribution of ¹⁴C in malate and aspartate following a 10-s pulse with ¹⁴CO₂ increases from 2% to 17% (P. milioides) and 4% to 16% (M. arvensis) when leaves are illuminated at the CO₂ compensation concentration (20 l CO₂/l, 21% O₂) versus air (340 l CO₂/l, 21% O₂). Chasing recently incorporated ¹⁴C for up to 5 min with ¹²CO₂ failed to show any substantial turnover of label in the C₄ acids or in carbon-4 of malate. The C₄-acid labeling patterns of leaves of the closely related C₃ species, P. laxum Sw. and M. moricandioides (Boiss.) Heywood, were found to be relatively unresponsive to changes in pCO₂ from air to . These data demonstrate that the C₃-C₄ intermediate species of Panicum and Moricandia possess an inherently greater capacity for CO₂ assimilation via phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) at the CO₂ compensation concentration than closely related C₃ species. However, even at , CO₂ fixation by PEP carboxylase is minor compared to that via ribulosebisphosphate carboxylase (EC 4.1.1.39) and the C₃ cycle, and it is, therefore, unlikely to contribute in a major way to the mechanism(s) facilitating reduced photorespiration in the C₃-C₄ intermediate species of Panicum and Moricandia.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - PEP phosphoenolpyruvate - CO₂ compensation concentration - 3PGA 3-phosphoglycerate - SuP sugar monophosphates - SuP₂ sugar bisphosphates Published as Paper No. 8249, Journal Series, Nebraska Agricultural Research Division     

The fixation probability of a beneficial allele in a population dividing by binary fission

We derive formulae for the fixation probability, P, of a rare benefical allele segregating in a population of fixed size which reproduces by binary fission, in terms of the selection coefficient for the beneficial allele, s. We find that an earlier result P 4s does not depend on the assumption of binary fission, but depends on an assumption about the ordering of events in the life cycle. We find that P 2s for mutations occurring during chromosome replication and P 2.8s for mutations occurring at random times between replication events.     

Redox modulation of reductase and phosphatase activities in human erythrocytes

Summary We have previously reported that ferricyanide reductase activity in human erythrocytes depended on glycolysis and could be modulated by several compounds including oxidants and hormones like insulin. Insulin could activate glycolysis, probably as a consequence of tyrosine phosphorylation of protein band 3, implicating phosphorylation reactions as an important signal for activation of the reductase by insulin. Reversible phosphorylation of cellular proteins is also believed to play a key role in the action of insulin. Cytosolic acid phosphatase activity has been found in human erythrocytes. To further extend initial reports, we studied the effect of modulators on the cytosolic erythrocyte acid phosphatase. Mild oxidants like ferricyanide (1 mM), vanadate (1 mM), Mn²⁺ (0.5 and 1 mM), and phenylarsine oxide (10 and 100 M) inhibited the phosphatase activity. Similarly, insulin at concentrations that stimulate ferricyanide reduction (500, 1000 IU/ml) inhibited the activity of the phosphatase enzyme. The overall results indicated that oxidants are able to inhibit the acid phosphatase and stimulate the redox enzyme. In addition, a significant negative correlation (r = –0.400; P = 0.006) was observed between phosphatase and reductase activities. The observations discussed here, together with previous ones, emphasize that a close association between reductase and phosphatase enzymes may exist and also suggest a role for redox reactions in tyrosine phosphorylation/dephosphorylation-mediated signal transduction pathways.     

Respiration and oxygen transport functions of the blood from an intertidal fish,Helcogramma medium (Tripterygiidae)

Synopsis Aquatic and aerial oxygen uptake (̇O₂), ventilation frequency, and oxygen transport properties of the blood were determined for the intertidal fish Helcogramma medium. Ventilation frequency increased in response to decreased environmental PO₂ and aquatic ̇O₂ was maintained down to a critical PO₂ of 30–40 mm Hg. Below PO₂ 30 mm Hg fish intermittently gulped air and finally emerged into air at PO₂ 18 mm Hg. After 1 h exposure to air ̇O₂ decreased to 60% of the aquatic rate and this was accompanied by an increase in blood lactate. Aerobic expansibility was reduced in air (×1.2) compared to water (× 5.5). The Hb concentration was 0.47 ± 0.13 mmol 1^–1 and hematocrit 11.55 ± 3.61% indicating a moderate O₂-carrying capacity. Oxygen affinity was not especially high (P₅₀ = 19 mm Hg at pH 7.7 and 15°C) and ATP was the predominant acid-soluble phosphate regulating P₅₀. The equilibrium curve was essentially hyperbolic (Hill's n = 1.2) with a marked Bohr effect = –1.06) and Root effect (saturation depressed by 50% at pH7.1). The pattern of respiration and the respiratory properties of the blood together with observations of the behaviour of the fish during aerial exposure indicated that Helcogramma is adapted to living in a well-aerated environment yet can adequately tolerate short term exposure to low aquatic PO₂ or air.     

