Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly

The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic activity of various Escherichia coli and Salmonella enterica strains. Soluble ClyA monomers spontaneously assemble into annular dodecameric pore complexes upon contact with membranes or detergent. At ClyA monomer concentrations above ∼100 nm, the rate-limiting step in detergent- or membrane- induced pore assembly is the unimolecular reaction from the monomer to the assembly-competent protomer, which then oligomerizes rapidly to active pore complexes. In the absence of detergent, ClyA slowly forms soluble oligomers. Here we show that soluble ClyA oligomers cannot form dodecameric pore complexes after the addition of detergent and are hemolytically inactive. In addition, we demonstrate that the natural cysteine pair Cys-87/Cys-285 of ClyA forms a disulfide bond under oxidizing conditions and that both the oxidized and reduced ClyA monomers assemble to active pores via the same pathway in the presence of detergent, in which an unstructured, monomeric intermediate is transiently populated. The results show that the oxidized ClyA monomer assembles to pore complexes about one order of magnitude faster than the reduced monomer because the unstructured intermediate of oxidized ClyA is less stable and dissolves more rapidly than the reduced intermediate. Moreover, we show that oxidized ClyA forms soluble, inactive oligomers in the absence of detergent much faster than the reduced monomer, providing an explanation for several contradictory reports in which oxidized ClyA had been described as inactive.     

The pentafunctional FAS1 genes of Saccharomyces cerevisiae and Yarrowia lipolytica are co-linear and considerably longer than previously estimated

Summary The fatty acid synthetase (FAS) gene FAS1 of the alkane-utilizing yeast Yarrowia lipolytica was cloned and sequenced. The gene is represented by an intron-free reading frame of 6228 by encoding a protein of 2076 amino acids and 229980 Da molecular weight. This protein exhibits a 58% sequence similarity to the corresponding Saccharomyces cerevisiae FAS -subunit. The sequential order of the five FAS1-encoded enzyme domains, acetyl transferase, enoyl reductase, dehydratase and malonyl/palmityl-transferase, is co-linear in both organisms. This finding agrees with available evidence that the functional organization of FAS genes is similar in related organisms but differs considerably between unrelated species. In addition, previously reported conflicting data concerning the 3 end of S. cerevisiae FAS1 were re-examined by genomic and cDNA sequencing of the relevant portion of the gene. Thereby, the translational stop codon was shown to lie considerably downstream of both published termination sites. The S. cerevisiae FAS1 gene thus has a corrected length of 6153 by and encodes a protein of 2051 amino acids and 228667 Da molecular weight.     

Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content

Top-down control analysis (TDCA) is a useful tool for quantifying constraints on metabolic pathways that might be overcome by biotechnological approaches. Previous studies on lipid accumulation in oilseed rape have suggested that diacylglycerol acyltransferase (DGAT), which catalyses the final step in seed oil biosynthesis, might be an effective target for enhancing seed oil content. Here, increased seed oil content, increased DGAT activity, and reduced substrate:product ratio are demonstrated, as well as reduced flux control by complex lipid assembly, as determined by TDCA in Brassica napus (canola) lines which overexpress the gene encoding type-1 DGAT. Lines overexpressing DGAT1 also exhibited considerably enhanced seed oil content under drought conditions. These results support the use of TDCA in guiding the rational selection of molecular targets for oilseed modification. The most effective lines had a seed oil increase of 14%. Moreover, overexpression of DGAT1 under drought conditions reduced this environmental penalty on seed oil content.     

Validation of candidate genes putatively associated with resistance to SCMV and MDMV in maize (Zea mays L.) by expression profiling

Background  

The potyviruses sugarcane mosaic virus (SCMV) and maize dwarf mosaic virus (MDMV) are major pathogens of maize worldwide. Two loci, Scmv1 and Scmv2, have ealier been shown to confer complete resistance to SCMV. Custom-made microarrays containing previously identified SCMV resistance candidate genes and resistance gene analogs were utilised to investigate and validate gene expression and expression patterns of isogenic lines under pathogen infection in order to obtain information about the molecular mechanisms involved in maize-potyvirus interactions.     

Synergistic antitumor effect of bispecific CD19 x CD3 and CD19 x CD16 diabodies in a preclinical model of non-Hodgkin's lymphoma

To target NK cells against non-Hodgkin's lymphoma, we constructed a bispecific diabody (BsDb) with reactivity against both human CD19 and FcgammaRIII (CD16). Bacterially produced CD19 x CD16 BsDb specifically interacted with both CD19(+) and CD16(+) cells and exhibited significantly higher apparent affinity and slower dissociation from the tumor cells than from effector cells. It was able to induce specific lysis of tumor cells in the presence of isolated human NK cells or nonfractionated PBLs. The combination of the CD19 x CD16 BsDb with a previously described CD19 x CD3 BsDb and CD28 costimulation significantly increased the lytic potential of human PBLs. Treatment of SCID mice bearing an established Burkitt's lymphoma (5 mm in diameter) with human PBLs, CD19 x CD16 BsDb, CD19 x CD3 BsDb, and anti-CD28 mAb resulted in the complete elimination of tumors in 80% of animals. In contrast, mice receiving human PBLs in combination with either diabody alone showed only partial tumor regression. These data clearly demonstrate the synergistic effect of small recombinant bispecific molecules recruiting different populations of human effector cells to the same tumor target.     

