John A. Chudek

The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis.

The effects of hypersaline treatment (osmotic upshock) on solute accumulation have been studied in the Gram-positive bacterium Bacillus subtilis. Natural abundance 13C NMR spectroscopy studies revealed only proline as a major organic osmoticum in cells grown in defined medium (no exogenous organic solutes) and this finding was confirmed by amino acid analysis. Intracellular concentrations of both K+ and proline rose markedly after osmotic upshock. K+ influx from the medium was rapid (less than 1 h) but proline synthesis was a slower process (5-9 h). Proline synthesis appeared to be dependent on the prior accumulation of K+ and it is possible that K+ serves in some manner as the signal for increased proline synthesis. In cells upshocked in medium enriched in glycine betaine the endogenous synthesis of proline was repressed and glycine betaine served as the sole organic osmoticum. K+ was also accumulated under these conditions.

Osmotic adjustment in cyanobacteria from hypersaline environments

The intracellular concentrations of the monovalent inorganic cations K+ and Na+, low molecular weight carbohydrates and quaternary ammonium compounds have been determined for 4 strains of cyanobacteria (Aphanothece halophytica, Coccochloris elabens, Dactylococcopsis salina and Synechocystis DUN52) originally isolated from hypersaline habitats (i.e. habitats with a salinity greater than that of seawater) over a range of external salt concentration (from 50% to 400% seawater). Intracellular cation levels (Na+ and K+) were determined to be within the range 80–320 mmol · dm-3 (cell volume), showing only minor changes in response to salinity. Intracellular carbohydrates were found to comprise a negligible component of the intracellular osmotic potential [at 2–19 mmol · dm-3 (cell volume)], throughout the salinity range. Quaternary ammonium compounds, however, were recorded in osmotically significant quantities [up to 1,640 mmol · dm-3 (cell volume)] in these strains, showing major variation in response to salinity. Thus Synechocystis DUN 52 showed an increase in quaternary ammonium compounds in the oder of 1,200 mmol · dm-3 between 50% and 400% seawater medium, accounting for a significant proportion of the change in external osmotic potential.Examination of intact cells and cell extracts using 13C and 1H nuclear magnetic resonance (NMR) spectroscopy confirmed the presence of the quaternary ammonium compound glycine betaine as the major osmoticum in the 4 strains; no other compounds were detected during NMR assays. These results suggest a common mechanism of osmotic adjustment, involving quaternary ammonium compounds, in cyanobacteria from hypersaline environments.

Osmotic adjustment in Spirulina platensis

The filamentous cyanobacterium Spirulina platensis has been examined for salt tolerance and osmotic adjustment. Salinities up to 150% seawater had little effect on growth yield or photosynthetic O2 evolution; higher salinities were markedly inhibitory. Osmotic adjustment was achieved by the intracellular accumulation of the low-molecular-weight carbohydrate glucosyl-glycerol in response to increased external salinity: in fullstrength (100%) seawater glucosyl-glycerol accounted for approximately 5.0% of the dry weight of the cyanobacterium. Trehalose was also present, particularly in cells at low salt concentration, and in 50% seawater medium accounted for up to 1.0% of the dry weight of the cyanobacterium. For cells grown in 100% seawater the ratio of trehalose to glucosyl-glycerol varied with temperature: at 37°C trehalose comprised 31% (w/w) of the low-molecular-weight carbohydrates while at 20°C only 9% of the total was trehalose. When subjected to hypo-osmotic shock the intracellular concentration of glucosyl-glycerol decreased and this was mirrored by an increase in glycogen. An understanding of the osmotic adjustment of S. platensis has implications both for the mass culturing of this and other strains of Spirulina and possibly also for the quality of the harvested product.

Organic solute accumulation in osmotically-stressed Enteromorpha intestinalis

The role of organic solutes in the osmotic adjustment processes of the marine macroalga Enteromorpha intestinalis (L.) Link was investigated in 1986, using fresh samples collected from mid-shore rock pools at Tayport, Fife, Scotland. Natural-abundance 13C nuclear magnetic-resonance spectroscopy revealed β-dimethylsulphoniopropionate (DMSP) to be the only major low molecular weight organic osmolyte present. However, on transfer to a hypersaline medium (300% sea water; 100%=35 S‰), tissue sucrose and proline levels increased markedly, while DMSP remained constant. Recovery of optimal photosynthetic activity and increases in inorganic ion levels occurred over a similar time scale to the changes in sucrose and proline (within 48 h), indicating that these two organic solutes are involved in hyperosmotic adjustment in E. intestinalis while DMSP is not. Freshly-collected plants transferred to 300% sea water medium in the dark showed no significant increases in organic osmolytes. In contrast, starch-enrichment (16 d continuous illumination) led to enhanced synthesis of sucrose and proline in the light and in darkness, but tissue DMSP levels showed no variation throughout. These observations suggest that DMSP is not involved in short-term osmoacclimation in E. intestinalis.

The Osmotic Responses of Penicillium ochro-chloron: Changes in Internal Solute Levels in Response to Copper and Salt Stress

The marked copper tolerance of Penicillium ochro-chloron has been confirmed as has its ability to grow in solutions of high salinity. The major low molecular weight organic solutes present in P. ochro-chloron were glycerol, erythritol, arabitol, mannitol, sorbitol and trehalose. Of these, glycerol increased significantly during growth in high concentrations of Na+ or Cu2+, a 15-fold increase, relative to the control, occurring in concentrations of 0.5 M. Cell K+ increased with elevated external Na+ but decreased slightly with elevated external Cu2+. With high external Cu2+ mycelium maintained a constant level of Cu2+, with low levels of Na+ in all treatments. It is concluded that the high concentrations of glycerol induced by high external Na+ or Cu2+ had a significant osmotic effect, allowing growth in media of high osmolality. The exclusion of ions may have a gratuitous role in the copper tolerance of P. ochro-chloron.

