EWS represses cofilin 1 expression by inducing nuclear retention of cofilin 1 mRNA.

In Ewing's sarcoma family tumors (ESFTs), the proto-oncogene EWS that encodes an RNA-binding protein is fused by chromosomal translocation to the gene encoding one of the E-twenty six (ETS) family of transcription factors, most commonly friend leukemia virus integration 1 (FLI-1). Although EWS/FLI-1 chimeric proteins are necessary for carcinogenesis, additional events seem to be required for transformation to occur. We have previously reported that a protein product of an EWS mRNA target, whose expression is negatively regulated by EWS but not by EWS/FLI-1, contributes to ESFT development. However, the mechanism by which EWS represses protein expression remains to be elucidated. Here, we report that overexpression of full-length EWS repressed protein expression and induced nuclear retention of reporter mRNAs in a tethering assay. In contrast, when a mutant lacking the EWS C-terminal nuclear localization signal (classified as a PY-NLS) was expressed, reporter protein expression was upregulated, and the number of cells exporting reporter mRNA to the cytoplasm increased. EWS binds to the 3'-untranslated region in another mRNA target, cofilin 1 (CFL1), and negatively regulates the expression of CFL1. Overexpression of EWS induced nuclear retention of CFL1 mRNA. Furthermore, ESFT cell proliferation and metastatic potential were suppressed by small interfering RNA-mediated CFL1 knockdown. Together, our findings suggest that EWS induces nuclear retention of CFL1 mRNA, thereby suppressing expression of CFL1, and that CFL1 promotes development of ESFT. Targeting CFL1 might therefore provide another novel approach for treatment of this aggressive disease.

Basal expression of c-Fos and Zif268 in the rat basal ganglia: immunohistochemical characterization of striatal Zif268-positive neurons.

Basal expression of the protein products of the inducible immediate early genes (IEGs), c-Fos and Zif268, was investigated in five regions of the rat basal ganglia using immunohistochemistry. In particular, high basal levels of Zif268 but very low levels of c-Fos were seen in the caudate-putamen (CPu). Double immunostaining revealed that many of the constitutively expressed Zif268-positive neurons were GABAergic but very few were cholinergic or neuronal nitric oxide synthase (nNOS)-positive, and some of the Zif268-positive neurons were also immunopositive for a glutamate NMDA receptor subunit NR1 or NR2A. No regional difference between the medial and lateral parts of the CPu was observed in the cellular phenotypes of Zif268-positive neurons. Almost no basal levels of Zif268 were seen in the other four regions: the globus pallidus, entopeduncular nucleus, subthalamic nucleus and substantia nigra pars reticulata. As in the CPu, negligible levels of c-Fos were seen in these four regions. Differential expression of these two IEGs may suggest gene-specific and region-specific functions of c-Fos and Zif268 in the basal ganglia. Constitutive expression of Zif268 existing mainly in the GABAergic neurons in the CPu may at least in part be maintained by glutamatergic afferents.

Development and application of a system for analysis of mixed cultures of microorganisms.

Development and application of a system for real-time quantitative assessment of individual cell activities in a mixed culture system was investigated. This was based on a concept that the activities of individual cells in a mixed culture can be assessed if the cells are physically separated (in separate compartments) in a vessel while the culture conditions, including the broth components, are maintained the same in all the compartments during the cultivation. On this basis, three different apparatus (M-1, M-2, and M-3) were constructed using various types of membranes. In terms of mass transfer characteristics and membrane fouling, the M-3 apparatus was the most effective system for analysis of mixed cultures at high cell densities. With the M-3 apparatus, the interrelationships between two alcohol-producing strains (Saccharomyces cerevisiae and Zymomonas mobilis) under anaerobic and aerobic conditions were studied. Under anaerobic condition, except for possible competition for nutrients, there were no significant effects of the activities of one microorganism on the other. However, under aerobic condition, amensalism was observed because acetaldehyde that was produced by Z. mobilis inhibited the growth of S. cerevisiae.

Regional differences in desensitization of c-Fos expression following repeated self-stimulation of the medial forebrain bundle in the rat.

