Anaerobic bioconversion of cellulose by Ruminococcus albus, Methanobrevibacter smithii, and Methanosarcina barkeri

A system is described that combines the fermentation of cellulose to acetate, CH₄, and CO₂ by Ruminococcus albus and Methanobrevibacter smithii with the fermentation of acetate to CH₄ and CO₂ by Methanosarcina barkeri to convert cellulose to CH₄ and CO₂. A cellulose-containing medium was pumped into a co-culture of the cellulolytic R. albus and the H₂-using methanogen, Mb. smithii. The effluent was fed into a holding reservoir, adjusted to pH 4.5, and then pumped into a culture of Ms. barkeri maintained at constant volume by pumping out culture contents. Fermentation of 1% cellulose to CH₄ and CO₂ was accomplished during 132 days of operation with retention times (RTs) of the Ms. barkeri culture of 7.5–3.8 days. Rates of acetate utilization were 9.5–17.3 mmol l⁻¹ day⁻¹ and increased with decreasing RT. The K _s for acetate utilization was 6–8 mM. The two-stage system can be used as a model system for studying biological and physical parameters that influence the bioconversion process. Our results suggest that manipulating the different phases of cellulose fermentation separately can effectively balance the pH and ionic requirements of the acid-producing phase with the acid-using phase of the overall fermentation. Received: 7 December 1999 / Received revision: 28 April 2000 / Accepted: 19 May 2000     

Two-step concerted mechanism for alkane hydroxylation on the ferryl active site of methane monooxygenase

 A two-step concerted mechanism for the conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO) is discussed. We propose that the enzymatic reaction mechanism is essentially the same as that of the gas-phase methane-methanol conversion by the bare FeO⁺ complex. In the initial stage of our mechanism, the ferryl (Fe—O) "iron" active site of intermediate Q and substrate methane come into contact to form the initial Q (CH₄) complex with an OFe—CH₄ bond. The C—H bonds of methane are significantly weakened by the formation of a five-coordinate carbon species, through orbital interactions between a C _3v - or D _2d -distorted methane and the Fe—O active site. The important transition state for an H atom abstraction exhibits a four-centered structure. The generated intermediate involves an HO—Fe—CH₃ moiety, and it is then converted into the final product complex including methanol as a ligand through a methyl migration that occurs via a three-centered transition state. The two-step concerted mechanism is consistent with recent experiments on regioselectivity of enzyme-catalyzed alkane hydroxylations. Received: 15 September 1997 / Accepted: 20 December 1997     

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

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

Polymerization of guaiacol by lignin-degrading manganese peroxidase from Bjerkandera adusta in aqueous organic solvents

 Lignin-degrading manganese (II) peroxidase (MnP) purified from the culture of a wood-rotting basidiomycete, Bjerkandera adusta, was used in the polymerization of guaiacol. MnP was found to catalyze polymerization of guaiacol in 50% aqueous acetone, dimethyl formamide, methanol, ethanol, dioxane, acetonitrile, ethylene glycol and methylcellosolve. Maximum yield of polyguaiacol was achieved in 50% aqueous acetone. The weight average molecular weight (M _w) of the polymer was estimated to be 30 300 by gel permeation chromatography. However, matrix-assisted laser desorption ionization time of flight mass spectroscopy (MALDI-TOF-MS) analysis gave a more reliable M _w of 1690. IR, ¹³C-NMR, MALDI-TOF-MS and pyrolysis GC-MS analyses showed the presence of C–C and C–O linkages and quinone structure in polyguaiacol. It was also indicated that polyguaiacol has a methoxy-phenyl group as the terminal moiety. This suggests that polyguaiacol is a branched polymer in which guaiacol units are cross-linked at the phenolic group. Thermal gravimetric and differential scanning calorimetric analyses were also carried out. MnP also catalyzed the polymerization of o-cresol, 2,6-dimethoxyphenol and other phenolic compounds and aromatic amines. M _w of these polymers ranged from around 1000 to 1500. Received: 2 August 1999 / Received revision: 10 December 1999 / Accepted: 4 January 2000     

Phase behaviour and crystallinity of plant cuticular waxes studied by Fourier transform infrared spectroscopy

