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.

Surface potential and surface charge density of the cerebral-cortex synaptic vesicle and stability of vesicle suspension.

Using a microelectrophoresis instrument employing the Lazer-Zee system, the electrophoretic mobility of synaptic vesicles isolated from Guinea-pig brain cortex was measured under conditions. The mobility was found to depend on both pH and ionic concentration of the solution. The surface of the synaptic vesicle was shown to be negatively charged under physiological conditions. The isoelectric point was observed at pH 4.0 in 0.01 M NaCl solution. Effects of divalent cations were examined and reversal of surface charge was observed in 0.1 M CaCl2 solution. Interaction of vesicles was also considered on the basis of the DLVO theory of colloid stability by using calculated values of surface charge density and surface potential of the synaptic vesicle.

Epitope mapping and characterization of monoclonal antibodies to human protein C.

Six monoclonal antibodies specific to human protein C were characterized. Epitopes of these antibodies were determined on isolated proteolytic peptides of protein C by immunological methods. Three antibodies bound light chain of protein C: PC01 bound the gamma-carboxyglutamic acid domain calcium-dependently, while PC02 and PC08 bound the first epidermal growth factor-like domain in calcium-dependent and independent manners, respectively. The other three antibodies bound the heavy chain of protein C: PC13 bound activation peptide, PC04 recognized the activation site and PC09 bound the region close to a disulfide bond connecting light and heavy chains. Activation of protein C with thrombin-thrombomodulin complex was inhibited strongly by PC04 and moderately by PC08, PC09 and PC13. PC04 and PC13 may directly block the activation site. On the other hand, epitopes of PC08 and PC09 may be involved in interaction between protein C and thrombin-thrombomodulin complex, or locate close to activation site on the tertiary structure of protein C. Anticlotting activity of protein C was inhibited strongly by PC01 and moderately by PC02, PC08 and PC09, while amidolytic activity was inhibited only by PC09. The epitopes described here may constitute part of protein-C-specific sites, which are important for the function of protein C.

Determination of effective and safe dose for intracoronary administration of nicorandil in dogs.

STUDY OBJECTIVE: The aim was to evaluate the effective and safe dosage for intracoronary administration of nicorandil (2-nicotinamidoethyl nitrate) in dogs. DESIGN: Five ml of saline with or without nicorandil (0.025, 0.25, 1.0 or 2.5 mg) was constantly infused into the left anterior descending coronary artery (LAD) in 1 min without blood flow reduction in group A (normoxic group, n = 5) and with 50% blood flow reduction of the control value in group B (ischaemic group, n = 5). SUBJECTS: 10 mongrel dogs of either sex were used, weight 14.6-20.5 kg. MEASUREMENTS AND MAIN RESULTS: The myocardial weight of the perfused area was 33.2(SD 7.9) g. In group A, LAD blood flow increased from 28.4(8.6) to 86.3(25.0) ml.min-1, p less than 0.05, following intracoronary infusion of 0.25 mg nicorandil. Higher doses caused no further increase. In both groups, high doses (1.0 mg or more) suppressed regional myocardial contraction, expressed as % segment shortening, from 20.0(4.7) to 10.0(9.0)%, p less than 0.05, in group A, and from 12.7(8.5) to 6.5(5.6)%, p less than 0.05, in group B. Multiple ventricular premature beats developed in both groups only at the dose of 2.5 mg: 6.8(5.5) beats.min-1 in group A and 4.5(4.2) beats.min-1 in group B. Ventricular fibrillation developed in two dogs in group B. Nicorandil did not affect the local bipolar electrocardiogram of the subendo- and subepicardium in the perfused area in either group. CONCLUSIONS: Intracoronary administration of 0.25 mg of nicorandil was effective for coronary vasodilatation without deleterious mechanical or electrical effects. Larger doses of the agent showed adverse effects depending on the dosages.

A new sandwich enzyme-linked immunosorbent assay (ELISA) for transforming growth factor alpha (TGF alpha) based upon conformational modification by antibody binding.

