Phosphorylation of Mycoplasma pneumoniae cytadherence-accessory proteins in cell extracts.

A cell-free system was used to characterize the phosphorylation of Mycoplasma pneumoniae proteins HMW1 and HMW2, which are involved in the adherence of this organism to human tracheal epithelium during infection. The pH and cation requirements for phosphorylation of HMW1 and HMW2 were determined, and the effects of glycolytic intermediates, cyclic AMP, and eukaryotic kinase-phosphatase inhibitors and stimulators on this process were examined. Phosphoamino acid analysis identified serine as the major phosphate acceptor for both HMW1 and HMW2 in this system.     

The in vitro effects of Triton WR-1339 on lipid synthesis by bone cells.

Triton WR-1339, administered parenterally, has long been known to be a potent hyperlipemic agent. In vitro lipid biosynthesis is stimulated in liver and brain preparations from animals injected with Triton. Only in a perfused isolated liver system has an in vitro effect of Triton on lipid synthesis been demonstrated. In the present study, lipid biosynthesis has been shown to increase in bone, a third organ system, under the influence of in vitro Triton WR-133. This stimulation affects most major lipid classes. Triton similarly stimulates lipid synthesis in tissue cultures of bone cells. This is the first report of an effect of Triton on lipid synthesis (1) in bone and (2) in any tissue culture system.     

Impacts of Nutrient Reduction on Coastal Communities

Eutrophication due to high anthropogenic nutrient loading has greatly impacted ecological processes in marine coastal waters and, therefore, much effort has been put into reducing nitrogen and phosphorus discharges into European and North-American waters. Nutrient enrichment usually resulted in increase of biomass and production of phytoplankton and microphytobenthos, often coinciding with shifts in species composition within the primary producer community. Consequences of increasing eutrophication for higher trophic levels are still being disputed, and even less is known about the consequences of nutrient reduction on coastal food webs. Here, we present 30-year concurrent field observations on phytoplankton, macrozoobenthos and estuarine birds in the Dutch Wadden Sea, which has been subject to decades of nutrient enrichment and subsequent nutrient reduction. We demonstrate that long-term variations in limiting nutrients (phosphate and silicon) were weakly correlated with biomass and more strongly with community structures of phytoplankton, macrozoobenthos and estuarine birds. Although we cannot conclusively determine if, and if so to what extent, nutrient enrichment and subsequent nutrient reduction actually contributed to the concurrent trends in these communities, it appears likely that part of the variance in the studied coastal communities is related to changes in nutrient loads. Our results imply that nutrient reduction measures should not ignore the potential consequences for policies aimed at bird conservation and exploitation of marine living resources. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of cysteines involved in S-nitrosylation, S-glutathionylation, and oxidation to disulfides in ryanodine receptor type 1

The skeletal muscle Ca(2+)-release channel (ryanodine receptor type 1 (RyR1)) is a redox sensor, susceptible to reversible S-nitrosylation, S-glutathionylation, and disulfide oxidation. So far, Cys-3635 remains the only cysteine residue identified as functionally relevant to the redox sensing properties of the channel. We demonstrate that expression of the C3635A-RyR1 mutant in RyR1-null myotubes alters the sensitivity of the ryanodine receptor to activation by voltage, indicating that Cys-3635 is involved in voltage-gated excitation-contraction coupling. However, H(2)O(2) treatment of C3635A-RyR1 channels or wild-type RyR1, following their expression in human embryonic kidney cells, enhances [(3)H]ryanodine binding to the same extent, suggesting that cysteines other than Cys-3635 are responsible for the oxidative enhancement of channel activity. Using a combination of Western blotting and sulfhydryl-directed fluorescent labeling, we found that two large regions of RyR1 (amino acids 1-2401 and 3120-4475), previously shown to be involved in disulfide bond formation, are also major sites of both S-nitrosylation and S-glutathionylation. Using selective isotopecoded affinity tag labeling of RyR1 and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, we identified, out of the 100 cysteines in each RyR1 subunit, 9 that are endogenously modified (Cys-36, Cys-315, Cys-811, Cys-906, Cys-1591, Cys-2326, Cys-2363, Cys-3193, and Cys-3635) and another 3 residues that were only modified with exogenous redox agents (Cys-253, Cys-1040, and Cys-1303). We also identified the types of redox modification each of these cysteines can undergo. In summary, we have identified a discrete subset of cysteines that are likely to be involved in the functional response of RyR1 to different redox modifications (S-nitrosylation, S-glutathionylation, and oxidation to disulfides).     