Prejunctional α2-Adrenoceptors and Peroxide-Induced Potentiation of Norepinephrine Release from the Bovine Iris

Peroxides can enhance field-stimulated [³H]norepinephrine ([³H]NE) release in isolated irides from several mammalian species. In the present study, we investigated the role of prejunctional ₂-adrenoceptors in peroxide-induced potentiation of sympathetic neurotransmission in bovine isolated irides. Isolated hemi-irides were incubated in a Krebs buffered-solution containing [³H]NE and prepared for studies of neurotransmitter release using the superfusion method. ₂-Adrenoceptor agonists, oxymetazoline, UK-14304 and clonidine inhibited field-stimulated [³H]NE overflow without affecting basal tritium efflux. Pretreatment of tissues with H₂O₂ (300 M) had no effect on inhibition of evoked [³H]NE release caused by the ₂-adrenergic agonists. However, H₂O₂ (300 M) caused significant (P < 0.01) leftward shifts of excitatory concentration-response curves to yohimbine (10 nM–1 M). In contrast, yohimbine (1 M) did not prevent the enhancement of evoked [³H]NE overflow induced by H₂O₂ (300 M). In conclusion, excitatory effects of peroxides on sympathetic neurotransmission in bovine irides are not mediated by prejunctional ₂-adrenoceptors.     

Effects of phenoxybenzamine on insulin secretion from isolated rat islets of Langerhans

In isolated rat islets the ₂-adrenergic antagonist phenoxybenzamine was found to be only partially effective at relieving the inhibition of glucose-induced insulin secretion mediated by noradrenaline. Further experiment revealed a direct inhibitory effects of phenoxybenzamine itself on the secretory response to glucose. At concentrations above 1 M the antagonist inhibited insulin secretion in a dose-dependent manner, with greater than 50% inhibition at 50 M. The inhibition of secretion developed rapidly in perifused islets, and was not altered when islets were also incubated with idazoxan or benextramine, suggesting that it did not reflect binding of phenoxybenzamine to the ₂-receptor. Paradoxically phenoxybenzamine significantly increased the basal secretion rate in the presence of 4 mM glucose. The results demonstrate that phenoxybenzamine can exert direct effects on insulin secretion which are unrelated to its -antagonist properties.     

Stimulation of Immunoreactive Insulin Release by Glucose in Rat Brain Synaptosomes

The effect of glucose on the release of immunoreactive insulin (IRI) in synaptosomes isolated from rat brain was studied. In the absence of glucose synaptosomes release about 4% (0.77 IU/mg protein) of total content. Glucose increases significantly the IRI released by synaptosomes. Addition of the glycolytic inhibitor iodoacetic acid (IAA), decreased the glucose-induced release of IRI by about 50%, suggesting that glucose metabolism is involved. The observation that glucose provides a concentration related signal for IRI release indicates that this synaptosomal preparation may be useful as a model for research on the mechanism of insulin release in brain.     

Ethanol fermentation by flocculating yeast: Performance and stability dependence on a critical fermentation rate

Summary A system coupling fermentor and decantor permitted strong accumulation of yeast flocs that were homogeneously suspended in the reactional volume. At 100–190 g/l glucose feed practically total substrate conversion was attained. At 130 g/l glucose feed the highest productivity (18.4 g.l.h) and the highest ethanol yield (90.6%) were reached with biomass levels of 80–90 g/l. We observed that the stability of this system is limited when a critical fermentation rate (D.S_o) close to 39–40 g/l.h (with corresponding ethanol productivities of 19–20 g/l.h) is reached. Higher fermentation rates provoked de-flocculation and lost of biomass.Symbols D dilution rate (h^–1) - E ethanol (g/l) - S_r residual substrate (g/l) - S_o substrate in the feed (g/l) - X biomass (g/l) - ethanol yield (%) - DS_o fermentation rate (g/l.h) (for S_r0) - P_E ethanol productivity (g/l.h)     

Studies of insulin release and rat pancreatic islet metabolism with diastereomers of D-glucose

Studies of insulin release with diastereomers and other analogues of D-glucose demonstrated that only sugars which undergo oxidation to CO₂ stimulate insulin release by the pancreatic islet. None of the non-metabolizable diastereomers of glucose stimulated insulin release in the presence of a substimulatory concentration of glucose for fuel. Although 5.5 mM glucose formed 77% as much CO₂ as 16.7 mM mannose and twice that of 16.7 mM fructose, 5.5 mM glucose did not stimulate insulin release whereas 16.7 mM mannose and fructose did stimulate insulin release. These results indicate that the important stimulus for glucose-induced insulin release involves metabolism of glucose, but that the stimulus does not involve solely a fuel function of glucose.     

