Acid sphingomyelinase activation requires caspase-8 but not p53 nor reactive oxygen species during Fas-induced apoptosis in human glioma cells

During apoptosis of human glioma cells induced by anti-Fas antibody, ceramide formation with activation of acid, but not neutral sphingomyelinase (SMase), was observed. A potent inhibitor of acid SMase, SR33557, effectively inhibited ceramide formation and apoptosis. Fas-induced apoptosis and ceramide formation proceeded regardless of p53 status. The agents, which modify intracellular levels of reactive oxygen species (ROS) and reduced glutathione (GSH), failed to modulate Fas-induced acid SMase activation and apoptosis. Moreover, expression of functional p53 protein using a temperature-sensitive human p53val(138) induced ceramide generation by activation of neutral SMase but not acid SMase through ROS formation. Peptide inhibitors for caspases-8 (z-IETD-fmk) and -3 (z-DEVD-fmk) suppressed Fas-induced apoptosis. However, activation of acid SMase was inhibited only by z-IETD-fmk. Thus, ceramide generated by acid SMase may take a part in Fas-induced apoptosis of human glioma cells and acid SMase activation may be dependent on caspase-8 activation, but not on p53 nor ROS.     

Reaction mechanism of the Co2+-activated multifunctional bromoperoxidase-esterase from Pseudomonas putida IF-3.

The reaction mechanism of the Co2+-activated bromoperoxidase-esterase of Pseudomonas putida IF-3 was studied. Site-directed mutagenesis suggested that the serine residue of the catalytic triad conserved in serine hydrolases participates in the bromination and ester hydrolysis reactions. The enzyme released a trace amount of free peracetic acid depending on the concentration of H2O2, which had been considered the intermediate in the reaction of nonmetal haloperoxidases to oxidize halide ions to hypohalous acid. However, the formation of free peracetic acid could not explain the enzyme activation effect by Co2+ ions which completely depleted the free peracetic acid. In addition, the kcat value of the enzymatic bromination was 900-fold higher than the rate constant of free peracetic acid-mediated bromination. Those results strongly suggested that the peracetic acid-like intermediate formed at the catalytic site is the true intermediate and that the formation of free peracetic acid is only a minor reaction involving the enzyme. We propose the possible reaction mechanism of this multifunctional enzyme based on these findings.     

Detection and induction of CTLs specific for SYT-SSX-derived peptides in HLA-A24(+) patients with synovial sarcoma

To investigate the immunogenic property of peptides derived from the synovial sarcoma-specific SYT-SSX fusion gene, we synthesized four peptides according to the binding motif for HLA-A24. The peptides, SS391 (PYGYDQIMPK) and SS393 (GYDQIMPKK), were derived from the breakpoint of SYT-SSX, and SS449a (AWTHRLRER) and SS449b (AWTHRLRERK) were from the SSX region. These peptides were tested for their reactivity with CTL precursors (CTLps) in 16 synovial sarcoma patients using HLA-A24/SYT-SSX peptide tetramers and also for induction of specific CTLs from four HLA-A24(+) synovial sarcoma patients. Tetramer analysis indicated that the increased CTLp frequency to the SYT-SSX was associated with pulmonary metastasis in synovial sarcoma patients (p < 0.03). CTLs were induced from PBLs of two synovial sarcoma patients using the peptide mixture of SS391 and SS393, which lysed HLA-A24(+) synovial sarcoma cells expressing SYT-SSX as well as the peptide-pulsed target cells in an HLA class I-restricted manner. These findings suggest that aberrantly expressed SYT-SSX gene products have primed SYT-SSX-specific CTLps in vivo and increased their frequency in synovial sarcoma patients. The identification of SYT-SSX peptides may offer an opportunity to design peptide-based immunotherapeutic approaches for HLA-A24(+) patients with synovial sarcoma.     

Coevolution of the S-locus genes SRK,SLG and SP11/SCR in Brassica oleracea and B. rapa

Brassica self-incompatibility (SI) is controlled by SLG and SRK expressed in the stigma and by SP11/SCR expressed in the anther. We determined the sequences of the S domains of 36 SRK alleles, 13 SLG alleles, and 14 SP11 alleles from Brassica oleracea and B. rapa. We found three S haplotypes lacking SLG genes in B. rapa, confirming that SLG is not essential for the SI recognition system. Together with reported sequences, the nucleotide diversities per synonymous and nonsynonymous site (pi(S) and pi(N)) at the SRK, SLG, and SP11 loci within B. oleracea were computed. The ratios of pi(N):pi(S) for SP11 and the hypervariable region of SRK were significantly >1, suggesting operation of diversifying selection to maintain the diversity of these regions. In the phylogenetic trees of 12 SP11 sequences and their linked SRK alleles, the tree topology was not significantly different between SP11 and SRK, suggesting a tight linkage of male and female SI determinants during the evolutionary course of these haplotypes. Genetic exchanges between SLG and SRK seem to be frequent; three such recent exchanges were detected. The evolution of S haplotypes and the effect of gene conversion on self-incompatibility are discussed.     

