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Seven triterpene saponins were isolated from n-butanol fractions of blue cohosh (Caulophyllum thalictroides) roots and rhizomes. Their structures were established by spectral ((1)H NMR, (13)C NMR, 2D-NMR, and APCI-MS) techniques and chemical reactions as hederagenin 3-O-alpha-L-arabinopyranoside (1); caulophyllogenin 3-O-alpha-L-arabinopyranoside (2); hederagenin 3-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranoside (3); 3-O-alpha-L-arabinopyranosyl-hederagenin 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (4); 3-O-alpha-L-arabinopyranosyl- caulophyllogenin 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (5); 3-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranosyl- echinocystic acid 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (6); 3-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-hederagenin 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (7). All seven compounds were identified in this species for the first time.

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Several complementary physical techniques have been used to characterize the aggregate structures formed in solutions containing dimyristoylphosphatidylcholine (DMPC)/dihexanoylphosphatidylcholine (DHPC) at ratios of < or =0.5 and to establish their morphology and lipid organization as that of bicelles. (31)P NMR studies showed that the DMPC and DHPC components were highly segregated over a wide range of DMPC/DHPC ratios (q = 0.05-0.5) and temperatures (15 degrees C and 37 degrees C). Only at phospholipid concentrations below 130 mM did the bicelles appear to undergo a change in morphology. These results were corroborated by fluorescence data, which demonstrated the inverse dependence of bicelle size on phospholipid concentration as well as a distinctive change in phospholipid arrangement at low concentrations. In addition, dynamic light scattering and electron microscopy studies supported the hypothesis that the bicellar phospholipid aggregates are disk-shaped. The radius of the planar domain of the disk was found to be directly proportional to the ratio of DMPC/DHPC and inversely proportional to the total phospholipid concentration when the DMPC/DHPC ratio was held constant at 0.5. Taken together, these results suggest that bicelles with low q retain the morphology and bilayer organization typical of their liquid-crystalline counterparts, making them useful membrane mimetics.

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One new iridoid glycoside and five known flavonol glycosides have been isolated from the leaves of Morinda citrifolia. The new iridoid exists as an epimeric mixture in solution. Complete assignments of the proton and carbon chemical shifts for the individual epimers were accomplished on the basis of high-resolution 1D and 2D NMR data. Their antioxidative activities were measured. All of these compounds showed DPPH free radical scavenging activity at the concentration of 30 microM.

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A new iridoid glucoside (1), named citrifolinoside A, was isolated from the leaves of Morinda citrifolia along with the known iridoids asperuloside and asperulosidic acid. The structure of 1 was established by interpretation and full assignments of NMR spectroscopic data.

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The cuticle of higher plants functions primarily as a protective barrier for the leaves and fruits, controlling microbial attack as well as the diffusion of water and chemicals from the outside environment. Its major chemical constituents are waxes (for waterproofing) and cutin (a structural support polymer). However, the insolubility of cutin has hampered investigations of its covalent structure and domain architecture, which are viewed as essential for the design of crop protection strategies and the development of improved synthetic waterproofing materials. Recently developed strategies designed to meet these investigative challenges include partial depolymerization using enzymatic or chemical reagents and spectroscopic examination of the intact polyesters in a solvent-swelled form. The soluble oligomers from degradative treatments of lime fruit cutin are composed primarily of the expected 10,16-dihydroxyhexadecanoic and 16-hydroxy-10-oxo-hexadecanoic acids; low-temperature HF treatments also reveal sugar units that are covalently attached to the hydroxyfatty acids. Parallel investigations of solvent-swollen cutin using 2D NMR spectroscopy assisted by magic-angle spinning yield well-resolved spectra that permit detailed comparisons to be made among chemical moieties present in the intact biopolymer, the soluble degradation products, and the unreacted solid residue.

