Cellular distribution of complement receptor type 4 (CR4): expression on human platelets

Neutrophils have been shown to express a receptor for C3dg that is distinct from CR2 and is termed complement receptor type 4 (CR4). In the present study, other peripheral blood cell types were examined by indirect immunofluorescence and flow cytometry for the presence of C3dg binding activity. Specific uptake of C3dg occurred with neutrophils, platelets, and B lymphocytes, but not with eosinophils or T lymphocytes. Monocytes, contained within a mixed cell population of peripheral blood mononuclear cells and platelets, also bound C3dg, whereas purified monocytes did not. Binding of 125I-labeled glutaraldehyde-cross-linked C3dg to platelets was saturable, with an average of 1940 C3dg molecules bound per platelet at saturation (n = 8), ranging in number from 660 to 3930 molecules bound. Activation of platelets with thrombin did not consistently cause an increase in the expression of CR4 sites. 125I-C3dg binding to platelets was competitively inhibited equally well by unlabeled C3dg and iC3b, and approximately fourfold less well by C3b. The addition of platelets to elutriated monocytes generated C3dg binding activity on these cells by the formation of platelet-monocyte complexes. Thus, the CR4 on platelets accounted for the C3dg binding activity initially observed with partially purified monocytes. The adherent property of platelets may enable them to confer on certain other cell types the ability to localize C3dg-coated immune complexes or particles.     

Effects of acorn masting on population dynamics of three forest-dwelling rodent species in Hokkaido,Japan

The effects of the abundance of acorns of the oak, Quercus crispula, on the population dynamics of three rodent species (Apodemus speciosus, A. argenteus, and Clethrionomys rufocanus) were analyzed using time series data (1992–2006). The data were obtained in a forest in northern Hokkaido, Japan, by live trapping rodents and directly counting acorns on the ground. Apodemus speciosus generally increased in abundance following acorn masting. However, the clear effect of acorn abundance was not detected for the other two rodent species. Acorns of Q. crispula contain tannins, which potentially have detrimental effects on herbivores. Apodemus speciosus may reduce the damage caused by acorn tannins with tannin-binding salivary proteins and tannase-producing bacteria, whereas such physiological tolerance to tannins is not known in the other two rodent species. The differences in the effects of acorns between the three species may be due to differences in their physiological tolerance to tannins.     

Mutagenesis of subunit N of the Escherichia coli complex I. Identification of the initiation codon and the sensitivity of mutants to decylubiquinone

The last gene in the nuo operon of Escherichia coli, nuoN, encodes a membrane-bound subunit of Complex I (NADH:ubiquinone oxidoreductase). In this report, the gene for subunit N was disrupted by a 163 bp deletion in the chromosome, resulting in the loss of Complex I function, as measured by deamino-NADH oxidase activity. This activity could be recovered after transformation of the mutant strain by a plasmid that contains the previously identified nuoN gene and the upstream intergenic region between nuoM and nuoN. Mutagenesis of the first ATG downstream of nuoM led to a loss of function, indicating that this is the likely initiation codon for nuoN, and predicting a protein of 485 amino acids and 52 044 Da. Thirty site-specific mutations in nuoN at 19 different positions were constructed in a vector that expresses the full-length subunit N with both an octahistidine tag and an HA epitope tag at the carboxyl terminus. Highly conserved charged and aromatic residues were selected for mutagenesis, as well as a substitution that occurs as a secondary mutation in Leber's hereditary optic neuropathy (LHON). Membranes from the mutant strains were tested for production of subunit N by immunoblots and for NADH-linked activities. Mutants with substitutions at six different positions (K158, K217, H224, K247, Y300, and K395) had rates of deamino-NADH oxidase activity that were no more than 50% of that of the wild type and had reduced rates of proton translocation. These mutants also showed enhanced inhibition by decylubiquinone, indicating that subunit N interacts with quinones. The mutation associated with LHON, G391S, had little effect on these functions.     

Characterization of the first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase by surface labeling,cross-linking,and mutagenesis

The first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase has been analyzed by cysteine substitution mutagenesis. 13 of the 26 residues tested were found to be accessible to the reaction with 3-(N-maleimidylpropionyl)-biocytin. The other 13 residues predominantly found in the central region of the polypeptide chain between the two transmembrane spans were more resistant to labeling by 3-(N-maleimidylpropionyl)-biocytin while in membrane vesicle preparations. This region of subunit a contains a conserved residue Glu-80, which when mutated to lysine resulted in a significant loss of ATP-driven proton translocation. Other substitutions including glutamine, alanine, and leucine were much less detrimental to function. Cross-linking studies with a photoactive cross-linking reagent were carried out. One mutant, K74C, was found to generate distinct cross-links to subunit b, and the cross-linking had little effect on proton translocation. The results indicate that the first transmembrane span (residues 40-64) of subunit a is probably near one or both of the b subunits and that a less accessible region of the first cytoplasmic loop (residues 75-90) is probably near the cytoplasmic surface, perhaps in contact with b subunits.     

What Is the Role of ε in the Escherichia coli ATP Synthase?

The ATP synthase from Escherichia coli is a prototype of the ATP synthases that are found in many bacteria, in the mitochondria of eukaryotes, and in the chloroplasts of plants. It contains eight different types of subunits that have traditionally been divided into F₁, a water-soluble catalytic sector, and F_o, a membrane-bound ion transporting sector. In the current rotary model for ATP synthesis, the subunits can be divided into rotor and stator subunits. Several lines of evidence indicate that is one of the three rotor subunits, which rotate through 360 degrees. The three-dimensional structure of is known and its interactions with other subunits have been explored by several approaches. In light of recent work by our group and that of others, the role of in the ATP synthase from E. coli is discussed.