Cees Erkelens

The nature of the primary electron acceptor in green sulfur bacteria

It was shown previously (Van de Meent, E.J., Kobayashi, M., Erkelens, C., Van Veelen, P.A., Amesz, J. and Watanabe, T. (1991) Biochim. Biophys. Acta 1058, 356–362) by means of HPLC, NMR and optical and mass spectroscopy that the primary electron acceptor of heliobacteria is 81-hydroxychlorophyll (Chl) a. In view of the spectral and functional similarities between this pigment and the primary electron acceptor of green sulfur bacteria, we have applied the same methods to various species of green sulfur bacteria (Prosthecochloris aestuarii, Chlorobium limicola, C. limicola f. thiosulfatophilum, C. vibrioforme and C. phaeovibrioides) in order to study the identity and the occurrence of the latter pigment. It was already shown from flash spectroscopic and reversed phase HPLC experiments on isolated membranes and solubilized membrane fractions of P. aestuarii that the most likely candidate for the primary acceptor is a pigment named bacteriochlorophyll (BChl) 663, which had been tentatively identified as a lipophilic from of BChl c. In this communication we will show by means of optical spectroscopy, 252Cf-plasma desorption mass spectroscopy and 1H-NMR that BChl 663 is an isomer of Chl a. This result again emphasizes the similarities between the reaction centers of green sulfur bacteria, heliobacteria and Photosystem I. By means of normal-phase HPLC analysis of the five species of green sulfur bacteria it is shown that BChl 663 is universally present and in comparable quantities in this group of photosynthetic bacteria. No other pigments with similar spectroscopic properties were detected.

A flow-through probe for in Vivo31P NMR spectroscopy of unanesthetized aquatic vertebrates at 9.4 tesla

Abstract A flow cell which fits in a modified bioprobe of a Bruker MSL-400 NMR spectrometer and allows the monitoring of the energy metabolism of an enclosed aquatic vertebrate at a selected temperature, water composition, and oxygen level ranging from 0 to 100% air saturation is described. The animal is pressed against the observation window and immobilized by an inflatable plastic bag. No anesthetics are used during the actual experiment. The signal of the tissue of interest is picked up with a surface coil, which is double-tuned to the phosphorus (162 MHz) and proton (400 MHz) frequencies. The flow cell can be moved vertically to the desired position. The usefulness of the fish probe is demonstrated by spectra of excellent resolution and signal-to-noise, obtained from the myotomal muscles of carp, goldfish, rainbow trout, and tilapia, by high phosphocreatine/ inorganic phosphate ratios, indicating a situation of low stress, and by stability of all NMR-observed parameters over periods of at least one working day (8 h).

Determination of high-energy phosphate compounds in fish muscle: 31P-NMR spectroscopy and enzymatic methods

Abstract 1. 1. High-energy phosphate compounds were measured in the lateral red and epaxial white muscles of goldfish by: (1) in vivo31P-NMR spectroscopy; (2) in vitro31P-NMR spectroscopy of excised tissue; (3) in vitro31P-NMR spectroscopy of perchloric acid extracts and (4) enzymatic analysis of perchloric acid extracts. 2. 2. A rapid breakdown of phosphocreatine and accumulation of inorganic phosphate was observed during handling, tissue excision and extraction. 3. 3. The lability of phosphocreatine may explain the well-known differences between in vivo NMR spectra and spectra of perchloric acid extracts. 4. 4. A modified procedure in described for extraction of frozen tissue samples, resulting in 100% yield of phosphocreatine, ATP and inorganic phosphate, as judged by the recovery of internal standards.

Characterisation of uniformly 13C, 15N labelled bacteriochlorophyll a and bacteriopheophytin a in solution and in solid state: complete assignment of the 13C, 1H and 15N chemical shifts

In this investigation we report a complete assignment of 13C, 1H and 15N solution and solid state chemical shifts of two bacterial photosynthetic pigments, bacteriochlorophyll (BChl) a and bacteriopheophytin (BPheo) a. Uniform stable‐isotope labelling strategies were developed and applied to biosynthetic preparation of photosynthetic pigments, namely uniformly 13C, 15N labelled BChl a and BPheo a. Uniform stable‐isotope labelling with 13C, 15N allowed performing the assignment of the 13C, 15N and 1H resonances. The photosynthetic pigments were isolated from the biomass of photosynthetic bacteria Rhodopseudomonas palustris 17001 grown in uniformly 13C (99%) and 15N (98%) enriched medium. Both pigments were characterised by NMR in solution (acetone‐d6) and by MAS NMR in solid state and their NMR resonances were recorded and assigned through standard liquid 2D 13C13C COSY, 1H13C HMQC, 1H15N HMBC and solid 2D 13C13C RFDR, 1H13C FSLG HETCOR and 1H15N HETCOR correlation techniques at 600 MHz and 750 MHz. The characterisation of pigments is of interest from biochemical to pharmaceutical industries, photosynthesis and food research. Copyright

Nitrogen metabolism in cultures of Tabernaemontana divaricata

Abstract A cell suspension culture from Tabernaemontana divaricata was fed with 15 N-labelled ammonium or nitrate. The incorporation of label in free amino acids, protein amino acids and indole alkaloids was determined. Ammonium was found to be used more extensively than nitrate in the biosynthesis of these compounds. For tryptamine considerably lower labelling percentages were found than for the indole alkaloid O -acetylvallesamine and the amino acids. This indicates a vacuolar pool of tryptamine, formed at the beginning of the culture-period and not available for further alkaloid biosynthesis.

Magnetic resonance microscopy for studying the development of chicken and mouse embryos

In this new era of transgenic mouse models, the need to evaluate the effects of gene manipulation during embryonic development is increasing. Traditional embryological studies are invasive, sacrificing embryos before processing in order to reveal specific information like morphology, histology, antigen distribution, gene expression patterns, or physiological parameters. Each objective requires a specific method, excluding the use of one specimen for multiple questions, while for temporal information each method has to be repeated on several specimens during development. The non-invasive character of magnetic resonance microscopy (MRM) allows the study of subsequent stages of normal development in a single embryo in utero over extended periods. In addition, MRM offers the possibility of studying the onset and course of a malformation during development. MRM has proven to be a powerful tool for fixed embryos [1] and living chicken embryos in ovo [2]. Recently, Smith and coworkers [3] managed to visualize and follow living rat embryos in utero in a 2.0T MR microscope by a 3D projection encoding technique with a total scanning time of 27 min The objectives of the present study are to visualize fixed chicken embryos and living mouse embryos in utero. Imaging of the embryo requires very high resolution in combination with excellent contrast. To achieve this, we used moderate to ultra high magnetic fields of 7.0 and 17.6T for the chicken material and 7.0T for the mouse embryos in utero and explored various fast imaging sequences. Fast imaging is necessary to avoid artifacts from embryonic movements that cannot be controlled by the researcher.