Nuclear magnetic resonance microscopy as a non-invasive tool to study the development of lepidopteran pupae

Abstract The application of NMR microscopy to the non-invasive study of internal structures in lepidopteran pupae is described and examples presented for an immature pupa of Pieris brassicae and a mature female pupa of Graphiphora augur. By using a chemical shift selective imaging procedure it was possible to determine separately the distributions of water and lipid. This technique makes it possible, in principle, to observe the relationship between the progress of metamorphosis and the utilization of lipid reserves and could thus represent a valuable new approach to the study of pupal developmental processes.

The chemical synthesis of Leishmania donovani phosphoglycan via polycondensation of a glycobiosyl hydrogenphosphonate monomer

A polycondensation of 2,3,6-tri-O-benzoyl-4-O-(2,3,4-tri-O-benzoyl-beta-D-galactopyranosyl)-al pha-D-mannopyranosyl hydrogenphosphonate in the presence of trimethylacetyl chloride has been used to synthesize a linear poly[beta-D-galactopyranosyl-(1-->4)-alpha-D-mannopyranosyl phosphate] representing the phosphoglycan part of the lipophosphoglycan from Leishmania donovani.

An NMR microscopic study of grape (Vitis vinifera L.)

SummaryMature healthy grape berries and berries wound-inoculated with the fungusBotrytis cinerea were examined by1H NMR microimaging using 2D and 3D spin echo and gradient echo procedures. These NMR images were compared with representations obtained by conventional histology, where possible using the same specimens. 3D imaging datasets from excised seeds were reconstructed by surface rendering and maximum intensity projection to allow interpretation of their internal structure. T2-weighted spin echo images revealed the major features of the pericarp, septum and loculi of whole berries. T1-weighted images were less discriminatory of parenchyma tissues in the fruit but revealed the endosperm in seeds as a chemically shifted feature. A non-invasive study by T1-weighted spin echo NMR imaging of infection byB. cinerea over a 6-day period showed that the disease spread throughout the exocarp but failed to spread in the mesocarp, a result confirmed by histological examination of the same specimen. Surface rendering of 3D datasets of excised seeds revealed the two ruminations of the endosperm and the distal location of the chalaza. The position of the embryonic axis was revealed in T2-weighted maximum intensity projections. This noninvasive study revealed the need to apply a range of imaging techniques and parameters to visualise the structural features of the different parts of the grape berry.

Ascorbic acid conjugates isolated from the phloem of Cucurbitaceae.

Analysis of phloem exudates from the fruit of Cucurbitaceae revealed the presence of several compounds with UV-visible absorption spectra identical to that of l-ascorbic acid. In Cucurbita pepo L. (zucchini), the compounds could be isolated from phloem exudates collected from aerial parts of the plant but were not detected in whole tissue homogenates. The compounds isolated from the phloem exudates of C. pepo fruit were eluted from strong anion exchange resin in the same fraction as l-ascorbic acid and were oxidised by ascorbate oxidase (E.C. 1.10.3.3). The major compound purified from C. pepo fruit exudates demonstrated similar redox properties to l-ascorbic acid and synthetic 6-O-glucosyl-l-ascorbic acid (6-GlcAsA) but differed from those of 2-O-glucosyl-l-ascorbic acid (2-GlcAsA) isolated from the fruit of Lycium barbarum L. Parent and fragment ion masses of the compound were consistent with hexosyl-ascorbate in which the hexose moiety was attached to C5 or C6 of AsA. Acid hydrolysis of the major C. pepo compound resulted in the formation of l-ascorbic acid and glucose. The purified compound yielded a proton NMR spectrum that was almost identical to that of synthetic 6-GlcAsA. A series of l-ascorbic acid conjugates have, therefore, been identified in the phloem of Cucurbitaceae and the most abundant conjugate has been identified as 6-GlcAsA. The potential role of such conjugates in the long-distance transport of l-ascorbic acid is discussed.

Magnetic resonance imaging study of a soft actuator element during operation

The physical structure and the actuation mechanism of a Nafion-based soft actuator – a Pt-containing ionic polymer–metal composite (IPMC) – were investigated using in situ magnetic resonance imaging (MRI) during application of four different electrical regimes. Importantly, the raw MRI data were used to generate spatial maps of both proton density (PD) and proton spin–spin relaxation time (T2) across the sample. These were successfully employed to study changes in the distribution and chemical environment of water molecules absorbed within the operating actuator device. The IPMC sample was mapped in this way during the application of a small d.c. potential across its thickness. Three main phenomena were observed in the results: initial rapid increase in T2 at both electrodes, without an observed change in PD; slower formation of a region of high T2 and high PD at the IPMC cathode; and contraction of the polymer along the anode and its expansion along the cathode, giving rise to bending actuation. Reversing the polarity of the applied potential resulted in the reversal of the direction of the bending deformation of the IPMC sample and of the distribution of PD and T2 within it. These phenomena were explained in terms of the unusual structure of Nafion and its interaction with host ions and the electric field. Up to 20% of the total water content of the IPMC was found to be involved in long-range electro-diffusion.