The phase behaviour of cuticular waxes from leaves of Hedera helix L. and Juglans regia L. was studied by Fourier transform infrared spectroscopy. For this purpose reconstituted waxes, isolated cuticular membranes, dewaxed polymer matrix membranes and whole leaves were studied in the horizontal attenuated total reflection and transmission modes. Melting curves of cuticular waxes were derived from temperature-dependent changes in the absorption maximum of the symmetric stretching mode of CH₂ groups (ν_s, at approx. 2856–2848 cm⁻¹). With increasing temperature absorption band doublets due to CH₂ scissoring (δ_sciss) and rocking (δ_rock) movements (at approx. 1473–1471 and 730–720 cm⁻¹, respectively) indicative of an orthorhombic arrangement of alkyl chains merged into a single peak. The area ratio of the peaks at approx. 720 and 730 cm⁻¹ was used as a measure for aliphatic crystallinity of plant cuticular waxes at a given temperature. The investigations of reconstituted cuticular waxes and those still embedded in isolated cuticles or in situ on the leaf produced comparable results. The findings are discussed in terms of the properties of the cuticular transport barrier. Received: 21 March 1997 / Accepted: 25 April 1997     

Fungal biosolubilization of Rhenish brown coal monitored by Curie-point pyrolysis/gas chromatography/mass spectrometry using tetraethylammonium hydroxide

Residues and coal fractions that remained after the biosolubilization of Rhenish brown coal by strains of Lentinula edodes and Trametes versicolor have been studied by Curie-point pyrolysis/gas chromatography/mass spectrometry using tetraethylammonium hydroxide (NEt₄OH) at 610 °C. To differentiate methyl derivatives of esters and ethers from free or bound hydroxyl and carboxyl groups NEt₄OH was used in the thermochemolysis experiments instead the commonly used tetramethylammonium hydroxide. A comparison of humic acid fractions before and after fungal attack shows considerable alteration of the soluble macromolecules of coal. Depending on the coal fraction studied and the fungi used, the assortment of fatty acid esters released during the pyrolysis varies significantly. Furthermore, dicarbonic acid ethyl diesters as well as ethyl derivatives of aromatic ethers and acids yield information about humic acid structure and the biosolubilization of brown coal. Variations in the mixture produced are possibly caused by differences in the pattern of extracellular enzymes secreted that attack the macromolecular structural elements of brown coal. Therefore pyrolysis of native and microbiologically altered geomacromolecules using NEt₄OH allows one to differentiate between free hydroxyl groups as well as substances that are attached to humic substances via ester or ether bridges, and their methylated counterparts. Received: 13 July 1998 / Received revision: 12 October 1998 / Accepted: 16 October 1998     

The geochemistry of methane in Lake Fryxell,an amictic,permanently ice-covered,antarctic lake

The abundance and distribution of dissolved CH₄ were determined from 1987–1990 in Lake Fryxell, Antarctica, an amictic, permanently ice-covered lake in which solute movement is controlled by diffusion. CH₄ concentrations were < 1 υM in the upper oxic waters, but increased below the oxycline to 936 μM at 18 m. Sediment CH₄ was 1100 μmol (1 sed)⁻¹ in the 0–5 cm zone. Upward flux from the sediment was the source of the CH₄, NH₄ ⁺, and DOC in the water column; CH₄ was 27% of the DOC+CH₄ carbon at 18 m. Incubations with surficial sediments indicated that H¹⁴CO₃ ⁻ reduction was 0.4 μmol (1 sed)⁻¹ day⁻¹ or 4× the rate of acetate fermentation to CH₄. There was no measurable CH₄ production in the water column. However, depth profiles of CH₄, NH₄, and DIC normalized to bottom water concentrations demonstrated that a significant CH₄ sink was evident in the anoxic, sulfate-containing zone of the water column (10–18 m). The δ¹³CH₄ in this zone decreased from −72 % at 18 m to −76% at 12 m, indicating that the consumption mechanism did not result in an isotopic enrichment of ¹³CH₄. In contrast, δ¹³CH₄ increased to −55 % at 9 m due to aerobic oxidation, though this was a minor aspect of the CH₄ cycle. The water column CH₄ profile was modeled by coupling diffusive flux with a first order consumption term; the best-fit rate constant for anaerobic CH₄ consumption was 0.012 yr⁻¹. On a total carbon basis, CH₄ consumption in the anoxic water column exerted a major effect on the flux of carbonaceous material from the underlying sediments and serves to exemplify the importance of CH₄ to carbon cycling in Lake Fryxell.     

