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Summary Pulmonary zygomycosis is a relatively uncommon complication of solid organ or peripheral blood stem cell transplantation and has a high associated mortality. Optimal therapy consists of complete resection of infected tissue and treatment with amphotericin B (AmB). We describe two patients, one of whom underwent orthotopic heart transplantation and the other who received a peripheral blood stem cell transplant, who were diagnosed with invasive pulmonary zygomycosis. Both patients were treated with a liposomal preparation of AmB and early partial resection of the infected structures followed by prolonged posaconazole maintenance therapy. Despite incomplete resection, this treatment regimen resulted in a favourable outcome in both patients, including survival of more than 17 months in one patient at last follow up. For patients in whom complete resection of pulmonary zygomycosis is not possible, subtotal resection and treatment with liposomal AmB followed by therapy with posaconazole may be an effective treatment option.

Tillage practices, crop rotations, and government regulations all play a part in whole profitability. Mixed integer programming models can evaluate all alternatives simultaneously. This article provides a profitability example modeled to reflect the actual farm program acreage, farm program payment yields, baseline acreage, crop mix, soils, and equipment investment. Results reveal land rental as the most profitable operation until farm size reaches a minimum of 1100 acres. The model selected reduced tillage for crop mixes beyond 1200 acres.

Analysis of extracts from two woad species (Isatis tinctoria and Isatis indigotica) and Polygonum tinctorium revealed that only one indigo precursor (indican) was present in Polygonum, but two precursors were found in Isatis spp. This was done using high performance liquid chromatography (HPLC), coupled to an evaporative light scattering detector (ELSD). In Isatis spp., the indigo precursors indican and a fraction representing isatan B were identified. The proportion of indican and isatan B was different between the two Isatis spp. tested. For the first time, it was possible to quantify the precursors in woad plant species, and the results were found to be in good agreement with those made from total indigo quantification using two different spectrophotometric methods or a derivatization technique.

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), made by Fab1p, is essential for vesicle recycling from vacuole/lysosomal compartments and for protein sorting into multivesicular bodies. To isolate PtdIns(3,5)P2 effectors, we identified Saccharomyces cerevisiae mutants that display fab1delta-like vacuole enlargement, one of which lacked the SVP1/YFR021w/ATG18 gene. Expressed Svp1p displays PtdIns(3,5)P2 binding of exquisite specificity, GFP-Svp1p localises to the vacuole membrane in a Fab1p-dependent manner, and svp1delta cells fail to recycle a marker protein from the vacuole to the Golgi. Cells lacking Svp1p accumulate abnormally large amounts of PtdIns(3,5)P2. These observations identify Svp1p as a PtdIns(3,5)P2 effector required for PtdIns(3,5)P2-dependent membrane recycling from the vacuole. Other Svp1p-related proteins, including human and Drosophila homologues, bind PtdIns(3,5)P2 similarly. Svp1p and related proteins almost certainly fold as beta-propellers, and the PtdIns(3,5)P2-binding site is on the beta-propeller. It is likely that many of the Svp1p-related proteins that are ubiquitous throughout the eukaryotes are PtdIns(3,5)P2 effectors. Svp1p is not involved in the contributions of FAB1/PtdIns(3,5)P2 to MVB sorting or to vacuole acidification and so additional PtdIns(3,5)P2 effectors must exist.

A method to quantify the indigo precursor indican (indoxyl-beta-D-glucoside) in Polygonum tinctorium L. has been developed. Plant material was extracted in deionized water, and indican was identified and quantified using high performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD). Results confirmed that with this method it is possible to measure indican content in a short time, obtaining reliable and reproducible data. Using this method, leaf indican content was quantified every 15 days during the growing season (from May to October) in P. tinctorium crops grown in a field experiment in Central Italy. Results showed that indican increased along the growing season until flowering and was positively affected by photosynthetic active radiation (PAR). Indican is naturally hydrolyzed by native beta-glucosidase to indoxyl and glucose, the indoxyl yielding indigo. The activity of two enzymes, sweet almond beta-glucosidase and Novarom G preparation, were compared with P. tinctorium native beta-glucosidase to evaluate indigo production. Results showed that the ability to promote indigo formation increased as follows: almond beta-glucosidase

