Visualization of microglia in living tissues using Iba1-EGFP transgenic mice.

Microglia are thought to play important roles not only in repairing injured tissue but in regulating neuronal activity, and visualizing the cells is very useful as a means of further investigating the function of microglia in vivo. We previously cloned the ionized calcium-binding adaptor molecule 1 (Iba1) gene, which is expressed selectively in microglia/microphages. To generate new transgenic mice to visualize microglia with enhanced green fluorescent protein (EGFP), we here constructed a plasmid carrying EGFP cDNA under control of the Iba1 promoter. This construct was injected into C57B/6 mouse zygotes, and the Iba1-EGFP transgenic line was developed. Fluorescent in-situ hybridization analysis revealed that the Iba1-EGFP transgene was located on chromosome 11D. No obvious defects were observed during development or in adulthood, and the EGFP fluorescence remained invariant over the course of at least four generations. Judging from the immunoreactivity with anti-Iba1 antibody, all EGFP-positive cells in the adult brain were ramified microglia. In the developing transgenic embryos, EGFP signals were detected as early as embryonic Day 10.5. The most prominent EGFP signals were found in forebrain, spinal cord, eye, foreleg, yolk sac, liver, and vessel walls. At postnatal Day 6, clear EGFP signals were observed in the supraventricular corpus callosum, known as "fountain of microglia", where ameboid microglia migrate into the brain parenchyma and mature into ramified microglia. Iba1-EGFP transgenic mice thus permit observation of living microglia under a fluorescence microscope and provide a useful tool for studying the function of microglia in vivo.

Sequence and genetic arrangement of the UL region of the monkey B virus (Cercopithecine herpesvirus 1) genome and comparison with the UL region of other primate herpesviruses.

The complete DNA sequence of the unique long (U(L)) region of monkey B virus (BV) was determined. Based on sequence homology and the presence of transcriptional control element motifs, homologues of every open reading frame present in the U(L) region of the Human herpesvirus 1 (herpes simplex virus 1, HSV-1) and Human herpesvirus 2 (herpes simplex virus 2, HSV-2) genomes were identified in BV. The BV genes are arranged in the same order and orientation as in HSV. These results demonstrate that the BV U(L) region is entirely co-linear with that of HSV-1 and HSV-2.

Urinary metabolites of genistein administered orally to rats.

In a study on the metabolism of flavonoids, the isoflavone genistein was administered orally to rats. Urine samples were collected and treated with beta-glucuronidase and arylsulfatase. Genistein and its metabolites, 4',5,7-trihydroxyisoflavanone (M1), 4',7-dihydroxyisoflavan (M2), and p-ethylphenol (M3) were isolated from the urine following treatment with enzymes. The structures of M1, M2, and M3 were determined on the basis of chemical and spectral data.

Mechanisms underlying extracellular ATP-evoked interleukin-6 release in mouse microglial cell line, MG-5.

Microglia play various important roles in the CNS via the synthesis of cytokines. The ATP-evoked production of interleukin-6 (IL-6) and its intracellular signals were examined using a mouse microglial cell line, MG-5. ATP, but not its metabolites, produced IL-6 in a concentration-dependent manner. Although ATP activated two mitogen-activated protein kinases, i.e. p38 and extracellular signal-regulated protein kinase, only p38 was involved in the IL-6 induction. However, the activation of p38 was not sufficient for the IL-6 induction because 2'- and 3'-O-(4-benzoylbenzoyl) ATP, an agonist to P2X7 receptors, failed to produce IL-6 despite the fact that it activated p38. Unlike in other cytokines in microglial cells, P2Y rather than P2X7 receptors seem to have a major role in the IL-6 production by the cells. The ATP-evoked IL-6 production was attenuated by Gö6976, an inhibitor of Ca(2+)-dependent protein kinase C (PKC). The P2Y receptor responsible for these responses was insensitive to pertussis toxin (PTX) and was linked to phospholipase C. Taken together, ATP acting on PTX-insensitive P2Y receptors activates p38 and Ca(2+)-dependent PKC, thereby resulting in the mRNA expression and release of IL-6 in MG-5. This is a novel pathway for the induction of cytokines in microglia.

Huge cystic lymphangioma of the pancreas: report of a case.