Identification of HN-methyl NOEs in large proteins using simultaneous amide-methyl TROSY-based detection

A pair of HN-methyl NOESY experiments that are based on simultaneous TROSY-type detection of amide and methyl groups is described. The preservation of cross-peak symmetry in the simultaneous ¹H–¹⁵N/¹³CH₃ NOE spectra enables straightforward assignments of HN-methyl NOE cross-peaks in large and complex protein structures. The pulse schemes are designed to preserve the slowly decaying components of both ¹H–¹⁵N and methyl ¹³CH₃ spin-systems in the course of indirect evolution (t ₂) and acquisition period (t ₃) of 3D NOESY experiments. The methodology has been tested on {U-[¹⁵N,²H]; Ileδ1-[¹³CH₃]; Leu,Val-[¹³CH₃,¹²CD₃]}-labeled 82-kDa enzyme Malate Synthase G (MSG). A straightforward procedure that utilizes the symmetry of NOE cross-peaks in the time-shared 3D NOE data sets allows unambiguous assignments of more than 300 HN-methyl interactions in MSG from a single 3D data set providing important structural restraints for derivation of the backbone global fold.     

Stability of metal ion complexes formed with methyl phosphate and hydrogen phosphate

 The acidity constants of methyl phosphoric acid, CH₃OPO(OH)₂, and orthophosphoric acid, HOPO(OH)₂, and the stability constants of the 1 : 1 complexes formed between Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, or Cd²⁺ and methyl phosphate, CH₃OPO₃ ^2–, or hydrogen phosphate, HOPO₃ ^2–, were determined by potentiometric pH titration in aqueous solution (25  °C;I = 0.1 M, NaNO₃). On the basis of previously established log K versus pK _a straight-line plots for the complexes of simple phosphate monoesters and phosphonate derivatives, R-PO₃ ^2–, where R is a noncoordinating residue, it is shown that the stability of the M(CH₃OPO₃) complexes is solely determined (as one might expect) by the basicity of the –PO₃ ^2– residue. It is emphasized that the mentioned reference lines may also be used to reveal increased complex stabilities, for example, for certain complexes formed with 8-quinolyl phosphate the occurrence of 7-membered chelates can be proven in this way; the same procedure is also applicable to complexes of nucleotides, etc. The M(HOPO₃) complexes are slightly more stable (on average by 0.08 log unit) than it is expected from the basicity of HPO₄ ^2–; this observation is attributed to a more effective solvation, including hydrogen bonding, than is possible with CH₃OPO₃ ^2– species. Received: 9 November 1995 / Accepted: 5 February 1996     

Methane Budget of a Black Forest Spruce Ecosystem Considering Soil Pattern

Study included seven soils, an adjacent spring and brook and was conducted to estimate CH₄ source and sink strengths of forest soils along a wetness gradient, i.e. their exchange with atmosphere (direct emission), and hydrosphere (indirect emission). Soils are represented by anaerobic Histosol, oxic Cambisols, Histosol with degraded peatlayers and Gleysols having intermediate redox state. They could be separated into three emission groups: CH₄ emitting (248–318 kg C ha⁻¹ a⁻¹), CH₄ uptake (−0.1 to −5 kg C ha⁻¹ a⁻¹), and soils on the edge of CH₄ uptake and release (−0.2–20 kg C ha⁻¹ a⁻¹). Although soils with CH₄ uptake were dominant (75%), the soil specific CH₄ budget identified the study field (6.53 ha) as CH₄ source (40.9 kg C ha⁻¹ a⁻¹). Not only CH₄ emissions, but also dissolved CH₄ in soil solution varied regularly with soil type. Individual soil solutions contained 0.008–151 μmol CH₄ l⁻¹. CH₄ vanished to negligible loads, when dissolved CH₄ passed an oxidative downslope soil zone, but promoted CH₄ uptake was measured at this soil. In turn, CH₄ was discharged to the atmosphere, when the soil solution left the pedosphere across an anaerobic soil zone. These measured indirect emissions were low (34 g C a⁻¹), but the values of individual soil solution indicate possible higher discharges (3.9 kg a⁻¹) at a different soil pattern. The results suggest that CH₄ uptake rates of temperate forests are overestimated.     

