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A panel of monoclonal antibodies (MAC204, MAC236, MAC265) which recognise extracellular matrix glycoproteins implicated in plant-microbe interactions has been used to study glycoprotein antigens in petioles of turnip (Brassica campestris L.). While MAC204 recognised two glycoproteins (gp120 and gp45) with apparent M(r) 120,000 and 45,000 in petiole extracts made with 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) buffer containing sodium dodecyl sulfate, MAC236 recognised gp120 but not gp45, and MAC265 gave no or only weak reactivity. Tissue dissection studies established that gp120 was predominantly associated with the vascular bundle whereas gp45 was largely associated with the pith. This was consistent with results from tissue prints probed with MAC204 and MAC236 which also suggested a vascular localisation for gp120. Immunoelectronmicroscopy showed that MAC204 and MAC236 both labelled three-way junctions between cells of the phloem and sclerid fibres. Both gp120 and gp45 were shown to carry epitopes in common with known hydroxyproline-rich glycoproteins. Unlike gp45, gp120 could be extracted from petioles with Tris buffer alone and then isolated from this extract by trichloroacetic acid treatment (which left gp120 soluble), followed by size-exclusion and ion-exchange chromatography. Amino acid analysis revealed gp120 to be a novel glycoprotein, particularly rich in proline, lysine, valine and threonine but relatively poor in hydroxyproline. The most abundant sugars were arabinose and galactose. The potential role of this very basic cell surface glycoprotein in plant defence against microbes is discussed.

Antimicrobial saponins are found in many plant species and it has been suggested that these compounds may contribute to the protection of plants against pathogen attack. The ability of a number of phytopathogenic fungi to produce saponin-detoxifying enzymes suggests that detoxification of these compounds may have widespread significance in determining the outcome of plant-fungus interactions. The contribution of saponins to plant defence is being investigated by studies of mechanisms of saponin resistance in pathogens, and also by the isolation of plant mutants which lack saponins.

A panel of monoclonal antibodies that recognize plant extracellular matrix glycoproteins previously implicated in plant-microbe interactions was used to study the effects of pathogen inoculation and wounding on glycoproteins in petioles of Brassica campestris. The panel of monoclonals comprised two sets: JIM11, JIM12, and JIM20 recognize epitopes carried on hydroxyproline-rich glycoproteins (HRGPs) (M. Smallwood, A. Beven, N. Donovan, S. J. Neill, J. Peart, K. Roberts, and J. P. Knox, Plant J. 5:237–246, 1994); MAC204 and MAC265 recognize glycoproteins of the Rhizobium infection thread (K. A. VandenBosch, D. J. Bradley, S. Perotto, G. W. Butcher, and N. J. Brewin, EMBO J. 8:335–342, 1989). Wounding or inoculation of petioles with avirulent strains of pathovars of Xanthomonas campestris induced the synthesis of two new groups of antigens: gp160 ran as a smear on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with apparent molecular mass from 120 to 200 kDa and was recognized by JIM20 and MAC204; gpS remained in the stacking gel on SDS-PAGE and was recognized by JIM11, JIM20, and MAC204. The response to virulent strains of pathovars of X. campestris was either less pronounced or absent. gpS comprised several components that were resolved by cation-exchange chromatography. Some of these components were characterized as extensin-like HRGPs. The level of induction of the gpS group of antigens by virulent strains was not altered by mutation of a number of genes required for basic pathogenicity or by heat-killing the bacteria.

The plasma membrane and the vacuolar membrane (tonoplast) contain water-selective channel proteins called aquaporins. These 27 kDa proteins function as monomers and facilitate osmotic pressure-driven water transport through the membranes. The presence of aquaporins in a membrane increases its water permeability 5 to 10-fold.

