Determination of deuterium-labeled tryptophan in proteins by means of high-performance liquid chromatography and thermospray mass spectrometry

In order to develop direct methods for determining the extent of metabolic incorporation of isotopically labeled amino acids into a protein, the determination of deuterated tryptophan in [2H5]tryptophan-bacteriorhodopsin was investigated. The isotopically modified protein was subjected to alkaline hydrolysis. After phenyl isothiocyanate derivatization of the hydrolysate, the mixture was separated by reversed-phase liquid chromatography. Field desorption mass spectrometry and thermospray mass spectrometry were investigated for their ability to determine the ratio between [2H5]tryptophan and total tryptophan in the collected fractions. In order to check the procedure a set of known tryptophan/[2H5]tryptophan mixtures were passed through the same derivatization, HPLC separation, and lyophilization procedure as used for the biological samples.     

Spatial Genetic Structure of the Abundant and Widespread Peatmoss Sphagnum magellanicum Brid.

Spore-producing organisms have small dispersal units enabling them to become widespread across continents. However, barriers to gene flow and cryptic speciation may exist. The common, haploid peatmoss Sphagnum magellanicum occurs in both the Northern and Southern hemisphere, and is commonly used as a model in studies of peatland ecology and peatmoss physiology. Even though it will likely act as a rich source in functional genomics studies in years to come, surprisingly little is known about levels of genetic variability and structuring in this species. Here, we assess for the first time how genetic variation in S. magellanicum is spatially structured across its full distribution range (Northern Hemisphere and South America). The morphologically similar species S. alaskense was included for comparison. In total, 195 plants were genotyped at 15 microsatellite loci. Sequences from two plastid loci (trnG and trnL) were obtained from 30 samples. Our results show that S. alaskense and almost all plants of S. magellanicum in the northern Pacific area are diploids and share the same gene pool. Haploid plants occur in South America, Europe, eastern North America, western North America, and southern Asia, and five genetically differentiated groups with different distribution ranges were found. Our results indicate that S. magellanicum consists of several distinct genetic groups, seemingly with little or no gene flow among them. Noteworthy, the geographical separation of diploids and haploids is strikingly similar to patterns found within other haploid Sphagnum species spanning the Northern Hemisphere. Our results confirm a genetic division between the Beringian and the Atlantic that seems to be a general pattern in Sphagnum taxa. The pattern of strong genetic population structuring throughout the distribution range of morphologically similar plants need to be considered in future functional genomic studies of S. magellanicum.     

Differentiation of the membrane system in the cells of imaginai discs

Summary Imaginal discs from developing larvae of the fly Calliphora erythrocephala fixed in permanganate or osmium tetroxide and embedded in Epon 812 were observed by electron microscopy. When the larval growth ceases, the differentiation manifests itself through an enlargement of the endoplasmic reticulum, by which continuous membrane contact is established between all cell organelles. During the same time mitochondria swell up and transform into lipid granules and the intercellular contacts weaken.I am indebted to Mrs Mariann Carleson and Miss Brita Nilsson for technical aid.     

Productivity-diversity relationships for plants, bryophytes, lichens, and polypore fungi in six northern forest landscapes

We investigated the relationship between site productivity and diversity of vascular plants, bryophytes, lichens, and polypore fungi in forests based on species richness data in 0.25 ha forest plots (grain size), selected from six 150–200 ha study areas (focus), and spanning over a latitudinal distance of 1350 km (extent) in Norway. We 1) searched for prevailing productivity-diversity relationships (PDRs), 2) compared PDRs among taxonomic groups and species found in different micro-habitats, and 3) investigated the effect of increasing plot (grain) size on PDRs. Using vegetation types as a surrogate for site productivity, we found a general pattern of increasing species richness with site productivity. On average total species richness doubled with a ten-fold increase in productivity. Lichens PDRs stood out as less pronounced and more variable than for other species groups investigated. PDRs of species associated with downed logs tended to level off at high-productive sites, a pattern interpreted as an effect of disturbance. Increasing the grain size >10-fold did not change the proportional difference in species richness between sites with high and low productivity.     

High-resolution solid-state 13C-NMR study of carbons C-5 and C-12 of the chromophore of bovine rhodopsin. Evidence for a 6-S-cis conformation with negative-charge perturbation near C-12

Solid-state 13C magic-angle spinning NMR spectroscopy has been employed to study the conformation of the 11-cis-retinylidene Schiff base chromophore in bovine rhodopsin. Spectra were obtained from lyophilized samples of bovine rhodopsin selectively 13C-labeled at position C-5 or C-12 of the retinyl moiety, and reconstituted in the fully saturated branched-chain phospholipid diphytanoyl glycerophosphocholine. Comparison of the NMR parameters for carbon C-5 presented in this paper with those published for retinyl Schiff base model compounds and bacteriorhodopsin by Harbison and coworkers [Harbison et al. (1985) Biochemistry 24, 6955-6962], indicate that in bovine rhodopsin the C-6-C-7 single bond has the unperturbed cis conformation. This is in contrast to the 6-S-trans conformation found in bacteriorhodopsin. The NMR parameters for bovine [12-13C]rhodopsin present evidence for the presence of a negative charge interacting with the retinyl moiety near C-12, in agreement with the model for the opsin shift presented by Honig and Nakanishi and coworkers [Kakitani et al. (1985) Photochem. Photobiol. 41, 471-479].     

