Kazushige Hamada

Characterization of a Novel Rat Brain Glycosylphosphatidylinositol-anchored Protein (Kilon), a Member of the IgLON Cell Adhesion Molecule Family

In the central nervous system, many cell adhesion molecules are known to participate in the establishment and remodeling of the neural circuit. Some of the cell adhesion molecules are known to be anchored to the membrane by the glycosylphosphatidylinositol (GPI) inserted to their C termini, and many GPI-anchored proteins are known to be localized in a Triton-insoluble membrane fraction of low density or so-called “raft.” In this study, we surveyed the GPI-anchored proteins in the Triton-insoluble low density fraction from 2-week-old rat brain by solubilization with phosphatidylinositol-specific phospholipase C. By Western blotting and partial peptide sequencing after the deglycosylation with peptide N-glycosidase F, the presence of Thy-1, F3/contactin, and T-cadherin was shown. In addition, one of the major proteins, having an apparent molecular mass of 36 kDa after the peptide N-glycosidase F digestion, was found to be a novel protein. The result of cDNA cloning showed that the protein is an immunoglobulin superfamily member with three C2 domains and has six putative glycosylation sites. Since this protein shows high sequence similarity to IgLON family members including LAMP, OBCAM, neurotrimin, CEPU-1, AvGP50, and GP55, we termed this protein Kilon (akindred of IgLON). Kilon-specific monoclonal antibodies were produced, and Western blotting analysis showed that expression of Kilon is restricted to brain, and Kilon has an apparent molecular mass of 46 kDa in SDS-polyacrylamide gel electrophoresis in its expressed form. In brain, the expression of Kilon is already detected in E16 stage, and its level gradually increases during development. Kilon immunostaining was observed in the cerebral cortex and hippocampus, in which the strongly stained puncta were observed on dendrites and soma of pyramidal neurons.

Identification of NAP-22 and GAP-43 (neuromodulin) as major protein components in a Triton insoluble low density fraction of rat brain

NAP-22 is a membrane-localized brain enriched acidic protein having a Ca(2+)-dependent calmodulin binding activity. Further fractionation of the NAP-22 containing membrane showed the localization of NAP-22 in a Triton insoluble fraction of low density. Besides NAP-22, this fraction was found to contain GAP-43 (neuromodulin), trimeric G proteins, and some GPI-anchored proteins such as Thy-1 and N-CAM-120. Presence of some protein tyrosine kinases, such as src and fyn, was also shown.

The DRE sequence TATCGATA, a putative promoter-activating element for Drosophila melanogaster cell-proliferation-related genes

We have confirmed that the DNA replication-related element (DRE) consisting of an 8-bp palindrome, TATCGATA, and not neighboring sequences, are responsible for activating promoters of the Drosophila melanogaster (Dm) PCNA (proliferating cell nuclear antigen)- and DNA polymerase alpha-encoding genes in both cultured cell and transgenic fly systems. We have so far found 153 copies of DRE in the Dm gene database. 73 of them are concentrated within the 600-bp upstream regions from the transcription start points of 61 genes. Interestingly, many of these genes are involved in either DNA replication, transcription, translation, signal transduction, cell cycle or other putative regulatory functions, and are possibly related to cell proliferation. It seems likely that DRE is an element common to the regulation of cell-proliferation-related genes, although their expression patterns may be different depending on which of regulatory elements other than the DRE are combined.

The clinical significance of core promoter and precore mutations during the natural course and interferon therapy in patients with chronic hepatitis B