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Atomic force microscopy and solid-state nuclear magnetic resonance have been used to investigate the effect of water absorption on the nanoscale elastic properties of the biopolyester, cutin, isolated from tomato fruit cuticle. Changes in the humidity and temperature at which fruits are grown or stored can affect the plant surface (cuticle) and modify its susceptibility to pathogenic attack by altering the cuticle's rheological properties. In this work, atomic force microscopy measurements of the surface mechanical properties of isolated plant cutin have been made as a first step to probing the impact of water uptake from the environment on surface flexibility. A dramatic decrease in surface elastic modulus (from approximately 32 to approximately 6 MPa) accompanies increases in water content as small as 2 wt %. Complementary solid-state nuclear magnetic resonance measurements reveal enhanced local mobility of the acyl chain segments with increasing water content, even at molecular sites remote from the covalent cross-links that are likely to play a crucial role in cutin's elastic properties.

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Although suberin in potato wound periderm is known to be a polyester containing long-chain fatty acids and phenolics embedded within the cell wall, many aspects of its molecular structure and polymer-polymer connectivities remain elusive. The present work combines biosynthetic incorporation of site-specifically (13)C-enriched acetates and phenylalanines with one- and two-dimensional solid-state (13)C NMR spectroscopic methods to monitor the developing suberin polymer. Exogenous acetate is found to be incorporated preferentially at the carboxyl end of the aliphatic carbon chains, suggesting addition during the later elongation steps of fatty acid synthesis. Carboxyl-labeled phenylalanine precursors provide evidence for the concurrent development of phenolic esters and of monolignols typical of lignin. Experiments with ring-labeled phenylalanine precursors demonstrate a predominance of sinapyl and guaiacyl structures among suberin's phenolic moieties. Finally, the analysis of spin-exchange (solid-state NOESY) NMR experiments in ring-labeled suberin indicates distances of no more than 0.5 nm between pairs of phenolic and oxymethine carbons, which are attributed to the aromatic-aliphatic polyester and the cell wall polysaccharide matrix, respectively. These results offer direct and detailed molecular information regarding the insoluble intermediates of suberin biosynthesis, indicate probable covalent linkages between moieties of its polyester and polysaccharide domains, and yield a clearer overall picture of this agriculturally important protective material.

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Cutin and suberin are support polymers involved in waterproofing the leaves and fruits of higher plants, regulating the flow of nutrients among various plant organs, and minimizing the deleterious impact of microbial pathogens. Despite the complexity and intractable nature of these plant biopolyesters, their molecular structure and development are amenable to study by suitable solid-state and solution-state NMR techniques. Interactions of tomato cutin with water were examined by solid-state 2H and 13C NMR, showing that water films enhance rapid segmental motions of the acyl chains and are associated with a fivefold increase in surface elasticity upon cutin hydration. The suberization of wounded potato tissues was studied by solid-state 13C NMR, revealing the likely phenylpropanoid structures that permit dense cross-linking of the suberin structure and their proximity to the cell-wall polysaccharides. Finally, two new approaches were developed to elucidate the molecular structures of these biopolymers: partial depolymerization followed by spectroscopic analysis of the soluble oligomers; and swelling of the intact materials followed by magic-angle spinning (MAS) NMR analysis.

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A recent method to obtain the number of water molecules of hydration of multilamellar lipid vesicles using magic angle spinning nuclear magnetic resonance has been re-examined. The previous interpretation divided the water into bulk and interlamellar water and ignored water in defects (lakes) that are intrinsic to multilamellar lipid vesicles; the result was inconsistent with x-ray results for the lipid DOPC. The new interpretation takes advantage of the reduction of lake water with increased spinning and it uses osmotic pressure measurements to determine the loss of interlamellar water. The new result for DOPC from magic angle spinning is consistent with x-ray results.