Acetobacter turbidans alpha-amino acid ester hydrolase: how a single mutation improves an antibiotic-producing enzyme

The alpha-amino acid ester hydrolase (AEH) from Acetobacter turbidans is a bacterial enzyme catalyzing the hydrolysis and synthesis of beta-lactam antibiotics. The crystal structures of the native enzyme, both unliganded and in complex with the hydrolysis product D-phenylglycine are reported, as well as the structures of an inactive mutant (S205A) complexed with the substrate ampicillin, and an active site mutant (Y206A) with an increased tendency to catalyze antibiotic production rather than hydrolysis. The structure of the native enzyme shows an acyl binding pocket, in which D-phenylglycine binds, and an additional space that is large enough to accommodate the beta-lactam moiety of an antibiotic. In the S205A mutant, ampicillin binds in this pocket in a non-productive manner, making extensive contacts with the side chain of Tyr(112), which also participates in oxyanion hole formation. In the Y206A mutant, the Tyr(112) side chain has moved with its hydroxyl group toward the catalytic serine. Because this changes the properties of the beta-lactam binding site, this could explain the increased beta-lactam transferase activity of this mutant.     

Clonality in South African isolates and evidence for a European origin of the root pathogen Thielaviopsis basicola

Thielaviopsis basicola is a soil-borne fungal pathogen with a wide host range and a cosmopolitan distribution. It causes disease on many agricultural crops, and in South Africa is the causal agent of black pod rot of groundnuts and black root rot on chicory. Knowledge of the population diversity of T. basicola could provide valuable information regarding management strategies, the possible movement, origin, and reproductive strategies of the fungus. The objective of this study was to determine the population diversity of T. basicola isolates from groundnuts and chicory in South Africa using co-dominant polymorphic markers. These markers were also used to compare isolates from South Africa with those from other hosts and geographic regions. Seven loci revealed nine alleles and two genotypes, one on groundnut and one on chicory, differing at only two loci. T. basicola isolates from eight different countries and ten different hosts revealed 17 genotypes across the seven loci with 39 different alleles. The lack of diversity for the two South African host-related populations of isolates suggests that T. basicola was introduced into South Africa. Some evidence is provided for a European origin of the pathogen, possibly linked to trade in root crops.     

Mitochondria Are Linked to Calcium Stores in Striated Muscle by Developmentally Regulated Tethering Structures

Bi-directional calcium (Ca²⁺) signaling between mitochondria and intracellular stores (endoplasmic/sarcoplasmic reticulum) underlies important cellular functions, including oxidative ATP production. In striated muscle, this coupling is achieved by mitochondria being located adjacent to Ca²⁺ stores (sarcoplasmic reticulum [SR]) and in proximity of release sites (Ca²⁺ release units [CRUs]). However, limited information is available with regard to the mechanisms of mitochondrial-SR coupling. Using electron microscopy and electron tomography, we identified small bridges, or tethers, that link the outer mitochondrial membrane to the intracellular Ca²⁺ stores of muscle. This association is sufficiently strong that treatment with hypotonic solution results in stretching of the SR membrane in correspondence of tethers. We also show that the association of mitochondria to the SR is 1) developmentally regulated, 2) involves a progressive shift from a longitudinal clustering at birth to a specific CRU-coupled transversal orientation in adult, and 3) results in a change in the mitochondrial polarization state, as shown by confocal imaging after JC1 staining. Our results suggest that tethers 1) establish and maintain SR–mitochondrial association during postnatal maturation and in adult muscle and 2) likely provide a structural framework for bi-directional signaling between the two organelles in striated muscle.     

Toll-like receptor expression and function in human dendritic cell subsets: implications for dendritic cell-based anti-cancer immunotherapy

Dendritic cells (DCs) are central players of the immune response. To date, DC-based immunotherapy is explored worldwide in clinical vaccination trials with cancer patients, predominantly with ex vivo-cultured monocyte-derived DCs (moDCs). However, the extensive culture period and compounds required to differentiate them into DCs may negatively affect their immunological potential. Therefore, it is attractive to consider alternative DC sources, such as blood DCs. Two major types of naturally occurring DCs circulate in peripheral blood, myeloid DCs (mDCs) and plasmacytoid (pDCs). These DC subsets express different surface molecules and are suggested to have distinct functions. Besides scavenging pathogens and presenting antigens, DCs secrete cytokines, all of which is vital for both the acquired and the innate immune system. These immunological functions relate to Toll-like receptors (TLRs) expressed by DCs. TLRs recognize pathogen-derived products and subsequently provoke DC maturation, antigen presentation and cytokine secretion. However, not every TLR is expressed on each DC subset nor causes the same effects when activated. Considering the large amount of clinical trials using DC-based immunotherapy for cancer patients and the decisive role of TLRs in DC maturation, this review summarizes TLR expression in different DC subsets in relation to their function. Emphasis will be given to the therapeutic potential of TLR-matured DC subsets for DC-based immunotherapy.