A sandwich-type enzyme-linked immunosorbent assay (ELISA) for human TGF alpha was established utilizing monoclonal and polyclonal anti-synthetic human TGF alpha antibodies with defined epitopes. A polyclonal antibody which was raised in a rabbit and affinity purified by C terminal peptide (hTGF alpha (34-50)) recognized both intact and denatured human TGF alpha. Murine monoclonal antibodies isolated bound only to the denatured form of TGF alpha at the second loop (hTGF alpha (16-33)). However, the rabbit antibody was found to induce a conformation change of intact TGF alpha and the resultant immunocomplex was recognized by monoclonal antibodies. By virtue of this property, the ELISA could detect both native and denatured TGF alpha with the same efficiency with a detection limit of 0.1 ng/ml. Human EGF did not interfere with the ELISA. Production of TGF alpha in several transformed human cell lines was quantitatively examined. Some cell lines were found to secrete TGF alpha, but the production rate was very low, except one melanoma, suggesting that TGF alpha may function only locally in a very limited area in vivo.

Microglia-specific localisation of a novel calcium binding protein, Iba1.

Recently it has been shown that mRNA of Iba1 (ionized calcium binding adaptor molecule 1), which was a novel calcium binding protein cDNA-cloned by our group, is specifically expressed in microglia in cultures of rat brain cells [Imai et al. Biophys. Biochem. Res. Commun., 224 (1996) 855-862]. In the present study, immunocytochemical and immunohistochemical examinations demonstrated that Iba1 protein is expressed in microglia alone both in cultured brain cells and in the brain, respectively. In a mixed cell culture of embryonic rat brain, immunocytochemically positive for Iba1 protein were the microglia but it was not detectable in neurons, astroglia, or oligodendroglia. Immunohistochemical staining of adult rat brain sections showed Iba1 protein to be specifically localised in ramified microglia. In addition, immunohistochemical staining and immunoblot analysis of activated microglia in the facial nucleus after facial nerve axotomy shows that expression of Iba1 protein was upregulated and peaked at 7 days. These results indicated that localisation of Iba1 protein is restricted to microglia both in vitro and in vivo, and that Iba1 protein plays a role in regulating the function of microglia, especially in the activated microglia.

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.

Immunological localization and ontogenetic development of inhibin alpha subunit in rat brain.

This study examined the immunolocalization and ontogeny of the inhibin-specific alpha subunit in the brain of male rats. Immunohistochemistry using antiserum directed against the mature region of porcine inhibin alpha (1-19, Tyr20) revealed positive reactions in process-bearing cells resembling astroglia in several regions, especially in the dorsal region of the third ventricle, medial and ventral arcuate nucleus, hippocampal dentate gyrus, and layers 1-3 of the cerebral cortex. Generally, inhibin alpha-positive cells in the limbic cortex had larger cell bodies and longer processes than those in the hypothalamus. These inhibin alpha-positive cells were verified to be positive for glial fibrillary acidic protein (GFAP), a differentiated astroglial marker, by double immunolabelling. The expression of inhibin alpha mRNA was higher in the brains of neonatal rats than in those of adult rats, as revealed by reverse transcription-competitive polymerase chain reaction, although the similar changes of immunoreactive inhibin alpha subunit in the brain was not observed. Orchiectomy did not affect expression of inhibin alpha mRNA in the hypothalamic area. This study suggests that inhibin-related peptide is produced by differentiated astrocytes, especially in the hypothalamic arcuate nucleus, the hippocampal dentate gyrus, and the cerebral cortex, and that the expression of inhibin alpha is regulated during brain development.

Complete nucleotide sequence of human vasoactive intestinal peptide/PHM-27 gene and its inducible promoter.