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Singly and doubly labeled delta-aminolevulinic acid derivatives were used to prepare rat liver outer mitochondrial membrane (OM) cytochrome b5 containing a 13C-labeled heme active site. A variety of NMR experiments, including HMBC and INADEQUATE in conjunction with the more commonly used HMQC, NOESY, and COSY, were conducted to make unambiguous assignments of protonated carbons and the quaternary pyrrole-alpha and -beta carbons in both isomeric forms of the paramagnetic active center of OM cytochrome b5. Because the long interpulse delays in the HMBC experiment have a detrimental effect on the detectability of fast relaxing paramagnetically affected resonances. INADEQUATE is proposed as the experiment of choice for assigning quaternary carbons in paramagnetic hemes with carefully chosen macrocycle labeling patterns. Furthermore, the applicability of the INADEQUATE experiment to paramagnetic heme active sites should be facilitated greatly by the availability of biosynthetic methods for producing isotopically labeled b-hemes and, more recently, isotopically labeled c-hemes.

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The utility of rotational echo double resonance (REDOR) NMR spectroscopy for determining the conformations of linear peptides has been examined critically using a series of crystalline and amorphous samples. The focus of the present work was the evaluation of long-distance (> 5 A) interactions using 13C-15N dephasing. Detailed studies of specifically labeled melanostatin and synthetic analogs of the alpha-factor yeast mating hormone show that nitrogen-dephased, carbon-observe REDOR measurements are reliable for distances up to 6.0 A, and that dipolar interactions can be detected for distances up to 7 A. By contrast, nitrogen-observe REDOR gives reliable results only for distances shorter than 5.0 A. To measure distances accurately, REDOR data must be corrected for the effects of natural-abundance spins. These corrections are particularly important for measuring long distances, which are of the greatest value for determining peptide secondary structure. We have developed a spherical shell model for calculating the effect of these background spins. The REDOR studies also indicate that in a lyophilized powder, the tridecapeptide alpha-factor mating pheromone from Saccharomyces cerevisiae (WHWLQLKPGQPMY) probably exists as a distribution of different turn structures around the KPGQ region. This finding revises previous solid-state NMR studies on this peptide, which concluded alpha-factor assumes a distorted type-I beta-turn in the Pro-Gly central region of the molecule [J.R. Garbow, M. Breslav, O. Antohi, F. Naider, Biochemistry, 33 (1994) 10094].

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A sample preparation method using spherical glass ampoules has been used to achieve 1.5-Hz resolution in 1H magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of aqueous multilamellar dispersions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), serving to differentiate between slowly exchanging interlamellar and bulk water and to reveal new molecular-level information about hydration phenomena in these model biological membranes. The average numbers of interlamellar water molecules in multilamellar vesicles (MLVs) of DOPC and POPC were found to be 37.5 +/- 1 and 37.2 +/- 1, respectively, at a spinning speed of 3 kHz. Even at speeds as high as 9 kHz, the number of interlamellar waters remained as high as 31, arguing against dehydration effects for DOPC and POPC. Both homonuclear and heteronuclear nuclear Overhauser enhancement spectroscopy (NOESY and HOESY) were used to establish the location of water near the headgroup of a PC bilayer. 1H NMR comparisons of DOPC with a lipid that can hydrogen bond (monomethyldioleoylphosphatidylethanolamine, MeDOPE) showed the following trends: 1) the interlamellar water resonance was shifted to lower frequency for DOPC but to higher frequency for MeDOPE, 2) the chemical shift variation with temperature for interlamellar water was less than that of bulk water for MeDOPE MLVs, 3) water exchange between the two lipids was rapid on the NMR time scale if they were mixed in the same bilayer, 4) water exchange was slow if they were present in separate MLVs, and 5) exchange between bulk and interlamellar water was found by two-dimensional exchange experiments to be slow, and the exchange rate should be less than 157 Hz. These results illustrate the utility of ultra-high-resolution 1H MAS NMR for determining the nature and extent of lipid hydration as well as the arrangement of nuclei at the membrane/water interface.

