Russell P. Newton

Cyclic AMP and higher plants

Abstract Despite the evidence in support, the extent of which is outlined in this review, the occurrence of cyclic AMP in tissues of higher plants has been doubted by a number of previous reviewers. Recent MS and other evidence vindicates earlier identification of an adenosine nucleotide from plant tissues as adenosine 3′:5′-cyclic monophosphate. The additional demonstration of 3′: 5′-cyclic nucleotide phosphodiesterases in higher plants, together with adenylate cyclase, a specific cyclic AMP binding protein, and calmodulin, means that plants possess all the necessary components for a functional cyclic AMP-regulated system. Whether such a system does function in plants is considered as are also the reported physiological effects of exogenously supplied cyclic AMP on plant tissues.

The properties and purification of a Blaberus craniifer plasma protein which enhances the activation of haemocyte prophenoloxidase by a β 1,3-glucan

Abstract A plasma factor has been detected in the cockroach, Blaberus craniifer , which, in haemocyte lysates, enhances the activation of a peptidase and prophenoloxidase (proPO) by laminarin (a β 1,3-glucan). The factor was isolated by affinity chromatography on laminarin-Sepharose and FPLC ion-exchange chromatography. It is a glycoprotein with a molecular weight ( M w ), as determined by SDS-electrophoresis, of ca 90,000. Amino acid analysis showed a very high content ( ca 65%) of hydrophilic amino acids. No peptidase or phenoloxidase (PO) activity was detected in the isolated plasma protein. After removal of the proPO-activating protease by chromatography on Blue Sepharose, the resulting partially purified proPO could no longer be activated by laminarin or laminarin plus purified plasma factor.

The antibacterial activity against MRSA strains and other bacteria of a <500Da fraction from maggot excretions/secretions of Lucilia sericata (Diptera: Calliphoridae).

The application of Lucilia sericata larvae to chronic, infected wounds results in the rapid elimination of infecting microorganisms, including MRSA. Previously, we demonstrated in vitro antibacterial activity of native excretions/secretions (nES) from L. sericata and partially purified two low mass antibacterial compounds with masses of 0.5-10kDa and <500Da. The present study reports the antibacterial effects of the <500Da fraction (ES<500) on the growth and morphology of a range of bacteria, including 12 MRSA strains. Distinct morphological changes were observed in Bacillus cereus and Escherichia coli following exposure to ES<500. Flow cytometry and confocal microscopy analyses, in conjunction with turbidometric and CFU assays, revealed bacteriostatic activity of nES against S. aureus and E. coli. ES<500 also demonstrated bacteriostatic activity against S. aureus, however, bactericidal activity and the induction of a viable but non-culturable state were observed with ES<500-treated E. coli.

Analysis of the free nucleotide pools of mammalian tissues by high-pressure liquid chromatography

Abstract Perchloric acid extracts of tissues were neutralized with tri-N-octylamine and, after removal of ClO4−, subjected to preliminary purification on a Cu2+-loaded column of Chelex 100. A high-pressure liquid chromatographic (HPLC) anion-exchange procedure was developed and gave good resolution of the naturally occurring free nucleotides on a single column. Where heterogeneous peaks eluted, an effective supplementary analysis was achieved by reverse-phase HPLC. An HPLC paired-ion technique was also evaluated for use in nucleotide analysis. Although anion-exchange was best for overall separation of nucleotides, both reverse-phase and paired-ion chromatography gave excellent separation of cyclic nucleotides. Reduced pyridine nucleotides were detected and measured in the form of their acid-decomposition products. The recovery of nucleotides was examined throughout the described analytical techniques and shown to be quantitative.

Fingerprint profile of Ginkgo biloba nutritional supplements by LC/ESI-MS/MS

Ginkgo biloba is one of the most popular herb nutrition supplements, with terpene lactones and flavonoids being the two major active components. A fingerprint profile method was developed using a capillary HPLC/MS method which can identify more than 70 components from the G. biloba product. The method allows the flavonoids and terpene lactones to be detected simultaneously and information of both the parent ion and its fragmentation can be obtained in just one HPLC/MS run. Targeted post-acquisition analysis allows mass spectrometric information regarding the identification of flavonoid components to be easily distinguished from other data, however the same approach for terpene lactones was less successful due to dimer formation and requires further development. The fingerprint profiles of five commercial G. biloba nutritional supplements were obtained and compared; variation of some components among the samples was observed and fortification could be detected. In the quality control analysis of the G. biloba product this method could be viewed as complementary to specific quantitative analysis of some bioactive components of the herb.

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.

Amino acid derivatives from Lucilia sericata excretions/secretions may contribute to the beneficial effects of maggot therapy via increased angiogenesis.

Background  Maggot therapy, utilizing the larvae of Lucilia sericata, has been reported to reduce the bacterial load within wounds and also to enhance wound healing. Maggot excretions/secretions (ES) have been shown to have a role in the success of maggot therapy. While the protein content of ES has been investigated, to date little research has focused on the small metabolites present in ES and their potential contribution to the therapy. Study of the molecular composition of the secretions and the potential bioactivities present will allow for a more detailed evaluation of the efficacy of maggot therapy.

Production of efrapeptins by Tolypocladium species and evaluation of their insecticidal and antimicrobial properties

This study shows for the first time that Tolypocladium species produce efrapeptins, a group of toxic peptides, in vivo but the quantities are too small to account for insect death, suggesting that these insecticidal compounds work in concert with other pathogenicity determinants. There is inter- and intraspecific variation in efrapeptin production in vitro by Tolypocladium species. T. parasiticum produced only efrapeptin E, in small quantities. Efrapeptins were detectable 48 h after inoculation and increased with biomass. The relative amounts of individual efrapeptins (C, D, E, F, G) produced by T. niveum in vitro were D>E>F>C>G but in vivo they were D>F>C>E>G. Efrapeptins were toxic to a wide range of insects when injected into the haemocoel. Mortality was dose-related. Efrapeptins also exhibited limited antifungal and antibacterial activity. Micrococcus luteus was considered an excellent indicator of efrapeptin presence in culture filtrate extracts because of its extreme sensitivity to these compounds.

Occurrence of guanosine 3′,5′-cyclic monophosphate (Cyclic GMP) and associated enzyme systems in Phaseolus vulgaris

Abstract Cyclic GMP, isolated from Phaseolus vulgaris , has been unequivocally identified by NMR and FAB-mass spectrometry with MIKES-scanning. Radioimmunoas

Mass spectrometric identification of adenosine 3′:5′-cyclic monophosphate isolated from a higher plant tissue

Abstract Mass spectrometric evidence is presented confirming the identification of the adenosine nucleotide previously isolated from tissues of Phaseolus vulgaris as adenosine 3′: 5′-cyclic monophosphate.