The acute self-stimulation of the medial forebrain bundle was reported to induce the expression of c-Fos, the protein product of c-fos, an immediate early gene, in the central nervous system. In the present study, we examined regional changes in c-Fos expression in several reward-related areas of rat brain in response to short- and long-term exposure to self-stimulation of the medial forebrain bundle. Short-term one-hour stimulation of the medial forebrain bundle for one day after training, which evoked steady self-stimulation behavior, significantly increased the number of c-Fos-positive neurons bilaterally in all of 15 brain structures assayed, as compared to the non-stimulation control. Among them, structures showing a larger number of the stained neurons on the stimulated side were the anterior olfactory nucleus, amygdala, medial caudate-putamen complex, lateral septum, bed nucleus of the stria terminals, ventral pallidum, substantia innominata, lateral preoptic area, medial preoptic area, lateral hypothalamus rostral to the stimulating electrodes, and substantia nigra. Long-term stimulation of the medial forebrain bundle once daily for five successive days, which maintained consistently stable self-stimulation behavior, also increased the number of c-Fos-positive neurons in the aforementioned structures, as compared to the control. However, the long-term rewarding stimulation diminished the increased number of labeled neurons, as compared to the short-term rewarding stimulation. Seven areas, medial caudate-putamen complex, ventral pallidum, substantia innominata, lateral preoptic area, medial preoptic area, rostral lateral hypothalamus and substantia nigra, showed asymmetrical, ipsilateral predominance after the short- and long-term stimulation. However, the stained neuron count in those areas after the long-term stimulation was reduced to less than 50% of that found after the short-term stimulation with the exception of lateral preoptic area and rostral lateral hypothalamus. The results suggest that the development of desensitization of c-Fos response may differ among the reward-relevant brain regions as a consequence of repeated self-stimulation. They also indicate that a larger portion of neurons in the lateral preoptic area and rostral lateral hypothalamus may be implicated in both short- and long-term self-stimulations of the medial forebrain bundle.

Methamphetamine induces fos expression in the striatum and the substantia nigra pars reticulata in a rat model of Parkinson's disease.

In rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion in the nigrostriatal pathway, methamphetamine (3 mg/kg, i.p.) induced Fos-like immunoreactivity (FLI) not only in the striatum on the intact side but also in the substantia nigra pars reticulata (SNr) on the lesioned side. The methamphetamine-induced hyperexpression of FLI in the SNr on the lesioned side was suppressed by pretreatment with either dopamine D1 receptor antagonist SCH-23390 (0.5 mg/kg, i.p.), D2 receptor antagonist raclopride (2 mg/kg, i.p.) or N-methyl-d-aspartate receptor antagonist MK-801 (1 mg/kg, i.p.), which was concomitant with inhibition of the methamphetamine-induced rotational behavior of each antagonist. However, the hyperexpression of FLI in the SNr was not suppressed by intrastriatal grafts of fetal ventral mesencephalon which could suppress the methamphetamine-induced rotation completely. These results indicate that opposite hemispheric asymmetries in FLI are induced by methamphetamine in the striatum and the SNr in the 6-OHDA rats. It is suggested that the FLIs in the two discrete sites are activated independently by different mechanisms, and furthermore, different neuronal pathways are involved in the methamphetamine-induced rotation and Fos expression in the SNr of 6-OHDA rats.

Peripherally administered tetrahydrobiopterin increases in vivo tryptophan hydroxylase activity in the striatum after transplantation of fetal ventral mesencephalon in six hydroxydopamine lesioned rats.

The intraperitoneal administration of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4), a natural cofactor for tyrosine hydroxylase and tryptophan hydroxylase (TRH), dose-dependently increased the extracellular concentration of 6R-BH4 itself in rat striatum. The concentration was investigated by in vivo microdialysis and measured simultaneously with 5-hydroxytryptophan (5-HTP), a precursor of serotonin, by high performance liquid chromatography with electrochemical detection. The 6R-BH4 (50 mg/kg, i.p.) administration increased the accumulation of 5-HTP as an index of in vivo TRH activity under the inhibition of aromatic L-amino acid decarboxylase by NSD-1015 in the striatum of both normal control and 6-hydroxydopamine lesioned rats with intrastriatal transplants of fetal ventral mesencephalon (VM). The results suggest that TRH in the striatum of both control and VM-grafted rats is activated by 6R-BH4 penetrating into the brain from the blood.

Methamphetamine-induced Fos expression in the substantia nigra pars reticulata in rats with a unilateral 6-OHDA lesion of the nigrostriatal fibers.

In rats with a unilateral 6-hydroxydopamine (6-OHDA)-induced lesion in the nigrostriatal fibers, methamphetamine (3 mg/kg, i.p.) induced Fos-like immunoreactivity (FLI), which was inhibited by pretreatment with N-methyl-D-aspartate antagonist MK-801 (1 mg/kg, i.p.), not only in the medial striatum contralateral to the lesion but also in the substantia nigra pars reticulata (SNr) ipsilateral to the lesion. Thus, hemispheric asymmetries in FLI were induced by methamphetamine in the medial striatum and the SNr in the 6-OHDA model of turning which may be related to the altered function of glutamatergic transmission.