The malonyl gramicidin channel: NMR-derived rate constants and comparison of calculated and experimental single-channel currents

Summary Malonyl gramicidin is incorporated into lysolecithin micelles in a manner which satisfies a number of previously demonstrated criteria for the formation of the transmembrane channel structure. By means of sodium-23 nuclear magnetic resonance, two binding sites are observed: a tight site and a weak site with binding constants of approximately 100m ^–1 and 1m ^–1, respectively. In addition, off-rate constants from the two sites were estimated from NMR analyses to bek _off ^t 3×10⁵/sec andk _off ^w 2×10⁷/sec giving, with the binding constants, the on-rate constants,k _on ^t 3×10⁷/msec andk _on ^w 2×10⁷/m sec.Five different multiple occupancy models with NMR-restricted energy profiles were considered for the purpose of calculating single-channel currents as a function of voltage and concentration utilizing the four NMR-derived rate constants (and an NMR-limit placed on a fifth rate constant for intrachannel ion translocation) in combination with Eyring rate theory for the introduction of voltage dependence.Using the X-ray diffraction results of Koeppe et al. (1979) for limiting the positions of the tight sites, the two-site model and a three-site model in which the weak sites occur after the tight site is filled were found to satisfactorily calculate the experimental currents (also reported here) and to fit the experimental currents extraordinarily well when the experimentally derived values were allowed to vary to a least squares best fit. Surprisingly the best fit values differed by only about a factor of two from the NMR-derived values, a variation that is well within the estimated experimental error of the rate constants.These results demonstrate the utility of ion nuclear magnetic resonance to determine rate constants relevant to transport through the gramicidin channel and of the Eyring rate theory to introduce voltage dependence.     

Ion channels in the membrane ofChara inflata

Summary Voltage-clamped steps in the electric potential difference (PD) across the membrane in cells of the green alga,Chara inflata, cause voltage- and time-dependent current flows, interpreted to arise from opening and closing of various types of ion channel in the membrane. With cells in the light, these channels are normally closed, and the resting PD is probably determined by the operation of an H⁺ efflux pump. Positive steps in PD from the resting level often caused the opening of K⁺ channels with sigmoid kinetics. The channels began to show opening when the PD–120 mV for an external concentration of K⁺ of 1.0mm. Return of the PD to the resting level caused closing of the channels with complex kinetics. Various treatments of the cell could cause these K⁺ channels to open, and remain open continuously, with the PD then lying closer to the Nernst PD for K⁺. The K⁺ channels have been identified by the blocking effects of TEA⁺. Another group of channels, probably Cl^– and Ca²⁺ associated with the action potential open when the PD is stepped to values less negative than –50 mV. Negative steps from the resting PD cause the slow opening, with a time course of seconds, of yet another type of channel, probably Cl^–.     

Density and apparent location of the sodium pump in frog sartorius muscle

Summary The binding of the cardiosteroid³H-ouabain to frog skeletal muscle was determined by studying the kinetics of its uptake and release.The amount of ouabain bound as a function of drug concentration in the external medium follows a hyperbolic relationship with a maximum binding (B _max) of the order of 2500 molecules per square micrometer of surface membrane and an affinity constant (K) of 2.2×10^–7 m. The data do not suggest a drug-receptor (Na pump site) relation other than one-to-one.Ouabain molecules are released from whole muscle into ouabain-free media very slowly. The release is a single exponential function of time (25 hr). When re-binding is prevented by the presence of unlabeled ouabain in the external medium, the loss of labeled ouabain is increased (15 hr). Increasing [K⁺]₀ from 2.5 to 10mm slows the time course of binding without any significant change in binding capacity of the muscle fibers.Experiments on detubulated muscles indicate that the density of pump sites is considerably higher in the surface than in the T-tubular membrane. These findings agree with the report by Narahara et al. [Narahara, H.T., Vogrin, V.G., Green, J.D., Kent, R.A., Gould, M.K. (1979)Biochim. Biophys. Acta 552:247] on the distribution of (Na⁺+K⁺)-ATPase among different cell membrane fractions from frog skeletal muscle.     