As an adaptation process to the growth retardation provoked by the presence of nonlethal concentrations of ergosterol biosynthesis inhibitors, Ustilago maydis alters the ratio of linoleic to oleic acid bound to plasma membrane complex lipids [Hernandez, A., Cooke, D.T., Lewis, M. & Clarkson, D.T. (1997) Microbiology143, 3165–3174]. This alteration increases plasma membrane H+-ATPase hydrolytic activity. Activation of H+-ATPase by the linoleic/oleic acid proportion is noncompetitive, nonessential and only involves changes in the maximum velocity of the pump. Optimum pH, affinity to MgATP and constants for the inhibition by vanadate and erythrosin B remain unchanged. This all indicates that activation of plasma membrane H+-ATPase by unsaturated fatty acids differs clearly from glucose-induced activation observed in yeast. Also, it is a physiologically relevant event similar to other, as yet uncharacterized, changes in plasma membrane H+-ATPase hydrolytic activity observed in plants and fungi, as part of an adaptation process to different stress conditions.

The hypothesis was tested that the negative effect of mineral nutrient deprivation (-N and -P) on the hydraulic conductance (Lo) of wheat roots may be relieved by increasing the fluidity of plasma membrane (PM) lipids through elevated temperature. An increase in root temperature from 20 to 30 degrees C increased the sap flow, Jv, from the excised roots of nutrient-deprived plants for 4 h, with a corresponding increase in Lo. In the same period, there was a decline in the flux of osmotically active solutes (Js) to the xylem. As the duration of the period at 30 degrees C increased, it was clear that the differential in Lo between control and nutrient-deprived roots was maintained, even though Lo was significantly greater than the initial (20 degrees C) value after 48 h. The lipid order parameter, determined by fluorescence polarization of 1,6 diphenyl-1,3,5-hexatriene (DPH), decreased markedly in two-phase purified PMs in the first 4 h of treatment at 30 degrees C, but thereafter remained steady. The differential between control and nutrient deprived roots was maintained throughout the 48 h period. The correlation between lowered Lo in nutrient deprived roots and increased PM lipid ordering remained unchanged in conditions where the overall membrane fluidity was increased by elevated temperature.

ATP and insulin stimulate increases in phosphatidylinositol (3,4,5)-trisphosphate levels in myeloid-derived U937 cells. Quantification of FITC-phalloidin binding by fluorescence-activated cell sorting reveals that both ATP and insulin stimulate actin polymerization with distinctive kinetics in U937 cells. The response to ATP is rapid and dose-dependent with an EC50 of 200 nM, and is abolished by pre-incubation with the Ca2+ chelator BAPTA-AM. At 800 nM concentration, wortmannin, a potent inhibitor of phosphoinositide 3-kinase (PI3K), blocks the late, but not the early phase of actin polymerization stimulated by 100 nM ATP. Responses elicited by 10 μg/ml insulin are slower, smaller and more transient than responses to ATP, and are inhibited by preincubation with 100 nM wortmannin. Actin polymerization can also be stimulated by thapsigargin, but not by phorbol ester, providing further evidence for a role for Ca2+ in actin polymerization. These data implicate distinct Ca2+ and PI3K-mediated pathways in the regulation of actin polymerization.

This paper discusses the application of a particular two-phase partitioning system to the isolation of plasma membranes from heterogeneous starting material, differing in physiological age. Plasma membranes were isolated from hypocotyl segments of mung beans (Vigna radiata L. Wilczek) on four successive days in order to examine the variation caused by ageing of the seedling. Additionally, the segments were cut at different positions of the hypocotyl to measure variation caused by position-related ageing. To assess purity and degree of contamination of the plasma membrane-enriched preparations, a series of membrane enzyme markers were screened for all isolated fractions. Glucan synthetase II activities were enriched in the plasma membrane fractions, but enrichment and recovery became less pronounced with increasing age. Plasma membrane ATPase activity affected by VO43-, Ca2+ and K+ was similar in all segments throughout the time-course of the experiment (4 days). However, control ATPase activity varied with segment origin: the physiologically oldest segments showed only 75% activity compared to the youngest ones. Km and Vmax values indicated a smaller proportion of active enzyme but higher substrate affinity as the age of the segments increased. Contamination by intracellular membranes was minimal and unrelated to tissue age.

