Rapid deposition of wheat cell wall structural proteins in response to Fusarium-derived elicitors.

Two novel cell wall structural proteins of spring wheat (Triticum aestivum L. em Thell.). undergo rapid deposition in the cell wall matrix in a H(2)O(2)-dependent reaction after the elicitation of cultures with Fusarium graminearum (L.)-derived elicitor. The amino acid compositions of these proteins were remarkably similar and indicated that they were highly acidic (pI 3.8). These proteins contained 13--17% each of Gly, Glx and Ser with lesser amounts (6--8%) of Ala, Asx and Thr, and it has been suggested that they are known as glycine- and serine-rich proteins (GSRPs). SELDI-MS ionization spectra demonstrated that these proteins have low molecular masses of 8590 and 4292 Da. These results are discussed in relation to the possible role of these novel proteins in rapid, cell wall defensive reactions to pathogenic attack.

Na+, K+-ATPase isozyme diversity; comparative biochemistry and physiological implications of novel functional interactions.

Na+, K+-ATPase is ubiquitously expressed in the plasma membrane of all animal cells where it serves as the principal regulator of intracellular ion homeostasis. Na+, K+-ATPase is responsible for generating and maintaining transmembrane ionic gradients that are of vital importance for cellular function and subservient activities such as volume regulation, pH maintenance, and generation of action potentials and secondary active transport. The diversity of Na+, K+-ATPase subunit isoforms and their complex spatial and temporal patterns of cellular expression suggest that Na+, K+-ATPase isozymes perform specialized physiological functions. Recent studies have shown that the alpha subunit isoforms possess considerably different kinetic properties and modes of regulation and the beta subunit isoforms modulate the activity, expression and plasma membrane targeting of Na+, K+-ATPase isozymes. This review focuses on recent developments in Na+, K+-ATPase research, and in particular reports of expression of isoforms in various tissues and experiments aimed at elucidating the intrinsic structural features of isoforms important for Na+, K+-ATPase function.

Role of extensin peroxidase in tomato (Lycopersicon esculentum Mill.) seedling growth.

It is proposed that inhibition of extensin peroxidase activity leads to a less rigid cell wall and thus promotes cell expansion and plant growth. A low-molecular-weight inhibitor derived from the cell walls of suspension-cultured tomato cells was found to completely inhibit extensin peroxidase-mediated extensin cross-linking in vitro at a concentration of 260 microg/ml. The inhibitor had no effect upon guaiacol oxidation catalyzed by extensin peroxidase or horseradish peroxidase. We have demonstrated that the light-irradiated inhibition of plant growth may be partially offset by inhibition of endogenous extensin peroxidase activity. Overall plant growth was enhanced by up to 15% in the presence of inhibitor relative to control plants. Inhibitor-treated and illuminated tomato hypocotyls grew up to 15% taller than untreated controls. The inhibitor had no effect upon etiolated plants over a 15-d period, suggesting that only low levels of peroxidase-mediated cross-linking can be found in the cell walls of etiolated plants. SDS-PAGE/Western blots of ionically bound protein from both etiolated and illuminated hypocotyls identified a doublet at 57/58.5 kDa which is immunoreactive with antibodies raised to tomato extensin peroxidase. Levels of the 58.5-kDa protein, determined by SDS-PAGE, were at least threefold higher in illuminated tomato hypocotyls than in etiolated hypocotyls. Three fold higher levels of extensin peroxidase, elevated in-vitro extensin cross-linking activity and 15% higher levels of cross-linked, non-extractable extensin were observed in illuminated tomato hypocotyls compared with etiolated tomato hypocotyls. This suggests that white-light inhibition of tomato hypocotyl growth appears to be mediated, at least partially, by deposition of cell wall extensin, a process regulated by Mr-58,500 extensin peroxidase. Our results indicate that the contribution of peroxidase-mediated extensin deposition to plant cell wall architecture may have an important role in plant growth.

Molecular aspects of cell wall modifications during fruit ripening.

Fruit ripening is associated with cell wall modifications. The present review focuses on cell wall components and the nature of noncovalent and covalent interactions in the primary cell wall. The role of structural protein cross-links are evaluated within the context of cell wall-mediated changes in texture during fruit ripening. The article discusses molecular approaches in fruit cell wall interactions to regulate processes in fruit ripening in order to improve post-harvest textural characteristics.

Extensin from suspension-cultured potato cells: a hydroxyproline-rich glycoprotein, devoid of agglutinin activity.

