Low Temperature Treatments Induce an Increase in the Relative Content of Both Linolenic and {lambda}3-Hexadecenoic Acids in Thylakoid Membrane Phosphatidylglycerol of Squash Cotyledons

The effect of low temperatures on the fatty acid compositionof phosphatidylglycerol (PG) in thylakoid membranes, in particularon the ratios of nmol% 16:1(3t) (mg fresh weight)^–1 ofcotyledons and nmol 16:1(3t) (mg chlo rophyll)^–1 weremeasured during squash seedling growth. Plants were germinatedand grown for one day at 30°C then were either kept at 30°C(control plants) or trans ferred to low temperatures (18, 14or 10°C). When plant were transferred from 30°C to lowtemperatures, the increase in fresh weight was gradually limited.The lowe the temperature, the smaller was the fresh weight.In contrast, the relative content of 16:1(3t) and 18:3, as wella the ratios of nmol 16:1(3t) (mg chlorophyll)^–1 and mol%16:1(3t) (mg cotyledon fresh weight)^–1 increased indicatingthat the increase of fresh weight and chlorophyll was mor sensitiveto low temperature than PG desaturation in thyla-koid membranes.Furthermore, low temperatures inducei an increase in 16:1(3t)and 18:3 (the final products of PC synthesis) at the expenseof 16:0 and 18:1 (the initial products of PG synthesis). However,within a range of temperature from 10 to 18°C, the extentof these changes (nmol% of 18:3 or 16:1(3t) per day) was graduallylimited by lower temperatures. We therefore propose that lowtemperature inhibit both fatty acid synthesis and desaturationactivities. However, at low temperatures the fatty acid synthesisis likely to be more strongly inhibited than the desaturationactivities, thus explaining the observed increase in the relativecontent of PG-18:3 and PG-16:l(3t). Results an discussed interms of the mechanism which could be in volved in the metabolismof PG in squash cotyledons. (Received July 5, 1996; Accepted March 10, 1997)     

Probable interaction between S100A7 and E-FABP in the cytosol of human keratinocytes from psoriatic scales

The overexpression of E-FABP and S100A7 in lesional psoriatic skin suggests a possible link with this hyperproliferative skin disease. In order to investigate a role for the proteins in this disease, the purifications for both proteins were re-analyzed. Moreover, a specific antiserum directed against purified human S100A7 was generated. By SDS-PAGE immunoblotting we show that E-FABP and S100A7 are expressed in cultured human differentiating keratinocytes and confirm their overexpression in psoriatic scales. Gel filtration and non-denaturing PAGE revealed that S100A7 co-purified with E-FABP, indicating an association between the two proteins. Ion-exchange chromatography resulted in the dissociation of the complex. Finally, immunoprecipitations using antiserum against E-FABP revealed that S100A7 co-immunoprecipitated with E-FABP from protein extracts of psoriatic scales. These data indicate that E-FABP and S100A7 might form a complex in the cytosol of human keratinocytes.     

The 26- and 14-kDa phosphoproteins associated with spinach chloroplast envelope membranes are distinct membrane-bound pools of the light-harvesting complex of photosystem II and of the small subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase

Two chloroplast envelope proteins from spinach (Spinacia oleracea L.) exhibiting relative molecular masses (M_rs) of 26 and 14 kDa are apparently phosphorylated by a unique Ca²⁺-dependent serine protein kinase. The activity of this enzyme shows the same sensitivity towards pH, Ca²⁺, Mg²⁺, H7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine] and ATP concentrations (Siegenthaler and Bovet 1993, Planta 190, 231–240). Autoradiographic analyses following two-dimensional-gel electrophoresis (isoelectric focusing and SDS-PAGE) associated with Western blotting experiments indicate that these two phosphoproteins appeared to be pools of the light-harvesting complex of photosystem II (LHCII) and of the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit, respectively. Immunoprecipitation of envelope-phosphorylated proteins, using immunoglobulins (IgG) directed to the apoprotein of LHCII and to the holoenzyme of Rubisco confirmed that LHCII and the Rubisco small subunit effectively incorporated ³²P from (-³²P)ATP in isolated envelope membranes. We propose that, in agreement with the fact that protein import is driven by ATP, the phosphorylation of LHCII and the Rubisco small subunit could take place after the processing of precursor proteins and could be an obligatory step for their internalization into chloroplasts.Abbreviations 2D two dimensional - IEF isoelectric focusing - IgG immunoglobulin G - LHCII light-harvesting chlorophyll a/b proteins of PSII - LHCII A apoprotein a of LHCII - LHCIIB apoprotein b of LHCII - LS Rubisco large subunit - Mops (3-[N-morpholino]propanesulfonic acid) - M_r relative molecular mass - PI isoelectric point - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SS Rubisco small subunit The authors are grateful to Delphine Herrmann and Xavier Denys for their technical assistance. They also greatly thank Prof. R. J. Ellis and Dr. L. Barnett (Warwick University, UK) and Dr. P. Schürmann (University of Neuchâtel, Switzerland) for providing them with antibodies directed to the pea and spinach Rubisco holoenzymes and Dr. M. Spangfort (Lund University, Sweden) for his gift of the antibody directed to the pea LHCII apoprotein. This study was supported by the Swiss National Science Foundation. This work was part of a doctoral program carried out by L.B. in the Laboratoire de Physiologie végétale, Université de Neuchâtel, Switzerland.     

Phenylmereuric Acetate and Phosphate Control of Light-Dependent Shrinkage and Reactions in Chloroplasts

An investigation of the action of phenylmereuric acetate (PMA) and phosphate on light-induced shrinkage (measured by light scattering and Coulter Counter techniques) and on photosynthetic reactions in spinach chloroplasts led to the following conclusions:

• 1) PMA stimulated light-induced shrinkage (under conditions of cyclic and non-cyclic electron flow) at concentrations which completely inhibited cyclic and non-cyclic photophosphorylation and nicotinamide adenine dinucleotide phosphate (NADP) reduction, though ferricyanide reduction was activated. Although PMA inhibited NADP reduction (probably because this sulfhydryl reagent interfered with the ferredoxin-NADP rednetase) it ean also be considered an uncoiipler (when ferricyanide is the electron acceptor).
• 2) Phosphate maximized light-induced shrinkage (under conditions of cyclic and non cyclic electron flow) at concentrations which did not affect ferricyanide reduction but caused a 40 to 50 per cent inhibition of NADP reduction.
• 3) The pattern of the light scattering response to these two compounds was quite different. In the presence of PMA, the forward (light on) and hack (light off) reactions went to completion rapidly. In the presence of phosphate, the back reaction was rapid but, in the light-induced reaction, three phases were discernible.
• 4) Compared with uncouplers such as NH₄Cl, carbonyl cyanide m-chlorophenyl-hydrazone, pentachlorophenol, and dicoumarol, all of which inhibited both photophosphorylation and conformational changes in chloroplasts, PMA (like quinacrine) had a specific action since it inhibited photophosphorylation while shrinkage was stimulated.
• 5) It appeared that PMA acted at a site beyond the formation of high energy inter-mediates and that, in the absence of photophosphorylation, more energy was diverted to mechanical work (shrinkage). It would seem that, in a cyclic electron flow system, in which ATP synthesis is blocked at a late step (e.g. by PMA), shrinkage may be an indirect method for measuring electron flow.