Serotonergic activity in the rat striatum after intrastriatal transplantation of fetal nigra as measured by microdialysis.

In vivo microdialysis was used to examine the effects of dopaminergic transplants on extracellular concentrations of dopamine (DA), serotonin (5-HT), and their precursors and major metabolites in the denervated rat striatum. Dialysis perfusates were collected from intact 6-hydroxydopamine (6-OHDA) lesion plus sham grafted, and lesion plus fetal substantia nigra (SN) grafted striata. The SN transplants ameliorated the reduction of striatal DA and dihydroxyphenylacetic acid (DOPAC) levels in rats with unilateral 6-OHDA lesions of the mesostriatal pathway. The transplants also increased extracellular levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in the denervated striatum. In response to NSD-1015 (an inhibitor of aromatic L-amino acid decarboxylase, AADC), 5-hydroxytryptophan (5-HTP) levels were substantially elevated in the SN grafted striata as compared with those in the sham grafted controls, which continued even after subsequent administration of L-3,4-dihydroxyphenylalanine (L-DOPA, 100 mg/kg i.p.). Immunohistochemical analysis showed hyperinnervation of 5-HT fibers in the grafted striatum, which was consistent with the results of microdialysis experiments. These results indicated that implantation of SN grafts into the 6-OHDA-lesioned striatum of rats induces hyperactivity of 5-HT synthesis, release and metabolism.

Cellular distributions of AMPA glutamate receptor subunits in fetal ventral mesencephalon transplants in the dopamine-depleted striatum of a rat.

To demonstrate the cellular distributions of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor subunits (GluR1, GluR2/3, and GluR4) in the intrastriatal grafts of a rat model of Parkinson's disease, immunocytochemistry was performed in 6-hydroxydopamine rats with intrastriatal transplants of fetal ventral mesencephalon (VM). In the fetal VM (at embryonic day 15) in which the tyrosine hydroxylase (TH) immunoreactivity was intensely observed, no GluR subunit immunoreactivity was detected. Within the intrastriatal fetal VM grafts containing TH-positive cells, a large number of cells immunoreactive for GluR1 and GluR2/3 were observed. However, the GluR1- and GluR2/3-positive cells tended to locate homogeneously within the grafts and were composed of various cell sizes and shapes, mainly medium-sized and aspiny cells. Weak GluR4-positive cells were seen in the grafts, although in some cases the staining was too faint to see any immunoreactive cells at all. Double immunostaining revealed that a part of TH-positive cells in the grafts was also immunopositive for GluR1 or GluR2/3. Both dopaminergic neurons and nondopaminergic neurons in the VM transplants appear to be modified functionally by glutamatergic afferents via various glutamate receptors, including GluR1 and GluR2/3 and, to a lesser extent, GluR4.

A case of liver metastasis from colon cancer masquerading as focal sparing in a fatty liver.

Focal sparing in diffusely fatty liver is a well recognized entity. However, it occasionally creates some problems in the diagnosis of hepatic mass lesions. We recently experienced a case of liver metastasis from colon cancer which appeared as a wedge-shaped hyperdense area on non-enhanced CT (computed tomography). Other imaging techniques also demonstrated a wedge-shaped area which was difficult to distinguish from mere focal sparing in the fatty liver. CT arteriography and dynamic magnetic resonance images were useful for diagnosing this metastatic tumor. CT during arterial portography showed a wedge-shaped ischemic area in the anterior segment caused by intrahepatic portal vein blockade. The histological findings eventually revealed that the tumor, an adenocarcinoma, was surrounded by fibrotic tissue that mimicked focal sparing. We present the radiological features of this case and discuss how to arrive at a correct diagnosis.

Dopaminergic transplants suppress L-DOPA-induced Fos expression in the dopamine-depleted striatum in a rat model of Parkinson's disease.

We examined the effects of MK-801, a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, and fetal ventral mesencephalic (VM) transplants on L-3,4-dihydroxyphenylalanine (L-DOPA)-induced Fos protein expression in the dopamine (DA)-depleted striatum. Unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway were produced in young adult female rats and grafting was performed 3 weeks later. Methamphetamine-induced rotational behavior recovered significantly on the 4th week after grafting. Immunohistochemical examinations of c-Fos and tyrosine hydroxylase (TH) were performed 3-4 months after grafting. L-DOPA (100 mg/kg, i.p.) markedly induced Fos-like immunoreactivity (FLI) in the DA-depleted striatum. Pretreatment with a large dose of MK-801 (3-4.5 mg/kg, i.p.) dose-dependently suppressed L-DOPA-induced FLI in the striatum. The stimulatory effect of L-DOPA on c-Fos expression observed within the lesioned striatum was suppressed by fetal VM transplants. It seemed that the graft-induced effect on FLI extended over a considerably larger area than that covered by the graft-derived TH-immunoreactive innervation. Taken together, these findings suggest that glutamatergic modulation is involved in the L-DOPA-induced c-Fos expression in the denervated striatum which is normalized by fetal VM transplants. It also seems likely that VM grafts suppress the L-DOPA-induced expression of transcriptional factors which might be involved in the mechanisms underlying various side effects of chronic L-DOPA therapy.

