Organization of a sterol-rich membrane domain by cdc15p during cytokinesis in fission yeast

Many membrane processes occur in discrete membrane domains containing lipid rafts, but little is known about how these domains are organized and positioned. In the fission yeast Schizosaccharomyces pombe, a sterol-rich membrane domain forms at the cell-division site. Here, we show that formation of this membrane domain is independent of the contractile actin ring, septation, mid1p and the septins, and also requires cdc15p, an essential contractile ring protein that associates with lipid rafts. cdc15 mutants have membrane domains in the shape of spirals. Overexpression of cdc15p in interphase cells induces abnormal membrane domain formation in an actin-independent manner. We propose that cdc15p functions to organize lipid rafts at the cleavage site for cytokinesis.     

DNA hybridization in nanostructural molecular assemblies enables detection of gene mutations without a fluorescent probe

We have developed a simple single nucleotide polymorphisms (SNPs) analysis utilizing DNA hybridization in nanostructural molecular assemblies. The novel technique enables the detection of a single-base mismatch in a DNA sequence without a fluorescent probe. This report describes for the first time that DNA hybridization occurs in the nanostructural molecular assemblies (termed reverse micelles) formed in an organic medium. The restricted nanospace in the reverse micelles amplifies the differences in the hybridization rate between mismatched and perfectly matched DNA probes. For a model system, we hybridized a 20-mer based on the p53 gene sequence to 20-mer complementary oligonucleotides with various types of mismatches. Without any DNA labeling or electrochemical apparatus, we successfully detected the various oligonucleotide mismatches by simply measuring the UV absorbance at 260 nm.     

Arresting and releasing Staphylococcal alpha-hemolysin at intermediate stages of pore formation by engineered disulfide bonds

alpha-Hemolysin (alphaHL) is secreted by Staphylococcus aureus as a water-soluble monomer that assembles into a heptamer to form a transmembrane pore on a target membrane. The crystal structures of the LukF water-soluble monomer and the membrane-bound alpha-hemolysin heptamer show that large conformational changes occur during assembly. However, the mechanism of assembly and pore formation is still unclear, primarily because of the difficulty in obtaining structural information on assembly intermediates. Our goal is to use disulfide bonds to selectively arrest and release alphaHL from intermediate stages of the assembly process and to use these mutants to test mechanistic hypotheses. To accomplish this, we created four double cysteine mutants, D108C/K154C (alphaHL-A), M113C/K147C (alphaHL-B), H48C/ N121C (alphaHL-C), I5C/G130C (alphaHL-D), in which disulfide bonds may form between the pre-stem domain and the beta-sandwich domain to prevent pre-stem rearrangement and membrane insertion. Among the four mutants, alphaHL-A is remarkably stable, is produced at a level at least 10-fold greater than that of the wild-type protein, is monomeric in aqueous solution, and has hemolytic activity that can be regulated by the presence or absence of reducing agents. Cross-linking analysis showed that alphaHL-A assembles on a membrane into an oligomer, which is likely to be a heptamer, in the absence of a reducing agent, suggesting that oxidized alphaHL-A is halted at a heptameric prepore state. Therefore, conformational rearrangements at positions 108 and 154 are critical for the completion of alphaHL assembly but are not essential for membrane binding or for formation of an oligomeric prepore intermediate.     

Glycation and post-translational processing of human interferon-gamma expressed in Escherichia coli

Until recently, nonenzymatic glycosylation (glycation) was thought to affect the proteins of long living eukaryotes only. However, in a recent study (Mironova, R., Niwa, T., Hayashi, H., Dimitrova, R., and Ivanov, I. (2001) Mol. Microbiol. 39, 1061-1068), we have shown that glycation takes place in Escherichia coli as well. In the present study, we demonstrate that the post-translational processing (proteolysis and covalent dimerization) observed with cysteineless recombinant human interferon-gamma (rhIFN-gamma) is tightly associated with its in vivo glycation. Our results show that, at the time of isolation, rhIFN-gamma contained early (but not advanced) glycation products. Using reverse phase high performance liquid chromatography in conjunction with fluorescence measurements, enzyme-linked immunosorbent assay, and mass spectrometry, we found that advanced glycation end products arose in rhIFN-gamma during storage. The latter were identified mainly in the Arg/Lys-rich C terminus of the protein, which was also the main target of proteolysis. Mass spectral analysis and N-terminal sequencing revealed four major (Arg140/Arg141, Phe137/Arg138, Met135/Leu136, and Lys131/Arg132) and two minor (Lys109/Ala110 and Arg90/Asp91) cleavage sites in this region. Tryptic peptide mapping indicated that the covalent dimers of rhIFN-gamma originating during storage were formed mainly by lateral cross-linking of the monomer subunits. Antiviral assay showed that proteolysis lowered the antiviral activity of rhIFN-gamma, whereas covalent dimerization completely abolished it.     

Radiation-induced chain reactions in alcohol solutions of diphenyliodonium salts: a high-sensitivity chemical dosimeter

Chain reactions in gamma-irradiated 2-propanol solutions of diphenyliodonium salts have been studied. Protonic acids were generated in the irradiated solutions with high yields, whereas acid formation as a result of thermal reactions was negligible. The solution can be used as a high-sensitivity chemical dosimeter. The G value of acidic protons increases with increasing concentration of diphenyliodonium salt at the lower concentrations because the reaction rate of a propagation reaction increases. However, the chain is limited by a termination reaction between phenyl radical and the iodonium salt: The G value shows a maximum value of 610 micromol J(-1) at the concentration of 0.08 mol dm(-3) and decreases at higher salt concentrations.     

Gas chromatographic-mass spectrometric determination of erythrocyte 3-deoxyglucosone in diabetic patients

To determine if the erythrocyte levels of 3-deoxyglucosone (3-DG) are increased in diabetic patients, and if they correlate with glycemic status, they were measured in diabetic patients without renal disease as well as in healthy subjects. The erythrocyte levels of 3-DG were measured by a selected ion monitoring method of gas chromatography-chemical ionization mass spectrometry using [(13)C(6)]-3-DG as an internal standard. The erythrocyte levels of 3-DG were significantly higher in diabetic patients than in healthy subjects. The erythrocyte concentration of 3-DG was significantly and positively correlated with HbA1c (r=0.84, P<0.001). However, no significant correlation could be found between erythrocyte 3-DG and age, onset age of diabetes, or duration of diabetes in our group of diabetic patients. In diabetes, the production of 3-DG in the erythrocytes is increased via the polyol pathway and/or the Maillard reaction due to hyperglycemia.     

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.