CD1 molecules efficiently present antigen in immature dendritic cells and traffic independently of MHC class II during dendritic cell maturation

Upon exposure to Ag and inflammatory stimuli, dendritic cells (DCs) undergo a series of dynamic cellular events, referred to as DC maturation, that involve facilitated peptide Ag loading onto MHC class II molecules and their subsequent transport to the cell surface. Besides MHC molecules, human DCs prominently express molecules of the CD1 family (CD1a, -b, -c, and -d) and mediate CD1-dependent presentation of lipid and glycolipid Ags to T cells, but the impact of DC maturation upon CD1 trafficking and Ag presentation is unknown. Using monocyte-derived immature DCs and those stimulated with TNF-alpha for maturation, we observed that none of the CD1 isoforms underwent changes in intracellular trafficking that mimicked MHC class II molecules during DC maturation. In contrast to the striking increase in surface expression of MHC class II on mature DCs, the surface expression of CD1 molecules was either increased only slightly (for CD1b and CD1c) or decreased (for CD1a). In addition, unlike MHC class II, DC maturation-associated transport from lysosomes to the plasma membrane was not readily detected for CD1b despite the fact that both molecules were prominently expressed in the same MIIC lysosomal compartments before maturation. Consistent with this, DCs efficiently presented CD1b-restricted lipid Ags to specific T cells similarly in immature and mature DCs. Thus, DC maturation-independent pathways for lipid Ag presentation by CD1 may play a crucial role in host defense, even before DCs are able to induce maximum activation of peptide Ag-specific T cells.     

Exploring beliefs behind support for and opposition to wildlife management methods: a qualitative study

Wildlife management methods such as culling (lethal control) and fencing can be controversial in some circumstances. Such controversy can be problematic for decision-makers or those managing decision-making processes and can lead to management delays or inertia. Understanding the reasons why people support or oppose specific management methods is therefore an important objective for researchers. Attitudes towards methods are in part based on individual beliefs about those methods, the species of wildlife being managed and other associated phenomena. This paper adopts a qualitative approach to develop understanding of these beliefs. We conducted 17 focus-groups on wild deer management at two locations in Britain, with both ‘professional’ land manager and ‘public’ participants (n?=?103). We identified a number of individual beliefs which are grouped into five categories: naturalness, overabundance, impacts, effectiveness and animal welfare. Our findings suggest that potentially controversial management methods will receive most support where the objective is to maintain a ‘natural’ environment, at sites where impacts are evident, and when using targeted and effective methods.     

Removal of PsaF alters forward electron transfer in photosystem I: evidence for fast reoxidation of QK-A in subunit deletion mutants of Synechococcus sp. PCC 7002

Recent studies of point mutations in photosystem I have suggested that the two kinetic phases of phylloquinone reoxidation represent electron transfer in the two branches of cofactors. This interpretation implies that changes in the relative amplitudes of the two kinetic phases represent a change in the extent of electron transfer in the two branches. Using time-resolved electron paramagnetic resonance (EPR), this issue is investigated in subunit deletion mutants of Synechococcus sp. PCC 7002. The spin-polarized EPR signals of P(700)(+)A(1)(-) and P(700)(+)FeS(-), both at room temperature and in frozen solution, are altered by deletion of PsaF and/or PsaE, and the differences from the wild type are much more pronounced in PS I complexes isolated from the mutants using Triton X-100 rather than n-dodecyl beta-d-maltopyranoside. The changes in the transient EPR data for the mutant complexes are consistent with a significant fraction of reaction centers showing (i) faster electron transfer from A(1)(-) to F(X), (ii) slower forward electron transfer from A(0)(-) to A(1), and (iii) slightly altered quinone hyperfine couplings, possibly as a result of a change in the hydrogen bonding. The fraction of fast electron transfer and its dependence on the isolation procedure are estimated approximately from simulations of the room temperature EPR data. The results are discussed in terms of possible models for the electron transfer. It is suggested that the detergent-induced fraction of fast electron transfer is most likely due to alteration of the environment of the quinone in the PsaA branch of cofactors and is not the result of a change in the directionality of electron transfer.     

The N-terminus of the intrinsically disordered protein α-synuclein triggers membrane binding and helix folding

Alpha-synuclein (αS) is a 140-amino-acid protein that is involved in a number of neurodegenerative diseases. In Parkinson's disease, the protein is typically encountered in intracellular, high-molecular-weight aggregates. Although αS is abundant in the presynaptic terminals of the central nervous system, its physiological function is still unknown. There is strong evidence for the membrane affinity of the protein. One hypothesis is that lipid-induced binding and helix folding may modulate the fusion of synaptic vesicles with the presynaptic membrane and the ensuing transmitter release. Here we show that membrane recognition of the N-terminus is essential for the cooperative formation of helical domains in the protein. We used circular dichroism spectroscopy and isothermal titration calorimetry to investigate synthetic peptide fragments from different domains of the full-length αS protein. Site-specific truncation and partial cleavage of the full-length protein were employed to further characterize the structural motifs responsible for helix formation and lipid-protein interaction. Unilamellar vesicles of varying net charge and lipid compositions undergoing lateral phase separation or chain melting phase transitions in the vicinity of physiological temperatures served as model membranes. The results suggest that the membrane-induced helical folding of the first 25 residues may be driven simultaneously by electrostatic attraction and by a change in lipid ordering. Our findings highlight the significance of the αS N-terminus for folding nucleation, and provide a framework for elucidating the role of lipid-induced conformational transitions of the protein within its intracellular milieu.     