We report herein the case of a 33-year-old woman who presented with palpable abdominal swelling found to be caused by a huge lymphangioma of the pancreas. An abdominal computed tomographic (CT) scan showed a large multilocular cystic mass with water-dense contents, which was derived from the pancreatic head. A pancreaticoduodenectomy (PD) was performed because the tumor had invaded the duodenum. The resected tumor, which was 23 x 12 x 23 cm in size with 21 of serous fluid, was pathologically diagnosed as a cystic lymphangioma. The endothelial cells lining the internal surface of the cystic spaces were immunohistochemically positive for factor VIII-R antigen and CD31. Our review of the literature revealed 45 reports of lymphangioma of the pancreas, including this one, but to the best of our knowledge this is only the fifth case that required a PD. Nevertheless, we recommend that a complete resection be performed to reduce the risk of recurrence.

Iba1 is an actin-cross-linking protein in macrophages/microglia.

Iba1 is a 17-kDa EF hand protein that is specifically expressed in macrophages/microglia and is upregulated during the activation of these cells. When exposed to macrophage colony-stimulating factor (M-CSF), microglia cell line MG5 immediately produces intense membrane ruffles in which Iba1 accumulates together with filamentous actin. In this study, we investigated the physical interaction between Iba1 and actin by centrifugation assay and electron microscopic examination and showed that Iba1 possesses actin-binding and -cross-linking activities. Inhibitory mutant Iba1 that suppresses M-CSF-induced membrane ruffling had lost the actin-cross-linking activity, and it inhibited the cross-linking activity of intact Iba1. These results indicate that Iba1 is a macrophage/microglia-specific actin-cross-linking protein essential for M-CSF-induced membrane ruffling.

Dermoid cyst of the colon.

Dermoid cysts are benign cystic teratomas lined by skin and epidermal appendages. We report a dermoid cyst occurring in a 26-year-old female whose chief complaint was irregular vaginal bleeding. Abdominal magnetic resonance image demonstrated a space-occupying lesion in the right lower abdomen. The mass showed hyperintensity on the T2 image and the signal was homogeneous for the interior. During abdominal surgery we made the diagnosis of subserous tumor of the colon and resected the ileocecal portion of the colon. The tumor measured 5.4 x 4.8 x 3.5 cm and was soft and elastic. On cross section, a unilocular cyst filled with atheromatous material was found. Pathological examination revealed a dermoid cyst. In the view of this diagnosis, a simple excision would have been an adequate treatment.

Tissue responses around polymethylmethacrylate particles implanted into bone: analysis of expression of bone matrix protein mRNAs by in situ hybridization.

Tissue responses around implanted polymethylmethacrylate (PMMA) particles were analyzed by in situ hybridization with digoxigenin-labeled procollagen alpha1(I) (COL), osteonectin, osteocalcin, and osteopontin (OPN) mRNA probes. PMMA particles (150-300 microm in diameter) were implanted into rat tibiae, and specimens were collected at 3, 5, 7, and 10 days after operation. New bone was formed centripetally, and bone-forming osteoblasts expressed all four kinds of mRNAs. A COL signal was expressed most strongly and widely. In the early stage, COL-positive cells were detected on and among particles sporadically. A COL signal was rarely detected in cells on the surfaces of the particles, suggesting that PMMA particles may suppress osteoblast differentiation. Osteonectin and osteocalcin mRNAs were expressed in bone-forming osteoblasts in a similar pattern by day 7. By contrast, an OPN signal was detected mainly on the particles, not only in COL-positive osteoblasts but also in COL-negative round cells. The latter cells had acid phosphatase activity, suggesting that they might be macrophages responding to a foreign body. At day 10, an OPN signal was detected continuously in multinucleated cells on PMMA particles, whereas new bone was formed away from particles. Our approach helped us to understand the initial cellular reaction to materials, which may determine their biocompatibility.

Co-induction of argininosuccinate synthetase, cationic amino acid transporter-2, and nitric oxide synthase in activated murine microglial cells.

Nitric oxide (NO) produced by activated microglia has been implicated in many pathophysiological events in the brain including neurodegenerative diseases. Cellular NO production depends absolutely on the availability of arginine, a substrate of NO synthase (NOS). Murine microglial MG5 cells were treated with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), and expression of inducible NO synthase (iNOS) and arginine-supplying enzymes was investigated by RNA blot analysis. iNOS mRNA was strongly induced after treatment and reached a maximum at 6-12 h. mRNA for argininosuccinate synthetase (AS), a citrulline-arginine recycling enzyme, increased at 6 h and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and AS proteins were also induced. In addition, mRNA encoding the cationic amino acid transporter-2 (CAT-2) was strongly induced shortly after treatment. Induction of mRNAs for iNOS, AS, and CAT-2 by LPS/IFN-gamma was also observed following stimulation of rat primary microglial cells. These results strongly suggest that both arginine transport by CAT-2 and citrulline-arginine recycling are important for high-output production of NO in activated microglial cells.

