The involvement of immunoproteasomes in induction of MHC class I-restricted immunity targeting Toxoplasma SAG1

The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8+ T cells and was exploited in the development of a DNA vaccine against the intracellular protozoan Toxoplasma gondii by constructing a chimeric DNA encoding a fusion protein between murine ubiquitin and the toxoplasma antigen SAG1. The SAG1 peptide was promptly degraded in antigen-presenting cells (APCs) transfected with the chimeric DNA. Degradation, however, was hampered by incubating the APCs with the proteasome inhibitor epoxomicin. Mice vaccinated with the DNA acquired potent protective immunity mediated by MHC class I-restricted CD8+ T cells against infection by the highly virulent Toxoplasma. The accelerated degradation and induction of immunity were dependent on the UPS since mice lacking an immuno-subunit of 20S proteasome, LMP7, lost these functions, although they were independent of the proteasome regulator PA28alpha/beta complex.     

Changes in bacterial community during fermentative hydrogen and acid production from organic waste by thermophilic anaerobic microflora

AIMS: Changes in fermentation pattern during the treatment of organic wastes containing solid materials by thermophilic anaerobic microflora were investigated with respect to product formation and bacterial community structure during hydrogen production. METHODS AND RESULTS: Anaerobic microflora enriched from sludge compost was cultivated using artificial garbage slurry in a continuous flow-stirred tank reactor. Product formation varied depending on pH and hydraulic retention time (HRT) applied. Community analysis by terminal restriction fragment length polymorphism and clone library analysis of polymerase chain reaction-amplified bacterial 16S rDNA indicated that difference in the fermentative product distribution could be caused by different populations of micro-organisms in the microflora. CONCLUSION: Hydrogen fermentation with acetate/butyrate formation was optimized at <1.0 d HRT at pH 5.0 and 6.0. Thermoanaerobacterium thermosaccharolyticum was the dominant hydrogen-producing micro-organism. Conversely, unidentified organisms became dominant after 4.0 d HRT at pH 7.0 and 8.0, where relatively high-solubilization efficiency of solid materials was observed with no production of hydrogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing product formation in the fermentation of solid organic wastes by a mixed population of micro-organisms. Various fermentation patterns including hydrogen fermentation were characterized and evaluated from engineering and microbial aspects.     

Control of metaphase-anaphase progression by proteolysis: cyclosome function regulated by the protein kinase A pathway, ubiquitination and localization

Ubiquitin-mediated proteolysis is fundamental to cell cycle progression. In the fission yeast Schizosaccharomyces pombe, a mitotic cyclin (Cdc13), a key cell cycle regulator, is degraded for exiting mitosis, while Cut2 has to be destroyed for the onset of sister chromatid separation in anaphase. Ubiquitination of these proteins requires the special destruction box (DB) sequences locating in their N-termini and the large, 20S complex called the anaphase-promoting complex or cyclosome. Here we show that cyclosome function during metaphase-anaphase progression is regulated by the protein kinase A (PKA) inactivation pathway, ubiquitination of the cyclosome subunit, and cellular localization of the target substrates. Evidence is provided that the cyclosome plays pleiotropic roles in the cell cycle: mutations in the subunit genes show a common anaphase defect, but subunit-specific phenotypes such as in G1/S or G2/M transition, septation and cytokinesis, stress response and heavy metal sensitivity, are additionally produced, suggesting that different subunits take distinct parts of complex cyclosome functions. Inactivation of PKA is important for the activation of the cyclosome for promoting anaphase, perhaps through dephosphorylation of the subunits such as Cut9 (Apc6). Cut4 (Apc1), the largest subunit, plays an essential role in the assembly and functional regulation of the cyclosome in response to cell cycle arrest and stresses. Cut4 is highly modified, probably by ubiquitination, when it is not assembled into the 20S cyclosome. Sds23 is implicated in DB-mediated ubiquitination possibly through regulating de-ubiquitination, while Cut8 is necessary for efficient proteolysis of Cdc13 and Cut2 coupled with cytokinesis. Unexpectedly, the timing of proteolysis is dependent on cellular localization of the substrate. Cdc13 enriched along the spindle disappears first, followed by decay of the nuclear signal, whereas Cut2 in the nucleus disappears first, followed by decline in the spindle signal during metaphase-anaphase progression.     

