The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

Sodium ions are necessary for growth and energy transduction in the marine cyanobacterium Oscillatoria brevis

The maximal growth rate of the marine cyanobacterium Oscillatoria brevis was reached at 200–400 mM NaCl and pH 9.0–9.6. NaCl was found (i) to stimulate the rate of the light-supported generation across the cytoplasmic membrane of the cells and (ii) to decrease the sensitivity of level and motility of the O. brevis trichomes to protonophorous uncouplers. The Na⁺/H⁺ antiporter, monensin, increased both and the uncoupler sensitivity of the cells. The data obtained agree with the assumption that O. brevis possesses a primary Na⁺ pump in its cytoplasmic membrane.Abbreviations ATP adenosine-5-triphosphate - TTFB tetrachlortrifluoromethylimidazol - CCCP carbonyl cyanide m-chlorophenylhydrazone - Na⁺ transmembrane electrochemical potential differences of Na⁺ - transmembrane electric potential difference - pNa transmembrane pNa difference     

The membrane potential in whole cells of Methanobacterium thermoautotrophicum

of whole cells of Methanobacterium thermoautotrophicum was estimated under varying conditions using an electrode sensitive to the lipophilic cation tetraphenylphosphonium chloride (TPP⁺). Since was found to be extremely sensitive to air, a special reaction vessel was developed to maintain strict anaerobiosis. The cells took up TPP⁺ under energization by H₂ and CO₂ thus allowing to calculate the from the distribution of TPP⁺ inside and outside the cells. The unspecific uptake of deenergized cells was around 10% of the total uptake of energized cells. TPP⁺ itself slightly diminished the , but had no effect on the formation of methane. Typical values of were in the range of-150 to-200 mV. showed a quantitative dependence on both the electron donor H₂ and the electron acceptor CO₂. NaCl stimulated the extent of the , whereas KCl slightly diminished it. Valinomycin resulted in a linear decline of , whereas the methane production rate was only slightly affected. In contrast, monensin reduced both methanogenesis and .Abbreviations pmf proton motive force - membrane potential - TPP⁺ tetraphenylphosphonium (chloride salt) - TPMP⁺ triphenylmethylphosphonium (chloride salt, if not otherwise indicated) - d.w. dry weight - t _d doubling time - PVC polyvinylchloride     

An investigation of the role of solutes in the xylem sap and in the xylem parenchyma as the source of root pressure

Summary Solute osmotic potentials (_x) in the vessels of hydroponically grown maize roots were measured to assess the osmotic-xylem-sap mechanism for generating root pressure (indicated by guttation). Solutes in vessels were measured in situ by X-ray microanalysis of plants frozen intact while guttating. Osmotic potentials outside the roots (_o) were changed by adding polyethylene glycol to the nutrient solution. Guttation rate fell when _o was decreased, but recovered towards the control value during 3–5 days when _o was greater than or equal to –0.3 MPa, but not when _o was equal to –0.4 MPa. In roots stressed to _o = –0.3 MPa, _x, was always more positive than _o, and _x changed only slightly (ca. 0.05 MPa). Thus the adjustment in the roots which increased root pressure cannot be ascribed to _x, contradicting the osmotic-xylem-sap mechanism. An alternative driving force was sought in the osmotic potentials of the vacuoles of the living cells (_v), which were analysed by microanalysis and estimated by plasmolysis. _v showed larger responses to osmotic stress (0.1 MPa). Some plants were pretreated with abundant KNO₃ in the nutrient solution. These plants showed very large adjustments in _v (0.4 MPa) but little change in _x (0.08 MPa). They guttated by 4 h after _o was lowered to –0.4 MPa. It is argued that turgor pressure of the living cells is a likely alternative source of root pressure. Published evidence for high solute concentrations in the xylem sap is critically assessed.Abbreviations _o external water potential - _x osmotic potential of xylem sap - _v osmotic potential of vacuolar sap - EDX energy dispersive X-ray microanalysis - CSEM cryo-scanning electron microscope - LN₂ liquid nitrogen - PEG polyethylene glycol     

Influence of Phosphorus Application on Water Relations, Biochemical Parameters and Gum Content in Cluster Bean Under Water Deficit

Relative water content (RWC), leaf water potential (_w) and osmotic potential (_s), contents of chlorophyll (Chl) a, Chl b, soluble sugars, and seed quality (gum content) were used to evaluate the role of phosphorus in alleviation of the deleterious effect of water deficit in clusterbean (Cyamopsis tetragonoloba L. Taub). Under water stress, _w, _s, and Chl and gum contents decreased and soluble sugar contents increased. Phosphorus application increased Chl and sugar contents in control plants and ameliorated negative effects of water stress.     