Molecular cloning and biological activity of a novel lysyl oxidase-related gene expressed in cartilage.

We cloned a cDNA encoding a novel lysyl oxidase-related protein, named LOXC, by suppression subtractive hybridization between differentiated and calcified ATDC5 cells, a clonal mouse chondrogenic EC cell line. The deduced amino acid sequence of mouse LOXC consists of 757 amino acids and shows 50% identity with that of mouse lysyl oxidase. Northern blot analysis showed a distinct hybridization band of 5.4 kilobases, and Western blot analysis showed an immunoreactive band at 82 kilodaltons. Expression of LOXC mRNA was detected in osteoblastic MC3T3-E1 cells and embryonic fibroblast C3H10T1/2 cells, whereas none of NIH3T3 fibroblasts and myoblastic C2C12 cells expressed LOXC mRNA in vitro. Moreover, the LOXC mRNA and protein levels dramatically increased throughout a process of chondrogenic differentiation in ATDC5 cells. In vivo, LOXC gene expression was localized in hypertrophic and calcified chondrocytes of growth plates in adult mice. The conditioned media of COS-7 cells transfected with the full-length LOXC cDNA showed the lysyl oxidase activity in both type I and type II collagens derived from chick embryos, and these activities of LOXC were inhibited by beta-aminopropionitrile, a specific inhibitor of lysyl oxidase. Our data indicate that LOXC is expressed in cartilage in vivo and modulates the formation of a collagenous extracellular matrix.

Mutation spectrum of o-aminoazotoluene in the cII gene of lambda/lacZ transgenic mice (MutaMouse).

The o-aminoazotoluene (AAT) has been evaluated as a possible human carcinogen by the International Agency for Research on Cancer. In rodents, it is carcinogenic mainly in the liver, and also in lung following long term administration. We previously examined in lambda/lacZ transgenic mice for the induction of lacZ mutations in liver, lung, urinary bladder, colon, kidney, bone marrow, and testis. AAT induced gene mutations strongly in the liver and colon. In the present report, we reveal the molecular nature of mutations induced by AAT in the lambda cII gene (the cII gene, a phenotypically selectable marker in the lambda transgene, has 294bp, which makes it easier to sequence than the original target, the 3kb lacZ gene). The cII mutant frequency in liver and colon was five and nine times higher, respectively, in AAT-treated mice than in control mice. Sequence analysis revealed that AAT induced G:C to T:A transversions, whereas spontaneous mutations consisted primarily of G:C to A:T transitions at CpG sites.

Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors.

The initial microglial responses that occur after brain injury and in various neurological diseases are characterized by microglial accumulation in the affected sites of brain that results from the migration and proliferation of these cells. The early-phase signal responsible for this accumulation is likely to be transduced by rapidly diffusible factors. In this study, the possibility of ATP released from injured neurons and nerve terminals affecting cell motility was determined in rat primary cultured microglia. Extracellular ATP and ADP induced membrane ruffling and markedly enhanced chemokinesis in Boyden chamber assay. Further analyses using the Dunn chemotaxis chamber assay, which allows direct observation of cell movement, revealed that both ATP and ADP induced chemotaxis of microglia. The elimination of extracellular calcium or treatment with pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, suramin, or adenosine-3'-phosphate-5'-phosphosulfate did not inhibit ATP- or ADP-induced membrane ruffling, whereas AR-C69931MX or pertussis toxin treatments clearly did so. As an intracellular signaling molecule underlying these phenomena, the small G-protein Rac was activated by ATP and ADP stimulation, and its activation was also inhibited by pretreatment with pertussis toxin. These results strongly suggest that membrane ruffling and chemotaxis of microglia induced by ATP or ADP are mediated by G(i/o)-coupled P2Y receptors.

Replication of enterotropic and polytropic murine coronaviruses in cultured cell lines of mouse origin.