Sequence-independent DNA binding activity of DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli

The DnaA protein specifically binds to the origin of chromosomal DNA replication and initiates DNA synthesis. In addition to this sequence-specific DNA binding, DnaA protein binds to DNA in a sequence-independent manner. We here compared the two DNA binding activities. Binding of ATP and ADP to DnaA inhibited the sequence-independent DNA binding, but not sequence-specific binding. Sequence-independent DNA binding, but not sequence-specific binding, required incubation at high temperatures. Mutations in the C-terminal domain affected the sequence-independent DNA binding activity less drastically than they did the sequence-specific binding. On the other hand, the mutant DnaA433, which has mutations in a membrane-binding domain (K327 to I344) was inert for sequence-independent binding, but could bind specifically to DNA. These results suggest that the two DNA binding activities involve different domains and perform different functions from each other in Escherichia coli cells.     

Stable expression of gastric proton pump activity at the cell surface

Stable cell lines expressing the gastric proton pump alpha- and/or beta-subunits were constructed. The cell line co-expressing the alpha- and beta-subunits showed inward Rb(+) transport, which was activated by Rb(+) in a concentration-dependent manner. In the alpha+beta-expressing cell line, rapid recovery of intracellular pH was also observed after acid load, indicating that this cell line transported protons outward. These ion transport activities were inhibited by a proton pump inhibitor, 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080). In a membrane fraction of the alpha+beta-expressing cell line, K(+)-stimulated ATPase (K(+)-ATPase) activity and the acylphosphorylation of the alpha-subunit were observed, both of which were also inhibited by SCH 28080. The specific activity and properties of the K(+)-ATPase were comparable to those found in the native gastric proton pump. In the stable cell lines, the alpha-subunit was retained in the intracellular compartment and was unstable in the absence of the beta-subunit, but it was stabilized and reached the cell surface in the presence of the beta-subunit. On the other hand, the beta-subunit was stable and able to travel to the cell surface in the absence of the alpha-subunit. These cell lines are ideal for the structure-function study of ion transport by the gastric proton pump as well as for characterization of the cellular regulation of surface expression of the functional proton pump.     

Ras activation in T cells determines the development of antigen-induced airway hyperresponsiveness and eosinophilic inflammation

The central role for Th2 cells in the development of Ag-induced airway hyperresponsiveness and eosinophilic inflammation is well documented. We have reported a crucial role for TCR-induced activation of the Ras/extracellular signal-regulated kinase mitogen-activated protein kinase cascade in Th2 cell differentiation. Here, we show that the development of both OVA-induced airway hyperresponsiveness and eosinophilic airway inflammation in a mouse asthma model are attenuated in transgenic mice by the overexpression of enzymatically inactive Ras molecules in T cells. In addition, reduced levels of IL-5 production and eosinophilic inflammation induced by nematode infection (Nippostrongylus brasiliensis or Heligmosomoides polygyrus) were detected. Thus, the level of Ras activation in T cells appears to determine Th2-dependent eosinophilic inflammation and Ag-induced airway hyperresponsiveness.     

Identification and characterization of the UL56 gene product of herpes simplex virus type 2

The UL56 gene product of herpes simplex virus (HSV) has been shown to play an important role in viral pathogenicity. However, the properties and functions of the UL56 protein are little understood. We raised rabbit polyclonal antisera specific for the UL56 protein of HSV type 2 (HSV-2) and examined its expression and properties. The gene product was identified as three polypeptides with apparent molecular masses ranging from 32 to 35 kDa in HSV-2-infected cells, and at least one species was phosphorylated. Studies of their origins showed that the UL56 protein of HSV-2 is also translated from the upstream in-frame methionine codon that is not present in the HSV-1 genome. Synthesis was first detected at 6 h postinfection and was not abolished by the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies revealed that the UL56 protein localized to both the Golgi apparatus and cytoplasmic vesicles in HSV-2-infected and single UL56-expressing cells. Deletion mutant analysis showed that the C-terminal hydrophobic region of the protein was required for association with the cytoplasmic membrane and that the N-terminal proline-rich region was important for its translocation to the Golgi apparatus and cytoplasmic vesicles. Moreover, the results of protease digestion assays and sucrose gradient fractionation strongly suggested that UL56 is a tail-anchored type II membrane protein associated with lipid rafts. We thus hypothesized that the UL56 protein, as a tail-anchored type II membrane protein, may be involved in vesicular trafficking in HSV-2-infected cells.