     

Retinol-binding protein in human serum: conformational changes induced by retinoic acid binding

Polyacrylamide gel electrophoresis and isoelectrofocusing followed by immunoblotting technique with an anti-human retinol-binding protein (RBP) serum were used to study holo-RBP and apo-RBP in human plasma. Three observations were made the technique allowed for the first time to directly and quantitatively analyse holo- and apo-RBP. Holo-RBP represented 97.86 +/- 0.78% and apo-RBP 1.94 +/- 0.73% of the total RBP. All-trans-retinoic acid (RA) was found to bind to apo-RBP and to significantly modify the tertiary structure of the protein; this raises the question of RBP involvement in the transport of RA. reconstitution of holo-RBP using apo-RBP from delipidized serum was achieved only after its incubation with natural all-trans-retinoids such as retinol, 3-dehydroretinol and retinoic acid but not with synthetic analogs of retinoic acid (13-cis-retinoic acid, TMMP, 13-cis-TMMP, TTNPB). It appears that RBP has a structure specificity for natural retinoids.     

Progression of HIV infection in misusers of injected drugs who stop injecting or follow a programme of maintenance treatment with methadone.

OBJECTIVE--To see whether misusers of injected drugs who stop injecting or switch to a programme of maintenance treatment with methadone have a reduced risk of progression of HIV infection when compared with a group of persistent misusers. DESIGN--Observational cohort study in HIV seropositive subjects with a current or past history of misusing injected drugs. SETTING--HIV outpatient clinic at the University Hospital of Zurich, Switzerland. PATIENTS--297 Current and former parenteral drug misusers (median age 27) with asymptomatic HIV infection. During the observation period 80 subjects adhered to a programme of maintenance treatment with methadone, 124 continued with parenteral drug misuse, and 93 former misusers remained free of illicit drugs. No antiretroviral treatment was given during the study. MAIN OUTCOME MEASURES--Probability of progression of HIV infection from asymptomatic to symptomatic (Centers for Disease Control stage IV) as calculated by life table analysis and compared in the three groups of patients by means of a log rank test, and predictors of disease progression as analysed with a Cox proportional hazards regression model. RESULTS--The 297 patients were followed up for a median of 16 months. The median duration of injecting drug misuse before enrollment was 7.1 years. There were no significant differences among the three groups with respect to CD4+ counts at the beginning of the study (median 0.44 x 10(9)/l). Life table analysis showed a significantly lower probability of progression of HIV disease in both the methadone treated group and former drug misusers than in persistent injecting drug misusers. Multivariate regression analysis showed a relative risk of progression of the disease of 1.78 (95% confidence interval 1.20 to 2.67; p less than 0.01) in persistent injecting drug misusers, 0.48 (0.29 to 0.77; p less than 0.01) in the methadone treated group, and 0.66 (0.41 to 1.06; p = 0.085) in former drug misusers. CONCLUSIONS--Stopping the misuse of injected drugs slows the progression of HIV disease in infected subjects. Drug treatment programmes are effective in secondary prevention of HIV associated morbidity.     

Roles for Drp1, a dynamin-related protein, and milton, a kinesin-associated protein, in mitochondrial segregation, unfurling and elongation during Drosophila spermatogenesis

Mitochondria undergo dramatic rearrangement during Drosophila spermatogenesis. In wild type testes, the many small mitochondria present in pre-meiotic spermatocytes later aggregate, fuse, and interwrap in post-meiotic haploid spermatids to form the spherical Nebenkern, whose two giant mitochondrial compartments later unfurl and elongate beside the growing flagellar axoneme. Drp1 encodes a dynamin-related protein whose homologs in many organisms mediate mitochondrial fission and whose Drosophila homolog is known to govern mitochondrial morphology in neurons. The milton gene encodes an adaptor protein that links mitochondria with kinesin and that is required for mitochondrial transport in Drosophila neurons. To determine the roles of Drp1 and Milton in spermatogenesis, we used the FLP-FRT mitotic recombination system to generate spermatocytes homozygous for mutations in either gene in an otherwise heterozygous background. We found that absence of Drp1 leads to abnormal clustering of mitochondria in mature primary spermatocytes and aberrant unfurling of the mitochondrial derivatives in early Drp1 spermatids undergoing axonemal elongation. In milton spermatocytes, mitochondria are distributed normally; however, after meiosis, the Nebenkern is not strongly anchored to the nucleus, and the mitochondrial derivatives do not elongate properly. Our work defines specific functions for Drp1 and Milton in the anchoring, unfurling, and elongation of mitochondria during sperm formation.     