Plasma membrane vesicles (ca 40% inside-out, after one freeze-thaw cycle) were extracted and purified from the shoots of oat (Avena sativa L.) and chickweed (Stellaria media L.) using the two-phase aqueous polymer technique. In the presence of ATP or GTP, a rapid uptake of 45Ca2+ occurred (0.77 and 0.62 nmol Ca2+ mg-1 protein, for ATP and GTP, respectively, in oat, and 0.53 and 0.51 nmol Ca2+ mg-1 protein, for ATP and GTP, respectively, in chickweed). Nucleotide-dependent Ca2+-transport was sensitive to 1 μM Erythrosin B (with ATP. inhibited by 52% in oat and in chickweed by 72%; with GTP, inhibition was similar in both species at ca 67%); ATP-dependent uptake was greater in oat than in chickweed, but not stimulated by calmodulin. Addition of the calcium ionophore A-23187 resulted in the release of label from the vesicles (41% and 63% release with ATP, and 24% and 52% release with GTP, in oat and chickweed, respectively). The results obtained suggest that Ca2+-transport is independent of the proton pump. In oat, kinetic data indicate a discontinuity in the absorption isotherm at 10 μM free calcium.

Plasma membrane vesicles were extracted from the shoots of 10-day-old oat, rye and rice plants and incubated with either cholesterol, stigmasterol or a mixture of sitosterol + campesterol (60:40). After ascertaining that the sterol composition of the vesicles had been altered by this treatment, the specific hydrolytic activity of the membrane-bound H+-ATPase (EC 3. 6. 1. 35) was measured. The results indicated that, although all sterols were taken up, cholesterol was best integrated into the plasma membrane of the species tested. After treatment, ATPase activity was altered in oat and rice, but not in rye. The results are discussed in the context of sterol/lipid and sterol/protein interactions in the plasma membrane.

A qualitative method has been devised to analyse the indigo precursors in leaf extracts of woad (Isatis spp.) using HPLC coupled to an evaporative light scattering detector and confirmed using HPLC coupled, on line, to a particle beam mass spectrometer. Using this technique, indican has been positively identified in shoot extracts of European (I. tinctoria) and Chinese (I. indigotica) woad. Greater amounts of indican were found in the Chinese compared with the European woad. Copyright © 2000 John Wiley & Sons, Ltd.

The plasma membrane cation-pumping ATPase is a key regulatory enzyme in cellular events. However, the mechanism by which its activity is controlled is still unclear. Lipid modulation is acknowledged to be a potentially important mechanism in this respect, although precisely how it is achieved remains controversial. This review examines the modulation of plant, animal, yeast and fungal plasma membrane-bound ATPases by sterols and phospholipids as well as discussing critically two putative modulating mechanisms, the ‘Annulus Hypothesis’ and membrane fluidity. The use of xenobiotics in helping to elucidate the role played by lipids in ATPase regulation is examined and a sequence of events by which lipid modulation may occur is suggested.

Objectives. To compare lipoprotein(a) [Lp(a)] and albumin concentrations in patients with chronic renal disease receiving different forms of treatment and to determine, if any, the relationship between these variables. Design. A prospective cross-sectional, case-controlled study. Setting. A tertiary referral nephrology and dialysis unit. Subjects. Forty-four consecutive non-diabetic patients with chronic renal failure treated by renal transplantation (n = 18), haemodialysis (n = 18), continuous ambulatory peritoneal dialysis (CAPD; n = 8), and 30 healthy controls from subjects drawn from University personnel were studied. Interventions. Fasting morning venous blood was analysed for Lp(a), albumin, total cholesterol and glucose concentrations. Main outcome measures. Comparison of plasma levels of these variables between the sub-groups. Results. Concentrations (median; 95% CI) of Lp(a) were significantly (P < 0.05) higher (38.4 mg dl−1; range 15.4–72.0) and of albumin lower (31.6 g l−1; range 28–35.2) in the CAPD group compared with both control subjects and other groups of chronic renal disease patients. Conclusions. The elevated Lp(a) concentrations seen only in association with reduced albumin concentrations in CAPD patients suggest a regulatory role for albumin with albumin losses stimulating production of Lp(a).

The eukaryotic cell membrane is thought to consist of a mobile bilayer of phospholipid, sometimes intercalated with sterols, in which peripheral and transmembrane proteins are embedded. This provides a model whereby the mode of action of many fungicidal and herbicidal compounds can be rationalised and understood. Some compounds, such as the polyene antibiotics, steroidal saponins and certain phytoalexins, induce membrane malfunction by direct insertion, often complexing with vital components. Others, such as paraquat and the nitrodiphenyl ether herbicides, cause membrane damage by inducing lipid peroxidations. Furthermore, there are many compounds, including the sterol-biosynthesis-inhibiting fungicides and the aryloxyphenoxypropionate and cyclohexanedione herbicides, which interfere with the biosynthesis of membrane components. Because membrane structure is fundamentally similar in eukaryotes, it is important to discover the reasons for any differential toxicity displayed by these compounds and, if necessary, to find ways of optimising desirable patterns of selectivity.