Summary: Intracellular ATP concentrations and energy charge were monitored during the growth of Spiroplasma citri and related to the number of colony-forming units, to the pH of the medium and to incorporation of radioactive precursors. Of three different methods used for extracting nucleotides - trichloroacetic acid, perchloric acid and boiling Tris/H2SO4 buffer - the trichloroacetic acid treatment was the most effective. During the active growth phase the ATP concentration increased exponentially, in the same way as other growth parameters. However, as soon as the metabolic activity slowed down, there was a rapid drop of ATP concentration and of total adenylate content, which was followed much later by a parallel decrease in the number of colony-forming units. During active growth, energy charge remained at around 0·9 and decreased only slowly during the stationary phase. Incorporation of labelled thymidine or phenylalanine followed a different pattern during the late phase of the culture and gave less information concerning the viability of the organisms.

By using the M-band technique we have shown that portions of the membranes of Bacillus megaterium and Escherichia coli vary in their affinity for magnesium-Sarkosyl crystals and in phospholipid composition. The portion to which deoxyribonucleic acid is attached comprises as little as 4% of the total cell membrane, has a particularly high degree of affinity for magnesium-Sarkosyl crystals, and is rich in phosphatidylethanolamine. The M-band fractionation does not depend on the use of lysozyme.

Summary: Electrophoresis of constituent proteins in polyacrylamide gels has become a useful aid in the identification of mycoplasmas. Razin & Rottem (1967) showed that mycoplasma proteins dissolved in phenol and acetic acid give reproducible, species-specific electro-phoretic patterns when separated in the presence of acetic acid and urea. We encountered difficulties when using this technique. Mycoplasmas often failed to dissolve completely in the phenol-acetic acid solution and a large fraction of the dissolved protein migrated as a single fast component. Also long periods of electrophoresis (at least 5 h) were necessary to give satisfactory separations. We have therefore investigated an alternative approach, using sodium dodecyl sulphate (SDS) to dissolve the cells prior to electrophoresis in the presence of SDS.

Lipid synthesis during the cell duplication cycle of Bacillus megaterium KM and Escherichia coli was studied by glycerol incorporation both in synchronized cultures and in unsynchronized exponentially growing populations subsequently fractionated according to size (and age). A large transient increase in the rate of incorporation per unit cell mass was observed around the time of cell division, probably reflecting the synthesis of the division septum.

Venous blood was mixed with varying amounts of 6% hydroxyethyl starch solution in vitro and also blood samples were obtained from leukapheresis donors who had received infusions of hydroxyethyl starch. These specimens were evaluated as to the accuracy of erythrocyte typing, direct antiglobulin-testing, and antibody detection using saline, albumin, low-ionic strength saline and papain media. Specimens were stored at 4 degrees C, and testing was repeated at intervals during one week. Moderate difficulties were encountered, due to rouleaux formation, in the interpretation of typing and antibody screening in some samples that contained greater than 30 percent of the hydroxyethyl starch solution. Rouleaux formation was readily dispersed with saline and could easily be distinguished from true agglutination by microscopic examination. Although the apparent difficulties were not extreme, blood bank personnel should be alerted to the potentially misleading effects of hydroxyethyl starch on test results.

SUMMARY: Procedures for the introduction of plasmid DNA into Gram-negative bacteria have been adapted and optimized to permit transformation of the plant pathogen Xanthomonas campestris pathovar campestris with the cloning vector pKT230 and other broad-host-range plasmids. The technique involves CaC12-induced competence and heat shock and is similar to that routinely used for Escherichia coli. Wild-type X. c. campestris strains appear to restrict incoming unmodified DNA, so that plasmid DNA for transformation must be prepared from X. c. campestris (into which it has previously been introduced by conjugation). To overcome this disadvantage a restriction-deficient mutant has been isolated.

Membranes of Bacillus megaterium and other bacteria were bound to crystals of cadmium lauroylsarcosinate and were resolved into nine lipoprotein fractions by elution with potassium chloride and sodium deoxycholate solutions. The fractions differed widely in protein/lipid ratio. Some major protein species were probably common to all the fractions, but the phospholipid composition showed some variation. Electron-microscopic examination after negative staining revealed that material in certain fractions was in the form of particles of diameter about 12nm, and other fractions consisted of amorphous aggregates. The composition of the membrane in terms of the nine fractions was influenced markedly by the conditions of culture of the bacteria. Radioactive-labelling experiments suggested that some fractions served as precursors of others, and one fraction appeared to contain the attachment points of the DNA.