Characterization of transgenic mice with targeted disruption of the catalytic domain of the double-stranded RNA-dependent protein kinase, PKR

The interferon-inducible, double-stranded RNA-dependent protein kinase PKR has been implicated in anti-viral, anti-tumor, and apoptotic responses. Others have attempted to examine the requirement of PKR in these roles by targeted disruption at the amino terminal-encoding region of the Pkr gene. By using a strategy that aims at disruption of the catalytic domain of PKR, we have generated mice that are genetically ablated for functional PKR. Similar to the other mouse model of Pkr disruption, we have observed no consequences of loss of PKR on tumor suppression. Anti-viral response to influenza and vaccinia also appeared to be normal in mice and in cells lacking PKR. Cytokine signaling in the type I interferon pathway is normal but may be compromised in the erythropoietin pathway in erythroid bone marrow precursors. Contrary to the amino-terminal targeted Pkr mouse, tumor necrosis factor alpha-induced apoptosis and the anti-viral apoptosis response to influenza is not impaired in catalytic domain-targeted Pkr-null cells. The observation of intact eukaryotic initiation factor-2alpha phosphorylation in these Pkr-null cells provides proof of rescue by another eukaryotic initiation factor-2alpha kinase(s).     

Presence of excess tetrahydrobiopterin during nitric oxide production from inducible nitric oxide synthase in LPS-treated rat aorta

Tetrahydrobiopterin (BH4) is one of the cofactors of nitric oxide synthase (NOS), and the synthesis of BH4 is induced as well as inducible NOS (iNOS) by lipopolysaccharide (LPS) and/or cytokines. BH4 has a protective effect against the cytotoxicity induced by nitric oxide (NO) and/or reactive oxygen species in various types of cells. The purpose of this study was to examine whether or not an excess of BH4 is present during the production of NO by iNOS in LPS-treated de-endothelialized rat aorta. Addition of LPS (10 microg/ml) to the aorta bath solution caused L-arginine (L-Arg)-induced relaxation from 1.5 hr after the addition of LPS in de-endothelialized rat aorta pre-contracted with 30 mM KCl. The L-Arg-induced relaxation was prevented by NOS inhibitors. BH4 content also increased from 3 hr after the addition of LPS. mRNAs of iNOS and GTP cyclohydrolase I (GTPCH), a rate-limiting enzyme of BH4 synthesis, were increased from 1.5 hr after addition of LPS. Although the expression of iNOS and GTPCH mRNAs was observed in the media, the expression levels in the media were much lower than those in the adventitia. Ten millimolar 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTPCH, strongly reduced L-Arg-induced relaxation, and decreased BH4 content to below the basal level in LPS-treated aorta, whereas 0.5 mM DAHP reduced the LPS-induced increase in BH4 content to the basal level but did not affect L-Arg-induced relaxation. The inhibition of L-Arg-induced relaxation by 10 mM DAHP was overcome by the addition of BH4 (10 microM). These results suggest that although BH4 is essential for NO production from iNOS, the increase in BH4 content above the basal level is not needed for eliciting L-Arg-induced relaxation by the treatment with LPS. Thus, an excess amount of BH4 may be synthesized during NO production by iNOS in LPS-treated rat aorta.     

Binding affinity of GM3 lactone for influenza virus

We investigated the interaction of GM3 lactone with influenza virus. The specific bindings of influenza virus and its hemagglutinin to GM3 lactone-containing mixed monolayers were studied by using a quartz-crystal microbalance. It has been known that gangliosides as receptors for influenza virus are also substrates for virus neuraminidase. GM3 lactone, however, was found to bind to influenza virus hemagglutinin, but not to be substrate for virus neuraminidase.     

