Phenol oxidising enzymes in the grain aphid’s saliva

Sucrose-agarose gels and sucrose liquid diets were used to study the phenol oxidising enzymes in the salivary secretions of the grain aphid, Sitobion avenae (Fabricius). Activity indicating the presence of two oxidoreductases, polyphenol oxidase (PPO) and peroxidase (Px), was found. Both enzymes were present in the aphids stylet sheath (gelling saliva) but only polyphenol oxidase activity was found in the halos around sheaths and thus in watery saliva. Electrical penetration graphs (EPG) revealed that the secretion of these enzymes into the gels, by an individual aphid, was associated with its probing activity observed during penetration of the epidermal and mesophyll tissues. The grain aphids PPO, secreted in its saliva reacted with a range of phenolic compounds. As most of these phenolics occur naturally in cereals, the grain aphid could modify its host-plants phenolic composition. The importance of the grain aphids polyphenol oxidase and peroxidase in detoxifying cereal phenolics is discussed.     

Endothelin-converting enzyme in human tissues

Our aim was to determine whether the expression of endothelin-converting enzyme in human tissues would correlate with the distribution of its substrate, big endothelin-1, and its product, the mature peptide. Site-directed antisera raised against the conserved C-terminus of the mammalian enzyme were used to measure the immunoreactive enzyme in microsomal fractions prepared from tissue homogenates and to localize staining to the endothelial cells lining large conduit and smaller resistance vessels within cardiac, adrenal, respiratory and brain tissue. The activity of endothelin-converting enzyme was measured and characterized in isolated endothelial cells. This pattern of staining in the vascular endothelium paralleled that of mature endothelin and big endothelin-1, and these peptides were detectable by radioimmunoassay in all tissues examined. Immunoreactive endothelin-converting enzyme localized to other cell types, including bronchial epithelial cells, and to fibres within the glial limitans, neuronal processes and cell bodies of the cerebral cortex. Although perivascular astrocytes in the subcortical white matter displayed intense endothelin-converting enzyme-like immunoreactivity, endothelin staining was not detected. The results suggest that endothelin-converting enzyme has a ubiquitous distribution within the human vascular endothelium and is positioned to catalyse the conversion of big endothelin-1 to the biologically active endothelin-1, which on release may contribute to the maintenance of basal tone in humans. Endothelin-converting enzyme localized to epithelial cells in peripheral tissues or astrocytes within the brain may be upregulated in pathophysiological conditions in which endothelin levels are increased and could represent a further target for therapeutic intervention by enzyme inhibitors. © 1998 Chapman & Hall     

Local Periocular Vaccination Protects against Eye Disease More Effectively Than Systemic Vaccination following Primary Ocular Herpes Simplex Virus Infection in Rabbits

Vaccination of experimental animals can provide efficient protection against ocular herpes simplex virus type 1 (HSV-1) challenge. Although it is suspected that local immune responses are important in protection against ocular HSV-1 infection, no definitive studies have been done to determine if local ocular vaccination would produce more efficacious protection against HSV-1 ocular challenge than systemic vaccination. To address this question, we vaccinated groups of rabbits either systemically or periocularly with recombinant HSV-2 glycoproteins B (gB2) and D (gD2) in MF59 emulsion or with live KOS (a nonneurovirulent strain of HSV-1). Three weeks after the final vaccination, all eyes were challenged with McKrae (a virulent, eye disease-producing strain of HSV-1). Systemic vaccination with either HSV-1 KOS or gB2/gD2 in MF59 did not provide significant protection against any of the four eye disease parameters measured (conjunctivitis, iritis, epithelial keratitis, and corneal clouding). In contrast, periocular vaccination with gB2/gD2 in MF59 provided significant protection against conjunctivitis and iritis, while ocular vaccination with live HSV-1 KOS provided significant protection against all four parameters. Thus, local ocular vaccination provided better protection than systemic vaccination against eye disease following ocular HSV-1 infection. Since local vaccination should produce a stronger local immune response than systemic vaccination, these results suggest that the local ocular immune response is very important in protecting against eye disease due to primary HSV-1 infection. Thus, for clinical protection against primary HSV-1-induced corneal disease, a local ocular vaccine may prove more effective than systemic vaccination.     