Cyclosporin A-sensitive decrease in the transmembrane potential across the inner membrane of liver mitochondria induced by low concentrations of fatty acids and Ca2+

At low Ca²⁺ concentrations the pore of the inner mitochondrial membrane can open in substates with lower permeability (Hunter, D. R., and Haworth, R. A. (1979) Arch. Biochem. Biophys., 195, 468-477). Recently, we showed that Ca²⁺ loading of mitochondria augments the cyclosporin A-dependent decrease in transmembrane potential () across the inner mitochondrial membrane caused by 10 M myristic acid but does not affect the stimulation of respiration by this fatty acid. We have proposed that in our experiments the pore opened in a substate with lower permeability rather than in the classic state (Bodrova, M. E., et al. (2000) IUBMB Life, 50, 189-194). Here we show that under conditions lowering the probability of classic pore opening in Ca²⁺-loaded mitochondria myristic acid induces the cyclosporin A-sensitive decrease and mitochondrial swelling more effectively than uncoupler SF6847 does, though their protonophoric activities are equal. In the absence of P_i and presence of succinate and rotenone (with or without glutamate) cyclosporin A either reversed or only stopped decrease induced by 5 M myristic acid and 5 M Ca²⁺. In the last case nigericin, when added after cyclosporin A, reversed the decrease, and the following addition of EGTA produced only a weak (if any) increase. In P_i-containing medium (in the presence of glutamate and malate) cyclosporin A reversed the decrease. These data show that the cyclosporin A-sensitive decrease in by low concentrations of fatty acids and Ca²⁺ cannot be explained by specific uncoupling effect of fatty acid. We propose that: 1) low concentrations of Ca²⁺ and fatty acid induce the pore opening in a substate with a selective cation permeability, and the cyclosporin A-sensitive decrease results from a conversion of to pH gradient due to the electrogenic cation transport in mitochondria; 2) the ADP/ATP-antiporter is involved in this process; 3) higher efficiency of fatty acid compared to SF6847 in the Ca²⁺-dependent pore opening seems to be due to its interaction with the nucleotide-binding site of the ADP/ATP-antiporter and higher affinity of fatty acids to cations.     

Osmoregulation and the control of phloem-sap composition in Ricinus communis L.

Phloem-sap composition was studied in plants of Ricinus communis L. grown on a waterculture medium. The sap possessed a relatively high K⁺:Na⁺ ratio and low levels of Ca²⁺ and free H⁺. Sucrose and K⁺ (together with its associated anions) accounted for 75% of the phloem-sap solute potential (_s). In plants kept in continuous darkness, a decrease in phloem-sap sucrose levels over 24h was accompanied by an increase in K⁺ levels. Measurements of phloem-sap _s and xylem water potential () indicated that this resulted in a partial maintenance of phloem turgor pressure _p. In darkness there was also a marked decrease in the malate content of the leaf tissue, and it is possible that organic carbon from this source was mobilized for export in the phloem. The results support the concept of the phloem sap as a symplastic phase. We interpret the increase in K⁺ levels in the phloem in darkness as an osmoregulatory response to conditions of restricted solute availability. This reponse can be explained on the basis of the sucrose-H⁺ co-transport mechanism of phloem loading.Abbreviations water potential - _s solute potential - _p pressure potential     

Osmotic adjustment and the inhibition of leaf,root, stem and silk growth at low water potentials in maize

