Opening windows into the cell: focused-ion-beam milling for cryo-electron tomography

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Lipid raft-based membrane compartmentation of a plant transport protein expressed in Saccharomyces cerevisiae

The hexose-proton symporter HUP1 shows a spotty distribution in the plasma membrane of the green alga Chlorella kessleri. Chlorella cannot be transformed so far. To study the membrane localization of the HUP1 protein in detail, the symporter was fused to green fluorescent protein (GFP) and heterologously expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In these organisms, the HUP1 protein has previously been shown to be fully active. The GFP fusion protein was exclusively targeted to the plasma membranes of both types of fungal cells. In S. cerevisiae, it was distributed nonhomogenously and concentrated in spots resembling the patchy appearance observed previously for endogenous H(+) symporters. It is documented that the Chlorella protein colocalizes with yeast proteins that are concentrated in 300-nm raft-based membrane compartments. On the other hand, it is completely excluded from the raft compartment housing the yeast H(+)/ATPase. As judged by their solubilities in Triton X-100, the HUP1 protein extracted from Chlorella and the GFP fusion protein extracted from S. cerevisiae are detergent-resistant raft proteins. S. cerevisiae mutants lacking the typical raft lipids ergosterol and sphingolipids showed a homogenous distribution of HUP1-GFP within the plasma membrane. In an ergosterol synthesis (erg6) mutant, the rate of glucose uptake was reduced to less than one-third that of corresponding wild-type cells. In S. pombe, the sterol-rich plasma membrane domains can be stained in vivo with filipin. Chlorella HUP1-GFP accumulated exactly in these domains. Altogether, it is demonstrated here that a plant membrane protein has the property of being concentrated in specific raft-based membrane compartments and that the information for its raft association is retained between even distantly related organisms.     

Untersuchungen Über faktoren,die sich bei folgekultivierung der pflanzen geltend machen

V práci byla sledována mo?nost allelopatického ovlivňování následných rostlin p?edplodinami p?i kultivaoi v odstupňovaných ?asových intervalech po sobě v té?e zemině u tě chto kombinací: mák — cukrovka, ho??ice — je?men, konopí — ?ito, cibule — ?epka. Pokusy byly prováděny v kvítiná?ích s kompostovou zeminou, umístěných během pokusu na zahradě a zapu? těných do p?dy. Byl sledován r?st p?edplodin a následných rostlin v po?áte?ních fá zích r?stu. P?ed vysetím následných rostlin byla stanovena u odebraných vzork? zemin intensita respirace, okam?itá vlhkost a obsah fyziologicky p?ístupného dusíku, fosforu a draslíku. Ve v?ech zkou?ených kombinacích byly následné rostliny ovlivněny kultivací p?edplodiny a následným ulo?ením zeminy. Změny r?stu následných rostlin ?áste? ně korelovaly s obsahem fyziologicky p?ístupného dusíku v zemině. Podle jejich charakteru v?ak bylo té? patrno, ?e se na nich podílely i allelopatické faktory. Zna?ně inhibi?ně p?sobil mák na cukrovku, mé ně inhibi?ně p?sobila ho??ice na je?men a cibule na ?epku. ??inek konopí na ?ito byl promě nný s dobou ulo?ení zeminy. Změny v obsahu fyziologicky p?ístupného dusíku, fosforu a draslíku v pokusné zemině neodpovídaly . mno?ství narostlé p?edplodiny, co? bylo podmíněno pou?itou kultiva?ní metodikou. Poměrně rychlé doplňování p?edplodinou vy?erpaných dusi?nan? v pokusné zemině s dobou jejího ulo?ení bylo pravděpodobně podmíněno nitrifikacními procesy. Podle stanovených změn intensity respirace pokusné zeminy se na allelopatickém ovlivnění mohla podílet i pudní mikroflora.     

The analogs of oxytocin and D-homoarginine vasopressin with bulky substituted phenylalanine in position 2

Solid phase technique on p-methylbenzhydrylamine resin wasused for the synthesis of eight analogs of oxytocin and 8-D-homoarginine vasopressin with the non-coded amino acids L- or D-2,3,4,5,6-pentamethylphenylalanine and L- or D-4-phenylphenylalanine in position 2. The preparation of theabove mentioned non-coded amino acids is described as well.All eight analogs were found to be potent inhibitors ofoxytocin activity in the uterotonic in vitro test in theabsence of Mg²⁺ ions. In the uterotonic test invitro in the presence of Mg²⁺ and in the test invivo, their potency is strongly decreased or completelyabolished. The substances are also weak pressor inhibitors.The L or D configuration does not seem to influence theactivity significantly.     

Novel type of red-shifted chlorophyll a antenna complex from Chromera velia: II. Biochemistry and spectroscopy

A novel chlorophyll a containing pigment–protein complex expressed by cells of Chromera velia adapted to growth under red/far-red illumination [1]. Purification of the complex was achieved by means of anion-exchange chromatography and gel-filtration. The antenna is shown to be an aggregate of ~ 20 kDa proteins of the light–harvesting complex (LHC) family, unstable in the isolated form. The complex possesses an absorption maximum at 705 nm at room temperature in addition to the main chlorophyll a maximum at 677 nm producing the major emission band at 714 nm at room temperature. The far-red absorption is shown to be the property of the isolated aggregate in the intact form and lost upon dissociation. The purified complex was further characterized by circular dichroism spectroscopy and fluorescence spectroscopy. This work thus identified the third different class of antenna complex in C. velia after the recently described FCP-like and LHCr-like antennas. Possible candidates for red antennas are identified in other taxonomic groups, such as eustigmatophytes and the relevance of the present results to other known examples of red-shifted antenna from other organisms is discussed. This work appears to be the first successful isolation of a chlorophyll a-based far-red antenna complex absorbing above 700 nm unrelated to LHCI.     

Red-shifted light-harvesting system of freshwater eukaryotic alga Trachydiscus minutus (Eustigmatophyta,Stramenopila)

Photosynthesis Research - Survival of phototrophic organisms depends on their ability to collect and convert enough light energy to support their metabolism. Phototrophs can extend their absorption...     

Hydrogen Sulfide Donor Protects Porcine Oocytes against Aging and Improves the Developmental Potential of Aged Porcine Oocytes

Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H₂S) in the process of porcine oocyte aging. H₂S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST). We demonstrated that H₂S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H₂S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H₂S from a donor (Na₂S.9H₂O) significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H₂S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H₂S on MPF and MAPK activities were detected and the intracellular mechanism underlying H₂S activity remains unclear, our study clearly demonstrates the role of H₂S in the regulation of porcine oocyte aging.