Interspecific variability of δ13C among trees in rainforests of French Guiana: functional groups and canopy integration

The interspecific variability of sunlit leaf carbon isotope composition (δ¹³C), an indicator of leaf intrinsic water-use efficiency (WUE, CO₂ assimilation rate/leaf conductance for water vapour), was investigated in canopy trees of three lowland rainforest stands in French Guiana, differing in floristic composition and in soil drainage characteristics, but subjected to similar climatic conditions. We sampled leaves with a rifle from 406 trees in total, representing 102 species. Eighteen species were common to the three stands. Mean species δ¹³C varied over a 6.0‰ range within each stand, corresponding to WUE varying over about a threefold range. Species occurring in at least two stands displayed remarkably stable δ¹³C values, suggesting a close genetic control of species δ¹³C. Marked differences in species δ¹³C values were found with respect to: (1) the leaf phenology pattern (average δ¹³C=–29.7‰ and –31.0‰ in deciduous-leaved and evergreen-leaved species, respectively), and (2) different types of shade tolerance defined by features reflecting the plasticity of growth dynamics with respect to contrasting light conditions. Heliophilic species exhibited more negative δ¹³C values (average δ¹³C=–30.5‰) (i.e. lower WUE) than hemitolerant species (–29.3‰). However, tolerant species (–31.4‰) displayed even more negative δ¹³C values than heliophilic ones. We could not provide a straightforward ecophysiological interpretation of this result. The negative relationship found between species δ¹³C and midday leaf water potential (Ψ_wm) suggests that low δ¹³C is associated with high whole tree leaf specific hydraulic conductance. Canopy carbon isotope discrimination (Δ _A ) calculated from the basal area-weighed integral of the species δ¹³C values was similar in the three stands (average Δ _A =23.1‰), despite differences in stand species composition and soil drainage type, reflecting the similar proportions of the three different shade-tolerance types among stands. Received: 30 November 1999 / Accepted: 23 March 2000     

Evidence for Negative Charge in the Conduction Pathway of the Cardiac Ryanodine Receptor Channel Provided by the Interaction of K+ Channel N-type Inactivation Peptides

We have investigated the interaction of two peptides (ShB — net charge +3 and ShB:E12KD13K — net charge +7) derived from the NH₂-terminal domain of the Shaker K⁺ channel with purified, ryanodine-modified, cardiac Ca²⁺-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a single-site binding scheme with 50% saturation occurring at 5.92 ± 1.06 μm for ShB and 0.59 ± 0.14 nm for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 ± 0.4 sec⁻¹μm ⁻¹ for ShB and 2000 ± 200 sec⁻¹μm ⁻¹ for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied potential with the bulk of the voltage-dependence residing in K_off. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As is the case with inactivation and block of K⁺ channels, this is mediated by a large increase in K_on. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites which will contribute to the ion handling properties of this channel. Received: 15 December 1997/Revised: 13 March 1998     

Kinetic, structural and electrostatic aspects of the reduction of pentacyanoferrate(III) complexes by myoglobin

 The mechanism of the reduction of pentacyanoferrate(III) complexes by oxymyoglobin has been studied by conventional and high-pressure kinetic methods, and also by structural modelling. The results of this and an earlier study show that an outer-sphere mechanism is operating for electron transfer between oxymyoglobin and Fe^III(CN)₅L ^{n –}, independent of the lability of the ligand L. The electron transfer process is preceded by precursor formation at a specific site on the protein close to the protein heme pocket. Received: 10 November 1998 / Accepted: 25 February 1999     

DNA-binding properties of antitumor-active cis-bis(pyridine)platinum(II) organoamides

 The interaction of the new antitumor-active platinum organoamide complexes [Pt{N(p–HC₆F₄)CH₂}₂(py)₂] and [Pt{N(C₆F₅)CH₂}₂(py)₂] (py = pyridine) with small G-containing (oligo)nucleotides [GMP, d(GpG)] has been studied to establish whether or not these compounds can bind to DNA in an analogous manner to cisplatin. The reaction products have been analyzed by ¹H, ¹⁹F and ³¹P NMR spectroscopy. From the NMR data it is concluded that the {Pt(py)₂}²⁺ moiety binds to the N7 position of the G base, analogously to cisplatin, with the organoamide ligand acting as the leaving group. For the GG-N7,N7 adduct, structural differences are found for the sugar conformation, compared with cisplatin. These differences may account for the activity of these new compounds in tumor cell lines resistant to cisplatin. Received: 25 September 1995 / Accepted: 7 March 1996     