Fumarate reduction inProteus mirabilis

1. Proteus mirabilis formed fumarate reductase under anaerobic growth conditions. The formation of this reductase was repressed under conditions of growth during which electron transport to oxygen or to nitrate is possible. In two of three tested chlorateresistant mutant strains of the wild type, fumarate reductase appeared to be affected.
2. Cytoplasmic membrane suspensions isolated from anaerobically grownP. mirabilis oxidized formate and NADH with oxygen and with fumarate, too.
3. Spectral investigation of the cytoplasmic membrane preparation revealed the presence of (probably at least two types of) cytochromeb, cytochromea ₁ and cytochromed. Cytochromeb was reduced by NADH as well as by formate to approximately 80%.
4. 2-n-Heptyl-4-hydroxyquinoline-N-oxide and antimycin A inhibited oxidation of both formate and NADH by oxygen and fumarate. Both inhibitors increased the level of the formate/oxygen steady state and the formate/fumarate steady state.
5. The site of inhibition of the respiratory activity by both HQNO and antimycin A was located at the oxidation side of cytochromeb.
6. The effect of ultraviolet-irradiation of cytoplasmic membrane suspensions on oxidation/reduction phenomena suggested that the role of menaquinone is more exclusive in the formate/fumarate pathway than in the electron transport route to oxygen.
7. Finally, the conclusion has been drawn that the preferential route for electron transport from formate and from NADH to fumarate (and to oxygen) includes cytochromeb as a directly involved carrier. A hypothetical scheme for the electron transport in anaerobically grownP. mirabilis is presented.

     

Ubiquitination of the glycosomal matrix protein receptor PEX5 in Trypanosoma brucei by PEX4 displays novel features

Trypanosomatids contain peroxisome-like organelles called glycosomes. Peroxisomal biogenesis involves a cytosolic receptor, PEX5, which, after its insertion into the organellar membrane, delivers proteins to the matrix. In yeasts and mammalian cells, transient PEX5 monoubiquitination at the membrane serves as the signal for its retrieval from the organelle for re-use. When its recycling is impaired, PEX5 is polyubiquitinated for proteasomal degradation. Stably monoubiquitinated TbPEX5 was detected in cytosolic fractions of Trypanosoma brucei, indicative for its role as physiological intermediate in receptor recycling. This modification's resistance to dithiothreitol suggests ubiquitin conjugation of a lysine residue. T. brucei PEX4, the functional homologue of the ubiquitin-conjugating (UBC) enzyme responsible for PEX5 monoubiquitination in yeast, was identified. It is associated with the cytosolic face of the glycosomal membrane, probably anchored by an identified putative TbPEX22. The involvement of TbPEX4 in TbPEX5 ubiquitination was demonstrated using procyclic ?PEX4 trypanosomes. Surprisingly, glycosomal matrix protein import was only mildly affected in this mutant. Since other UBC homologues were upregulated, it might be possible that these have partially rescued PEX4's function in PEX5 ubiquitination. In addition, the altered expression of UBCs, notably of candidates involved in cell-cycle control, could be responsible for observed morphological and motility defects of the ?PEX4 mutant.     

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.     

Autolytic Activity and Butanol Tolerance of Clostridium acetobutylicum

The effects of acetone and butanol on the growth of vegetative cells and the stability of swollen-phase bright-stationary-phase cells (clostridial forms) of Clostridium acetobutylicum P262 and an autolytic deficient mutant (lyt-1) were investigated. There was little difference in the sensitivity of strain P262 and the lyt-1 mutant vegetative cells and clostridial forms to acetone. The stability of the different morphological stages was unaffected by acetone concentrations far in excess of those encountered in factory fermentations. Butanol concentrations between 7 and 16 g/liter, which are within the range obtained in industrial fermentations, increased the degeneration of strain P262 clostridial forms but had no effect on the stability of lyt-1 clostridial forms which never underwent autolysis. Vegetative cells of the lyt-1 mutant were able to grow in higher concentrations of butanol than strain P262 vegetative cells. It was concluded that there is a relationship between butanol tolerance and autolytic activity.     

Proteins synthesized by inducer T cells: evidence for a mitogenic peptide shared by inducer molecules that stimulate different cell types

Inducer T lymphocytes synthesize and secrete peptides that stimulate growth and differentiation of many cell types, including lymphocytes and monocytes that kill foreign organisms, B lymphocytes, mast cells and hematopoietic precursor cells. To define these inducer molecules more precisely, we have generated clones of these T cells as a source of homogeneous material for biochemical analysis. These clones synthesize peptides that stimulate T and B cells to divide and that also induce the latter cells to secrete immunoglobulin. Inducer cells synthesize a 14 kilodalton growth polypeptide that stimulates T and B lymphocytes, as well as other cell types, to divide. This 14 kilodalton peptide is normally associated with different, larger peptides that appear to focus its mitogenic activity to one or another target cell.