Phosphate uptake in an obligately marine fungus: a specific requirement for sodium.

Phosphate uptake in the obligately marine fungus Thraustochytrium roseum is maximally stimulated by sodium chloride in a range of concentrations (0.2 to 0.4 molar) similar to those commonly encountered in littoral habitats. The effectiveness of sodium chloride for phosphate transport extends beyond its osmotic function and can be attributed specifically to sodium. Increases in respiration in the presence of the salt can be ascribed primarily to an osmotic effect.

Cyclodextrins: a new tool for the controlled lipid depletion of thylakoid membranes.

Cyclodextrins (CDs) have been used in controlled lipid depletion of thylakoid membranes avoiding the use of either detergents or lipolytic enzymes. Spinach thylakoid membranes were first treated with different CDs under various conditions. After removal of the CDs by washing, the amounts of mono-- and digalactosyldiacylglycerol (MGDG and DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), protein, pigment and plastoquinone remaining in the membranes were determined. The main results, obtained with alpha-CD and heptakis-(2,6-di-O-methyl)-beta-CD (DM-beta-CD), were as follows. (1)Acyl lipids were removed from thylakoid membranes by both CDs (DM-beta-CD being more efficient than alpha-CD; the extent of removal depended on both CD and chlorophyll concentrations. (2) alpha-CD presented a higher selectivity towards lip classes than did DM-beta-CD, but in both cases the removal order was SQDG > PG > MGDG > DGDG. (3) alpha-CD showed a preference for those lipids containing saturated 16-carbon acyl chains whereas DM-beta-CD was essentially insensitive to the fatty acid composition of the lipids. (4) The protein, chlorophyll and carotenoid contents of thylakoids were not affected by CD treatments. (5) Plastoquinones were removable but in small amounts only and with a low efficiency (DM-beta-CD > alpha-CD). (6) For all lipid classes, the extent of lipid removal was higher at 0 degrees than at 20 degrees C. (7) The presence of MgCl(2) reduced the removal of PG and SQDG but not affect galactolipid depletion levels. (8) Staple lipid depletion levels in thylakoid membranes were reached after 5-10 min of CD treatment at 0 degrees C. (9) Of the four CDs tested, only three (alpha-CD, beta-CD, and DM-beta-CD) promoted lipid depletion whereas one (hydroxypropyl-beta-CD) failed completely to do so. It is concluded that CD-mediated lipid removal provides a valuable and versatile tool to achieve controlled and specific lipid depletions in biological membranes. A few examples of the consequences of a CD-induced lipid depletion on fluorescence and electron transport properties of thylakoids are given to show the usefulness of CDs in the investigation of structure-function relationship in photosynthetic membranes.

Transmembrane distribution of phospholipids and their involvement in electron transport, as revealed by phospholipids A2 treatment of spinach thylakoids.

Thylakoid membranes were treated with either pancreatic or snake venom phospholipase A2, and the residual phospholipid content of these membranes was determined and compared to the rates of Photosystem II and/or Photosystem I electron transports. The hydrolysis curves of both phosphatidylglycerol and phosphatidylcholine displayed a first, rapid phase which was almost temperature-insensitive, followed by a second, slower phase which depended strongly on the temperature. When pancreatic phospholipase A2 had access either to the outer face or to both faces of the thylakoid membrane, either only part of or all the phospholipids, respectively, could be hydrolysed. These results were interpreted as indicating an asymmetric distribution of phospholipids across the thylakoid membrane, phosphatidylglycerol and phosphatidylcholine being preferentially located in the outer and the inner layer, respectively. When acting on uncoupled thylakoid membranes, phospholipase A2 exerted an inhibitory effect on Photosystem II activity and a stimulatory effect on Photosystem I activity. The involvement of phosphatidylcholine and of phosphatidylglycerol in electron transport activities of Photosystem II and of Photosystem I are discussed with special reference to the role of the external and internal pools of these phospholipids.

A single and continuous spectrophotometric assay for various lipolytic enzymes, using natural, non-labelled lipid substrates.