C-band polymorphisms of chromosome 9: quantification by Ce-bands

Summary C-band polymorphisms of chromosome 1 can be quantified by C_e bands visualized using oblique epi-illumination. In this paper the polymorphic region of chromosome 9, including variants such as 9qh+, inversions, and translocations, was analyzed in a total of 1860 chromosomes from 20 individuals and 8 fetuses. In this sample we found between zero and five C_e bands on 9q and between zero and four C_e bands on 9p with a maximum of five C_e bands per chromosome. On the basis of these observations at least 19 different polymorphic patterns can be expected theoretically, 15 of which were observed in our sample. Among these, five polymorphic C_e-band classes were distinguishable by the method presented. Considering both homologues, 5²=25 quantitatively discernible chromosome 9 pairs may exist.     

13C-n.m.r. study of citrate metabolism in rabbit renal proximal-tubule cells.

U.v.-visible-absorption and e.p.r. spectroscopy were used to study the type 2 and type 3 copper centres in the mercury derivative of laccase. After treatment with peroxide the mercury derivative of laccase exhibits a fully developed absorption band at 330 nm (delta epsilon = 2900 +/- 100 M-1.cm-1, which is characteristic of type 3 copper in the oxidized state. In addition, there is a weak ligand-field absorption at 740 nm (epsilon = 380 +/- 30 M-1.cm-1), which can be assigned to the type 3 pair. Because the e.p.r. spectrum of the type 2 copper is well resolved in the case of the mercury derivative of laccase, for the first time we have been able to observe spectroscopic evidence for a pH-dependent structural transition that has been invoked to explain the kinetics of enzyme reduction [Andréasson & Reinhammar (1979) Biochim. Biophys. Acta 568, 145-156]. According to the e.p.r. data the pKa lies in the range 6-7, and comparisons with a model compound show that the spectral changes can plausibly be interpreted in terms of the deprotonation of a water molecule in the co-ordination sphere of the type 2 copper.     

Genotype frequencies associated with incompatibility systems in tristylous plants

A general procedure has been given previously for calculating frequencies of the morphs Long, Mid, and Short in equilibrium populations of tristylous plants. It is now demonstrated that an equilibrium state actually exists at the genotype level if this procedure produces admissible morph frequencies. This result holds in a diploid model, with or without linkage between the two loci involved. It is shown how the genotype frequencies may be determined for any set of mating probabilities. It is also explained how these frequencies may be calculated in a tetraploid model incorporating double reduction. The general theory is applied to a particular situation where the Mid morph is at a selective disadvantage as a seed parent.     

Continuity of the membrane systems in the cells of imaginal discs

Summary Imaginal discs from mature larvae of the fly Calliphora erythrocephala fixed in permanganate and embedded in Epon 812 were observed by electron microscopy. Irrespective of the type of the disc, the endoplasmic reticulum in the cells was found to be in unbroken contact with the outer nuclear membrane, Golgi areas, the mitochondria and the cell membrane. The imaginai discs are syncytial in that all cells are in cytoplasmic communication through secondary breaks in the cell membranes.The investigation was facilitated by grants from the Nordic Insulin Foundation.I am indebted to Mrs. Mariann Carleson and Miss Brita Nilsson for technical aid.     

Time-resolved ultraviolet resonance Raman studies of protein structure: application to bacteriorhodopsin.

Time-resolved ultraviolet resonance Raman spectra of bacteriorhodopsin are used to study protein structural changes on the nanosecond and millisecond time scales. Excitation at 240 nm is used to selectively enhance vibrational scattering from tyrosine so that changes in its hydrogen bonding and protonation state can be examined. Both nanosecond and millisecond UV Raman difference spectra indicate that none of the tyrosine residues change ionization state during the BR----K and BR----M transitions. However, intensity changes are observed at 1172 and 1615 cm-1 in the BR----M UV Raman difference spectra. The 1615-cm-1 feature shifts down 25 cm-1 in tyrosine-d4-labeled BR, consistent with its assignment as a tyrosine vibration. The intensity changes in the BR----M UV Raman difference spectra most likely reflect an increase in resonance enhancement that occurs when one or more tyrosine residues interact more strongly with a hydrogen-bond acceptor in M412. The frequency of the v7a feature (1172 cm-1) in the BR----M UV Raman difference spectra supports this interpretation. The proximity of Tyr-185 and Asp-212 in the retinal binding pocket suggests that deprotonation of the Schiff base in M412 causes Tyr-185 to stabilize ionized Asp-212 by forming a stronger hydrogen bond.