Methane-Oxidizing Bacteria in a Finnish Raised Mire Complex: Effects of Site Fertility and Drainage

Methane-oxidizing bacteria (MOB) are the only biological sinks for methane (CH₄). Drainage of peatlands is known to decrease overall CH₄ emission, but the effect on MOB is unknown. The objective of this work was to characterize the MOB community and activity in two ecohydrologically different pristine peatland ecosystems, a fen and a bog, and their counterparts that were drained in 1961. Oligotrophic fens are groundwater-fed peatlands, but ombrotrophic bogs receive additional water and nutrients only from rainwater. The sites were sampled in August 2003 down to 10 cm below the water table (WT), and cores were divided into 10-cm subsamples. CH₄ oxidation was measured by gas chromatography (GC) to characterize MOB activity. The MOB community structure was characterized by polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) and sequencing methods using partial pmoA and mmoX genes. The highest CH₄ oxidation rates were measured from the subsamples 20–30 and 30–40 cm above WT at the pristine oligotrophic fen (12.7 and 10.5 μmol CH₄ dm⁻³ h⁻¹, respectively), but the rates decreased to almost zero in the vicinity of WT. In the pristine ombrotrophic bog, the highest oxidation rate at 0–10 cm was lower than in the fen (8.10 μmol CH₄ dm⁻³ h⁻¹), but in contrast to the fen, oxidation rates of 4.5 μmol CH₄ dm⁻³ h⁻¹ were observed at WT and 10 cm below WT. Drainage reduced the CH₄ oxidation rates to maximum values of 1.67 and 5.77 μmol CH₄ dm⁻³ h⁻¹ at 30–40 and 20–30 cm of the fen and bog site, respectively. From the total of 13 pmoA-derived DGGE bands found in the study, 11, 3, 6, and 2 were observed in the pristine fen and bog and their drained counterparts, respectively. According to the nonmetric multidimensional scaling of the DGGE banding pattern, the MOB community of the pristine fen differed from the other sites. The majority of partial pmoA sequences belonged to type I MOB, whereas the partial mmoX bands that were observed only in the bog sites formed a distinct group relating more to type II MOB. This study indicates that fen and bog ecosystems differ in MOB activity and community structure, and both these factors are affected by drainage.     

Theoretical study on the functionalization of BC2N nanotube with amino groups

Using density functional theory calculations, we investigated properties of a functionalized BC₂N nanotube with NH₃ and five other NH₂-X molecules in which one of the hydrogen atoms of NH₃ is substituted by X = ?CH₃, ?CH₂CH₃, ?COOH, ?CH₂COOH and ?CH₂CN functional groups. It was found that NH₃ can be preferentially adsorbed on top of the boron atom, with adsorption energy of ?12.0 kcal mol^?1. The trend of adsorption-energy change can be correlated with the trend of relative electron-withdrawing or -donating capability of the functional groups. The adsorption energies are calculated to be in the range of ?1.8 to ?14.2 kcal mol^?1, and their relative magnitude order is found as follows: H₂N(CH₂CH₃) > H₂N(CH₃) > NH₃ > H₂N(CH₂COOH) > H₂N(CH₂CN) > H₂N(COOH). Overall, the functionalization of BC₂N nanotube with the amino groups results in little change in its electronic properties. The preservation of electronic properties of BC₂N coupled with the enhancement of solubility renders their chemical modification with either NH₃ or amino functional groups to be a way for the purification of BC₂N nanotubes.     

Saccharide production from methanol by transposon 5 mutants derived from the extracellular polysaccharide-producing bacterium Methylobacillus sp. strain 12S

