Membrane-associated phosphoinositides-specific phospholipase C forms from <Emphasis Type="Italic">Catharanthus roseus</Emphasis> transformed roots

We have previously reported that Catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC) activities, one soluble and the other membrane associated. Detergent, divalent cations, and neomycin differentially regulate these activities and pure protein is required for a greater understanding of the function and regulation of this enzyme. In this article we report a partia purification of membrane-associated PLC. We found that there are at least two forms of membrane-associated PLC in transformed roots of C. roseus. These forms were separated on the basis of their affinity for heparin. One form shows an affinity for heparin and elutes at approx 600 mM KCl. This form has a molecular mass of 67 kDa by size exclusion chromatography and Western blot analysis, whereas the other form does not bind to heparin and has a molecular mass of 57 kDa. Possible differential regulation of these forms during transformed root growth is discussed.     

Targeted stimulation of meiotic recombination

Meiotic recombination in Saccharomyces cerevisiae is initiated by programmed DNA double-strand breaks (DSBs), a process that requires the Spo11 protein. DSBs usually occur in intergenic regions that display open chromatin accessibility, but other determinants that control their frequencies and non-random chromosomal distribution remain obscure. We report that a Spo11 construct bearing the Gal4 DNA binding domain not only rescues spo11Delta spore inviability and catalyzes DSB formation at natural sites but also strongly stimulates DSB formation near Gal4 binding sites. At GAL2, a naturally DSB-cold locus, Gal4BD-Spo11 creates a recombinational hotspot that depends on all the other DSB gene functions, showing that the targeting of Spo11 to a specific site is sufficient to stimulate meiotic recombination that is under normal physiological control.     

Chemotaxonomic study on Thymus villosus from Portugal

The composition of the essential oils of four populations of Thymus villosus subsp. lusitanicus (Boiss.) Coutinho from Portugal was investigated by GC and GC-MS. To study the chemical polymorphism the results obtained from GC analyses of the volatile oils from individual plants from four populations were submited to Principal Component and Cluster analyses. A comparision with the essential oil of T. villosus subsp. villosus, previously studied by us was done. Important differences with regard to the major constituents in these two taxa were found. Linalool, geranyl acetate, geraniol and terpinen-4-ol were the main components of the essential oils of T. villosus subsp. lusitanicus, whereas in the oil of T. villosus subsp. villosus p-cymene, myrcene and alpha-terpineol were the major ones. Although, both taxa showed chemical polymorphism, different types of essential oils were characterized in each one: linalool; linalool/ terpinen-4-ol/trans-sabinene hydrate; linalool/1,8-cineole; geranyl acetate/geraniol; geranyl acetate/geraniol/1,8-cineole in T. villosus subsp. lusitanicus and p-cymene/camphor/linalool; p-cymene/borneol; linalool/geraniol/geranyl acetate; alpha-terpineol/camphor/myrcene in T. villosus subsp. villosus. Thus, the two subspecies of T. villosus can be easely differenciated by the composition of their essential oils.     

EGb761 Blocks MPP+-Induced Lipid Peroxidation in Mouse Corpus Striatum

EGb761 has been suggested to be an antioxidant and free radical scavenger. Excess generation of free radicals, leading to lipid peroxidation (LP), has been proposed to play a role in the damage to striatal neurons induced by 1-methyl-4-phenylpyridinium (MPP⁺). We investigated the effects of EGb761 pretreatment on MPP⁺ neurotoxicity. C-57 black mice were pretreated with EGb761 for 17 days at different doses (0.63, 1.25, 2.5, 5 or 10 mg/kg) followed by administration of MPP⁺, (0.18, 0.36 or 0.72 mg/kg). LP was analyzed in corpus striatum at 30 min, 1 h, 2 h and 24 h after MPP⁺ administration. Striatal dopamine content was analyzed by HPLC at the highest EGb761 dose at 2 h and 24 h after MPP⁺ administration. MPP⁺-induced LP was blocked (100%) by EGb761 (10 mg/kg). Pretreatment with EGb761 partially prevented (32%) the dopamine-depleting effect of MPP⁺ at 24 h. These results suggest that supplements of EGb761 may be effective at preventing MPP⁺-induced oxidative stress.     

