Protein tyrosine phosphatase receptor type Q in cerebrospinal fluid reflects ependymal cell dysfunction and is a potential biomarker for adult chronic hydrocephalus.

BACKGROUND AND PURPOSE: Protein tyrosine phosphatase receptor type Q (PTPRQ) was extracted from the cerebrospinal fluid (CSF) of patients with probable idiopathic normal-pressure hydrocephalus (iNPH) by proteome analysis. We aimed to assess the feasibility of using CSF PTPRQ concentrations for the additional diagnostic criterion of iNPH in Japanese and Finnish populations. METHODS: We compared PTPRQ concentrations among patients with probable iNPH and neurologically healthy individuals (normal control [NC] group), patients with normal-pressure hydrocephalus (NPH) of acquired and congenital/developmental aetiologies, patients with Alzheimer's disease and patients with Parkinson's disease in a Japanese analysis cohort. A corresponding iNPH group and NC group in a Finnish cohort was used for validation. Patients in the Finnish cohort who underwent biopsy were classified into two groups based on amyloid and/or tau deposition. We measured PTPRQ expression levels in autopsied brain specimens of iNPH patients and the NC group. RESULTS: Cerebrospinal fluid PTPRQ concentrations in the patients with NPH of idiopathic, acquired and congenital/developmental aetiologies were significantly higher than those in the NC group and those with Parkinson's disease, but iNPH showed no significant differences when compared with those in the Alzheimer's disease group. For the patients with iNPH, the area under the receiver-operating characteristic curve was 0.860 in the Japanese iNPH and 0.849 in the Finnish iNPH cohorts. Immunostaining and in situ hybridization revealed PTPRQ expression in the ependymal cells and choroid plexus. It is highly possible that the elevated PTPRQ levels in the CSF are related to ependymal dysfunction from ventricular expansion. CONCLUSIONS: Cerebrospinal fluid PTPRQ levels indicated the validity of this assay for auxiliary diagnosis of adult chronic hydrocephalus.

Structural snapshots of V/A-ATPase reveal the rotary catalytic mechanism of rotary ATPases.

V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with FoF1 ATP synthase. When powered by ATP hydrolysis, the V1 domain rotates the central rotor against the A3B3 hexamer, composed of three catalytic AB dimers adopting different conformations (ABopen, ABsemi, and ABclosed). Here, we report the atomic models of 18 catalytic intermediates of the V1 domain of V/A-ATPase under different reaction conditions, determined by single particle cryo-EM. The models reveal that the rotor does not rotate immediately after binding of ATP to the V1. Instead, three events proceed simultaneously with the 120˚ rotation of the shaft: hydrolysis of ATP in ABsemi, zipper movement in ABopen by the binding ATP, and unzipper movement in ABclosed with release of both ADP and Pi. This indicates the unidirectional rotation of V/A-ATPase by a ratchet-like mechanism owing to ATP hydrolysis in ABsemi, rather than the power stroke model proposed previously for F1-ATPase.

Inhibition of angiogenic factor production from murine mast cells by an antiallergic agent (epinastine hydrochloride) in vitro.

Angiogenesis is an important event both in the development of allergic inflammatory responses and in the pathophysiology of tissue remodeling in allergic diseases. In the present study, therefore, we examined the influence of antihistamines on angiogenesis through the choice of epinastine hydrochloride (EP) and murine mast cells in vitro. Mast cells (5 x 10(5) cells/mL) presensitized with murine IgE specific for ovalbumin (OVA) were stimulated with 10 ng/mL OVA in the presence of various concentrations of EP for 4 hours. The levels of angiogenesis factors, keratinocyte-derived chemokine (KC), tumor necrosis factor-alpha (TNF), and vascular endothelial growth factor (VEGF) in culture supernatants, were examined by ELISA. We also examined mRNA expression for the angiogenesis factors by RT-PCR. EP significantly inhibited the production of KC, TNF, and VEGF induced by IgE-dependent mechanism at more than 25 ng/mL. Semiquantitative analysis using RT-PCR showed that EP also significantly reduced mRNA expressions for KC, TNF, and VEGF. These results strongly suggest that EP suppresses angiogenesis factor production through the inhibition of mRNA expression in mast cells and results in favorable modification of clinical conditions of allergic diseases.