The expansion growth of plant organs is inhibited at low water potentials ( _w), but the inhibition has not been compared in different organs of the same plant. Therefore, we determined elongation rates of the roots, stems, leaves, and styles (silks) of maize (Zea mays L.) as soil water was depleted. The _w was measured in the region of cell expansion of each organ. The complicating effects of transpiration were avoided by making measurements at the end of the dark period when the air had been saturated with water vapor for 10 h and transpiration was less than 1% of the rate in the light. Growth was inhibited as the _w in the region of cell expansion decreased in each organ. The _w required to stop growth was-0.50,-0.75, and-1.00 MPa, in this order, in the stem, silks, and leaves. However, the roots grew at these _w and ceased only when _w was lower than-1.4 MPa. The osmotic potential decreased in each region of cell expansion and, in leaves, roots and stems, the decrease was sufficient to maintain turgor fully. In the silks, the decrease was less and turgor fell. In the mature tissue, the _w of the stem, leaves and roots was similar to that of the soil when adequate water was supplied. This indicated that an equilibrium existed between these tissues, the vascular system, and the soil. At the same time, the _w was lower in the expanding regions than in the mature tissues, indicating that there was a _w disequilibrium between the growing tissue and the vascular system. The disequilibrium was interpreted as a _w gradient for supplying water to the enlarging cells. When water was withheld, this gradient disappeared in the leaf because _w decreased more in the xylem than in the soil, indicating that a high flow resistance had developed in the xylem. In the roots, the gradient did not decrease because vascular _w changed about the same amount as the soil _w. Therefore, the gradient in _w favored water uptake by roots but not leaves at low _w. The data show that expansion growth responds to low _w differently in different growing regions of the plant. Because growth depends on the maintenance of turgor for extending the cell walls and the presence of _w gradients for supplying water to the expanding cells, several factors could have been responsible for these differences. The decrease of turgor in the silks and the loss of the _w gradient in the leaves probably contributed to the high sensitivity of these organs. In the leaves, the gradient loss was so complete that it would have prevented growth regardless of other changes. In the roots, the maintenance of turgor and _w gradients probably allowed growth to continue. This difference in turgor and gradient maintenance could contribute to the increase in root/shoot ratios generally observed in water-limited conditions.Symbols _s osmotic potential - _w water potential     

The effect of humidity and light on cellular water relations and diffusion conductance of leaves ofTradescantia virginiana L.

Turgor (_p) and osmotic potential (_s) in epidermal and mesophyll cells, in-situ xylem water potential (-xyl) and gas exchange were measured during changes of air humidity and light in leaves ofTradescantia virginiana L., Turgor of single cells was determined using the pressure probe. Sap of individual cells was collected with the probe for measuring the freezing-point depression in a nanoliter osmometer. Turgor pressure was by 0.2 to 0.4 MPa larger in mesophyll cells than in epidermal cells. A water-potential gradient, which was dependent on the rate of transpiration, was found between epidermis and mesophyll and between tip and base of the test leaf. Step changes of humidity or light resulted in changes of epidermal and mesophyll turgor (_p-epi, _p-mes) and could be correlated with the transpiration rate. Osmotic potential was not affected by a step change of humidity or light. For the humidity-step experiments, stomatal conductance (g) increased with increasing epidermal turgor.g/_p-epi appeared to be constant over a wide range of epidermal turgor pressures. In light-step experiments this type of response was not found and stomatal conductance could increase while epidermal turgor decreased.Symbols E transpiration - g leaf conductance - w leaf/air vapour concentration difference - -epi water potential of epidermal cells - -mes water potential of mesophyll cells - -xyl water potential of xylem - _p-epi turgor pressure of epidermal cells - _p-mes turgor pressure of mesophyll cells - _s-epi osmotic potential of epidermal cells - _s-mes osmotic potential of mesophyll cells     

Effects of moisture stress on the multiplication and expansion of cells in leaves of sugar beet

Summary Sugar beets were subjected to moisture stress by decreasing the water potential of the culture solution osmotically with polyethylene glycol by a known amount, , and, alternatively by applying matric potential, , at the plant roots. Lowering the water potential at the root surface less than 200 millibars by either method resulted in significant decreases in the rate of cell multiplication. The final number of cells per leaf at = -372 mb the final was 165% of that at = -473 mb ( = –101 mb); similarly at = –15 mb the final cell number was 198% of that at = –196 mb ( = –181 mb). The mean cell volume of leaves was not significantly affected by these levels of moisture stress.     