A rapid, continuous spectrophotometric assay for measuring the amount and activity of several lipolytic enzymes is described. It is based on the metachromatic properties of the cationic dye safranine, and makes use of the fact that an adequate combination of a lipolytic enzyme with one of its substrates leads to a change in the net negative charge at the lipid/water interface, which is monitored by the absorbance change of safranine. Utilizing this method, most lipolytic enzymes can be detected in very low amounts (milliunit or less) in about 1 min without employing radiolabelled lipids or synthetic lipid analogues. Over a wide range of enzyme concentrations, there is a good linearity between the initial hydrolysis rate (determination by the safranine method) and the amount of enzyme. The versatility of the assay is illustrated by examples showing how phospholipase A2, triacylglycerol hydrolase, phospholipase D or phospholipase C (either general or phosphatidylinositol-specific) activities can be detected, either separately or sequentially. Due to its high sensitivity, simplicity, and rapidity, this assay should find its main application in monitoring column effluents during the purification steps of lipolytic enzymes.

(Galacto) lipid export from envelope to thylakoid membranes in intact chloroplasts. II. A general process with a key role for the envelope in the establishment of lipid asymmetry in thylakoid membranes.

The transfer of organelle of newly synthesized lipid molecules from inner envelope to thylakoid membranes, as well as their subsequent transbilayer distribution in these membranes, have been studied in intact chloroplasts isolated from young and mature spinach, young pea and mature lettuce leaves, using a recently developed methodology (Rawyler, A., Meylan, M. and Siegenthaler, P.A. (1992) Biochim. Biophys. Acta 1104, 331-341). Three radiolabelled precursors were used. UDP-[14C]galactose allowed to follow the fate of mono- and digalactosyldiacylglycerol (MGDG and DGDG) made from polyunsaturated, preexisting diacylglycerol (DAG), whereas [14C]acetate and [14C]glycerol 3-phosphate were used to follow the fate of MGDG and phosphatidylglycerol (PG), respectively, after de novo synthesis. MGDG, DGDG and PG molecules assembled at the envelope level were found to be exportable to thylakoids in amounts strictly proportional to the amounts synthesized, provided that the necessary substrates were not limiting. Lipid export was class-selective; under our conditions, as much as 50-80% of the MGDG, 87% of the PG and 20-30% of the DGDG synthesized were exported to thylakoids. However, within the MGDG class labelled from [14C]acetate, there was hardly any selectivity in the export of its various molecular species. For MGDG, the proportionality coefficient, which reflects the efficiency of the export process, was higher in chloroplasts from young than from mature leaves, and higher in spinach than in pea and lettuce. Temperature affected the efficiency of galactolipid export in a class-dependent way. MGDG synthesis and export had similar Q10 values of about 4 in young and 3 in mature spinach leaves, while the Q10 of DGDG export was higher than that of its synthesis. In most cases, the transmembrane distribution of labelled lipids in thylakoids was found to match closely the corresponding distribution of mass, regardless of plant age and species and of incubation time and temperature. In some cases however, small but significant differences occurred between the label and the mass transbilayer distributions of MGDG (labelled molecules more inwardly oriented), DGDG and PG (more outwardly oriented). We propose a general model in which the thylakoid lipid asymmetry is primarily preestablished in the chloroplast envelope by the topography of its lipid-synthesizing enzymes, together with the occurrence of relatively fast lateral diffusion and translocation rates of the newly synthesized lipids. Transient fusions between inner envelope and thylakoid membranes would allow lipid export by lateral diffusion and build the observed lipid asymmetry in the latter.

The topology of phosphatidylglycerol populations is essential for sustaining photosynthetic electron flow activities in thylakoid membranes.

The transmembrane distribution of phosphatidylglycerol (PG) was determined in rightside-out (RO) and inside-out vesicles (IO) obtained by fragmentation of spinach thylakoids in a Yeda press, followed by partition in an aqueous dextran-polyethyleneglycol two-phase system. Using the phospholipase A(2) from porcine pancreas to digest selectively PG molecules in the outer monolayer (exposed to the incubation medium) of the membrane, we found the molar outside/inside distribution to be 70/30+/-5 in RO and 40/60+/-3 in IO. The transmembrane distribution of PG in IO was the opposite of that in intact thylakoids (molar ratio 58/42+/-3). The phospholipid population which sustained most of the uncoupled photosystem II electron flow activity was localized in the inner monolayer (exposed to the thylakoid lumen) of both thylakoid and RO membranes. In contrast, the activity in IO membranes was highly dependent on the PG population located in the outer monolayer. This finding brings the first direct demonstration of the dependence of the photosynthetic electron flow activity on the integrity of the inner topological pool of PG in the thylakoid membrane.