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In paramagnetic heme proteins, it is often problematic to make proton resonance assignments for heme substituents that do not have large isotropic shifts and consequently lie under the large envelope of polypeptide resonances. Furthermore, assignments that would normally be performed with the aid of HMBC experiments in diamagnetic molecules can prove difficult in the active site of paramagnetic heme proteins if T2(-1) > 2JCH. To circumvent this problem, a new method is presented to selectively detect 1H in 1Hn-13C-13C fragments biosynthetically introduced into the active site of heme proteins. The pulse sequence combines well-known building blocks such as INEPT to transfer 1H spin magnetization to bonded 13C nuclei, followed by INADEQUATE to generate 13C-13C double-quantum coherence that is selected with pulsed field gradients, and finally reverse-INEPT to transfer magnetization back to 1H nuclei for subsequent observation. The new 1Hn-13C-13C edited experiment takes advantage of the relatively large values of 1JCH and 1JCC, avoiding the long interpulse delays in HMBC that compromise the detectability of rapidly relaxing nuclei. The potential applicability of the pulse sequence is demonstrated by its contribution to the unambiguous assignment of the carbonyl carbons in the heme propionates of ferricytochrome b5.

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Two hemispherectomized girls, one operated on the right, the other on the left, were followed from time of surgery until 9 and 10 years of age and compared with respect to course of language acquisition following surgery. At conclusion of follow-up, receptive and expressive language, phoneme perception and production, and sentence processing of the two hemispherectomized children were compared with those of two control groups of similar age, one developing language normally, the other language-impaired. The left-hemispherectomized child's abilities were similar to those of the language-impaired children; the right-hemispherectomized child's abilities resembled those of the language-normal children. Implications for localization of developmental anomalies in language-impaired children are discussed.

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It has been suggested that at physiological pH, the trypsin-catalyzed activation of the lipase cofactor, procolipase, to colipase has no consequence for intestinal lipolysis and serves primarily to release the N-terminal pentapeptide, enterostatin, a satiety factor (Larsson, A., and C. Erlanson-Albertsson 1991. The effect of pancreatic procolipase and colipase on pancreatic lipase activation. Biochim. Biophys. Acta 1083:283-288). This hypothesis was tested by measuring the adsorption of [14C]colipase to monolayers of 1-stearoyl-2-oleoyl-sn-3-glycerophosphocholine and 13, 16-cis, cis-docosadienoic acid in the presence and absence of procolipase. With saturating [14C]colipase in the subphase, the surface excess of [14C]colipase is 29% higher than that of procolipase, indicating that colipase packs more tightly in the interface. With [14C]colipase-procolipase mixtures, the proteins compete equally for occupancy of the argon-buffer interface. However, if a monolayer of either or both lipids is present, [14C]colipase dominates the adsorption process, even if bile salt is present in the subphase. If [14C]colipase and procolipase are premixed for > 12 h at pH approximately 8, this dominance is partial. If they are not premixed, procolipase is essentially excluded from the interface, even if procolipase is added before [14C]colipase. These results suggest that the tryptic cleavage of the N-terminal pentapeptide of procolipase may be of physiological consequence in the intestine.

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The role played by the outer mitochondrial membrane (OM) cytochrome b5 heme propionate groups in the electrostatic binding between OM cytochrome b5 and horse heart cytochrome c was investigated by 13C NMR spectroscopy and X-ray crystallography. To achieve these aims, 13C-labeled heme OM cytochrome b5 was expressed in Escherichia coli as previously described [Rivera M., Walker, F.A. (1995) Anal. Biochem. 230, 295-302]. Assignment of the resonances arising from the heme propionate carbons in ferricytochrome b5 was carried out by a combination of one- and two-dimensional NMR experiments. Titrations of [13C]heme-labeled OM cytochrome b5 with horse heart cytochrome c were carried out in order to monitor the resonances arising from the heme propionate carbonyl carbons in OM cytochrome b5. The results from these titrations clearly show that only the heme propionate located on the exposed heme edge in OM cytochrome b5 participates in the electrostatic stabilization of the complex between OM cytochrome b5 and horse heart cytochrome c. Similar experiments carried out monitoring 13C resonances arising from several other heme substituents demonstrated that the stoichiometry of the complex is 1:1. A conditional binding constant, K which equals 3.8 x 10(4) +/- 1.4 x 10(4) at mu = 0.02 M, was obtained for the formation of the complex by fitting the binding curves obtained experimentally to a model based on this stoichiometry. The X-ray crystal structure of rat liver OM cytochrome b5 solved to 2.7 A resolution shows that the structures of bovine liver microsomal cytochrome b5 and rat liver OM cytochrome b5 are almost identical when compared at medium resolution. The similarity between the two structures, combined with the findings that only the heme propionate located on the exposed heme edge of OM cytochrome b5 participates in the electrostatic binding to cytochrome c and that the stability of this complex is similar to that measured for the association between microsomal cytochrome b5 and cytochrome c, clearly indicates that the site of interaction on OM cytochrome b5 is almost identical to the one elucidated for microsomal cytochrome b5. It is therefore possible to conclude that the large body of information gathered by many investigators for the nonphysiological interaction between microsomal cytochrome b5 and cytochrome c (recently reviewed) [Mauk, A. G. Mauk, M. R., Moore, G. R., & Northrup, S. H. (1995) Bioenerg. Biomembr. 27, 311-330] has indeed biological as well as pedagogical validity.

