Toc159- and Toc75-independent import of a transit sequence-less precursor into the inner envelope of chloroplasts

Chloroplast envelope quinone oxidoreductase (ceQORH) is an inner plastid envelope protein that is synthesized without cleavable chloroplast transit sequence for import. In the present work, we studied the in vitro-import characteristics of Arabidopsis ceQORH. We demonstrate that ceQORH import requires ATP and is dependent on proteinaceous receptor components exposed at the outer plastid surface. Competition experiments using small subunit precursor of ribulose-bisphosphate carboxylase/oxygenase and precursor of ferredoxin, as well as antibody blocking experiments, revealed that ceQORH import does not involve the main receptor and translocation channel proteins Toc159 and Toc75, respectively, which operate in import of proteins into the chloroplast. Molecular dissection of the ceQORH amino acid sequence by site-directed mutagenesis and subsequent import experiments in planta and in vitro highlighted that ceQORH consists of different domains that act concertedly in regulating import. Collectively, our results provide unprecedented evidence for the existence of a specific import pathway for transit sequence-less inner plastid envelope membrane proteins into chloroplasts.     

Enhancement of bioconversion of high-molecular mass polycyclic aromatic hydrocarbons in contaminated non-sterile soil by litter-decomposing fungi

With the focus on alternative microbes for soil-bioremediation, 18 species of litter-decomposing basidiomycetous fungi were screened for their ability to grow on different lignocellulosic substrates including straw, flax and pine bark as well as to produce ligninolytic enzymes, namely laccase and manganese peroxidase. Following characteristics have been chosen as criteria for the strain selection: (i) the ability to grow at least on one of the mentioned materials, (ii) production of either of the ligninolytic enzymes and (iii) the ability to invade non-sterile soil. As the result, eight species were selected for a bioremediation experiment with an artificially contaminated soil (total polycyclic aromatic hydrocarbon (PAH) concentration 250 mg/kg soil). Up to 70%, 86% and 84% of benzo(a)anthracene, benzo(a)pyrene, and dibenzo(a,h)anthracene, respectively, were removed in presence of fungi while the indigenous microorganisms converted merely up to 29%, 26% and 43% of these compounds in 30 days. Low molecular-mass PAHs studied were easily degraded by soil microbes and only anthracene degradation was enhanced by the fungi as well. The agaric basidiomycetes Stropharia rugosoannulata and Stropharia coronilla were the most efficient PAH degraders among the litter-decomposing species used.     

DNA methylation affecting the expression of murine leukemia proviruses.

The endogenous, vertically transmitted proviral DNAs of the ecotropic murine leukemia virus in AKR embryo fibroblasts were found to be hypermethylated relative to exogenous AKR murine leukemia virus proviral DNAs acquired by infection of the same cells. The hypermethylated state of the endogenous AKR murine leukemia virus proviruses in these cells correlated with the failure to express AKR murine leukemia virus and the lack of infectivity of cellular DNA. Induction of the endogenous AKR murine leukemia virus proviruses with the methylation antagonist 5-azacytidine suggested a causal connection between DNA methylation and provirus expression. Also found to be relatively hypermethylated and noninfectious were three of six Moloney murine leukemia virus proviral DNAs in an unusual clone of infected rat cells. Recombinant DNA clones which derived from a methylated, noninfectious Moloney provirus of this cell line were found to be highly active upon transfection, suggesting that a potentially active proviral genome can be rendered inactive by cellular DNA methylation. In contrast, in vitro methylation with the bacterial methylases MHpaII and MHhaI only slightly reduced the infectivity of the biologically active cloned proviral DNA. Recombinant DNA clones which derived from a second Moloney provirus of this cell line were noninfectious. An in vitro recombination method was utilized in mapping studies to show that this lack of infectivity was governed by mechanisms other than methylation.     

Measurement of 2J(H,C)- and 3J(H,C)-coupling constants by α/β selective HC(C)H-TOCSY

A new heteronuclear NMR pulse sequence for the measurement of ⁿJ(C,H) coupling constants, the /selective HC(C)H-TOCSY, is described. It is shown that the S³E element (Meissner et al., 1997a,b) can be used to obtain spin state selective coherence transfer in molecules, in which adjacent CH moieties are labeled with ¹³C. Application of the / selective HC(C)H-TOCSY to a 10nt RNA tetraloop 5-CGCUUUUGCG-3, in which the four uridine residues are ¹³C labeled in the sugar moiety, allowed measurement of two bond and three bond J(C,H) coupling constants, which provide additional restraints to characterize the sugar ring conformation of RNA in cases of conformational averaging.     

Role of the membrane skeleton in preventing the shedding of procoagulant-rich microvesicles from the platelet plasma membrane

The platelet plasma membrane is lined by a membrane skeleton that appears to contain short actin filaments cross-linked by actin-binding protein. Actin-binding protein is in turn associated with specific plasma membrane glycoproteins. The aim of this study was to determine whether the membrane skeleton regulates properties of the plasma membrane. Platelets were incubated with agents that disrupted the association of the membrane skeleton with membrane glycoproteins. The consequences of this change on plasma membrane properties were examined. The agents that were used were ionophore A23187 and dibucaine. Both agents activated calpain (the Ca2(+)-dependent protease), resulting in the hydrolysis of actin-binding protein and decreased association of actin with membrane glycoproteins. Disruption of actin-membrane interactions was accompanied by the shedding of procoagulant-rich microvesicles from the plasma membrane. The shedding of microvesicles correlated with the hydrolysis of actin-binding protein and the disruption of actin-membrane interactions. When the calpain-induced disruption of actin-membrane interactions was inhibited, the shedding of microvesicles was inhibited. These data are consistent with the hypothesis that association of the membrane skeleton with the plasma membrane maintains the integrity of the plasma membrane, preventing the shedding of procoagulant-rich microvesicles from the membrane of unstimulated platelets. They raise the possibility that the procoagulant-rich microvesicles that are released under a variety of physiological and pathological conditions may result from the dissociation of the platelet membrane skeleton from its membrane attachment sites.     

Preferential utilization of inorganic polyphosphate over other bioavailable phosphorus sources by the model diatoms Thalassiosira spp.

Polyphosphates and phosphomonoesters are dominant components of marine dissolved organic phosphorus (DOP). Collectively, DOP represents an important nutritional phosphorus (P) source for phytoplankton growth in the ocean, but the contribution of specific DOP sources to microbial community P demand is not fully understood. In a prior study, it was reported that inorganic polyphosphate was not bioavailable to the model diatoms Thalassiosira weissflogii and Thalassiosira pseudonana. However, in this study, we show that the previous finding was a misinterpretation based on a technical artefact of media preparation and that inorganic polyphosphate is actually widely bioavailable to Thalassiosira spp. In fact, orthophosphate, inorganic tripolyphosphate (3polyP), adenosine triphosphate (ATP) and adenosine monophosphate supported equivalent growth rates and final growth yields within each of four strains of Thalassiosira spp. However, enzyme activity assays revealed in all cultures that cell-associated hydrolysis rates of 3polyP were typically more than ~10-fold higher than degradation of ATP and the model phosphomonoester compound 4-methylumbelliferyl phosphate. These results build on prior work, which showed the preferential utilization of polyphosphates in the cell-free exudates of Thalassiosira spp., and suggest that inorganic polyphosphates may be a key bioavailable source of P for marine phytoplankton.