The nature and degree of the biological activity shown by triadimenol and paclobutrazol are influenced considerably by the absolute configuration of the two asymmetric carbon atoms present in the molecules. The order of the activity of triadimenol enantiomers was found to be: fungitoxicity—1S, 2R > 1R, 2R > 1R, 2S > 1S, 2S; inhibition of gibberellin biosynthesis—1R, 2S > 1S, 2S > 1R, 2R > 1S, 2R; and inhibition of plant sterol biosynthesis—1R, 2S≈1S, 2R > 1R, 2R > 1S, 25. The relative activity of paclobutrazol enantiomers was: fungitoxicity— 2R, 3R > 2S, 3R > 2R, 3S≈2S, 3S; inhibition of gibberellin biosynthesis—2S, 3S > 2R, 3S > 2R, 3R > 2S, 3R; and inhibition of plant sterol biosynthesis—2R, 3S > 2R, 3R > 2S, 3S > 2S, 3R. These data indicate that the R configuration at the chiral carbon bearing the hydroxyl group is the prime determinant for fungitoxicity whereas enantiomers having the S configuration at this carbon are effective inhibitors of gibberellin biosynthesis. This agrees well with published data for the structurally related vinylazoles. Plant sterol C-14a demethylation was not always inhibited by enantiomers having high fungicidal activity thus indicating that the structural features necessary for binding to the plant C-14a demethylase may differ from those needed for the analogous fungal enzyme.

Sterols and their derivatives promote and maintain growth and development in plants and fungi by acting as membrane constituents and probably also as hormones, engaged in control of metabolism. Inhibitors of sterol biosynthesis, operating at various stages in the pathway, are useful probes for investigating these functions. Some of the inhibitors have assumed considerable commercial importance as agricultural fungicides and antimycotic drugs in medicine. There is also potential for using similar compounds to regulate plant growth. The work reviewed here, much of it done within the last few years, points unmistakably to a fundamental role for sterols in promoting growth and development in both plants and fungi. A clear target enzyme in the biosynthetic pathway for growth inhibition in both plants and fungi is the 14α-methylsterol demethylase. Work with triazoles, particularly with closely related analogues and separated enantiomers, has provided an insight into the affinity of these inhibitors for the different forms of cytochrome P-450 which catalyse fungal sterol 14α-demethylation, plant sterol 14α-demethylation and plant ent -kaurene oxidation. It may become increasingly necessary to evaluate the relative contribution of these processes to optimise performance of fungicides, herbicides and PGRs, both with regard to their activity at the primary site of action and with a view to controlling side effects. There is clear scope here for extending and improving in vitro enzyme assay methods. The effectiveness of the morpholines as agricultural fungicides contrasts with the high tolerance of plants to cyclopropyl and Δ 8 -sterols which are readily induced by application of these compounds. Further study of the basic reason for morpholine toxicity is, therefore, an area well worthy of further investigation. Inhibition of squalene epoxidase by allylamines has proved successful in the development of medical antimycotics but no agricultural fungicides, based on this mode of action, have been forthcoming. Other enzymes of the pathway have yet to be commercially exploited but the C-24 alkylation process seems clearly linked to growth in both plants and fungi and hence would appear to be a particularly promising site for new inhibitors. The pre-squalene enzyme HMG-CoA reductase seems to offer potential for regulating or stopping plant growth. Above all there is a need to evaluate precisely how sterols and their derivatives control growth and developmental processes. The judicious use of inhibitors of the type described here may help in this undertaking which, in turn, could generate new ideas for the design and discovery of more active and specific compounds.

Plasma membrane vesicles were extracted from the shoots of 10-day-old oat, rye and rice plants and incubated with either cholesterol, stigmasterol or a mixture of sitosterol + campesterol (60:40). After ascertaining that the sterol composition of the vesicles had been altered by this treatment, the specific hydrolytic activity of the membrane-bound H+-ATPase (EC 3.6.1.35) was measured. The results indicated that, although all sterols were taken up, cholesterol was best integrated into the plasma membrane of the species tested. After treatment, ATPase activity was altered in oat and rice, but not in rye. The results are discussed in the context of sterol/lipid and sterol/protein interactions in the plasma membrane.