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Biological semisolids such as aqueous phospholipid dispersions are amenable to experiments that combine magic-angle spinning (MAS) to yield high-resolution 1H NMR-spectra and two-dimensional nuclear Overhauser spectroscopy (NOESY) to estimate proton-proton distances. Using several selectively deuterated dimyristoylphosphatidylcholines, a systematic investigation has been made into the origin of an anomalous NOESY crosspeak between methyl groups of the lipid headgroup and the acyl chains. Although the spin-relaxation and line-narrowing behavior in these multilamellar systems argue against efficient spin diffusion, interdigitated or chain-bendback structural models that bring these 1H nuclei into close proximity are not implicated by the MAS-NOESY results for this phospholipid system. A mechanism has been proposed for spin communication between the two types of methyl protons within a conventional lipid bilayer structure: through-space interactions link the headgroup and backbone protons on adjacent molecules, and then spin diffusion occurs along the acyl chains.

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The performance of 32 children with language impairment-11 with expressive language impairment only (LI-E subgroup) and 21 with both receptive and expressive language impairment (LI-ER subgroup)-and of 22 children without language impairment (LN subgroup) was examined in a study of perception and imitation of synthesized /ba/ and /da/ syllables. Formant transition duration and task difficulty were varied in the perceptual tasks. The LI-E children were able to identify the syllables as well as the LN; the LI-ER were not. Of the children who succeeded on an identification task and proceeded to a serial ordering task incorporating the same stimuli, the LI-E children were the least successful on the second task. The ability to label the stimuli perceptually was highly correlated with absence of speech articulation errors in the LI children and with performance on the imitation task in all subjects. The findings are examined in relation to the hypotheses that rapid-rate perceptual processing is the sole basis of language impairment in children and that, in these children, production skill may predict phoneme perception rather than the reverse.

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Twenty-four children with language impairment (LI) and 22 children without language impairment (LN) participated in a study of discrimination, identification, and serial ordering of the highly dissimilar vowels/a/ versus /i/, and the highly similar vowels /epsilon/ versus /ae/. The vowel pairs were presented to the subjects in long- and short-duration sets. Both groups had greater difficulty in identifying /epsilon/ versus /ae/ than /a/ versus /i/. Neither group had greater difficulty with the short- than the long-duration vowel sets. The LI children were less efficient than the LN in identifying /a/ versus /i/, but could identify them accurately. They were significantly less accurate than the LN in identifying /epsilon/ versus /ae/. The majority of the children who could identify the /a/ and /i/ vowels were able to order them serially as well, although this second task appeared to be more difficult than identification. Fewer LI than LN children were able to proceed to the serial ordering task with /epsilon/ and /ae/. The children who could not identify the vowels within a set were almost always able to discriminate them. It was concluded that LI children have an auditory perceptual learning deficit and consequently a less robust central representation for steady state vowels than LN.

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