Abstract OBJECTIVE: We aimed to determine the clinical significance of mutations in core promoter and precore regions in chronic hepatitis B. We investigated changes in these mutations during the natural course and interferon therapy in patients with chronic hepatitis B. METHODS: A total of 93 patients with hepatitis B virus surface antigen were divided into four groups according to hepatitis B e antigen (HBeAg)/anti-HBe status and serum aminotransferase levels. Group I (n = 16) comprised HBeAg-positive patients with normal aminotransferase levels, group II (n = 31) HBeAg-positive patients with elevated aminotransferase levels, group III (n = 30) anti-HBe–positive patients with normal aminotransferase levels, and group IV (n = 16) anti-HBe–positive patients with elevated aminotransferase levels. All patients of group II and seven of group IV were treated with interferon. Three serial serum samples per untreated patient and eight samples per treated patient were tested for HBV DNA levels and core promoter and precore mutations by polymerase chain reaction combined with restriction fragment length polymorphism, and some were cloned and sequenced. RESULTS: Core promoter mutation was found in 38% of group I, 74% of group II, 97% of group III, and 100% of group IV. Precore mutation was found in 6% of group I, 90% of group II, and 100% of groups III and IV. The HBV DNA levels were significantly higher in groups I, II, IV, and III, in that order. Serial determination of these two mutations and viral levels showed that the core promoter mutation appeared to occur first, followed by a completion of the precore mutation along with a decrease in viral levels in patients who seroconverted to anti-HBe after interferon therapy. Interferon therapy suppressed both precore wild- and mutated-type viral levels equally. However, it did not induce any specific mutations. CONCLUSIONS: Core promoter mutation appeared to develop or complete first, followed by completion of the precore mutation, and the virus with these two mutations seemed to be the form to persist in the natural course of chronic hepatitis B. The clinical significance of these mutations appeared to be profoundly associated with the viral levels.

Localization of 2′,5′-oligoadenylate synthetase and the enhancement of its activity with recombinant interferon-α A/D in the mouse brain

Abstract Although the expression of 2′,5′-oligoadenylate synthetase (2–5AS) is induced by interferon (IFN), low constitutive levels can be detected in animals that have not been treated with IFN. In order to clarify which cells express 2-5AS in the mouse brain, the distribution of this enzyme in the brains of both normal healthy mice and mice treated with recombinant human IFN-α A/D was studied by Western blotting and immunohistochemistry, using a specific monoclonal antibody. On the Western blots, the antibody to 42-kD 2–5AS reacted slightly with extracts from the telencephalon, cerebellum, diencephalon, and medulla oblongata of normal mouse brain. 42-kD 2–5AS was predominantly found in the ependymal cells and epithelium of the choroid plexus, and to a lesser degree in neurons and glial cells. Injection of recombinant human IFN-α A/D into the left lateral ventricle enhanced the activity of the enzyme in the telencephalon, cerebellum, diencephalon, and medulla oblongata, but did not change the immunohistochemical localization of the enzyme. Direct injection of the IFN into the cortex of the telencephalon enhanced the activity of 2–5AS in all parts of the brain and immunoreactivity was observed in the neurons and glial cells surrounding the injection site. These data indicate that 42-kD 2–5AS activity in the mouse brain is enhanced by the injection of recombinant human IFN-α A/D either into the left lateral ventricle or cortex of the telencephalon. Expression of 42-kD 2–5AS in ependymal cells and epithelium of the choroid plexus may prevent viral infections in the brain.

In vivo interferon system assessed by 2′‐5′ oligoadenylate synthetase activity in chronic hepatitis C virus patients treated with pegylated interferon and ribavirin

Aim:  2′,5′ oligoadenylate synthetase (2‐5AS), an enzyme induced by interferon, is an accurate indicator of the antiviral effect of interferon. We measured it during pegylated interferon based therapies in patients with chronic hepatitis C virus (HCV) in order to determine the dynamics of antiviral status in vivo and the relationship between the response to exogenous interferon and the outcome of therapy.

The predictive value of liver fibrosis in determining the effectiveness of interferon and lamivudine therapies for chronic hepatitis B

BackgoundTo determine the best indicator of the effective use of interferon and lamivudine for the treatment of hepatitis B e antigen-positive chronic hepatitis, we retrospectively analyzed histologic and virologic status in 200 patients who were treated with interferon and 45 patients who were treated with lamivudine.MethodsHistological grading and staging scores were determined by international criteria and the METAVIR scoring system. The YMDD motif associated with lamivudine resistance was analyzed by the sequencing of hepatitis B virus (HBV) DNA.ResultsOf 200 interferon-treated patients, 62 (31%) seroconverted to anti-hepatitis B e (anti-HBe). Multivariate analysis showed that the significantly important predictors of response were a higher grading score (P = 0.0056) and lower staging score (P = 0.0010). Twenty (44%) of the 45 lamivudine-treated patients seroconverted to anti-HBe, and multivariate analysis showed that the significantly important predictors of response were a higher alanine aminotransferase (ALT) level (P = 0.0034) and lower hepatitis B e antigen levels (P = 0.0128). YMDD mutations occurred during therapy in 12 patients (27%). The significantly important predictor of the development of mutation was a higher staging score (P = 0.0226).ConclusionsBoth interferon and lamivudine were effective for patients with high ALT levels, but interferon’s efficacy appeared to be limited by the degree of fibrosis. Lamivudine appeared to be effective irrespective of the degree of fibrosis, but YMDD mutations seemed to develop sooner in patients with advanced liver fibrosis.

