Terence J. Walton

Evidence that cyclic AMP is involved in the hypersensitive response of Medicago sativa to a fungal elicitor

Abstract In response to challenge with a glycoprotein elicitor from the phytopathogenic fungus, Verticillium alboatrum , synthesis of the phytoalexin medicarpin is induced in Medicago sativa . Treatment of seedlings with the cell-permeating cyclic AMP analogue, dibutyryl cyclic AMP, resulted in the stimulation of phenylalanine ammonia lyase activity and induction of medicarpin synthesis. Challenge of M. sativa cell suspension cultures with fungal elicitor resulted in a significant, transient increase in intracellular cyclic AMP content, together with a pulse of adenylyl cyclase activity, both within a few minutes of the elicitor challenge, and a subsequent increase in cyclic AMP phosphodiesterase activity. Incubation of a membrane fraction from the M. sativa cells with the fungal elicitor resulted in a dose-dependent stimulation of the adenylyl cyclase activity. These observations are discussed in the context of the signal transduction mechanism with the M. sativa cellular defence system.

Evidence that generation of inositol 1,4,5-trisphosphate and hydrolysis of phosphatidylinositol 4,5-biphosphate are rapid responses following addition of fungal elicitor which induces phytoalexin synthesis in lucerine (Medicago sativea) suspension culture cells

Treatment of lucerne suspension culture cells with glycoprotein elicitor from the phytopathogenic fungus Verticillium albo-atrum R & B triggers Ca(2+)-mediated induction of antimicrobial secondary metabolites termed phytoalexins. The present study investigated the possible role of polyphosphoinositide signal transduction in phytoalexin elicitation. Within 1 min of addition of elicitor to lucerne suspension culture cells we found a 100-160% (15-25 pmol/g fresh wt) increase in the level of compound with chromatographic and electrophoretic properties expected for an inositol trisphosphate (InsP3) and which was strongly bound by an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-specific binding protein; after 3 min the level of this compound had fallen below that observed prior to elicitor challenge. In 32P-prelabelled cells, the relative proportion of radioactivity which cochromatographed with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) was found to have decreased by 48% 1 min after elicitor addition and that rapid depletion of membrane lipid radioactivity was specific to this lipid fraction. The rapid, transient increase in level of Ins(1,4,5)P3 and concomitant fall in PtdIns(4,5)P2 suggests that Ins(1,4,5)P3 generated by hydrolysis of PtdIns(4,5)P2 may provide a Ca(2+)-mobilizing signal in phytoalexin elicitation in lucerne.

Identification of cyclic nucleotide constituents of meristematic and non-meristematic tissue of Pisum sativum roots

Abstract The extraction of endogenous 3′,5′-cyclic-AMP, 3′,5′-cyclie-GMP, 3′,5′-cyclic-CMP, 3′,5′-cyclic-UMP, 3′,5′-cyclic-IMP and 3′,5′-cyclic-deoxyTMP from meristematic and non-meristematic regions of Pisum roots is described. Extracts were subjected to a sequential purification procedure involving adsorption chromatography on alumina, ion-exchange chromatography, and preparative electrophoresis. Purified samples were compared with cyclic nucleotide standards by HPLC and UV absorbance spectrophotometry; further analysis was carried out by fast atom bombardment mass spectrometry and mass-analysed ion kinetic energy spectrometry. 3′,5′-Cyclic-AMP, -GMP, -CMP and -UMP were found to be present in Pisum meristematic tissue and 3′,5′-cyclic-AMP, -GMP, -CMP, -IMP and -DTMP are present in the non-meristematic regions. Data indicate that there is a significantly higher concentration of cyclic CMP in meristematic tissue relative to that in non-meristematic regions.