Enhanced deposition and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in the plant cell wall is acknowledged to contribute to the formation of a resistant barrier against pathogen infection. We have isolated, from suspension-cultured potato (Solanum tuberosum L. cv. Desiree) cells, two forms of soluble HRGP, a cross-linked and a monomeric form; the latter can be converted to the cross-linked form by incubation with tomato extensin peroxidase and H2O2. The monomeric form was purified by Sephacryl S-200 gel-filtration, reverse-phase high-performance liquid chromatography and Mono-S cation-exchange chromatography into two isoforms (A, a minor form; B, a major form). The properties of the B isoform were further investigated. A quantitative enzyme-linked immunosorbent assay of the B isoform, using tomato extensin antiserum, showed a titration curve at a high antibody-dilution range comparable to that of purified tomato extension monomer (M.D. Brownleader and P.M. Dey, 1993, Planta 191: 457-469). The amino acid and carbohydrate compositions were similar to those of tomato extensin, but did not match well with the other two HRGPs from potato, potato lectin and potato bacterial agglutinin. These observations demonstrate the similarities of the B isoform to extensin. The homogeneity of the B isoform was demonstrated by its ability to be fully cross-linked in vitro, leaving no residual protein, into a high-molecular-weight form by the action of extensin peroxidase. The trifluoroacetic acid-deglycosylated sample migrated as a single protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, SDS-PAGE and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry indicated a molecular weight of approximately 67 kDa. Circular-dichroism spectroscopy demonstrated that the molecule possess an extended polyproline II helix conformation with no evidence of alpha-helix or beta- sheet secondary structure. In conclusion, we refer to this HRGP as potato extensin. As proposed for other extensins, potato extensin is likely to play a role in cell wall architecture and plant disease resistance.

Investigations into the molecular size and shape of tomato extensin.

The molecular characteristics of soluble extensin from tomato have been investigated. An apparent molecular mass greater than 240 kDa has been previously observed with the shape-dependent method of gel-filtration chromatography [Brownleader and Dey (1993) Planta (Berlin) 191, 457-469]. Tomato extensin is a heavily glycosylated protein that does not migrate into SDS/polyacrylamide gels. This shape-dependent behaviour raises doubts about agreement between the observed apparent mass and the absolute value. The molecular mass measured with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was 72.3 kDa, with no evidence of any other species except a doubly charged ion. The sample was therefore considered to be monodisperse under the conditions used. Electron microscopy of soluble extensin showed the presence of particles 40-50 nm in length and 2.0-2.5 nm in width. A minority of these particles showed a central 'kink'. A number of smaller and generally wider particles (20 nm x 2-4 nm) were considered to be folded monomers and larger particles were thought to be dimers. Sedimentation analysis showed that extensin exists in a rapid monomer-dimer equilibrium in the concentration range and buffer used. Sedimentation equilibrium data gave a Kd of 8.5 microM and sedimentation velocity data generated a Kd between 1 and 10 microM. The concentration dependence of the measured sedimentation coefficient was used, together with hydrodynamic bead modelling, to define plausible shapes for monomer and dimer. This suggests that monomeric extensin is an elongated rod of length 40 nm and width 2 nm, which forms staggered dimers of average length 50 nm and width 3 nm. Extensin is an integral component of the primary cell wall. The physical characteristics (size, shape and form) of the rod-like extensin have been evaluated in this paper so that the role that extensin plays in primary cell wall architecture and during plant disease resistance can be more fully understood.

Purification and Partial Characterization of Tomato Extensin Peroxidase.

Early plant defense response is characterized by elevation of activity of peroxidases and enhanced insolubilization of hydroxyproline-rich glycoproteins, such as extensin, in the cell wall. The insolubilization process (cross-linking between soluble extensin precursor molecules) is catalyzed by extensin peroxidases. We have ionically eluted extensin peroxidases from intact water-washed suspension-cultured tomato (hybrid of Lycopersicon esculentum Mill. and Lycopersicon peruvianum L. [Mill.]) cells and purified them to homogeneity by molecular sieve and cation-exchange chromatography. Four ionic forms of peroxidase (PI,PII,EPIII, and EPIV) were resolved; only the latter two cross-linked tomato soluble extensin. The molecular weight (34,000-37,000), amino acid composition, and isoelectric point (9.0) of the extensin peroxidases were determined. Substrate specificities of the enzymes were investigated: soluble extensin and potato lectin (a hydroxyproline-rich glycoprotein with a domain that strongly resembles extensin) were cross-linked by only two forms of the enzyme, whereas bovine serum albumin, aldolase, insulin, a number of other marker proteins, and proteins eluted from tomato cells (except extensin) could not be cross-linked. We have also isolated a yeast elicitor that enhances total peroxidase activity and extensin insolubilization within 1 h of challenge in cultured cells of tomato. A highly sensitive enzyme-linked immunosorbent assay technique using polyclonal antiserum raised against soluble tomato extensin was used to demonstrate extensin insolubilization in vivo. A tomato cell-wall peroxidase that cross-links extensin has been purified and may have a role in plant defense.

