Another brick in the wall? Recent developments concerning the yeast cell envelope

To a yeast, the cell wall is an important living organelle performing a number of vital functions, including osmotic and physical protection, selective permeability barrier, immobilized enzyme support and cell-cell recognition and adhesion. Our basic model of wall structure involves attachment of secreted mannoproteins to a fibrillar inner layer of β-glucan. Recent work has emphasised the importance of chitin in lateral walls, examined the mechanisms of attachment of mannoproteins to the various cell wall glucan fractions and elucidated the pathway of β-glucan synthesis, by means of resistance to glucan-binding killer toxins. The conventional view of wall structure has been challenged by the discovery of a class of GPI-anchored, serine/threonine-rich wall-proteins. It has been suggested, that these proteins are anchored in the plasma membrane, spanning the wall with extended O-glycosylated structures and protruding out into the medium. Examination of these proteins shows a diversity of structures, sizes and behaviour that makes it improbable that these represent a new class of wall proteins. The possible roles of one of these proteins associated with flocculation, Flo1p, are discussed.     

Phospholipid coatings for the prevention of membrane fouling

The aim of the present work was the development of phosphorylcholine-based treatments for biofiltration membranes and the demonstration that such treatments prevent or inhibit protein fouling. Microfiltration membranes of cellulose triacetate, polyether sulphone and polyvinylidene fluoride were etched with oxygen in a plasma chamber to generate surface hydroxyl groups and were then treated with the monomer 2-methacryloyloxyethyl phosphorylcholine. These membranes were evaluated with water, buffer, bovine serum albumin (BSA), yeast fermentation broth, beer and orange juice. The treatment of cellulose triacetate membranes reduced both the initial flux and the extent of water fouling. In terms of the integrated flux, these factors tended to cancel each other out. For protein, the membranes gave similar or higher fluxes but worse fouling. The cellular feed (yeast) reacted more favourably to the coating than the BSA. The polyether sulphone was scarcely affected by the coating; fouling remaining high with most ‘real’ feeds. There was lower initial flux but less flux decline with water and beer. Washing with water and cleaning with Tergazyme did not restore the initial flux. Polyvinylidene fluoride membranes gave the most positive results. In most cases, the coating both increased initial flux and decreased the rate of fouling. The coating was particularly effective for BSA and for beer and orange juice, where fouling is probably caused by a polysaccharide rather than by a protein. Electron microscopy showed, nonetheless, that fouling by proteins was accompanied by protein adsorption primarily on the upper surface of the membrane and that coated membranes showed less deposition and in different places than did untreated membranes.     

Strengthening fire-damaged concrete by confinement with fibre-reinforced polymer wraps

Extensive research has shown that fibre-reinforced polymer (FRP) wraps are very effective for strengthening concrete columns for increased axial and flexural load and deformation capacity, and this technique is now widely used around the world. The study reported in this paper extends the FRP confinement technique to strengthening fire-damaged circular concrete columns. An experimental programme was undertaken to study the compressive strength and stress-strain behaviour of both unconfined and FRP-confined plain concrete cylinders after being heated to various elevated temperatures for up to four hours and cooled to room temperature. The results show that FRP confinement is highly effective for enhancing the load-carrying capacity of even severely fire-damaged concrete columns. The results also yield insights into the mechanics of FRP confinement of concretes of similar composition but with varying compressive strengths. A modified version of a pre-existing confinement model is proposed for use in designing FRP strengthening schemes for fire-damaged concrete columns.     

Yeast flocculation: Cationic inhibition

Yeast flocculation was inhibited by high concentrations of a number of salts. Calcium and magnesiun salts were potent inhibitors and cesium salts were least effective. Partial inhibitors by different salts additive and were completely reversible by salt removal. Inhibition by salts was time dependent; prolonged incubation increased the degree of inhibition. Salt inhibition was partly caused by the action of salts lowering the buffer pH value, and partly caused by chaotropic inhibition of proteins on the surfaces of flocculent cells. Flocculation receptors on non-flocculent cells were ubaffected by high salt concentration. At sub-inhibitory salt concentrations, there was an enhancement of flocculation in both rate and extent.     

Yeast flocculation: a dynamic equilibrium

The steady state in yeast flocculation is a dynamic equilibrium between flocculated and dispersed yeast cells. The free cell concentration is directly proportional to the total cell concentration and may be expressed as an equilibrium constant. Increased agitation decreases floc size and equilibrium constant whilst increasing floc-surface area and free-cell concentration. Values of equilibrium constant are influenced by agitation in a complex relationship probably involving the floc-surface area and floc momentum. Inhibition of flocculation by mannose and low pH is reversible and becomes greater with increased agitation. Both these inhibitions appear consistent with a weakening of flocculent bond strength by these inhibitors.     

Activities of the semisynthetic glycopeptide LY191145 against vancomycin-resistant enterococci and other gram-positive bacteria

LY191145 is the prototype of a series of compounds with activities against vancomycin-resistant enterococci derived by modification of the glycopeptide antibiotic LY264826. LY191145 had MICs for vancomycin- and teicoplanin-resistant enterococci of < or = 4 micrograms/ml for 50% of isolates and < or = 16 micrograms/ml for 90% of isolates. Its MICs for vancomycin-resistant, teicoplanin-susceptible enterococci were 1 to 8 micrograms/ml. LY191145 retains the potent activities of its parent compound against staphylococci and streptococci. In vivo studies in a mouse infection model confirmed these activities. This compound indicates the potential of semisynthetic glycopeptides as agents against antibiotic-resistant gram-positive bacteria.     