Novel emission properties of melem caused by the heavy metal effect of lanthanides(III) in a LB film.

A novel emissive molecular system is constructed by the intercalation of the fluorophore melem (triamino-tri-s-triazine) within a Langmuir-Blodgett (LB) film of stearic acid with the periodic arrangement of lanthanides (Ln(III)), mainly Pr(III) with supporting of Eu(III). From emission spectra, decay curves, quantum yields and XPS measurements, it is clarified that the external heavy metal effect of Pr(III) on melem is much stronger in the film than in the bulk solid state, resulting in producing an unusual triplet state of melem. The triplet state of melem in the LB film donates the excitation energy to Pr(III) in the LB film, which is completely different from the energy transfer pathway of Pr-melem complex in the solid state through the singlet state of melem.     

Relationship between Phylogenetic Groups, Genotypic Clusters, and Virulence Gene Profiles of Escherichia coli Strains from Diverse Human and Animal Sources

Escherichia coli strains in water may originate from various sources, including humans, farm and wild animals, waterfowl, and pets. However, potential human health hazards associated with E. coli strains present in various animal hosts are not well known. In this study, E. coli strains from diverse human and animal sources in Minnesota and western Wisconsin were analyzed for the presence of genes coding for virulence factors by using multiplex PCR and biochemical reactions. Of the 1,531 isolates examined, 31 (2%) were found to be Shiga toxin-producing E. coli (STEC) strains. The majority of these strains, which were initially isolated from the ruminants sheep, goats, and deer, carried the stx_1c and/or stx_2d, ehxA, and saa genes and belonged to E. coli phylogenetic group B1, indicating that they most likely do not cause severe human diseases. All the STEC strains, however, lacked eae. In contrast, 26 (1.7%) of the E. coli isolates examined were found to be potential enteropathogenic E. coli (EPEC) strains and consisted of several intimin subtypes that were distributed among various human and animal hosts. The EPEC strains belonged to all four phylogenetic groups examined, suggesting that EPEC strains were relatively widespread in terms of host animals and genetic background. Atypical EPEC strains, which carried an EPEC adherence factor plasmid, were identified among E. coli strains from humans and deer. DNA fingerprint analyses, done using the horizontal, fluorophore-enhanced repetitive-element, palindromic PCR technique, indicated that the STEC, potential EPEC, and non-STEC ehxA-positive E. coli strains were genotypically distinct and clustered independently. However, some of the potential EPEC isolates were genotypically indistinguishable from nonpathogenic E. coli strains. Our results revealed that potential human health hazards associated with pathogenic E. coli strains varied among the animal hosts that we examined and that some animal species may harbor a greater number of potential pathogenic strains than other animal species.     

TMX, a human transmembrane oxidoreductase of the thioredoxin family: the possible role in disulfide-linked protein folding in the endoplasmic reticulum

Various proteins sharing thioredoxin (Trx)-like active site sequences (Cys-Xxx-Xxx-Cys) have been found and classified in the Trx superfamily. Among them, transmembrane Trx-related protein (TMX) was recently identified as a novel protein possessing an atypical active site sequence, Cys-Pro-Ala-Cys. In the present study, we describe the properties of this membranous Trx-related molecule. Endogenous TMX was detected as a protein of approximately 30 kDa with a cleavable signal peptide. TMX was enriched in membrane fractions and exhibited a similar subcellular distribution with calnexin localized in the endoplasmic reticulum (ER). The examination of membrane topology of TMX suggested that the N-terminal region containing the Trx-like domain was present in the ER lumen, where protein disulfide isomerase (PDI) was found to assist protein folding. Recombinant TMX showed PDI-like activity to refold scrambled RNase. These results indicate the possibility that TMX can modify certain molecules with its oxidoreductase activity and be involved in the redox regulation in the ER.