To understand the virus-cell interactions that occur during murine coronavirus infection, six murine cell lines (A3-1M, B16, CMT-93, DBT, IC-21 and J774A.1) were inoculated with eight murine coronaviruses, including prototype strains of both polytropic and enterotropic biotypes, and new isolates. All virus strains produced a cytopathic effect (CPE) with cell-to-cell fusion in B16, DBT, IC-21 and J774A.1 cells. The CPE was induced most rapidly in IC-21 cells and was visible microscopically in all cell lines tested. In contrast, the coronaviruses produced little CPE in A3-1M and CMT-93 cells. Although most virus-infected cells, except KQ3E-infected A3-1M, CMT-93 and J774A.1 cells, produced progeny viruses in the supernatants when assayed by plaque formation on DBT cells, the kinetics of viral replication were dependent on both the cell line and virus strain; replication of prototype strains was higher than that of new isolates. There was no significant difference in replication of enterotropic and polytropic strains. B16 cells supported the highest level of viral replication. To determine the sensitivity of the cell lines to murine coronaviruses, the 50% tissue culture infectious dose of the coronaviruses was determined with B16, DBT, IC-21 and J774A.1 cells, and compared to that with DBT cells. The results indicate that IC-21 cells were the most sensitive to murine coronaviruses. These data suggest that B16 and IC-21 cells are suitable for large-scale preparation and isolation of murine coronaviruses, respectively.

Urinary metabolites of gallic acid in rats and their radical-scavenging effects on 1,1-diphenyl-2-picrylhydrazyl radical.

As a part of our studies on the metabolism of natural compounds, gallic acid was orally administered to rats. The urinary metabolites were analyzed by high-performance liquid chromatography, and their structures were determined to be pyrogallol (M1), pyrogallol-1-O-beta-D-glucuronide (M2), 4-O-methylgallic acid-3-O-sulfate (M3), 2-O-methylpyrogallol-1-O-beta-D-glucuronide (M4), 2-O-methylpyrogallol (M5), 4-O-methylgallic acid (M6), and unchanged gallic acid on the basis of chemical and spectral data. The radical scavenging effects of gallic acid and its urinary metabolites were evaluated using 1,1-diphenyl-2-picrylhydrazyl radical.

Genotoxicity of o-aminoazotoluene (AAT) determined by the Ames test, the in vitro chromosomal aberration test, and the transgenic mouse gene mutation assay.

o-Aminoazotoluene (AAT) has been evaluated as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). The Ames test found it to be mutagenic in the presence of a metabolic activation system, whereas it has little clastogenicity either in vitro or in vivo in the chromosomal aberration assay. AAT is also carcinogenic in the lung or liver of mice and rats given long-term administrations. Therefore, metabolites generated in the liver etc. may have gene mutation activity, and carcinogenesis would occur. We examined the mutagenicity of AAT in a gene mutation assay, using lacZ transgenic mice (MutaMice) and a positive selection method. AAT showed positive results for organs with metabolic functions, such as liver and colon and other organs. Positive results were also seen in an Ames test in the presence of metabolic activation and negative results seen in a chromosomal aberration test. Therefore, AAT had the potential to cause gene mutation in the presence of metabolic activation systems in vitro and the same reaction was confirmed in vivo with organs with metabolic function, such as liver and colon, but little clastogenicity in vitro or in vivo. Thus, metabolites with gene mutation activity may be responsible for the carcinogenicity of AAT. The transgenic mouse mutation assay proved to be useful for concurrent assessment of in vivo mutagenicity in multiple organs and to supplement the standard in vivo genotoxicity tests, such as the micronucleus assay which is limited to bone marrow as the only target organ.

The expression of bone matrix protein mRNAs around beta-TCP particles implanted into bone.

Tissue response around beta-tricalcium phosphate (beta-TCP) particles (150-300 microm in diameter) implanted into rat tibiae was analyzed by in situ hybridization with digoxigenin-labeled procollagen alpha1(I) (COL), osteonectin, osteocalcin, and osteopontin (OPN) RNA probes. Specimens were collected at 3, 5, 7, and 10 days after the operation. Holes without implantation were used as control. In both the beta-TCP implanted and control groups, new bone was formed centripetally and all four kinds of mRNA were expressed in activated osteoblasts. A COL signal was expressed most strongly and widely, and was detected at the peripheral region of the hole at day 3. The other three mRNAs were also expressed in bone forming osteoblasts by day 7. However, in the earlier cell reaction stage, OPN expression in the beta-TCP implanted group was different than that in the control group: OPN mRNA was seen exclusively in the cells on the particles, and an OPN signal was detected not only in COL-positive cells, but also in COL-negative cells. The former cells may be osteoblasts and reflect the early process of bone formation on biomaterials. The latter cells may be macrophages and reflect foreign body reactions. Expression of these OPN mRNAs induced by implantation of beta-TCP may play a role in bone formation on the materials and in determining their biocompatibility.

Involvement of Iba1 in membrane ruffling and phagocytosis of macrophages/microglia.

