Masami Morimatsu

MAIL, a novel nuclear IκB protein that potentiates LPS-induced IL-6 production

We have identified and characterized a novel member of the ankyrin‐repeat family named ‘molecule possessing ankyrin‐repeats induced by lipopolysaccharide’ (MAIL). The C‐terminal portion of MAIL shared high sequence homology with the IκB family. Intraperitoneal injection of lipopolysaccharide (LPS) into mice rapidly (<0.5 h) induced MAIL mRNA in various tissues, particularly in the spleen, lymph node, and lung. Ectopically expressed MAIL was localized in the nucleus, and remarkably potentiated the LPS‐induced mRNA expression and secretion of interleukin (IL)‐6 in Swiss 3T3 cells. These findings indicate that MAIL is one of the nuclear IκB proteins and an activator of IL‐6 production.

Expression and localization of insulin-regulatable glucose transporter (GLUT4) in rat brain

The localization of glucose transporters (GLUTs) was examined in various regions of the rat brain. The mRNA of GLUT1 and GLUT3 were found ubiquitously in every brain region (cortex, hippocampus, midbrain, striatum, hypothalamus, medulla oblongata and cerebellum). The mRNA and protein of GLUT4, an insulin-regulatable glucose transporter in peripheral tissues, were also identified, particularly abundantly in the cerebellum. In situ hybridization analysis revealed that GLUT4 mRNA was present in some discrete cells, such as Purkinje cells in the cerebellum, the vestibular nucleus in the medulla oblongata and also in ependymal cells along the cerebral ventricles. The GLUT4 mRNA level in the cerebellum changed little in fasted or experimentally induced diabetic rats while those in adipose tissues decreased much. The results suggest that insulin-sensitive glucose uptake may occur in some specific cells of the brain but is regulated in a different manner from those in peripheral cells.

Isolation of canine C-reactive protein and characterization of its properties.

C-reactive protein (CRP) was isolated from the acute phase serum of dogs subjected to surgical stimulation. Its properties were characterized. Canine CRP was isolated by ion-exchange chromatography using DEAE-Sephacel and DEAE-Sephadex A-50 and affinity chromatography using protein A-Sepharose CL 4B in combination with agar-block electrophoresis. In immunoelectrophoresis, canine CRP had the same gamma-mobility as human gamma-type CRP. The molecular weight of purifined canine CRP was estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis to be approximately 157,000 and 155,000 respectively. This CRP was a thermolabile protein which completely lost its antigenicity by heating at 70 degrees C for 15 min. The serum concentration of CRP in normal beagle dogs ranged from 0.198 to 0.826 micrograms ml-1 (0.486 +/- 0.170 micrograms ml-1). The concentration was acutely increased by surgery as it was in man and was rapidly decreased with convalescence. Dogs can be a useful animal model for investigation of the mechanism of CRP production and the function of CRP.

Bovine haptoglobin: Single radial immunodiffusion assay of its polymeric forms and dramatic rise in acute-phase sera

Using purified bovine haptoglobin (Hp) and specific antisera, a single radial immunodiffusion (SRID) assay method has been developed to measure the serum Hp level in cattle. Bovine Hp is a highly polymerized protein showing heterogeneous molecular forms in serum. After treatment with cysteine or glutathione, Hp was partially reduced to a homogeneous form, suitable for SRID assay. This method gives values comparable to those obtained by hemoglobin-binding capacity assay, and has the advantage of being simple and convenient. Although serum Hp was not detectable in healthy cattle, it was found more than 50-fold after invasive surgery, indicating that Hp is a characteristic acute-phase protein in cattle.

The Multimerization of Hantavirus Nucleocapsid Protein Depends on Type-Specific Epitopes

ABSTRACT Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers. Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers. The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization. The NP-NP interaction was also detected by using a yeast two-hybrid assay. Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast. The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast. However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA. Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA. Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.

Induction of acute phase protein by recombinant human interleukin-6 (IL-6) in calves

Interleukin-6 (IL-6) is a major inducer of acute phase proteins in human and murine species. However, the effects of IL-6 have not yet been investigated in cattle. Following continuous infusion of recombinant human IL-6, serum concentrations of bovine haptoglobin and fibrinogen increased in a manner similar to those in cattle with acute phase reaction. In contrast, C-reactive protein and alpha 1-acid glycoprotein, as well as the other hematologic parameters, did not change significantly. Intravenous administration of recombinant human IL-6 resulted in only a mild and transient increase of bovine haptoglobin. These results suggest that the regulation of acute phase protein production in cattle is similar, but not identical, to that observed in human and murine species.

Targeted disruption of MAIL, a nuclear IκB protein, leads to severe atopic dermatitis-like disease

MAIL (molecule-possessing ankyrin repeats induced by lipopolysaccharide) is a nuclear IκB protein that is also termed interleukin-1-inducible nuclear ankyrin repeat protein or inhibitor of nuclear factor κB (IκB) ζ. In this study, we generated Mail–/– mice to investigate the roles of MAIL in whole organisms. Mail–/– mice grew normally until 4–8 weeks after birth, when they began to develop lesions in the skin of the periocular region, face, and neck. MAIL mRNA and protein were constitutively expressed in the skin of wild type controls, especially in the keratinocytes. Serum IgE was higher in Mail–/– mice than in normal. Histopathological analysis indicated that the Mail–/– skin lesions appeared to be atopic dermatitis (AD) eczema with inflammatory cell infiltration. In addition, markedly elevated expression of some chemokines such as thymus and activation-regulated chemokine was detected in the Mail–/– skin lesions, similar to that observed in the skin of patients with AD. In Mail–/– mice, MAIL-deficient keratinocytes might be activated to produce chemokines and induce intraepidermal filtration of inflammatory cells, resulting in the onset of the AD-like disease. These findings suggest that MAIL is an essential molecule for homeostatic regulation of skin immunity. The Mail–/– mouse is a valuable new animal model for research on AD.