Sex identification by male-specific growth hormone pseudogene (GH-Ψ) in Oncorhynchus masou complex and a related hybrid

It is often difficult to identify sexes of many fish species by conventional cytological method because of the lack of heteromorphic sex chromosomes. Isolation of sex-specific molecular markers is thus important for sexing and for understanding sex chromosome evolution in these species. We have identified genetic sexes by PCR-based male-specificity of a growth hormone pseudogene (GH-) in masu and Biwa salmon, two subspecies of the Oncorhynchus masou complex, and their hybrid Honmasu. PCRs with primers designed from sequences of chinook salmon GH genes amplified GH-I and GH-II fragments in both sexes, but a third GH- fragment was detected in predominant proportion of males and very few phenotypic females. The consistency of phenotypic sex with genetic sex identified by GH- for masu salmon, Biwa salmon and Honmasu was 93.1, 96.7 and 94%, respectively. The remaining individuals showed inconsistency or deviation from sex-specificity: a few phenotypic males lacked the GH-, whereas a few phenotypic females possessed the GH-. Sequence of the putative GH- fragment from such females was identical to that from genetic males, and shared about 95% homology with the corresponding GH- fragment from chinook salmon. This result confirmed that these females were really GH--bearing individuals. PCR analyses with primers designed from masu salmon GH- gave identical results, indicating that the absence of GH- in a few males was not resulted from primer mismatching. These GH--bearing females and GH--absent males were more likely to originate from spontaneous sex reversion than from crossing-over between GH- and the sex determination gene/region.     

Leaf conductances and xylem pressures of the host/mistletoe pair Eucalyptus behriana F. Muell. and Amyema miquelii (Lehm. ex Miq.) Tiegh. at permanently low plant water status in the field

Summary Over several days at permanently low plant water status in the field, where predawn xylem pressures () were never higher (less negative) than –1.2 MPa even after extended rain, leaf conductances (g) and transpiration rates of host trees, Eucalyptus behriana F. Muell., were higher than in mistletoes, Amyema miquelii (Lehm. ex Miq.) Tiegh., which contrasts with most studies known from the literature. Mistletoes influenced but not g of host leaves distal to the haustorium. Releasing xylem tension by cutting a host stem under water raised from about –3.5 MPa to about –0.5 MPa in both plants indicating that factors in the root zone were responsible for the low in the host. In all cases, with a freely transpiring or non-transpiring parasite at low and at artificially raised , mistletoe xylem pressure was lower than that of the host. Possible reasons are discussed.     

The effect of potassium application on leaf water relations characteristics of field grown barley plants (Hordeum distichum L.)

Leaf water relations characteristics were studied in spring barley fertilized at low (50 kg ha^-1) or high (200 kg ha^-1) levels of potassium applied as KCl. The leaf water relations characteristics were determined by the pressure volume (PV) technique.Seasonal analysis in fully irrigated plants showed that within 2 weeks from leaf emergence the leaf osmotic potential at full turgor ( ¹⁰⁰) decreased from about –0.9 to –1.6 MPa in leaf No 7 (counting the first leaf to emerge as number one) and from about –1.1 to –1.9 MPa in leaf No 8 (the flag leaf) due to solute accumulation. ¹⁰⁰ was 0.05 to 0.10 MPa lower in high K than in low K plants. Thus, an ontogenetically determined accumulation of solutes occurred in the leaves independent of K application. The ratio of leaf weight at full turgor to dry weight (TW/DW) decreased from about 5.5 in leaf No 6 to 4.5 in leaf No 7 and 3.8 in leaf No. 8. The TW/DW ratio was 4 to 10% higher in high K than in low K plants indicating larger leaf cell size in the former. The tissue modulus of elasticity () was increased in high K plants. The main effect of high K application on water relations was an increase in leaf water content and a slight decrease in leaf During drought limited osmotic adjustment and increase in elasticity of the leaf tissue mediated turgor maintenance. These effects were only slightly modified by high potassium application.     

<Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> strains isolated from moisture-damaged buildings produced surfactin and a substance toxic to mammalian cells