Ionized calcium binding adaptor molecule 1, Iba1, is an EF hand calcium binding protein whose expression is restricted to macrophages/microglia. In this study, Iba1 was shown to colocalize with F-actin in membrane ruffles induced by macrophage colony-stimulating factor and in phagocytic cups formed during zymosan phagocytosis. Expression of mutant Iba1 carrying either N- or C-terminal deletions or carrying a substitution in the calcium binding domain, suppressed the membrane ruffling and the phagocytosis. These results indicate that Iba1 is a key molecule in membrane ruffling and the phagocytosis of macrophages/microglia. Furthermore, Iba1 colocalized with a small GTPase Rac in the membrane ruffles and the phagocytic cups. The Iba1 mutants also suppressed membrane ruffling induced by dominant active Rac1V12, but do not affect microspikes by Cdc42V12 and stress fibers by RhoAV14. These observations suggest that Iba1 is involved in Rac and calcium signaling pathways.

Variation in the frequency of micronucleated hepatocytes following time course treatment in the mouse: liver micronucleus assay of partially hepatectomized tissues.

The time-course pattern of the frequency of micronucleated hepatocytes in vivo after partial hepatectomy (PH) was studied in mice using N-nitrosodimethylamine (DMN), N-nitrosodiethylamine (DEN), and 1,2-dimethylhydrazine (DMH), which are rodent liver carcinogens with potent clastogenic activity in the liver. With all chemicals, production of micronucleated hepatocytes was not clearly observed at 3 d after PH, but was clear 4 or 5 d after PH. We propose that it is preferable to perform a preliminary assay prior to the main assay when estimating the clastogenic potential of certain chemicals towards hepatocytes in vivo.

Synthesis, crystal structure and antimicrobial activities of two isomeric gold(I) complexes with nitrogen-containing heterocycle and triphenylphosphine ligands, [Au(L)(PPh3)] (HL = pyrazole and imidazole).

Two isomeric gold(I)-triphenylphosphine complexes with nitrogen-containing heterocycles, [Au(L)(PPh3) (HL = pyrazole (1), imidazole (2)) were isolated as colorless cubic crystals for 1 and colorless plate crystals for 2, respectively. The crystal structures of 1 and 2 were determined by single-crystal X-ray diffraction. These complexes were also fully characterized by complete elemental analyses, thermogravimetric/differential thermal analyses (TG/DTA) and FT-IR in the solid state and by solution NMR (31P, 1H and 13C) spectroscopy and molecular weight measurements in acetone solution. These complexes consisted of a monomeric 2-coordinate AuNP core both in the solid state and in solution. The molecular structures of 1 and 2 were compared with those of related gold(I) complexes, [Au(1,2,3-triz)(PPh3)] (3, Htriz = triazole), [Au(1,2,4-triz)(PPh3)]2 (4) as a dimer through a gold(I)-gold(I) bond in the solid state, and [Au(tetz)(PPh3)] (5, Htetz = tetrazole). Selective and effective antimicrobial activities against two gram-positive bacteria (B. subtilis, S. aureus) and modest activities against one yeast (C. albicans) found in these gold(I) complexes 1-4 are noteworthy, in contrast to poor activities observed in the corresponding silver(I) complexes.

Metabolites of orally administered Perilla frutescens extract in rats and humans.

As a part of our search for bioactive substances from the leaves of Perilla frutescens BRITTON var. acuta KUDO (Perillae Herba, Labiatae), the aqueous extract was orally administered to rats and humans, and metabolites in the urine, plasma, and/or bile were analyzed by a high-performance liquid chromatograph (HPLC) equipped with a photodiode array detector. When the extract was administered to rats, 10 metabolites, trans-caffeic acid-4-O-sulfate (1), trans-p-coumaric acid-4-O-sulfate (2), trans-ferulic acid-4-O-sulfate (3), trans-m-coumaric acid-3-O-sulfate (4), trans-caffeic acid (5), m-hydroxyphenylpropionic acid (6), trans-p-coumaric acid (7), trans-m-coumaric acid (8), luteolin (9), and apigenin (10) were detected in the urine, whereas four metabolites, scutellarein-6, 7-di-O-beta-glucuronide (11), apigenin-4'-O-sulfate-7-O-beta-glucuronide (12), apigenin-7-O-beta-glucuronide (13), and diosmetin-7-O-beta-glucuronide (14) were found in the bile. Compounds 1-8 and 11-14 were also found in the plasma. When the extract was given to humans, however, two metabolites, 1-O-(2,4,5-trimethoxycinnamoyl)-beta-glucuronic acid (15) and apigenin-4'-O-beta-glucuronide (16), were found in the urine and plasma. Thus, a species difference in the metabolism of the extract constituents was observed between rats and humans. Structures 1-16 were identified based on their chemical and spectral data.