Distribution of immunoreactive 2′,5′-oligoadenylate synthetase in mouse reproductive organs

Although 2′,5′-oligoadenylate synthetase (25AS) is an enzyme induced by inferferon (IFN) or viral infections and mediates one of the principal antiviral pathways turned on by IFN, low constitutive levels of the enzyme can be detected in various “normal” animals that have not been treated with IFN or virus. The distribution of this enzyme in the female and male reproductive organs of normal healthy mice was studied by Western blotting and by an immunohistochemical method, using a specific monoclonal antibody. On Western blotting, an antibody to 42-kD 2-5AS reacted with extracts from the ovary, oviduct, uterus, vagina, and placenta among the female reproductive organs, and testis, epididymis, and ductus deferens in the male. Immunohistochemically, the 2-5AS was localized on the following cells in the female reproductive organs: oocytes in the ovary; epithelium in the oviduct, uterus, and vagina; and trophoblasts in the placenta. Furthermore, the 2-5AS was localized on the epithelium and muscular layer in the ductus deferens and epithelium in the penis of the male mice, whereas the epithelium of the testis, epididymis, and seminal vesicle were stained faintly. It is well known that IFN is produced continuously in normal mice, so the 2-5AS in the tissues of normal mice is considered to be induced by such IFN produced under physiological conditions. Expression of the 2-5AS on the epithelium and trophoblasts in the reproductive organs may be responsible for the prevention of viral infections. However, the enzyme in oocytes may have some functions other than as an antiviral agent, since the enzyme was not detectable in embryos during early development.

Early reduction of infected hepatocytes by activated immunity at the time of interferon withdrawal hepatitis followed by lamivudine administration resulted in higher seroconversion in hepatitis Be antigen-positive patients with chronic hepatitis B.

BackgroundIt has been found that the efficacy of lamivudine (LAM) therapy can be improved by preceding administration with a short course of corticosteroid that induces a flare of the disease upon its withdrawal. Because of the side effects of corticosteroid, we tested the effect of a short course of interferon (IFN) as the primer instead of prednisolone, which was followed by LAM when the hepatitis flare occurred. The incidence of LAM resistance mutations and the effect of core promoter and precore mutations on the durability of the responses were also studied.MethodsPatients treated with interferon (IFN)-LAM therapy (n = 73) were compared to those treated with IFN alone (n = 117). The IFN-LAM group received IFN-α MU/day, t.i.w. for a 3-month period. LAM (10mg/day during 1 year) was started when IFN withdrawal hepatitis occurred during 2–10 months after stopping IFN. The LAM-resistant, core promoter, and precore mutations were examined by sequencing.Results(1) The IFN-LAM group developed exacerbated hepatitis following IFN withdrawal in 63 patients before starting LAM therapy. The seroconversion (SC) rate was significantly higher in the IFN-LAM group than in the IFN-alone group (61% vs 26%, P = 0.0001). (2) The LAM resistance mutation rate was 31% at 1 year after initiating LAM therapy. (3) In a stepwise discriminant-function analysis, decreased level of HBeAg determined at 4 weeks after LAM administration and increased level of HBeAb before the start of LAM administration contributed significantly on seroconversion to anti-HBe (P = 0.0073 and 0.004, respectively). (4) The reappearance rate of HBeAg within 6 months after the therapy (relapse) was 33% in the IFN-LAM group and 10% in the IFN-alone group. The prevalence of core promoter and precore mutations did not change before and after the therapy, nor did these mutations correlate with the relapse after stopping IFN-LAM therapy.Conclusions(1) Our findings suggest that early reduction of infected hepatocytes expressed by HBeAg by LAM may contribute to a high SC rate of IFN-LAM therapy. (2) The emergence of LAM-resistant mutations was similar to the previously reported rate, and neither core promoter nor precore mutations correlated with relapse of seroconverters after IFN-LAM withdrawal.