We have previously shown that the VIP precursor contains a novel PHI-27-like peptide, PHM-27, and that the synthesis of the prepro-VIP/PHM-27 mRNA is induced with cAMP and TPA in human neuroblastoma cells. In this study, we have determined the complete nucleotide sequence of the human VIP/PHM-27 gene. The gene spans 8,837 bp and consists of seven exons and six introns. Exon I of 165 bp consists of the 5' untranslated region of the gene, exon II of 117 bp encodes the signal peptide of prepro-VIP/PHM-27, exon III of 123 bp encodes the amino-terminal region, exon IV of 105 bp encodes PHM-27, exon V of 132 bp encodes VIP, exon VI of 89 bp contains the termination codon of the prepro-VIP/PHM-27 mRNA, and exon VII of 724 bp consists of the 3' untranslated region of the gene. VIP and its structurally related peptide, PHM-27, were encoded in different exons V and IV, and the sequences around the splice junctions between these exons and their adjacent introns were highly conserved, suggesting that the VIP-encoding and PHM-27-encoding exons have been duplicated from an ancestral exon over a broad area containing its adjacent introns. We also determined the 1,929-bp sequence of the 5' flanking region of the human VIP/PHM-27 gene and found that four TATA-box sequences were present at 28 bp, 145 bp, 772 bp, and 900 bp upstream of the cap site. Primer extension, exon mapping, and mung bean nuclease mapping analyses revealed that only the TATA-box sequence 28 bp upstream of the cap site was the promoter that is inducible by cAMP and TPA in the human neuroblastoma cells. An 18-bp sequence 52 bp upstream from the TATA-box sequence was suggested to be a cAMP/phorbol esters-responsive element of the human VIP/PHM-27 gene.

Immunohistochemical characterization of the suprachiasmatic nucleus and the intergeniculate leaflet in the hereditary bilaterally microphthalmic rat.

Immunohistochemical observation was performed in the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) of hereditary bilaterally microphthalmic rats without the optic nerve on both sides. In the microphthalmic rats, volume of the SCN reduced to ca. 70% of the normal and numbers of the vasoactive intestinal polypeptide (VIP)-like immunoreactive (lir) neurons were significantly decreased. Although the arginine vasopressin (aVP)- and the VIP-lir neurons distributed in the dorsomedial and the ventrolateral part of the SCN, respectively, as reported in the normal one, somatostatin-lir neurons, localizing mainly in a border area between the dorsomedial and the ventrolateral region of the normal SCN, were shifted to the ventral part of the SCN in the microphthalmic rats. The ventral part of the SCN was covered with neuropeptide Y (NPY)-lir fibers in both normal and mutant rats. The IGL was hardly delineated cytologically in the lateral geniculate nucleus (LGN) of the mutant rats. NPY-lir neurons were found in the dorsal part of the ventral LGN, in contrast to their even distribution in the normal IGL. These findings suggest that the IGL-SCN tract remains in the hereditary microphthalmic rats without the retinal projections.

ATP content in isolated mammalian nerve cells assayed by a modified luciferin-luciferase method.

ATP content in a nerve cell isolated from dorsal root ganglia of adult guinea-pigs by collagenase was measured by a newly developed technique modified from the conventional luciferin-luciferase methods. A small volume (4 microliters) of the nerve cell suspension, which contained 10-300 nerve cells (3-100 X 10(-4) microliters of cellular volume) under view of an inverted, phase-contrast microscope, was heat-treated for about 1 s by flame of an alcohol lamp. This heat-treated cell suspension was then reacted with a luciferin-luciferase solution. Light flux from the bioluminescence thus elicited gave an ATP content in single nerve cell, 27 pg (mean) +/- 10 pg (S.D.). ATP concentration in a nerve cell was calculated as 1.7 mM (mean) +/- 0.6 mM. The ATP content in a nerve cell was reduced when the nerve cells were exposed to KCN (5 microM) or dinitrophenol (20 microM), respectively.

The lipid and protein content of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata purified to constant composition: implications for vesicle structure.