Fungicidic Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings contained heat-stable, methanol-soluble substances that inhibited motility of boar spermatozoa within 15 min of exposure and killed feline lung cells in high dilution in 1 day. Boar sperm cells lost motility, cellular ATP, and NADH upon contact to the bacterial extract (0.2 g dry wt/ml). Two bioactive substances were purified from biomass of the fungicidal isolates. One partially characterized substance, 1,197 Da, was moderately hydrophobic and contained leucine, proline, serine, aspartic acid, glutamic acid and tyrosine, in addition to chromophore(s) absorbing at 365 nm. In boar sperm and human neural cells (Paju), the compound depolarized the transmembrane potentials of mitochondria (_m) and the plasma membrane (_p) after a 20-min exposure and formed cation-selective channels in lipid membranes, with a selectivity K⁺:Na⁺:Ca²⁺ of 26:15:3.5. The other substance was identified as a plasma-membrane-damaging lipopeptide surfactin. Plate-grown biomass of indoor Bacillus amyloliquefaciens contained ca. 7% of dry weight of the two substances, 1,197 Da and surfactin, in a ratio of 1:6 (w:w). The in vitro observed simultaneous collapse of both cytosolic and mitochondrial ATP in the affected mammalian cell, induced by the 1,197-Da cation channel, suggests potential health risks for occupants of buildings contaminated with such toxins.Abbreviations RP-HPLC Reversed-phase high-performance liquid chromatography - BLM Black lipid membrane - DAD Diode-array detector - _m Mitochondrial membrane potential - _p Plasma membrane potential - JC-1 5,5,6,6-Tetrachloro-1,1,3,3-tetraethylbenz-imidazolo carbocyanine iodide - AM Calcein acetoxymethyl ester - PI Propidium iodide - MALDI-TOF-MS Matrix-assisted laser desorption ionization time-of-flight mass spectrometry - ESI-IT-MS Electrospray ionization ion trap mass spectrometry - EC₅₀ Endpoint concentration which caused 50% change in the viability parameters - FCCP Carbonyl cyanide 4-trifluoromethoxyphenylhydrazone     

Effects of Osmotic Drought Stress Induced by a Combination of NaCl and Polyethylene Glycol on Leaf Water Status,Photosynthetic Gas Exchange,and Water Use Efficiency of Pistacia khinjuk and P. mutica

Leaf water potential, leaf osmotic potential, chlorophyll a and b contents, stomatal conductance, net photosynthetic rate, and water use efficiency were determined in two pistachio species (Pistacia khinjuk L. and P. mutica L.) grown under osmotic drought stress induced by a combination of NaCl and polyethylene glycol 6000. A decrease in values for all mentioned variables was observed as the osmotic potential of the nutrient solution (_s) decreased. The osmotic adjustment () of the species increased by decreasing _s. Thus P. khinjuk had a higher osmotic drought stress tolerance than P. mutica.     

ATP is required for K+ active transport in the archaebacterium Haloferax volcanii

The Archaebacterium Haloferax volcanii concentrates K⁺ up to 3.6 M. This creates a very large K⁺ ion gradient of between 500- to 1,000-fold across the cell membrane. H. volcanii cells can be partially depleted of their internal K⁺ but the residual K⁺ concentration cannot be lowered below 1.5 M. In these conditions, the cells retain the ability to take up potassium from the medium and to restore a high internal K⁺ concentration (3 to 3.2 M) via an energy dependent, active transport mechanism with a K _m of between 1 to 2 mM. The driving force for K⁺ transport has been explored. Internal K⁺ concentration is not in equilibrium with m suggesting that K⁺ transport cannot be accounted for by a passive uniport process. A requirement for ATP has been found. Indeed, the depletion of the ATP pool by arsenate or the inhibition of ATP synthesis by N,N-dicyclohexylcarbodiimide inhibits by 100% K⁺ transport even though membrane potential m is maintained under these conditions. By contrast, the necessity of a m for K⁺ accumulation has not yet been clearly demonstrated. K⁺ transport in H. volcanii can be compared with K⁺ transport via the Trk system in Escherichia coli.Abbreviations CCCP Carbonylcyanide m-chlorophenyl-hydrazone - DCCD N,N-dicyclohexylcarbodiimide - MES 2-[N-morpholino] ethane sulfonic acid - MOPS 3-[N-morpholino] propane sulfonic acid - TRIS Tris (hydroxymethyl) aminomethane - TPP tetraphenyl phosphonium     

Phloem turgor and the regulation of sucrose loading in Ricinus communis L.