Dopaminergic transplants alter in vivo activity of tryptophan hydroxylase in the striatum in a rat model of Parkinson's disease.

L-3,4-Dihydroxyphenylalanine (L-DOPA) inhibits the activity of tryptophan hydroxylase (TRH) and thus serotonin synthesis. This inhibitory effect of L-DOPA may be related to some side effects in the patients under L-DOPA therapy. The effects of transplantation of fetal ventral mesencephalon (VM) on extracellular 5-hydroxytryptophan (5-HTP) accumulation was examined by microdialysis as an index of in vivo activity of TRH in the striatum of 6-hydroxydopamine (6-OHDA)-lesioned rats. In the rat striatum perfused with m-hydroxybenzylhydrazine (NSD-1015; an inhibitor of aromatic L-amino acid decarboxylase), L-DOPA and 5-HTP in dialysate were measured simultaneously. In response to NSD-1015, 5-HTP levels were substantially elevated in the lesion plus VM-grafted striata as compared with those in the lesion plus sham-grafted striata. The results indicate that implantation of dopamine-rich VM grafts into the 6-OHDA-lesioned striatum of rats induces hyperactivity of TRH.

Variant von Willebrand disease with defective binding to factor VIII: the first case from Japan.

This is the first case of variant von Willebrand disease (vWD) with defective binding of von Willebrand factor (vWF) to factor VIII (F.VIII) to be diagnosed in Japan. An 8-year-old Japanese girl, who had had recurrent episodes of subcutaneous hematomas, showed a prolonged A-PTT, low F.VIII (F.VIII:C 4 U/dl, FVIII:Ag 4 U/dl), and normal level of vWF (RCof 80 U/dl, vWF:Ag 60 U/dl). The patient's vWF-multimeric structure on SDS agarose gel electrophoresis was similar to that in normal subjects. A F.VIII binding assay was performed, as described by Nishino et al. (1989). F.VIII binding (y) of vWF was expressed as a function of the amount of immobilized vWF (x) on the wells of a polystyrene plate. Regression lines from normal subjects and the patient had a high correlation coefficient. F.VIII binding capacity was estimated by the slope of the regression lines. The slope for normal subjects showed y = 0.002 + 0.653x, while, in contrast, the slope for the patient showed y = 0.005 + 0.009x, indicating that the capacity of vWF from the patient to bind F.VIII was markedly decreased. Exons 18-20 of the vWF gene, covering the first 132 amino acids of mature vWF subunit from the patient, were sequenced, using the PCR amplification method. A point mutation C-->T at codon 816 in exon 19, predicting a substitution of Trp for Arg(53), was characterized; this was inherited by the patient from her mother.

Effects of bucolome and related barbiturate derivatives on the displacement of bilirubin from plasma albumin in vivo and in vitro.

Bucolome, one of barbiturate derivatives, lowers plasma bilirubin in Gilbert's syndrome without inductive effect on liver microsomal enzymes. To clarify the mechanism of this action, the study was performed concerning the effects of bucolome and five other closely related barbiturate derivatives including phenobarbital (PB) on plasma bilirubin in homozygous Gunn rats and on albumin-bilirubin binding in vitro. When 15 mg/100 g of bucolome was administered to Gunn rats, remarkable drop in plasma bilirubin continued for more than 48 hrs. This decrease of bilirubin was returned to preinjection level after intravenous injection of albumin. Three Gunn rats died as a result of administration of 30 mg/100 g. This dose caused no pathological change in control rats. In in vitro study, bucolome displaced bilirubin from human albumin strongly. PB had almost no effect on the plasma bilirubin in Gunn rats and the in vitro action was very small. Among the barbiturate derivatives, compounds which have cyclohexyl radicals in N position showed stronger plasma bilirubin decreasing effects in Gunn rats. From these results, a strong action to displace bilirubin from plasma albumin is concluded as the mechanism of bucolome to decrease plasma bilirubin.