The Path from the RNA World

We describe a sequential (step by step) Darwinian model for the evolution of life from the late stages of the RNA world through to the emergence of eukaryotes and prokaryotes. The starting point is our model, derived from current RNA activity, of the RNA world just prior to the advent of genetically-encoded protein synthesis. By focusing on the function of the protoribosome we develop a plausible model for the evolution of a protein-synthesizing ribosome from a high-fidelity RNA polymerase that incorporated triplets of oligonucleotides. With the standard assumption that during the evolution of enzymatic activity, catalysis is transferred from RNA → RNP → protein, the first proteins in the ``breakthrough organism' (the first to have encoded protein synthesis) would be nonspecific chaperone-like proteins rather than catalytic. Moreover, because some RNA molecules that pre-date protein synthesis under this model now occur as introns in some of the very earliest proteins, the model predicts these particular introns are older than the exons surrounding them, the ``introns-first' theory. Many features of the model for the genome organization in the final RNA world ribo-organism are more prevalent in the eukaryotic genome and we suggest that the prokaryotic genome organization (a single, circular genome with one center of replication) was derived from a ``eukaryotic-like' genome organization (a fragmented linear genome with multiple centers of replication). The steps from the proposed ribo-organism RNA genome → eukaryotic-like DNA genome → prokaryotic-like DNA genome are all relatively straightforward, whereas the transition prokaryotic-like genome → eukaryotic-like genome appears impossible under a Darwinian mechanism of evolution, given the assumption of the transition RNA → RNP → protein. A likely molecular mechanism, ``plasmid transfer,' is available for the origin of prokaryotic-type genomes from an eukaryotic-like architecture. Under this model prokaryotes are considered specialized and derived with reduced dependence on ssRNA biochemistry. A functional explanation is that prokaryote ancestors underwent selection for thermophily (high temperature) and/or for rapid reproduction (r selection) at least once in their history. Received: 14 January 1997 / Accepted: 19 May 1997     

Prostaglandin E2 and 4-Aminopyridine Prevent the Lipopolysaccharide-Induced Outwardly Rectifying Potassium Current and Interleukin-1β Production in Cultured Rat Microglia

Abstract: Brain inflammation includes microglial activation and enhanced production of diffusible chemical mediators, including prostaglandin E₂. Prostaglandin E₂ is generally considered a proinflammatory molecule, but it also promotes neuronal survival and down-regulates some aspects of microglial activation. It remains unknown, however, if and how prostaglandin E₂ prevents microglial activation. In primary culture, microglial activation is predicted by a characteristic pattern of whole-cell potassium currents and interleukin-1β production. We investigated if prostaglandin E₂ could alter these currents and, if so, whether these currents are necessary for microglial activation. Microglia were isolated from mixed cell cultures prepared from neonatal rat brains and exposed to 0–10 µ M prostaglandin E₂ and lipopolysaccharide for 24 h. Currents were elicited by using standard patch-clamp technique, and interleukin-1β production was measured by ELISA. Peak outward current densities in microglia treated with lipopolysaccharide plus prostaglandin E₂ (10 n M ) were reduced significantly from those of cells treated with lipopolysaccharide alone. Prostaglandin E₂ and 4-aminopyridine (a blocker of outward potassium currents) also significantly reduced interleukin-1β production. Thus, although prostaglandin E₂ is classified generally as a proinflammatory chemical, it has complex roles in brain inflammation that include preventing microglial activation, perhaps by reducing the outward potassium current.     

cis-Isomers of Cytokinins Predominate in Chickpea Seeds throughout Their Development

Trans-isomers of cytokinins (CK) are thought to predominate and have greater biological activity than corresponding cis-isomers in higher plants. However, this study demonstrates a system within which the predominant CK are cis-isomers. CK were measured at four developmental stages in developing chickpea (Cicer arietinum L. cultivar Kaniva) seeds by gas chromatography-mass spectrometry. Concentrations were highest at an early endospermic fluid stage and fell considerably when the cotyledons expanded. The cis-isomers of zeatin nucleotide ([9R-MP]Z), zeatin riboside ([9R]Z), and zeatin (Z) were present in greater concentrations than those of corresponding trans-isomers: (trans)[9R-MP]Z, (trans)[9R]Z, (trans)Z, or dihydrozeatin riboside. Dihydrozeatin, dihydrozeatin nucleotide, and the isopentenyl-type CK concentrations were either low or not detectable. Root xylem exudates also contained predominantly cis-isomers of [9R-MP]Z and [9R]Z. Identities of (cis)[9R]Z and (cis)Z were confirmed by comparison of ion ratios and retention indices, and a full spectrum was obtained for (cis)[9R]Z. Tissues were extracted under conditions that minimized the possibility of RNase hydrolysis of tRNA following tissue disruption, being a significant source of the cis-CK. Since no isomerization of (trans)[²H]CK internal standards occurred, it is unlikely that the cis-CK resulted from enzymic or nonenzymic isomerization during extraction. Although quantities of total CK varied, similar CK profiles were found among three different chickpea cultivars and between adequately watered and water-stressed plants. Developing chickpea seeds will be a useful system for investigating the activity of cis-CK or determining the origin and metabolism of free CK.     