Influence of unsaturated fatty acids in chloroplasts. Shift of the pH optimum of electron flow and relations to deltapH, thylakoid internal pH and proton uptake.

Linolenic acid (C18:3) is the main endogenous unsaturated fatty acid of thylakoid membrane lipids, and seems in its free form to exert significant effects on the structure and function of photosynthetic membranes. In this investigation the effect of linolenic acid was studied at various pH values on the electron flow rate in isolated spinach chloroplasts and related to deltapH, the proton pump and the pH of the inner thylakoid space (pHi). The deltapH and pHi were estimated from the extent of the fluorescence quenching of 9-aminoacridine. Linolenic acid caused a shift (approximately one unit) of the pH optimum for electron flow toward acidity in the following systems: (a) photosystems II + I (from H2O to NADP+ or to 2,6-dichlorophenolindophenol) coupled or non-coupled; (b) photosystem II (from H2O to 2,6-dichlorophenolindophenol in the presence of dibromothymoquinone). In photosystem I conditions (phenazine methosulphate), the deltapH of the control increased as a function of external pHo with a maximum around pH 8.8. When linolenic acid was added, the deltapH dropped, but its optimum was shifted toward more acidic pHo. The same phenomena were also observed in photosytems II + I (from H2O to ferricyanide) and in photosystem II conditions (from H2O to ferricyanide in the presence of dibromothymoquinone). However, the deltapH was smaller and the sensitivity of the proton gradient toward linolenic acid was eventually higher than for photosystem I electron flow activity. The proton pump which might be considered as a measure of the internal buffering capacity of thylakoids was optimum at pHo, 6.7 in the controls. An addition of linolenic acid diminished the proton pump and shifted its optimum toward higher pHo. As a consequence, pHi increased when pHo was raised. At the optimal pHo 8.6 to 9, pHi were 5 to 5.5. Additions of increasing concentrations of linolenic acid displaced the curves toward higher pHi. A decrease of pHo was therefore required to maintain the pHi in the range of 5-5.5 for maximum electron flow. In conclusion, the electron flow activity seems to be delicately controlled by the proton pump (buffer capacity), deltapH, pHi and pHo. Fatty acids damage the membrane integrity in such a way that the subtile equilibrium between the factors is disturbed.     

Effects of microwave and radio frequency electromagnetic fields on lichens

The effects of electromagnetic fields on lichens were investigated. Field experiments of long duration (1–3 years) were combined with laboratory experiments and theoretical considerations. Samples of the lichen species Parmelia tiliacea and Hypogymnia physodes were exposed to microwaves (2.45 GHz; 0.2, 5, and 50 m W/cm²; and control). Both species showed a substantially reduced growth rate at 50 m W/cm². A differentiation between thermal and nonthermal effects was not possible. Temperature measurements on lichens exposed to microwaves (2.45 GHz, 50 m W/cm²) showed a substantial increase in the surface temperature and an accelerated drying process. The thermal effect of microwave on lichens was verified. The exposure of lichens of both species was repeated near a short-wave broadcast transmitter (9.5 MHz, amplitude modulated; maximum field strength 235 V/m, 332 mA/m). No visible effects on the exposed lichens were detected. At this frequency, no thermal effects were expected, and the experimental results support this hypothesis. Theoretical estimates based on climatic data and literature showed that the growth reductions in the initial experiments could very likely have been caused by drying of the lichens from the heating with microwaves. The results of the other experiments support the hypothesis that the response of the lichens exposed to microwaves was mainly due to thermal effects and that there is a low probability of nonthermal effects. © 1996 Wiley-Liss, Inc.