Comparative study of human steroid 5α-reductase isoforms in prostate and female breast skin tissues: sensitivity to inhibition by finasteride and epristeride

Steroid 5alpha-reductase (5-AR) catalyses the reduction of testosterone (T) to dihydrotestosterone (DHT). The 5alpha-reductase found in human benign prostatic hyperplasia (BPH) has been compared with that found in human breast skin tissue in respect of sensitivity to inhibition by Finasteride and Epristeride. Kinetic studies showed the presence of two isoforms of 5alpha-reductase in benign prostatic hyperplasia indicated by low and high Km isoforms for testosterone, while female breast skin tissue contained only one isoform. The isoforms differ in their affinity for the inhibitors Finasteride and Epristeride, both compounds being more effective for the low Km 5alpha-reductase isoform than the high Km 5alpha-reductase of prostatic tissue, with Finasteride displaying competitive inhibition and Epristeride uncompetitive. Finasteride and Epristeride are also inhibitors of skin 5alpha-reductase, which possesses a comparable Ki for Finasteride to that of the low Km prostatic enzyme, but Epristeride was a less potent inhibitor of the skin enzyme relative to the prostate isoform. These results suggest that the inhibitors have therapeutic potential, other than for treatment of benign prostatic hyperplasia, for treating skin disorders influenced by the action of dihydrotestosterone and warrant further investigation.

Influence of light on cyclic nucleotide metabolism in plants; effect of dibutyryl cyclic nucleotides on chloroplast components

Abstract -Dark-grown Phaseolus seedlings exposed to light for 18 hr contain more than × 3 the concentration of cyclic AMP in controls kept in an 18 hr light/6 hr dark cycle. Chloroplast cyclic AMP concentrations behaved similarly. Cyclic AMP phosphodiesterase activity of spinach chloroplasts showed a light-dependent response. The phosphodiesterase activity of chloroplasts from light-grown and dark-grown spinach seedlings exhibit similar V max but different K m values. The K m of the enzyme from light-exposed plants was more than × 30 that of the corresponding activity from dark-treated plants. The two enzymes showed different responses to the combined addition of Ca 2+ and calmodulin; a × 3 stimulation of the enzymic activity from light-treated plants was observed. The light-effects were not directly related to general photosynthetic effects, reflected in protein and chlorophyll concentrations. Light regimes also markedly modified the effects of dibutyryl cyclic AMP and dibutyryl cyclic GMP on terpenoid concentrations. Dibutyryl cyclic AMP doubled the α-tocopherol content and reduced by 40% the γ-tocopherol and chlorophyll concentrations of chloroplasts from light-grown plants; simultaneously their ubiquinone content increased by 48%. The main effect on dark-treated plants was that of dibutyryl cyclic AMP which gave a 107% increase in ubiquinone concentration. The main response to dibutyryl cyclic GMP was seen in dark-grown plants with substantial increases in the concentration of α-tocopherol, γ-tocopherol, and chlorophyll. Plants grown in darkness and then given 15 min illumination showed an 83% increase in α-tocopherol concentration and a 168% increase in ubiquinone concentration following treatment with dibutyryl cyclic GM Under these conditions the cyclic AMP analogue increased chlorophyll and ubiquinone concentrations by 57 and 78%, respectively.

Characterisation of membrane phospholipids and glycolipids from a halophilic archaebacterium by high‐performance liquid chromatography/electrospray mass spectrometry