The lipid, protein, acetylcholine and ATP content of cholinergic synaptic vesicles isolated from the richly innervated electric organ of Torpedo marmorata and purified to constant composition has been determined. The number of vesicles present in the preparations has been estimated by quantitative electron microscopy and the mean composition of the vesicle deduced. The acetylcholine content of the purest preparations was considerably greater than that previously attained and reached a mean of 6.10 mmole/g of protein and 2.6 X 10(5) molecules/vesicle; the mean values, for all determinations, were 4.1 +/- S.E.M. 0.6 and 2.6 X 10(5) +/- S.E.M. 0.6 X 10(5) respectively. The lipid and protein content of the vesicle (about 140 and 80 ag/vesicle respectively) is relatively low, indicating a thin, lipid-rich membrane and a highly hydrated core of which not more than 1-2% can be occupied by protein. These findings are consistent with conclusions drawn from recent density determinations made at different osmotic pressures using penetrating and non-penetrating gradients.

Neuronal adhesion molecule telencephalin induces rapid cell spreading of microglia.

Telencephalin (TLCN) is a neuronal surface glycoprotein whose expression is restricted to the telencephalon, the most rostral segment of the brain. TLCN binds to lymphocyte function-associated antigen-1 (LFA-1) integrin. In the central nervous system, LFA-1 is selectively and constitutively expressed by microglia, suggesting that TLCN/LFA-1 binding may mediate cell-cell interactions between telencephalic neurons and microglia. In the present study, we investigated the effects of recombinant TLCN protein on the morphology of microglia. TLCN induced an intensive spreading of lamellipodia, causing a rapid change in microglial morphology. In contrast, TLCN induced no significant change in morphology of neuroblastoma and fibroblasts. Furthermore, the TLCN-induced spreading of microglia was accompanied by a clustering of LFA-1 on cell surface membrane. These results provide evidence that TLCN binding to the surface of microglia transduces signals into microglia that mediate or accelerate cell spreading and LFA-1 redistribution, implying that neuronal TLCN may control the state and/or function of microglia in both physiological and pathological conditions.

Neurotrophic effects of annexin V on cultured neurons from embryonic rat brain.

Recombinant human annexin V showed survival promoting activity in embryonic rat neocortical and mesencephalic neurons in vitro. The neurotrophic effect was observed from a relatively low dose and in a dose-dependent manner. The neurotrophic activity of annexin V was completely blocked by anti-annexin V antibody. Northern blot analysis demonstrated that annexin V mRNA is expressed in non-neuronal cells in the CNS. These results suggest that annexin V plays certain roles in the CNS as a paracrine-type neurotrophic factor.

Purification of rabbit, rat and mouse protein C with the use of monoclonal antibody to human protein C, PC01.

Ca(++)-dependent monoclonal antibody specific to gamma-carboxyglutamic acid (Gla) domain of protein C was produced. It did not cross-react to the other vitamin K-dependent plasma proteins but to protein C of the other species. Using this monoclonal antibody, PC01, rabbit (170 micrograms), rat (60 micrograms) and mouse (40 micrograms) protein Cs were isolated from 100 ml of their plasma by affinity chromatography. All of these protein Cs were two chain form linked by disulfide bond as well as human protein C and activated by thrombin-thrombomodulin complex. Rat and mouse protein Cs showed similar characters to human protein C. On the other hand rabbit protein C had different M(r) of heavy and light chains and showed lower anticoagulant activity compared with human protein C.

Elevated serum levels of soluble vascular cell adhesion molecule-1 in NIDDM patients with proliferative diabetic retinopathy.