Galactolipid export from envelope to thylakoid membranes in intact chloroplasts. I. Characterization and involvement in thylakoid lipid asymmetry.

The galactolipid transfer from inner envelope to thylakoid membranes has been studied in intact spinach chloroplasts. Plastids, isolated from mature leaves, were dark-incubated in the presence of UDP-[14C]galactose. After various synthesis periods at 5 or 25 degrees C, intact plastids were reisolated and osmotically lysed. Thylakoid membranes were then prepared by a special procedure which removed greater than or equal to 99% of the envelope amount initially present. Under these conditions, purified thylakoids were found to contain radiolabelled MGDG and DGDG, indicating that galactolipids were exported from the inner envelope. The amounts exported were proportional to the amounts synthesized. About 55% of the MGDG and 25% of the DGDG synthesized in plastids were transferred to thylakoids, irrespectively of incubation time or temperature. The MGDG/DGDG radioactivity ratio was 7 in intact plastids and 18 in thylakoids, suggesting a preferential export of MGDG. Purified thylakoid membranes were then submitted to a lipolytic treatment designed to discriminate between the MGDG and DGDG pools belonging to the outer (stroma-facing) or to the inner monolayer. The radioactivity present in the lyso-products (corresponding to the outer pools) and in the residual parent lipids (corresponding to the inner pools) was measured. The labelled MGDG showed a transmembrane outside:inside distribution (mol%) of 50:50, which differed from the native (mass) MGDG asymmetry of 64:36. In contrast, the label and mass asymmetries of DGDG gave the same value of 15:85. These label distributions were affected neither by incubation time (from 5 to 90 min) nor by temperature (from 5 to 25 degrees C). We discuss the possibilities that transient fusions between the stroma-facing monolayers of the inner envelope and of the thylakoid membrane, and/or galactolipid transfer protein(s), together with lipid translocating activities in thylakoids, may account for the galactolipid export observed in mature spinach chloroplasts.

Adjuvant chemotherapy in operable breast cancer: cyclophosphamide, methotrexate, and fluorouracil versus chlorambucil, methotrexate, and fluorouracil--11-year results of Swiss Group for Clinical Cancer Research trial SAKK 27/82.

PURPOSE: To compare two adjuvant combination chemotherapies, cyclophosphamide, methotrexate, and fluorouracil (CMF) and chlorambucil, methotrexate, and fluorouracil (LMF), for patients who had undergone potentially curative surgery for unilateral breast cancer, in terms of relapse, survival, and toxicity. PATIENTS AND METHODS: Selection criteria was as follows: stage pT1-3a, N+ or N-, M0, less than 72 years of age. Eligible patients were randomized to receive either CMF (cyclophosphamide 100 mg/m2 orally on days 1 to 14, methotrexate 40 mg/m2 intravenously (I.V.) on days 1 and 8, fluorouracil 600 mg/m2 I.V. on days 1 and 8) or LMF (Leukeran [Wellcome A.G., Bern, Switzerland] 5 mg/m2 orally on days 1 to 14 with the some I.V. cytostatic drugs). Follow-up examinations were performed every 3 months during the first 3 years after mastectomy, and every 6 months thereafter. RESULTS: A total of 246 patients were randomized, of whom 232 who were fully eligible and contribute to the analyses presented here. No statistically significant difference in favor of adjuvant CMF over LMF emerges after a median follow-up duration of 11.2 years, for either overall survival (P = .15) or disease-free survival (P = .14). A consistent trend suggestive of a possible relative benefit associated with CMF should be pointed out. However, CMF presents a significantly worse toxicity profile as concerns hematologic parameters as well as alopecia, nausea, and vomiting. CONCLUSION: This prospective trial has not identified a statistically significant difference in disease-free survival or overall survival between the two adjuvant regimens LMF and CMF. Although a trend in favor of CMF has been observed in premenopausal patients, this has to be weighted against its definitely more pronounced toxicity profile.

