Schistosoma mekongi: a prominent neutrophil chemotactic activity of egg antigen with reference to that of Schistosoma japonicum

Schistosoma mekongi causes granulomatous lesions around eggs deposited in the liver with neutrophil-rich inflammatory reactions in the early stage of the egg laying. To define the aspects of the typical pathogenesis of S. mekongi infection, we determined the difference between soluble egg antigen (SEA) from S. mekongi and S. japonicum with a focus on chemotactic factors for neutrophils or eosinophils. Mean volume and protein amount of S. mekongi eggs was 71 and 58% of those of Schistosoma japonicum eggs, respectively. Neutrophil chemotactic activity of S. mekongi SEA was about two times higher than that of S. japonicum. In contrast, eosinophil chemotactic activity of S. mekongi SEA was about half of that of S. japonicum SEA. Molecular analysis revealed that S. mekongi SEA contains higher molecular-weight components with a lower level of glycosylation, and this is likely to be related to the intense neutrophil chemotactic activity in comparison with S. japonicum SEA. The prominent chemotactic reactivity for neutrophils is likely to be involved in the typical pathogenesis of mekongi schistosomiasis.     

The NH(2)-terminal transmembrane and lumenal domains of LGP85 are needed for the formation of enlarged endosomes/lysosomes

LGP85 is a lysosomal membrane protein possessing a type III topology and is also known as a member of the CD36 superfamily of proteins, such as CD36 and the scavenger-receptor BI (SR-BI). We have recently demonstrated that overexpression of LGP85 in various mammalian cell lines causes the enlargement of endosomal/lysosomal compartments (ELCs). Using chimeras and deletion mutants, we show here that the lumenal region of LGP85 is necessary, but not sufficient, for the development of ELCs. Effective formation of enlarged ELC was largely dependent on the presence of a preceding NH₂-terminal transmembrane segment. Analyses of deletion mutants within the lumenal domain further revealed a requirement of the NH₂-terminal transmembrane proximal lumenal region, with high sequence similarity with SR-BI for the enlargement of ELC. These results suggest that an interaction of the NH₂-terminal transmembrane proximal lumenal domain of LGP85 with the inner leaflet of endosomal/lysosomal membranes through the connection with the transmembrane domain is an essential determinant for the regulation of endosomal/lysosomal membrane traffic. Interestingly, although the NH₂-terminal transmembrane domain itself was not sufficient for the enlargement of ELCs, it appeared to be required for direct targeting of LGP85 from the trans -Golgi network to late endosomes/lysosomes. Taken together, these results indicate the involvement of distinct domain of LGP85 in the targeting to, and biogenesis and maintenance of, ELC.     

Identification of a 521-kilodalton protein (Gli521) involved in force generation or force transmission for Mycoplasma mobile gliding

Several mycoplasma species are known to glide on solid surfaces such as glass in the direction of the membrane protrusion, but the mechanism underlying this movement is unknown. To identify a novel protein involved in gliding, we raised monoclonal antibodies against a detergent-insoluble protein fraction of Mycoplasma mobile, the fastest glider, and screened the antibodies for inhibitory effects on gliding. Five monoclonal antibodies stopped the movement of gliding mycoplasmas, keeping them on the glass surface, and all of them recognized a large protein in immunoblotting. This protein, named Gli521, is composed of 4,738 amino acids, has a predicted molecular mass of 520,559 Da, and is coded downstream of a gene for another gliding protein, Gli349, which is known to be responsible for glass binding during gliding. Edman degradation analysis indicated that the N-terminal region is processed at the peptide bond between the amino acid residues at positions 43 and 44. Analysis of gliding mutants isolated previously revealed that the Gli521 protein is missing in a nonbinding mutant, m9, where the gli521 gene is truncated by a nonsense mutation at the codon for the amino acid at position 1170. Immunofluorescence and immunoelectron microscopy indicated that Gli521 localizes all around the base of the membrane protrusion, at the "neck," as previously observed for Gli349. Analysis of the inhibitory effects of the anti-Gli521 antibody on gliding motility revealed that this protein is responsible for force generation or force transmission, a role distinct from that of Gli349, and also suggested conformational changes of Gli349 and Gli521 during gliding.     

Identification of the region responsible for fibril formation in the CAD domain of caspase-activated DNase

Caspase-activated DNase (CAD) has a compact domain at its N-terminus (CAD domain, 87 amino acid residues), which comprises one alpha-helix and five beta-strands forming a single sheet. The CAD domain of CAD (CAD-CD) forms amyloid fibrils containing alpha-helix at low pH in the presence of salt. To obtain insights into the mechanism of amyloid fibril formation, we identified the peptide region essential for fibril formation of CAD-CD and the region responsible for the salt requirement. We searched for these regions by constructing a series of deletion and point mutants of CAD-CD. Fibril formation by these CAD-CD mutants was examined by fluorescence analysis of thioflavin T and transmission electron microscopy. C-Terminal deletion and point mutation studies revealed that an aromatic residue near the C-terminus (Trp81) is critical for fibril formation. In addition, the main chain conformation of the beta5 strand, which forms a hydrophobic core with Trp81, was found to be important for the fibril formation by CAD-CD. The N-terminal 30 amino acid region containing two beta-strands was not essential for fibril formation. Rather, the N-terminal region was found to be responsible for the requirement of salt for fibril formation.     