Breast cancer angiogenesis--new approaches to therapy via antiangiogenesis, hypoxic activated drugs, and vascular targeting

Several groups have shown that quantitation of tumor angiogenesis by counting blood vessels in primary breast cancer gives an independent assessment of prognosis. Poor prognosis is associated with high blood vessel counts. We have shown that the rate of cell division in endothelial cells is much higher in breast tumours than in normal breast. Breast cancer cell lines and primary human breast tumours express a wide range of vascular growth factors, including VEGF, placenta growth factor, pleiotrophin, TGF beta 1, acidic and basic FGF, and platelet-derived endothelial cell growth factor. Inhibiting angiogenesis by blocking vascular growth factors would be difficult with highly specific agents, but drugs with a broader spectrum of antagonism may be effective. We have developed several suramin analogues which are less toxic than suramin in vivo but more potent in inhibiting angiogenesis, and these have been developed for Phase I. A combination of anti-angiogenesis agents with drugs activated by hypoxia may also be useful, because anti-angiogenesis alone may not kill cells, whereas activation of hypoxic drugs could synergize. New endpoints may be necessary because inhibition of new blood vessel formation may not cause tumour regression. Thus, the endpoint of stable disease and biochemical assessment of inhibition of angiogenesis may be much more important in therapeutic studies and for drug development in the future. The prognostic importance of angiogenesis suggests that this should be a major new therapeutic target.     

Yeast flocculation: receptor definition by mnn mutants and concanavalin A.

Yeast flocculation involves the binding of surface lectins on flocculent yeasts, to carbohydrate receptors present as constituents of yeast cell walls. Receptors were investigated by coflocculation of flocculent strains of Saccharomyces cerevisiae, of both Flo 1 and NewFlo phenotypes, to known mnn mutants which vary in the wall mannan structure. Strong coflocculation was found with mnn1, mnn4, mnn9 and control strains, while very little coflocculation was found with mnn2 and mnn5 strains. In contrast, aggregation of these mutants by concanavalin A, a lectin with similar sugar inhibition to NewFlo phenotype flocculation, showed strong aggregation of mnn1, mnn4 and mnn5 strains and poor aggregation of mnn2 and mnn9 strains. The mmn mutant data suggested that flocculation receptors were the outer-chain mannan side-branches, two or three mannose residues in length, confirming an earlier theory based on sugar inhibition data. The similarities and differences between flocculation and concanavalin A aggregation are discussed.     

Accelerated radiotherapy, carbogen and nicotinamide (ARCON) in locally advanced head and neck cancer: a feasibility study

BACKGROUND AND PURPOSE: ARCON (Accelerated Radiotherapy, CarbOgen, Nicotinamide) achieves a large therapeutic gain in rodents. A phase I/II study was therefore undertaken to determine its feasibility in patients with locally advanced head and neck cancer. MATERIALS AND METHODS: The accelerated regime CHART was used in 35 patients given carbogen and/or nicotinamide with 11 small volume fractions. Eight patients received carbogen, 12 received nicotinamide and 15 were treated with ARCON. Treatment compliance, side-effects and acute mucositis were monitored in all cases. RESULTS: All patients underwent CHART as intended. In the 23 patients receiving carbogen, two failed to complete treatment. Compliance with nicotinamide was much lower. Out of 25 patients, only 52% received 10-11 doses of the 80 mg/kg/day of the drug. The most common side-effect was nausea and vomiting, which responded to standard anti-emetics in almost half of the patients. Historical comparisons with the CHART head and neck trials indicate that there was no increase in the severity of acute mucositis in any of these patients. Although the observation period is not sufficiently long to be definitive (median 20 months) there is no evidence of an increase in late normal tissue reactions. CONCLUSIONS: ARCON using CHART as the radiotherapy protocol is feasible in patients with advanced head and neck cancer. However, we are concerned about the low compliance rate in our patients, which is far lower than that reported elsewhere. The implications are discussed together with identifying strategies for increasing compliance.     

Yeast flocculation: Calcium specificity

Expression of flocculation in yeast requires the presence of multi-charged cations. Calcium ions fulfil this role over a broad pH range. At near neutral pH, magnesium and a variety of transition elements can induce flocculation. Calcium-induced flocculation was competitively inhibited by excess sodium ions whereas magnesium-induced flocculation was not competitively inhibited. Potassium and lithium ions caused similar inhibition but to a lesser extent. ⁴⁵Ca effux from yeast cells was greatly increased by the external presence of buffer and cations. Inhibition of flocculation by chelating agents, EDTA or EGTA, was overcome by excess calcium ions. Flocculation could not be induced, under these conditions, by excess magnesium or transition-element salts. It was concluded that yeast flocculation has a direct specific requirement for calcium ions. Other ions cause flocculation indirectly by effecting calcium ion leakage from cells, which is then able to initiate flocculation. Low calcium concentrations were susceptible to competitive inhibition by sodium ions present in most acidic buffers. In addition, citrate ions inhibited flocculation, probably by sequestration of leaked calcium. Flocculation by salts, other than calcium, was thus restricted to a neutral or high pH range.