Contribution of Archaea to total prokaryotic production in the deep Atlantic Ocean

Fluorescence in situ hybridization (FISH) in combination with polynucleotide probes revealed that the two major groups of planktonic Archaea (Crenarchaeota and Euryarchaeota) exhibit a different distribution pattern in the water column of the Pacific subtropical gyre and in the Antarctic Circumpolar Current system. While Euryarchaeota were found to be more dominant in nearsurface waters, Crenarchaeota were relatively more abundant in the mesopelagic and bathypelagic waters. We determined the abundance of archaea in the mesopelagic and bathypelagic North Atlantic along a south-north transect of more than 4,000 km. Using an improved catalyzed reporter deposition-FISH (CARD-FISH) method and specific oligonucleotide probes, we found that archaea were consistently more abundant than bacteria below a 100-m depth. Combining microautoradiography with CARD-FISH revealed a high fraction of metabolically active cells in the deep ocean. Even at a 3,000-m depth, about 16% of the bacteria were taking up leucine. The percentage of Euryarchaeota and Crenarchaeaota taking up leucine did not follow a specific trend, with depths ranging from 6 to 35% and 3 to 18%, respectively. The fraction of Crenarchaeota taking up inorganic carbon increased with depth, while Euryarchaeota taking up inorganic carbon decreased from 200 m to 3,000 m in depth. The ability of archaea to take up inorganic carbon was used as a proxy to estimate archaeal cell production and to compare this archaeal production with total prokaryotic production measured via leucine incorporation. We estimate that archaeal production in the mesopelagic and bathypelagic North Atlantic contributes between 13 to 27% to the total prokaryotic production in the oxygen minimum layer and 41 to 84% in the Labrador Sea Water, declining to 10 to 20% in the North Atlantic Deep Water. Thus, planktonic archaea are actively growing in the dark ocean although at lower growth rates than bacteria and might play a significant role in the oceanic carbon cycle.     

Significantly greater antioxidant anticancer activities of 2,3-dehydrosilybin than silybin

Silybin or silymarin extract has been used to treat liver diseases, and has now been entered into clinical trials for cancer treatment. Here, we compared antioxidant and anticancer activities between silybin and its oxidized form 2,3-dehydrosilybin (DHS). With IC50 at three-fold lower concentrations than silybin, DHS inhibited reactive oxygen species generation in glucose-glucose oxidase system and HepG2 cells. Compared with silybin, DHS elicited greater protection against H2O2-induced HepG2 cell death and galactosamine-induced liver injury in vivo. It is known that oxidants induce releases of metalloproteinases (MMP)-2,-9 which are responsible for invasive and metastasis potentials of transformed cells. DHS at 10 microM markedly inhibited MMP-2,-9 releases as well as invasiveness, while silybin at 90 microM had marginal effects. DHS but not silybin at 30 microM induced apoptosis and loss of mitochondrial membrane potentials. LD50 of DHS was five-fold lower than that of silybin. Our data suggest that DHS may be more useful therapeutically than silybin.     

Effects of age and altrenogest treatment on conceptus development and secretion of LH, progesterone and eCG in early-pregnant mares

The treatment of early pregnant mares with a history of repeated early embryonic loss with the progestin altrenogest has become routine; however no controlled studies on the efficiency of altrenogest to prevent embryonic losses are available so far. In the present study, we have investigated effects of altrenogest treatment in mares on conceptus development and the secretion of LH, progesterone, and eCG until day 100 of pregnancy. In addition, differences related to age of mares were assessed. Mares were treated with altrenogest (0.044 mg/kg per os once daily) or sunflower oil (10 ml per os once daily) from day 6 to day 100 after ovulation. Blood samples for analysis of LH, progesterone, and eCG were collected. The size of the embryonic vesicle and embryo/fetus was determined by ultrasound. No difference in the per cycle pregnancy rate between altrenogest-treated (75%) and sunflower oil-treated mares (74%) was detected (n.s.). A significant effect of age but not of altrenogest treatment on mean diameter of the embryonic vesicle was found between days 12 and 22 of pregnancy (e.g. day 15: control, 4-8 years: 22.9 ± 1.0 mm, >8 years: 22.0 ± 1.7 mm, altrenogest, 4-8 years: 26.1 ± 2.0 mm, >8 years: 20.4 ± 1.0 mm, P < 0.05). A significant effect of age and treatment on size of the embryo proper between days 30 and 45 was detected (P < 0.05). In the control group but not in the altrenogest group, size of the embryo proper respective fetus was negatively correlated with age of the mares (day 30: r = −0.834, P < 0.05; day 35: r = −0.506, P < 0.05). Plasma concentrations of LH and progesterone were neither effected by age nor by treatment of mares, but significant effects of age and altrenogest treatment on eCG concentrations between days 40 and 130 were detected (P < 0.05). The present study demonstrates for the first time a positive influence of altrenogest-treatment on a retarded development of the embryo respective fetus around the beginning of placentation in mares older than 8 years.