Hallmarks of Hepatitis C Virus in Equine Hepacivirus

ABSTRACT Equine hepacivirus (EHcV) has been identified as a closely related homologue of hepatitis C virus (HCV) in the United States, the United Kingdom, and Germany, but not in Asian countries. In this study, we genetically and serologically screened 31 serum samples obtained from Japanese-born domestic horses for EHcV infection and subsequently identified 11 PCR-positive and 7 seropositive serum samples. We determined the full sequence of the EHcV genome, including the 3′ untranslated region (UTR), which had previously not been completely revealed. The polyprotein of a Japanese EHcV strain showed approximately 95% homology to those of the reported strains. HCV-like cis-acting RNA elements, including the stem-loop structures of the 3′ UTR and kissing-loop interaction were deduced from regions around both UTRs of the EHcV genome. A comparison of the EHcV and HCV core proteins revealed that Ile190 and Phe191 of the EHcV core protein could be important for cleavage of the core protein by signal peptide peptidase (SPP) and were replaced with Ala and Leu, respectively, which inhibited intramembrane cleavage of the EHcV core protein. The loss-of-function mutant of SPP abrogated intramembrane cleavage of the EHcV core protein and bound EHcV core protein, suggesting that the EHcV core protein may be cleaved by SPP to become a mature form. The wild-type EHcV core protein, but not the SPP-resistant mutant, was localized on lipid droplets and partially on the lipid raft-like membrane in a manner similar to that of the HCV core protein. These results suggest that EHcV may conserve the genetic and biological properties of HCV. IMPORTANCE EHcV, which shows the highest amino acid or nucleotide homology to HCV among hepaciviruses, was previously reported to infect horses from Western, but not Asian, countries. We herein report EHcV infection in Japanese-born horses. In this study, HCV-like RNA secondary structures around both UTRs were predicted by determining the whole-genome sequence of EHcV. Our results also suggest that the EHcV core protein is cleaved by SPP to become a mature form and then is localized on lipid droplets and partially on lipid raft-like membranes in a manner similar to that of the HCV core protein. Hence, EHcV was identified as a closely related homologue of HCV based on its genetic structure as well as its biological properties. A clearer understanding of the epidemiology, genetic structure, and infection mechanism of EHcV will assist in elucidating the evolution of hepaciviruses as well as the development of surrogate models for the study of HCV.

IκBζ Is a Transcriptional Key Regulator of CCL2/MCP-1

CCL2, also referred to as MCP-1, is critically involved in directing the migration of blood monocytes to sites of inflammation. Consequently, excessive CCL2 secretion has been linked to many inflammatory diseases, whereas a lack of expression severely impairs immune responsiveness. We demonstrate that IκBζ, an atypical IκB family member and transcriptional coactivator required for the selective expression of a subset of NF-κB target genes, is a key activator of the Ccl2 gene. IκBζ-deficient macrophages exhibited impaired secretion of CCL2 when challenged with diverse inflammatory stimuli, such as LPS or peptidoglycan. These findings were reflected at the level of Ccl2 gene expression, which was tightly coupled to the presence of IκBζ. Moreover, mechanistic insights acquired by chromatin immunoprecipitation demonstrate that IκBζ is directly recruited to the proximal promoter region of the Ccl2 gene and is required for transcription-enhancing histone H3 at lysine-4 trimethylation. Finally, IκBζ-deficient mice showed significantly impaired CCL2 secretion and monocyte infiltration in an experimental model of peritonitis. Together, these findings suggest a distinguished role of IκBζ in mediating the targeted recruitment of monocytes in response to local inflammatory events.

Immunohistochemical and in situ hybridization analysis of galectin-3, a β-galactoside binding lectin, in the urinary system of adult mice

Galectin is an animal lectin that has high affinity to β-galactoside of glycoconjugates. In the present study, cellular expression of galectin subtypes in the urinary system of adult mice was examined by in situ hybridization and immunohistochemistry. The major subtype expressed in the murine urinary system was galectin-3, which was expressed continuously from the kidney to the distal end of the urethra. The renal cortex expressed galectin-3 more intensely than the medulla. Renal galectin-3 immunoreactivity was strongest in the cortical collecting ducts, where principal cells were the sole cellular source. All cell layers of the transitional epithelium from the renal pelvis to the urethra strongly expressed galectin-3 at the mRNA and protein levels. An electron microscopic study demonstrated diffuse cytoplasmic localization of galectin-3 in principal cells of the collecting ducts and in the bladder epithelial cells. Urethral galectin-3 expression at the pars spongiosa decreased in intensity near the external urethral orifice, where the predominant subtype of galectin was substituted by galectin-7. The muscular layer of the ureter and urinary bladder contained significant signals for galectin-1. Taken together, the observations indicate that the adult urinary system shows intense and selective expression of galectin-3 in epithelia of the uretic bud- and cloaca-derivatives.