Phase II study of doxifluridine in advanced colorectal adenocarcinoma.

Doxifluridine, a new fluoropyrimidine derivative, was tested in a cooperative phase II trial by the Swiss Group for Clinical Cancer Research in advanced measurable colorectal cancer. The drug was given in a five consecutive day schedule by a bolus intravenous injection at a dose of 4 g/m2 per day and repeated every three to four weeks. Of 42 eligible patients, 40 had no previous chemotherapy. Response was defined in 27 patients having received two or more courses of doxifluridine. Seven responses (26%) were observed. Responses were seen only in sigmoid and rectum primary tumors. Toxicity consisted mainly of leukopenia (53% of the evaluable patients), nausea and vomiting (38%). Other toxicities such as dermatitis, myocardial injury, and hair loss were also observed. Doxifluridine has therapeutic activity, albeit limited, in advanced rectosigmoid adenocarcinoma.

Direct gas-phase detection of nerve and blister warfare agents utilizing active capillary plasma ionization mass spectrometry.

Ultrasensitive direct gas-phase detection of chemical warfare agents (CWAs) is demonstrated utilizing active capillary plasma ionization and triple quadrupole mass spectrometry (MS) instrumentation. Four G- agents, two V-agents and various blistering agents [including sulfur mustard (HD)] were detected directly in the gas phase with limits of detection in the low parts per trillion (ng m(-3)) range. The direct detection of HD was shown for dry carrier gas conditions, but signals vanished when humidity was present, indicating a possible direct detection of HD after sufficient gas phase pretreatment. The method provided sufficient sensitivity to monitor directly the investigated volatile CWAs way below their corresponding minimal effect dose, and in most cases even below the eight hours worker exposure concentration. In general, the ionization is very soft, with little to no in-source fragmentation. Especially for the G-agents, some dimer formation occurred at higher concentrations. This adds complexity, but also further selectivity, to the corresponding mass spectra. Our results show that the active capillary plasma ionization is a robust, sensitive, "plug and play" ambient ionization source suited (but not exclusively) to the very sensitive detection of CWAs. It has the potential to be used with portable MS instrumentation.

Evidence for lipid kinase activities in spinach chloroplast envelope membranes.

Three spinach chloroplast envelope membrane preparations (i.e. whole, outer and inner membranes) were incubated in the presence of [gamma-32P]ATP. After lipid extraction and separation by TLC, four main phosphorylated lipids were detected by autoradiography in whole envelope preparations. These phospholipids were identified by comparing their Rf with that of lipid markers and by a deacylation procedure. They were found to be phosphatidic acid (PA) and lyso-PA, L-alpha-phosphatidyl-inositol 4-monophosphate (PIP) and lyso-PIP. These lipids were not equally distributed in the outer and inner envelope membranes. Chloroplast envelope membranes were verified not to be contaminated by plasma membranes. It is concluded that lipid kinase activities are associated with spinach chloroplast envelope membranes.

Phase II trial of anaxirone (TGU) in advanced colorectal cancer: an EORTC Early Clinical Trials Group (ECTG) study.

Anaxirone, a rationally synthesised triepoxide derivative, was given to 46 patients with metastatic colorectal cancer. Good risk patients received 800 mg/m2 as a rapid intravenous injection every 4 weeks, whereas poor risk patients received 650 mg/m2. Of 46 patients, 45 were evaluable for toxicity and 42 for efficacy analysis. There were 37/45 patients with poor risk, showing no difference in toxicity as compared to good risk patients. The major toxic effect was myelosuppression with 34% of all patients experiencing grade 3 or 4 leucopenia; thrombocytopenia was less frequent. Locoregional phlebitis occurred in 66% of the patients. There was no objective tumour response to anaxirone in 42 evaluable patients. Only 4 patients achieved stabilisation of the disease lasting maximally up to 248 days. Anaxirone is inactive in metastatic colorectal cancer.

Phase II study of 5'-deoxy-5-fluorouridine (doxifluridine) in advanced malignant melanoma.