We studied 68 Japanese NIDDM patients (38 men and 30 women), aged 56.9+/-1.2 years (range 33-75 years), with a BMI of 23.1+/-0.5 kg/m2 without hypertension, dyslipidemia, and diabetic macroangiopathy for evaluating the relationship between serum soluble vascular cell adhesion molecule-1 (sVCAM-1) levels and the severity of diabetic retinopathy. Fundus examination was performed by an ophthalmologist using an ophthalmoscope, and the findings were graded as: (1) no signs of diabetic retinopathy (NDR), (2) background diabetic retinopathy (BDR), or (3) proliferative diabetic retinopathy (PDR). Serum sVCAM-1 levels were measured in duplicate by enzyme-linked immunosorbent assay using the soluble VCAM-1 KIT (R&D Systems Ltd., Ablingdon, Oxfordshire, UK). There was no difference in serum sVCAM-1 levels between patients with BDR (n = 17) and patients with NDR (n = 40) (1035.3+/-104.4 and 978.8+/-48.9 ng/ml, respectively, P = 0.8), but patients with PDR (n = 11) showed a significant increase of serum sVCAM-1 levels compared with patients with NDR (1281.8+/-166.3 and 978.8+/-48.9 ng/ml, respectively, P = 0.02). Although serum sVCAM-1 levels were correlated, not only with age but also with the known diabetic duration (r = 0.39, P = 0.001, and r = 0.40, P = 0.0007, respectively), age-adjusted sVCAM-1 levels were still significantly higher in the PDR group than in the NDR group. In contrast. serum sVCAM-1 levels were not related to the presence of diabetic nephropathy or HbA1c levels. Our results suggest that sVCAM-1 might be implicated in the development of the diabetic retinopathy, and measurement of serum sVCAM-1 levels in NIDDM patients maybe clinically useful for assessing the severity and possibly the activity of diabetic retinopathy.

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.

Ability of N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitroquinoline 1-oxide, dimethylnitrosamine, and NaCl to induce unscheduled DNA synthesis, stimulate replicative DNA synthesis, and produce DNA single-strand breaks in pyloric mucosa of rat stomach.

Male F344 rats were given test chemicals orally, and samples of their pyloric mucosa were incubated in vitro. Induction of unscheduled DNA synthesis (UDS) and stimulation of replicative DNA synthesis in the pyloric mucosa were then examined by addition of [3H]thymidine and simultaneous determinations of DNA synthesis in the presence and absence of hydroxyurea, an inhibitor of replicative DNA synthesis. DNA damage was also examined by the alkaline elution method with DNA single-strand scission as a marker. The results showed four types of abilities of the chemicals to affect UDS and replicative DNA synthesis in the pyloric mucosa of rat stomach 1-2 h after their administration: (1) induction of UDS and stimulation of replicative DNA synthesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a glandular stomach carcinogen, (2) induction of only UDS by 4-nitroquinoline 1-oxide (4NQO), a glandular stomach carcinogen, (3) stimulation of only replicative DNA synthesis by NaCl, a glandular stomach tumor promoter, and (4) neither induction of UDS nor stimulation of replicative DNA synthesis by dimethylnitrosamine (DMN), a liver carcinogen. DNA single-strand scission was induced by MNNG and 4NQO, being maximal 2 h after their administration, but was not induced by NaCl or DMN. Thus it correlated well with the induction of UDS. The present results indicate four types of inductive abilities of chemicals on UDS and replicative DNA synthesis in rat stomach pyloric mucosa and show that this method can detect differences in the action mechanisms and organ specificities of glandular stomach carcinogens.

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.

Mutagenicities of xanthone derivatives in Salmonella typhimurium TA100, TA98, TA97, and TA2637.

The mutagenicities of naturally occurring xanthones were tested in Salmonella typhimurium TA100, TA98, TA97, and TA2637 by the preincubation method. Xanthydrol, gentisein, gentisin, isogentisin, 1-hydroxy-3,7-dimethoxyxanthone, 1,3,7-trimethoxyxanthone, desmethylbellidifolin, bellidifolin and dimethylbellidifolin were mutagenic, but unsubstituted xanthone was not mutagenic to TA100, TA98, TA97 and TA2637 with or without a metabolic activation system. The beta-O-glucosides, norswertianolin and swertianolin, were only mutagenic when a metabolic activation system containing beta-glucosidase was used, and the C-glucoside mangiferin was not mutagenic even with this system.