Gastrointestinal symptoms and diet of members of an irritable bowel self-help group.

A self-administered questionnaire was completed by 103 members of a self-help group for irritable bowel syndrome (IBS) and 130 controls, to compare the presence of IBS symptoms. Dietary fibre prescription, compliance and efficacy were documented in the IBS group. There were significant differences in the frequency of defaecation and number of symptoms present between the two groups. Most of the IBS subjects had been prescribed a high fibre diet but adherence to the diet and symptomatic improvement were poor. In contrast many IBS sufferers were following a range of diets not prescribed by their doctor. The seriousness of the IBS condition was highlighted by the fact that 8% of the IBS group had retired early due to their condition.

Enzyme immobilization by adsorption.

Much has already been written on the subject of biocatalyst (cell/enzyme) immobilization, and it is not the function of a text such as this to go into all the various aspects of the field. For general reviews on the area, the reader is referred to refs. 1-3. The introduction to this chapter is also meant, however, to serve as an introduction to the succeeding three chapters, and is thus more general than is usual in this series.

Enzyme immobilization by entrapment.

Entrapment methods of immobilization are mostly used in cell immobilization procedures, but have found some application with enzymes, particularly when the enzyme is essentially a dead cell or crude homogenate(1). In theory the entrapped enzyme is not attached to the polymer; its free diffusion is merely restrained. In practice, however, some or all of the enzyme may be an integral part of the polymer matrix; for example, chain termination in polyacrylamide gel formation may occur onto the enzyme (see also ref. 2).

Enzyme immobilization by covalent bonding.

Enzyme immobilization by covalent bonding to an insoluble polymer has the supposed advantage of irreversible binding of the enzyme to the support matrix. Many of the bonds commonly employed are not 100% stable in use, however. Unless some care is taken to ensure that the enzyme can withstand the reaction conditions, substantial loss of enzyme activity will occur. Even under favorable conditions, 60% loss of activity is not uncommon.

Cell immobilization.

We will be concerned in this chapter with methods that may be used to immobilize live rather than dead cells. The use of immobilized viable cells is seen as an alternative approach to extracellular metabolite production by traditional fermentation technology. There are a number of reasons why this may be advantageous. First, immobilized cells are easily utilized in continuous flow reactors without, theoretically, the contamination of the product stream by cellular material. Second, cell densities in the reactor of at least one order of magnitude greater than with traditional fermentation techniques are possible. Third, immobilization has been reported to enhance the longevity of cells, in particular plant cells, which is therefore useful in extending the productive life of slow-growing cells. Fourth, it has been suggested that immobilization may enhance the release of secondary metabolites from cells, particularly when (as in plant cells) these metabolites are normally stored intracellularly.

Stability of alginate-immobilized algal cells.

Investigations were carried out using immobilized Chlorella cells to determine the diameter, compressibility, tolerance to phosphate chelation, and ability to retain algal cells during incubation of various alginate beads. These physical bead characteristics were found to be affected by a variety of interactive factors, including multivalent cation type (hardening agent) and cell, cation, and alginate concentration, the latter exhibiting a predominant influence. The susceptibility of alginate beads to phosphate chelation was found to involve a complex interaction of cation type, concentration, and pH of phosphate solution. A scale of response ranging from gel swelling to gel shrinking was observed for a range of conditions. However, stable calcium alginate beads were maintained in incubation media with a pH of 5.5 and a phosphate concentration of 5 microM. A preliminary investigation into cell leakage from the beads illustrated the importance of maintaining a stable gel structure and limiting cell growth to reduce leakage.

An electron microscope study of hepatocellular changes in the rat during chronic treatment with acetamide. Parenchyma, foci and neoplasms.

Male Leeds strain rats were fed a diet containing 5.0% by weight of acetamide (AA), for up to 35 weeks, inducing a high incidence of hepatic cell neoplasms. The sequential morphological changes induced by AA in the hepatic parenchymal cells were studied by electron microscopy and were compared with those observed in foci of cellular alteration, neoplastic nodules and hepatocellular carcinomata. The observed fine structural changes including early glycogen depletion, dispersal and dislocation of the rough endoplasmic reticulum (ER), smooth ER proliferation and nuclear irregularity and nucleolar abnormalities. Glycogenosis developed in some cells during treatment and was characteristic of the 'clear cell' foci, as well as being common in the neoplasms. The most consistent alteration, observed in parenchymal cells and the cells of foci, nodules and carcinomas, was that involving the rough ER. This persisted after withdrawal of AA from the diet, whereas many other changes were transient, and may represent a change in differentiation associated with neoplastic induction.