Temporary upregulation of anti-inflammatory cytokine IL-13 expression in the brains of CD14 deficient mice in the early stage of prion infection

CD14 deficient (CD14^−/−) mice survived longer than wild-type (WT) C57BL/6J mice when inoculated with prions intracerebrally, accompanied by increased expression of anti-inflammatory cytokine IL-10 by microglia in the early stage of infection. To assess the immune regulatory effects of CD14 in detail, we compared the gene expression of pro- and anti-inflammatory cytokines in the brains of WT and CD14^−/− mice infected with the Chandler strain. Gene expression of the anti-inflammatory cytokine IL-13 in prion-infected CD14^−/− mice was temporarily upregulated at 75 dpi, whereas IL-13 gene expression was not upregulated in prion-infected WT mice. Immunofluorescence staining showed that IL-13 was mainly expressed in neurons of the thalamus at 75 dpi. These results suggest that CD14 can suppress IL-13 expression in neurons during the early stage of prion infection.     

OX40 ligand regulates splenic CD8 dendritic cell-induced Th2 responses in vivo

In mice, splenic conventional dendritic cells (cDCs) can be separated, based on their expression of CD8α into CD8⁻ and CD8⁺ cDCs. Although previous experiments demonstrated that injection of antigen (Ag)-pulsed CD8⁻ cDCs into mice induced CD4 T cell differentiation toward Th2 cells, the mechanism involved is unclear. In the current study, we investigated whether OX40 ligand (OX40L) on CD8⁻ cDCs contributes to the induction of Th2 responses by Ag-pulsed CD8⁻ cDCs in vivo, because OX40–OX40L interactions may play a preferential role in Th2 cell development. When unseparated Ag-pulsed OX40L-deficient cDCs were injected into syngeneic BALB/c mice, Th2 cytokine (IL-4, IL-5, and IL-10) production in lymph node cells was significantly reduced. Splenic cDCs were separated to CD8⁻ and CD8⁺ cDCs. OX40L expression was not observed on freshly isolated CD8⁻ cDCs, but was induced by anti-CD40 mAb stimulation for 24 h. Administration of neutralizing anti-OX40L mAb significantly inhibited IL-4, IL-5, and IL-10 production induced by Ag-pulsed CD8⁻ cDC injection. Moreover, administration of anti-OX40L mAb with Ag-pulsed CD8⁻ cDCs during a secondary response also significantly inhibited Th2 cytokine production. Thus, OX40L on CD8⁻ cDCs physiologically contributes to the development of Th2 cells and secondary Th2 responses induced by Ag-pulsed CD8⁻ cDCs in vivo.     

Aspergillus nidulans class V and VI chitin synthases CsmA and CsmB, each with a myosin motor-like domain, perform compensatory functions that are essential for hyphal tip growth

The polarized synthesis of cell wall components such as chitin is essential for the hyphal tip growth of filamentous fungi. The actin cytoskeleton is known to play important roles in the determination of hyphal polarity in Aspergillus nidulans. Previously, we suggested that CsmA, a chitin synthase with a myosin motor-like domain (MMD), was involved in polarized chitin synthesis in a manner dependent on the interaction between the MMD and the actin cytoskeleton. The genome database indicates that A. nidulans possesses another gene encoding another chitin synthase with an MMD. In this study, we characterized this gene, which we designated csmB. The csmB null mutants examined were viable, although they exhibited defective phenotypes, including the formation of balloons and intrahyphal hyphae and the lysis of subapical regions, which were similar to those obtained with csmA null mutants. Moreover, csmA csmB double null mutants were not viable. Mutants in which csmB was deleted and the expression of csmA was under the control of the alcA promoter were viable but severely impaired in terms of hyphal growth under alcA-repressing conditions. We revealed that CsmB with three copies of a FLAG epitope tag localized at the hyphal tips and forming septa, and that the MMD of CsmB was able to bind to actin filaments in vitro. These results suggest that CsmA and CsmB perform compensatory functions that are essential for hyphal tip growth.     

Substitution of the N-glycan function in glycosyltransferases by specific amino acids: ST3Gal-V as a model enzyme

The sialyltranferase ST3Gal-V transfers a sialic acid to lactosylceramide. We investigated the role of each of the N-glycans modifying mouse ST3Gal-V (mST3Gal-V) by measuring the in vitro enzyme activity of Chinese hamster ovary (CHO) cells transfected with ST3Gal-V cDNA or its mutants. By examining mutants of mST3Gal-V, in which each asparagine was replaced with glutamine (N180Q, N224Q, N334Q), we determined that all three sites are N-glycosylated and that each N-glycan is required for enzyme activity. Despite their importance, N-glycosylation sites in ST3Gal-V are not conserved among species. Therefore, we considered whether the function in the activity that is performed in mST3Gal-V by the N-glycan could be substituted for by specific amino acid residues selected from the ST3Gal-V of other species or from related sialyltransferases (ST3Gal-I, -II, -III, and -IV), placed at or near the glycosylation sites. To this end, we constructed a series of interspecies mutants for mST3Gal-V, specifically, mST3Gal-V-H177D-N180S (medaka or tetraodon type), mST3Gal-V-N224K (human type), and mST3Gal-V-T336Q (zebrafish type). The ST3Gal-V activity of these mutants was quite similar to that of the wild-type enzyme. Thus, we have demonstrated here that the N-glycans on mST3Gal-V are required for activity but can be substituted for specific amino acid residues placed at or near the glycosylation sites. We named this method SUNGA (substitution of N-glycan functions in glycosyltransferases by specific amino acids). Furthermore, we verified that the ST3Gal-V mutant created using the SUNGA method maintains its high activity when expressed in Escherichia coli thereby establishing the usefulness of the SUNGA method in exploring the function of N-glycans in vivo.     