Combined high-performance liquid chromatography and electrospray mass spectrometry (LC/ES-MS) has been used for direct characterisation of the polar membrane lipids in total lipid extracts from Halobacterium salinarium, a species of halophilic archaebacterium. The principle phospholipids found were the diphytanyl archaeol phosphatidylglycerol and diphytanyl archaeol phosphatidylglycerolphosphate methyl ester. The application of LC/ES-MS revealed the additional presence of diphytanyl archaeol phosphatidylglycerol sulphate The extracts also contained an archaeol glycolipid, initially detected in preliminary offline ES-MS studies, which was further characterised by LC/ES-MS and by product ion tandem mass spectrometry (MS/MS) as a sulphate ester of diglycosyl-2,3-di-O-phytanyl-sn-glycerol. Whilst archaeol phospho- and glycolipids containing a (C(20)-C(20))-isopranyl glycerol ether core predominated, LC/ES-MS of the extracts from Halobacterium salinarium indicated the presence of an analogue containing one double bond in its isoprenyl ether core as a minor component of the phosphatidylglycerolphosphate methyl ester fraction, providing a further example of the previously recognised existence of isoprenologues of diphytanyl archaeols which occur as minor components of archaebacterial membrane lipids. The value of these techniques in compositional analysis of archaebacterial lipid extracts is discussed.

High-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry of phospholipids in Natronobacterium magadii

A novel method of coupled liquid chromatography/atmospheric pressure ionization mass spectrometry (LC/APCI-MS) has been used to analyze archaebacterium phospholipids in Natronobacterium magadii. Four major lipids, C20-C20 and C20-C25 PGP-Me and PG were found to exist in this organism. APCI mass spectra provided ample information for elucidation of the lipid structures. As an example, the positive-ion fragmentation of C20-C20 PGP-Me is discussed. In our study, LC/APCI-MS was shown to be an effective method in both the positive- and negative-ion modes for the direct mass spectrometric analysis of archaebacterium phospholipids.

Application of high-performance liquid chromatography/electrospray mass spectrometry for the characterization of membrane lipids in the haloalkaliphilic archaebacterium Natronobacterium magadii

The membrane lipid composition of Natronobacterium magadii, a species of haloalkaliphilic archaebacterium, has been studied by electrospray mass spectrometry (ES-MS), reversed phase high-performance liquid chromatography/electrospray mass spectrometry (LC/ES-MS), and tandem mass spectrometry. The negative-ion and positive-ion electrospray mass spectra and product ion mass spectra of the polar lipids are reported. LC/ES-MS of the total lipid extract in conjunction with product ion mass spectrometry established diphytanyl- and phytanyl, sesterterpanylglycerol-diether analogues of phosphatidylglycerolphosphate methyl ester as the major archaeols, with much lower levels of diphytanyl- and phytanyl, sesterterpanylglycerol-diether analogues of phosphatidylglycerol. In addition to these saturated, isopranyl archaeols, the presence of unsaturated archaeol analogues containing up to three double bonds was detected in Nb. magadii, representing the first description of isoprenyl ether lipids in a haloalkaliphilic archaebacterium.

Qualitative and quantitative mass spectrometric analysis of cyclic nucleotides and related enzymes

Abstract The application of fast atom bombardment mass spectrometry (MS) and collisionally induced dissociation with mass-analysed kinetic energy spectrum scanning to biochemical studies of cyclic nucleotides is reviewed. MS analysis of partially purified tissue extracts has enabled the demonstration of the natural occurrence in mammals and higher plants of five cyclic nucleotides in addition to the previously established adenosine 3′,5′-cyclic monophosphate; analysis of the products of a putative cytidylate cyclase enzyme-catalyzed reaction has demonstrated the biosynthesis of cytidine 3′,5′-cyclic monophosphate and four novel analogues as side products, thereby enabling the development of specific assays. Quantitative applications have included kinetic analysis of phosphodiesterase and cyclic nucleotide-responsive protein kinase activity, while further quantitative applications have included the structural elucidation of synthetic cyclic nucleotide derivatives used in pharmacological studies. The impact of MS upon cyclic nucleotide research and its future potential are discussed.

Differentiation of Isomeric Purine and Pyrimidine Mononucleotides by Fast Atom Bombardment Tandem Mass Spectrometry

Abstract The use of positive ion fast atom bombardment mass-analysed ion kinetic energy (FAB/MIKE) spectroscopy to differentiate the 2′, 3′-and 5′-monophosphate isomers of adenosine, guanosine and cytidine is described.