Bi-directional regulation of Ser-985 phosphorylation of c-met via protein kinase C and protein phosphatase 2A involves c-Met activation and cellular responsiveness to hepatocyte growth factor

Previous studies indicated that treatment of cells with 12-O-tetradecanoylphorbol-13-acetate induced phosphorylation of Ser-985 at the juxtamembrane of c-Met, the receptor tyrosine kinase for hepatocyte growth factor (HGF), and this was associated with decreased tyrosine phosphorylation of c-Met. However, the regulatory mechanisms and the biological significance of the Ser-985 phosphorylation in c-Met remain unknown. When A549 human lung cancer cells were exposed to oxidative stress with H(2)O(2), H(2)O(2) treatment induced phosphorylation of Ser-985, but this was abrogated by an inhibitor for protein kinase C (PKC). Likewise, treatment of cells with NaF (an inhibitor of protein phosphatases) allowed for phosphorylation of Ser-985, and a protein phosphatase responsible for dephosphorylation of Ser-985 was identified to be protein phosphatase 2A (PP2A). The effects of PKC inhibitors revealed that PKCdelta and -epsilon were responsible for the Ser-985 phosphorylation of c-Met, and pull-down analysis indicated that associations of PKCdelta and -epsilon with c-Met may be involved in the regulation of Ser-985 phosphorylation of c-Met. Instead, PP2A was constitutively associated with c-Met, whereas its activity to dephosphorylate Ser-985 of c-Met was decreased when cells were exposed to H(2)O(2). Addition of HGF to A549 cells in culture induced c-Met tyrosine phosphorylation, the result being mitogenic response and cell scattering. In contrast, in the presence of H(2)O(2) stress, HGF-dependent tyrosine phosphorylation of c-Met was largely suppressed with a reciprocal relationship to Ser-985 phosphorylation, and this event was associated with abrogation of cellular responsiveness to HGF. These results indicate that Ser-985 phosphorylation of c-Met is bi-directionally regulated through PKC and PP2A, and the Ser-985 phosphorylation status may provide a unique mechanism that confers cellular responsiveness/unresponsivenss to HGF, depending on extracellular conditions.     

6-S-cysteinyl flavin mononucleotide-containing histamine dehydrogenase from Nocardioides simplex: molecular cloning, sequencing, overexpression, and characterization of redox centers of enzyme

Histamine dehydrogenase from Nocardioides simplex is a homodimeric enzyme and catalyzes oxidative deamination of histamine. The gene encoding this enzyme has been sequenced and cloned by polymerase chain reactions and overexpressed in Escherichia coli. The sequence of the complete open reading frame, 2073 bp coding for a protein of 690 amino acids, was determined on both strands. The amino acid sequence of histamine dehydrogenase is closely related to those of trimethylamine dehydrogenase and dimethylamine dehydrogenase containing an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-flavin mononucleotide, and one 4Fe-4S cluster as redox active cofactors in each subunit of the homodimer. The presence of the identical redox cofactors in histamine dehydrogenase has been confirmed by sequence alignment analysis, mass spectral analysis, UV-vis and EPR spectroscopy, and chemical analysis of iron and acid-labile sulfur. These results suggest that the structure of histamine dehydrogenase in the vicinity of the two redox centers is almost identical to that of trimethylamine dehydrogenase as a whole. The structure modeling study, however, demonstrated that a putative substrate-binding cavity in histamine dehydrogenase is quite distinct from that of trimethylamine dehydrogenase.     

Evaluation of mutation effects on UGT1A1 activity toward 17beta-estradiol using liquid chromatography-tandem mass spectrometry

Mutations in the gene encoding UDP-glucuronosyltransferase 1A1 (UGT1A1) may reduce the glucuronidation of estradiol, bilirubin, etc. In the present study, we used a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method to assay the activities of recombinant mutated UGT1A1 toward 17beta-estradiol (E2), by determining its glucuronide (E2G) content. Direct evidence for glucuronide formation was provided by E2G-specific ion peaks. The UGT1A1 activities of G71R (exon 1), F83L (exon 1), I322V (exon 2) and G493R (exon 5) mutants were 24, 30, 18 and 0.6% of the normal UGT1A1 activity, respectively. In conclusion, our study showed that LC/MS/MS enabled accurate evaluation of the effects of mutations on recombinant UGT1A1 activity towards E2.     