A re-evaluation of species limits in Chaetobromus (Danthonieae: Poaceae)

Past changes in the taxonomy of Chaetobromus , a Cape grass with considerable fodder potential, reflect a history of instability due to inadequate past sampling and intergradation among taxa in most characters. This biosystematic study differs from earlier investigations by its much more intensive approach to sampling, taking into consideration inter- and intrapopulational variation in 75 anatomical, morphological and cytological characters, across a total of 169 samples. Both phenetic methods and population aggregation analysis revealed the existence of three major groups, approximating three formerly described taxa and reflecting divergent ecological strategies in Chaetobromus . However, continuity among groups in most characters and a lack of clear-cut diagnostic field characters argues against their recognition at species level. Thus, Chaetobromus is here described as monotypic, the type species, C. involucratus , comprising three subspecies C. involucratus ssp. involucratus, C. involucratus ssp. sericeus and C. involucratus ssp. dregeanus .     

Rapid screening of solvents and carrier compounds for lactic acid recovery by emulsion liquid extraction and toxicity on Lactobacillus casei (ATCC 11443)

This paper describes a rapid method to identify the best solvent and carrier compound combinations with the highest extraction capability and the lowest microbial toxicity characteristics for product recovery from microbial fermentation. The extraction system has an aqueous phase, and an emulsion phase, which was a blend of sodium carbonate and organic phase [91% (v/v) organic solvent, 5% (v/v or wt/v) carrier compound, and 4% (v/v) surfactant Span 80]. Alamine 336, or tri-n-octylamine in n-heptane; Alamine 336, Alamine 304, or tributyl phosphate in hexane; and Alamine 304 or tributyl phosphate in iso-octane; Alamine 304 or Amberlite in xylene demonstrated high lactic acid extraction. For determination of bacterial toxicity of selected solvent and carrier compounds, Lactobacillus casei subsp. rhamnosus (ATCC 11443) was grown in LAF medium containing one of the selected organic solvent, carrier compound, and Span 80 in 250 ml flask at 37 °C and 125 rpm. Samples were collected regularly during 48 hour incubation, and measured for changes in cell density by absorbance at 620 nm, cell count using a fluorescent dye with flow cytometry, and lactic acid, and glucose concentrations by HPLC. Hexadecane:tributyl phosphate, n-dodecane:tri-n-octylamine, and kerosene:tri-n-octylphosphine oxide demonstrated the least microbial toxicity among the tested blends with excess solvent media. Whereas, hexanes:Alamine 304 and xylenes:Alamine 304 were nontoxic in solvent saturated media.This revised version was published online in October 2005 with corrections to the Cover Date.     

Distribution of calmodulin protein and mRNA in growing pollen tubes

 Pollen tube growth is a vital process for angiosperm fertilisation and is dependent on the presence of a tip-focused gradient of cytosolic free calcium ([Ca²⁺]_c). In order to clarify some of the target molecules which convey the Ca²⁺ signal information, we investigated calmodulin distribution during tube growth. Fluorescently labelled calmodulin was pressure microinjected into pollen tubes and its distribution monitored by confocal microscopy. Calmodulin distributes evenly throughout the cell, but some of its binding sites form a V-shaped collar behind the apical region. This specific association dissipates upon growth arrest, and suggests an interaction of calmodulin with cytoskeletal-bound target proteins. The distribution of calmodulin mRNA was also analysed by microinjection of fluorescently labelled mRNA. No specific pattern was observed, with an even localisation in the body of tube and a lower concentration in the cell apex. Studies with localised application of inhibitors/activators indicate that calmodulin plays a crucial role in tip elongation but does not direct tube orientation. Received: 6 March 1998 / Revision accepted: 20 April 1998