Forty-two patients with malignant melanoma were treated with doxifluridine, 4000 mg/m2 daily X 5, repeated every 3 weeks. The daily dose was reduced to 3000 mg/m2 in patients who had experienced severe myelosuppression with prior chemotherapy. A total of 35 patients were evaluable for response, and 25 of these received two or more courses. Two responses were observed. Toxicity mainly took the form of nausea, vomiting, stomatitis, dizziness, ataxia, and fatigue. Mild leukopenia was frequent (43%). Nadir counts less than 1.5 X 10(9)/l leukocytes or 50 X 10(9)/l platelets were seen in 7% and 2% of the courses respectively. Doxifluridine has no useful activity against malignant melanoma.

Rapid analysis of membrane lipids using a combination of thin-layer chromatography and scanning of photographic negatives.

A simple method is proposed for the analysis of the distribution and changes in membrane lipids subjected to different treatments (lipolytic, aging, etc.). This technique involves only one thin-layer chromatographic step followed by a scanning of the photographic negative of the charred thin layer. This method is time saving, inexpensive and does not require the technical skill usually demanded in lipidology. The precision of this method is compared with that obtained with the classical TLC-GC method: its variability is roughly twice that of the TLC-GC method.

Phosphatidylglycerol molecular species of photosynthetic membranes analyzed by high-performance liquid chromatography: theoretical considerations.

A reversed-phase high-performance liquid chromatography technique was developed to separate, identify, and quantify individual phosphatidylglycerol (PG) molecular species in thylakoid membranes isolated from higher plant leaves. PG was first separated by thin-layer chromatography; then the dinitrobenzoyl derivatives of diacylglycerols produced after phospholipase C hydrolysis of PG were separated by a C18 reversed-phase column and detected at 254 nm. A linear response of the detector was observed in the range of 0.025 to 12 nmol of PG molecular species. It was established that there was an excellent correlation (r = 0.996) between the carbon and double-bond number in the aliphatic residues and the relative retention time of dinitrobenzoyl derivatives. A new equivalent carbon number value (ECN*) which takes into consideration the number of cis-(nc) and trans-(nt) double bonds per molecular species was defined as ECN* = CN - 2nc - nt, where CN is the number of carbon atoms in the aliphatic residues. The logarithm of the retention time increased linearily as a function of ECN* value. However, in this type of correlation, it may happen that two molecular species of PG having distinct relative retention times had the same ECN* value. In this case, the two molecular species can be identified by the linear correlation (r = 1) existing between the reciprocal of the relative retention time and the number of double bonds (0 < or = n < or = 3) in the separate 18:n/delta 3-trans-hexadecenoic acid -16:1(3t)- and 18:n/16:0 molecular species series. The advantages of this method are good separation, cohort elution time, quantitative precision, and predictable retention times of PG molecular species from chloroplast membranes. The method has been used routinely to identify the ten PG molecular species of thylakoid membranes in squash, potato, lettuce, and spinach leaf: 18:3/16:1(3t), 18:3/16:0, 18:2/16:1(3t), 18:2/16:0, 18:1/16:1(3t), 18:1/16:0, 18:0/16:1(3t), 18:0/16:0, 16:0/16:1(3t), and 16:0/16:0.

Do glycerolipids display lateral heterogeneity in the thylakoid membrane?

The lateral heterogeneity of lipids in the thylakoid membrane has been questioned for over 20 yrs. It is generally believed that glycerolipids are asymmetrically distributed within the plane of the membrane. In the present investigation, we isolated several thylakoid membrane domains by using sonication followed by separation in an aqueous dextran-polyethylene glycol two-phase system. This technique, which avoids detergent treatments, allowed us to obtain stroma and grana lamellae vesicles as well as grana central core and grana margin vesicles from thylakoids. The relative distribution of the four lipid classes, i.e., monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, and phosphatidylglycerol, was found to be statistically identical in all four thylakoid fractions and in whole thylakoids. Similarly, the relative amount of fatty acids in each individual lipid and the eight main phosphatidylglycerol molecular species was identical in all thylakoid membrane fractions tested as well as in the intact thylakoid membrane. Based on presently available procedures for obtaining thylakoid subfractions that are unable to discriminate microdomains within the membrane, it is concluded that glycerolipids are evenly distributed within the plane of the thylakoid membrane. These data are discussed in terms of "bulk" and "specific" lipids.