Kinesin-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons

To address questions about mechanisms of filament-based organelle transport, a system was developed to image and track mitochondria in an intact Drosophila nervous system. Mutant analyses suggest that the primary motors for mitochondrial movement in larval motor axons are kinesin-1 (anterograde) and cytoplasmic dynein (retrograde), and interestingly that kinesin-1 is critical for retrograde transport by dynein. During transport, there was little evidence that force production by the two opposing motors was competitive, suggesting a mechanism for alternate coordination. Tests of the possible coordination factor P150(Glued) suggested that it indeed influenced both motors on axonal mitochondria, but there was no evidence that its function was critical for the motor coordination mechanism. Observation of organelle-filled axonal swellings ("organelle jams" or "clogs") caused by kinesin and dynein mutations showed that mitochondria could move vigorously within and pass through them, indicating that they were not the simple steric transport blockades suggested previously. We speculate that axonal swellings may instead reflect sites of autophagocytosis of senescent mitochondria that are stranded in axons by retrograde transport failure; a protective process aimed at suppressing cell death signals and neurodegeneration.     

Applicability of bacterial endotoxins test to various blood products by the use of endotoxin-specific lysates

Endotoxin contamination is a serious threat to the safety of parenteral drugs, and the rabbit pyrogen test has played a crucial role in controlling this contamination. Although the highly sensitive endotoxin test has replaced the pyrogen test for various pharmaceuticals, the pyrogen test is still implemented as the control test for most blood products in Japan. We examined the applicability of the endotoxin test to blood products for reliable detection and quantification of endotoxin. Nineteen types of blood products were tested for interfering factors based on spike/recovery of endotoxin by using 2 types of endotoxin-specific lysate reagents for photometric techniques. Interfering effects on the endotoxin test by the products could be eliminated by diluting from 1/2 to 1/16, with the exception of antithrombin III. However, conventional lysate reagents that also react with non-pyrogenic substances, such as (1–3)-β-d-glucan, produced results that were not relevant to endotoxin content or pyrogenicity. Our results showed that the endotoxin test would be applicable to most blood products if used with appropriate endotoxin-specific lysate reagents.     

Alternative trans-splicing: a novel mode of pre-mRNA processing

Alternative splicing is an important process contributing to proteome diversity without involving an increase in the number of genes. In some cases, alternative splicing is carried out under 'trans-mode', called alternative trans-splicing, in which exons located on separate pre-mRNA molecules are selectively joined to produce mature mRNAs encoding proteins with distinct structures and functions. However, it is not known how widespread or how frequently trans-splicing occurs in vivo. Recently, trans-allelic trans-splicing has been unambiguously demonstrated in Drosophila using a SNP (single nucleotide polymorphism) as a marker. In this review, we provide an overview of alternative trans-splicing in Drosophila and mammals, and discuss its mechanisms.     

Molecular design of LPS-binding peptides

Lipopolysaccharide (LPS), a major constituent of the outer membrane of Gram-negative bacteria, is highly toxic and can cause sepsis or septic shock. Therefore, detection of LPS and the ability to neutralize its toxicity is important. We previously obtained a strong LPS-binding peptide, Li5-001, using the phage display method (Matsumoto et al., 2010. J. Microbiol. Methods. 82, 54-58). We modified the sequence the amino acid sequence of this peptide (KNYSSSISSIHAC), by replacing and deleting amino acids to obtain higher LPS-binding affinity and greater resistance to protease digestion. Consequently we obtained a dodecapeptide, Li5-025 (K′YSSSISSIRAC′, K′ and C′ are D-forms of K and C, respectively) which showed a high affinity for LPS, approximately 1000 folds higher affinity than Li5-001 and Kd value of 0.01 nM. By replacing both N- and C-terminal amino acids from L-type to D-type, the peptide was rendered resistant to protease digestion without altering its overall binding capacity.     

Sequence and expression of rat ICAM-1.

We have isolated cDNA clones-coding for rat intercellular adhesion molecule-1 (RICAM-1) from a cDNA library constructed from rat Ax cells stimulated with IL-1 beta using the mouse ICAM-1 cDNA as a hybridization probe. The RICAM-1 sequence shows 79.1% homology with mouse ICAM-1 and 55.6% homology with human ICAM-1 at the nucleic acid level. In order to examine the expression of RICAM-1 on Chinese hamster ovary (CHO) cells, we constructed the vector, pSV-RICAM1-neo, containing the SV40 promoter. Flowcytometric analysis showed that CHO-K1 cells transfected with pSV-RICAM1-neo expressed high amounts of RICAM-1 on their surfaces.