Cadmium-copper antagonism in the activation of periplasmic nitrous oxide reductase of copper-deficient cells fromPseudomonas stutzeri

Summary Copper-deficient cells ofPseudomonas stutzeri strain ZoBell synthesize catalytically inactive nitrous oxide (N₂O) reductase which is activated by added Cu(II) in the absence of de novo protein synthesis. The apparentK _m for the activation process is 0.13 M. Activation is temperature-dependent and is inhibited by Cd(II)(K _i 1.27 M) and less strongly by Zn(II), Ni(II), and Co(II). The same metal ions at 20 M have little or no effect on N₂O reduction of intact cells. Apo-N₂O reductase of transposon Tn5-inducednos ^– mutants with defective Cu-chromophore biosynthesis is not reactivated by Cu(II). N₂O reductase of Cu-sufficient and Cu-deficient wild type, and ofnos ^– mutants is localized in the periplasm, the latter providing the likely site of metal incorporation into the apoenzyme.     

Carbon isotope composition of biochemical fractions isolated from leaves of Bryophyllum daigremontianum berger,a plant with crassulacean acid metabolism: Some physiological aspects related to CO2 dark fixation

Isotope analysis of the biochemical fractions isolated quantitatively from young and mature leaves of Bryophyllum daigremontianum Berger have been carried out before and after a dark period of accumulation of organic acids. The mature leaf is enriched in ¹³C compared to the young leaf. The ¹³C values of the different leaf constituents vary between the ¹³C values of C₄ plants (-11) and those of C₃ plants (-27). During the dark period, the two types of leaves store organic acids with ¹³C values of -15 and lose insoluble sugars, including starch with a ¹³C value of -12. Furthermore, young leaves store phosphorylated compounds with ¹³C values of -11 and lose weakly polymerised sugars with ¹³C values of -18. These results led to the formulation of a hypothesis of the origin of the two substrates of -carboxylation: phosphoenolpyruvate arises from the glycolytic breakdown of the insoluble sugars rich in ¹³C, and the major portion of the CO₂ is the result of the complete breakdown (respiration) of the soluble sugars rich in ¹²C. The existence of two independent sugar pools leads to the assumption that there are two separate glycolytic pathways. The ¹³C enrichment of the stored products of the young leaves in the day seems to be the result of a weak discrimination for ¹³C by ribulose diphosphate carboxylase, which reassimilates to a great extent the CO₂ released from malate accumulated in the night.Abbreviations CAM crassulacean acid metabolism - C₃ metabolism metabolism with primary carbon fixed by the Calvin and Benson pathway - C₄ metabolism metabolism with primary carbon fixed by the Hatch and Slack pathway - ¹³C() (R_sample-R_PDB) 10³/R_PDB where PDB=Pee Dee belemnite (belemite from the Pee Dee formation South Carolina) and R=¹³C/¹²C - NAD-MDH(EC1.1.1.37) NAD-malate dehydrogenase - NADP-ME (EC1.1.1.40) NADP-malic enzyme - PEP phosphoenolpyruvate - PEPC (EC4.1.1.31) PEP carboxylase - PGA phosphoglyceric acid - Py.di-PK(EC2.7.9.1) pyruvate, Pi-dikinase - RuDP ribulose diphosphate - RuDPC (EC4.1.1.39) RuDP carboxylase     

Unambiguous through-bond sugar-to-base correlations for purines in 13C, 15N-labeled nucleic acids: The HsCsNb,HsCs(N)bCb,and HbNbCb experiments

Summary A set of three 3D (¹H, ¹³C, ¹⁵N) triple-resonance correlation experiments has been designed to provide H1-H8 intraresidue sugar-to-base correlations in purines in an unambiguous and efficient manner. Together, the H_sC_sN_b, H_sC_s(N)_bC_b, and H_bN_bC_b experiments correlate the H1 sugar proton to the H8 proton of the attached base by means of the {H1, C1, N9, C8, H8} heteronuclear scalar coupling network. The assignment strategy presented here allows for unambiguous H1-H8 intraresidue correlations, provided that no two purines have both the same H1 and C1 chemical shifts and the same C8 and N9 chemical shifts. These experiments have yielded H1-H8 intraresidue sugar-to-base correlations for all five guanosines in the [¹³C, ¹⁵N] isotopically labeled RNA duplex r(GGCGCUUGCGUC)₂.