The influence of plant water relations on phloem loading was studied in Ricinus communis L. Phloem transport was maintained in response to bark incisions even at severe water deficits. Water stress was associated with a net increase in the solute content of the sieve tubes, which resulted in maintenance of a positive phloem turgor pressure _p. There was a significant increase in solute flux through the phloem with decreasing xylem water potential (). In addition, sugar uptake by leaf discs was examined in media adjusted to different water potentials with either sorbitol (a relatively impermeant solute) or ethylene glycol (a relatively permeant solute). The limitations in this experimental system are discussed. The results nevertheless indicated that sucrose uptake can be stimulated by a reduction in cell _p, but that it is little affected by cell or solute potential _s. On the basis of these data we suggest that sucrose loading is turgor-pressure dependent. This may provide the mechanism by which transport responds to changes in sink demand in the whole plant.Abbreviations water potential - _s solute potential - _p pressure potential     

<Superscript>13</Superscript>C-<Superscript>13</Superscript>C NOESY: A constructive use of <Superscript>13</Superscript>C-<Superscript>13</Superscript>C spin-diffusion

¹³C-¹³C NOESY experiments were performed under long mixing time conditions on reduced human superoxide dismutase (32 kDa, ¹⁵N, ^13C and 70% ²H labeled). ¹³C-¹³C couplings were successfully eliminated through post-processing of in-phase-anti-phase (IPAP) data. It appears that at mixing time _m of 3.0 s the spin diffusion mechanism allows the detection of 96% of the two-bond correlations involving C and C. The interpretation was confirmed by simulations. This approach broadens the range of applicability of ¹³C-¹³C NOESY spectroscopy.     

Significance of rooting depth in mire plants: Evidence from natural <Superscript>15</Superscript>N abundance

Variation in stable nitrogen isotope ratios (¹⁵N) was assessed for plants comprising two wetland communities, a bog-fen system and a flood plain, in central Japan. ¹⁵N of 12 species from the bog-fen system and six species from the flood plain were remarkably variable, ranging from –5.9 to +1.1 and from +3.1 to +8.7, respectively. Phragmites australis exhibited the highest ¹⁵N value at both sites. Rooting depth also differed greatly with plant species, ranging from 5cm to over 200cm in the bog-fen system. There was a tendency for plants having deeper root systems to exhibit higher ¹⁵N values; plant ¹⁵N was positively associated with rooting depth. Moreover, an increasing gradient of peat ¹⁵N was found along with depth. This evidence, together with the fact that inorganic nitrogen was depleted under a deep-rooted Phragmites australis stand, strongly suggests that deep-rooted plants actually absorb nitrogen from the deep peat layer. Thus, we successfully demonstrated the diverse traits of nitrogen nutrition among mire plants using stable isotope analysis. The ecological significance of deep rooting in mire plants is that it enables those plants to monopolize nutrients in deep substratum layers. This advantage should compensate for any consequential structural and/or physiological costs. Good evidence of the benefits of deep rooting is provided by the fact that Phragmites australis dominates as a tall mire grass.     

The solution conformation of sialyl-α(2→6)-lactose studied by modern NMR techniques and Monte Carlo simulations

Summary We present a comprehensive strategy for detailed characterization of the solution conformations of oligosaccharides by NMR spectroscopy and force-field calculations. Our experimental strategy generates a number of interglycosidic spatial constraints that is sufficiently large to allow us to determine glycosidic linkage conformations with a precision heretofore unachievable. In addition to the commonly used {¹H,¹H} NOE contacts between aliphatic protons, our constraints are: (a) homonuclear NOEs of hydroxyl protons in H₂O to other protons in the oligosaccharide, (b) heteronuclear {¹H,¹³C} NOEs, (c) isotope effects of O¹H/O²H hydroxyl groups on¹³C chemical shifts, and (d) long-range heteronuclear scalar coupling across glycosidic bonds.We have used this approach to study the trisaccharide sialyl-(26)-lactose in aqueous solution. The experimentally determined geometrical constraints were compared to results obtained from force-field calculations based on Metropolis Monte Carlo simulations. The molecule was found to exist in 2 families of conformers. The preferred conformations of the (26)-linkage of the trisaccharide are best described by an equilibrium of 2 conformers with angles at –60° or 180° and of the 3 staggered rotamers of the angle with a predominantgt conformer. Three intramolecular hydrogen bonds, involving the hydroxyl protons on C8 and C7 of the sialic acid residue and on C3 of the reducing-end glucose residue, contribute significantly to the conformational stability of the trisaccharide in aqueous solution. Supplementary material available from the corresponding author: Table containing values for the dihedral angles , , , , and for bond angles , for the six lowest-energy conformations of sialyl-(26)-lactose (1 page).