A CH₃OH-utilizing bacterium that has the ability to produce extracellular polysaccharide (EPS) was isolated from a soil sample, and was identified as the obligate methylotroph Methylobacillus sp. strain 12S on the basis of its 16S rDNA sequence and growth-substrate specificity. The EPS produced by strain 12S was purified and the sugar composition was analysed by GC-MS and HPLC to reveal that the EPS was a heteropolymer composed of glucosyl, galactosyl, and mannosyl residues in the molar ratio 3:1:1. In order to produce mono- and/or oligosaccharides by single-step fermentation from CH₃OH, stain 12S was mutagenized by transposon 5. Among eleven EPS-deficient mutants, three strains were found to accumulate significant amounts of reducing sugars in the media. The amounts of the reducing sugars produced by the mutants (>ca. 700 mg glucose equivalent/l) were >11–22 times higher than those produced by the wild-type strain (<ca. 60 mg glucose equivalent/l). The GC-MS analysis showed that all the mutants accumulated glucose, erythrose, threose and a disaccharide-like compound in the media. Received: 25 August 1999 / Received revision: 15 March 2000 / Accepted: 24 March 2000     

Efficient production of androgenic doubled-haploid mutants in barley by the application of sodium azide to anther and microspore cultures

 The aim of this study was to establish a protocol for an efficient production of agronomical and/or physiological mutants from model (cvs. Igri and Cobra) and low-androgenic-responding (cv. Volga) cultivars of barley through the application of a mutagenic agent, sodium azide, to anthers and isolated microspores cultured in vitro. This technology offers the possibilities of screening for recessive mutants in the first generation, selecting for novel genotypes from very large haploid populations, avoiding chimerism and rapidly fixing selected genotypes as fertile true breeding lines. The mutagenic treatment, 10^–3–10^–5 M sodium azide, was applied during the anther induction pre-treatment or immediately after the microspore isolation procedure. Out of 616 M₂ doubled-haploid lines characterised under field conditions, a total of 63 morphological and developmental independent mutant lines were identified. The percentage of M₂ doubled-haploid lines carrying mutations per line analysed was 3.8% when 10^–4 M sodium azide was applied to anthers from the low-responding cv. Volga; this increased to 8.6% and 15.6% when 10^–5 and 10^–4 M sodium azide were applied to freshly isolated microspores from model cultivars. Received: 18 April 2000 / Revision received: 28 September 2000 / Accepted: 28 September 2000     

Membrane permeability of formate in human erythrocytes: NMR measurements

The rate of the rapid exchange of formate mediated by band 3 in human erythrocytes, under equilibrium exchange conditions, was measured by using a T ₁ relaxation method with ¹³C-labelled formate and ¹³C NMR, and a pulsed field-gradient spin-echo (PFGSE) method using ¹H NMR. The former analysis was based on large differences in T ₁ between the inside and the outside of the cells brought about by added Mn²⁺; the latter was based on large differences in the apparent diffusion coefficient inside and outside the cells. There was close agreement in the estimates of the membrane permeabilities made using both methods, suggesting a lack of interference of the exchange process by Mn²⁺. Regression analysis yielded estimates (under the specified conditions, including 37°C) of V _max of 3.5±0.3×10^–9 and 3.8±0.4×10^–9 mol cm^–2 s^–1, and K _m of 9.8±0.2 and 8.1±0.2 mM, for the T ₁ and the PFGSE methods, respectively. These are new estimates made using methodology that has not previously been applied to measuring rapid (sub-second time scale) formate exchange in cells. Received: 8 May 1998 / Accepted: 16 July 1998     

Simultaneous α/β spin-state selection for <Superscript>13</Superscript>C and <Superscript>15</Superscript>N from a time-shared HSQC-IPAP experiment

Two novel HSQC-IPAP approaches are proposed to achieve α/β spin-state editing simultaneously for ¹³C and ¹⁵N in a single NMR experiment. The pulse schemes are based on a time-shared (TS) 2D ¹H,¹³C/¹H,¹⁵N-HSQC correlation experiment that combines concatenated echo elements for simultaneous J(CH) and J(NH) coupling constants evolution, TS evolution of ¹³C and ¹⁵N chemical shifts in the indirect dimension and heteronuclear α/β-spin-state selection by means of the IPAP principle. Heteronuclear α/β-editing for all CH _n (n = 1–3) and NH _n (1–2) multiplicities can be achieved in the detected F2 dimension of a single TS-HSQC-F2-IPAP experiment. On the other hand, an alternative TS-HSQC-F1-IPAP experiment is also proposed to achieve α/β-editing in the indirect F1 dimension. Experimental and simulated data is provided to evaluate these principles in terms of sensitivity and performance simultaneously on backbone and side-chain CH, CH₂, CH₃, NH, and NH₂ spin systems in uniformly ¹³C/¹⁵N-labeled proteins and in small natural-abundance peptides.