Different In Vivo Effects of HIV-1 Immunodominant Epitope-Specific Cytotoxic T Lymphocytes on Selection of Escape Mutant Viruses

HIV-1 escape mutants are well known to be selected by immune pressure via HIV-1-specific cytotoxic T lymphocytes (CTLs) and neutralizing antibodies. The ability of the CTLs to suppress HIV-1 replication is assumed to be associated with the selection of escape mutants from the CTLs. Therefore, we first investigated the correlation between the ability of HLA-A*1101-restricted CTLs recognizing immunodominant epitopes in vitro and the selection of escape mutants. The result showed that there was no correlation between the ability of these CTLs to suppress HIV-1 replication in vitro and the appearance of escape mutants. The CTLs that had a strong ability to suppress HIV-1 replication in vitro but failed to select escape mutants expressed a higher level of PD-1 in vivo, whereas those that had a strong ability to suppress HIV-1 replication in vitro and selected escape mutants expressed a low level of PD-1. Ex vivo analysis of these CTLs revealed that the latter CTLs had a significantly stronger ability to recognize the epitope than the former ones. These results suggest that escape mutations are selected by HIV-1-specific CTLs that have a stronger ability to recognize HIV-1 in vivo but not in vitro.HIV-1-specific cytotoxic T lymphocytes (CTLs) have an important role in the control of HIV-1 replication during acute and chronic phases of an HIV-1 infection (5, 28, 33). On the other hand, HIV-1 can escape from the host immune system by various mechanisms. These may include the appearance of HIV-1 carrying escape mutations in its immunodominant CTL epitopes as well as Nef-mediated downregulation of HLA class I molecules. There is a growing body of evidence for the former mechanism, i.e., that CTLs targeting immunodominant HIV-1 epitopes select escape mutants in chronically HIV-1-infected individuals (18, 20, 36), whereas the latter mechanism was proved by demonstrating that HIV-1-specific CTLs fail to kill Nef-positive-HIV-1-infected CD4⁺ T cells but effectively kill Nef-defective-HIV-1-infected ones or that they suppress the replication of Nef-defective HIV-1 much more than that of Nef-positive HIV-1 (12, 13, 42, 45).It is speculated that HIV-1 immunodominant epitope-specific CTLs have the ability to suppress HIV-1 replication and effectively select escape mutants. However, the correlation between this ability of the CTLs and the appearance of escape mutants is still unclear, because it is not easy to evaluate the ability of HIV-1-specific CTLs to exert a strong immune pressure in vivo. To examine this ability, most previous studies measured the number of HIV-1-specific CTLs or CD8⁺ T cells and the CTL activity against target cells prepulsed with the epitope peptide or those infected with HIV-1 recombinant vaccinia virus (6, 7, 23, 46). However, the results obtained from such experiments do not reflect the ability of the CTLs to exert immune pressure in vivo. We and other groups previously utilized an assay to directly evaluate the ability of the CTLs to suppress HIV-1 replication in vitro (1, 17, 18, 42, 43). This assay may be better for evaluation of immune pressure by HIV-1-specific CTLs than other assays, because the ability of the CTLs to suppress HIV-1 replication is directly measured in cultures of HIV-1-infected CD4⁺ T cells incubated with HIV-1-specific CTL clones. But it still remains unknown whether this assay reflects immune pressure in vivo.In the present study, we investigated whether HIV-1-specific CTLs having a strong ability to suppress HIV-1 replication could positively select escape mutants. Since HLA-A*1101 is known to be an HLA allele relatively associated with a slow progression to AIDS (32), it is speculated that some HLA-A*1101-restricted CTLs would have a strong ability to suppress HIV-1 replication in vitro. Therefore, we first focused on 4 well-known HLA-A*1101-restricted CTL epitopes in the present study. We investigated the frequency of CTLs specific for these epitopes in chronically HIV-1-infected individuals, the ability of these CTLs to suppress HIV-1 replication in vitro, and whether the escape mutants were selected by the CTLs. Furthermore, we analyzed the expression of Programmed Death-1 (PD-1) on these CTLs ex vivo and antigen recognition of them.     

Vertebrate DNA transposon as a natural mutator: the medaka fish Tol2 element contributes to genetic variation without recognizable traces

DNA-based transposable elements, or DNA transposons, transpose in a cut-and-paste fashion, involving excision from the chromosome. If this process affects the function of a host gene and the excision rate is high, any gene associated with such an element would clearly be in a genetically "unstable" state, and there are many examples of unstable genes in various organisms. However, none have hitherto been reported in vertebrates. We here document the finding of an unstable mutant gene in the medaka fish, Oryzias latipes, a useful model animal for vertebrate genetics and evolutionary studies. In an inbred strain, excision of the Tol2 element inserted in a pigmentation gene occurs spontaneously, giving rise to different heritable phenotypes and new mutant genes that carry different excision footprint sequences. The phenotypic mutation rate is as high as 2% per gamete, representing a 1000-fold increase from spontaneous mutation rates so far determined with the same organism. With mutations caused by insertion, and then excision, of transposons, one can no longer recognize participation of transposons in their generation. Thus, the impact of DNA transposons on vertebrate genomes may be, and may have been, larger than commonly supposed.