Partially folded states of the cytolytic protein sticholysin II

Sticholysin II (Stn II) is a cytolytic protein produced by the sea anemone Stichodactyla helianthus, its effect being related to pore formation. The conformation of the protein and its temperature-induced transitions, in the 1.5-12.0 pH range and in the 0-0.5 M NaCl concentration interval, have been studied by circular dichroism and fluorescence spectroscopy. At temperature < 35 degrees C, the protein maintains the same, high beta-structure content, folded conformation in the 1.5-11.0 pH range and ionic strength up to 0.5 M. In the 1.5-3.5 pH range and ionic strength > or = 0.1 M, Stn II shows a thermal transition, resulting in a partially folded state characterized by: (i) a native-like content of regular secondary structure, as detected by far-UV CD; (ii) a largely disordered tertiary structure, as detected by near-UV CD, with partially exposed tryptophan residues according to their fluorescence emission; and (iii) ability to bind the hydrophobic probe 2-anilinonaphthalene-6-sulfonic acid. In the pH range 4.0-10.5, thermally-induced protein aggregation occurs. The obtained results demonstrate the existence of partially folded state of Stn II, which may contribute to the pore formation ability of this cytolysin.     

The type of competition modulates the ecophysiological response of grassland species to elevated CO2 and drought

The effects of elevated CO₂ and drought on ecophysiological parameters in grassland species have been examined, but few studies have investigated the effect of competition on those parameters under climate change conditions. The objective of this study was to determine the effect of elevated CO₂ and drought on the response of plant water relations, gas exchange, chlorophyll a fluorescence and aboveground biomass in four grassland species, as well as to assess whether the type of competition modulates that response. Elevated CO₂ in well‐watered conditions increased aboveground biomass by augmenting CO₂ assimilation. Drought reduced biomass by reducing CO₂ assimilation rate via stomatal limitation and, when drought was more severe, also non‐stomatal limitation. When plants were grown under the combined conditions of elevated CO₂ and drought, drought limitation observed under ambient CO₂ was reduced, permitting higher CO₂ assimilation and consequently reducing the observed decrease in aboveground biomass. The response to climate change was species‐specific and dependent on the type of competition. Thus, the response to elevated CO₂ in well‐watered grasses was higher in monoculture than in mixture, while it was higher in mixture compared to monoculture for forbs. On the other hand, forbs were more affected than grasses by drought in monoculture, while in mixture the negative effect of drought was higher in grasses than in forbs, due to a lower capacity to acquire water and mineral nutrients. These differences in species‐level growth responses to CO₂ and drought may lead to changes in the composition and biodiversity of the grassland plant community in future climate conditions.     

Human Peroxin PEX3 Is Co‐translationally Integrated into the ER and Exits the ER in Budding Vesicles

The long‐standing paradigm that all peroxisomal proteins are imported post‐translationally into pre‐existing peroxisomes has been challenged by the detection of peroxisomal membrane proteins (PMPs) inside the endoplasmic reticulum (ER). In mammals, the mechanisms of ER entry and exit of PMPs are completely unknown. We show that the human PMP PEX3 inserts co‐translationally into the mammalian ER via the Sec61 translocon. Photocrosslinking and fluorescence spectroscopy studies demonstrate that the N‐terminal transmembrane segment (TMS) of ribosome‐bound PEX3 is recognized by the signal recognition particle (SRP). Binding to SRP is a prerequisite for targeting of the PEX3‐containing ribosome?nascent chain complex (RNC) to the translocon, where an ordered multistep pathway integrates the nascent chain into the membrane adjacent to translocon proteins Sec61α and TRAM. This insertion of PEX3 into the ER is physiologically relevant because PEX3 then exits the ER via budding vesicles in an ATP‐dependent process. This study identifies early steps in human peroxisomal biogenesis by demonstrating sequential stages of PMP passage through the mammalian ER.