Molecular Characterization and Diagnosis of QoI Resistance in Cucumber and Eggplant Fungal Pathogens.

ABSTRACT The molecular mechanism of QoI fungicide resistance was studied using isolates of cucumber Corynespora leaf spot fungus (Corynespora cassiicola) and the eggplant leaf mold (Mycovellosiella nattrassii). In both pathogens, a mutation at position 143 from glycine to alanine (G143A) was detected in the cytochrome b gene that encodes for the fungicide-targeted protein. Moreover, the nucleotide sequence at amino acid position 143 was converted from GGT or GGA in sensitive (wild-type) to GCT or GCA in resistant (mutant-type) isolates. The methods of polymerase chain reaction restriction fragment length polymorphism commonly used for QoI resistance monitoring were employed successfully, leading to the amplified gene fragment from resistant isolates being cut with the restriction enzyme ItaI. However, heteroplasmy (the coexistence of wild-type and mutated alleles) was found when the resistant isolates of C. cassiicola, M. nattrassii, and Colletotrichum gloeosporioides (strawberry anthracnose fungus) were subcultured in the presence or absence of QoI fungicides. QoI resistance of cucumber powdery and downy mildew isolates persisted for a few years following the removal of the selection pressure imposed by the fungicide under both laboratory and commercial greenhouse conditions. The proportion of mutated sequences in cytochrome b gene decreased over time in the pathogen population. The protective efficacy of the full dose of azoxystrobin decreased when the populations of powdery and downy mildews contained resistant isolates at 10%. Using FMBIO, a fluorescence bio-imaging analyzer, the mutant allele from the QoI-resistant isolates could be detected at the level of 1%, whereas the detection sensitivity of ethidium-bromide-stained gels was approximately 10 times lower.

Accumulation of 8-oxo-2'-deoxyguanosine and increased expression of hMTH1 protein in brain tumors.

Oxidative DNA damage generated by an attack of reactive oxygen species causes mutation or cell death that may lead to various diseases and may be related to initiation or progression of carcinogenesis. 8-Oxo-2'-deoxyguanosine (8-oxo-dG) is a major oxidative DNA damage product that can result in mutation, and hMTH1, human MutT homolog protein 1, has been identified as an enzyme that hydrolyzes 8-oxo-dGTP to the monophosphate, thus preventing accumulation of 8-oxo-dG in DNA. With immunohistochemical approaches, we investigated accumulation of 8-oxo-dG and expression of hMTH1 in brain tumor tissues obtained from surgical and autopsy cases, including 42 neuroepithelial tumors, 5 meningiomas, 2 metastatic brain tumors, and 1 schwannoma. 8-Oxo-dG accumulation and hMTH1 expression were increased in various brain tumors. Nuclei of brain tumor cells were immunoreactive for 8-oxo-dG in all cases. In most cases, both nuclei and cytoplasm of the tumor cells were immunoreactive for hMTH1. Both 8-oxo-dG accumulation and hMTH1 expression were most evident in high-grade gliomas, indicating that oxidative stress was high in these gliomas. Thus, the defense mechanism against such oxidative stress may be enhanced as well. These results suggest that oxidative stress may play a role in tumor progression.

Limbic encephalitis associated with recurrent thymoma: a postmortem study.

The authors report an autopsied patient with limbic encephalitis and recurrent thymoma. The immunohistochemical study showed selective depositions of immunoglobulin G on the neurons in the limbic system and the tumor cells of the recurrent thymoma. The immunoblotting study detected two types of antibodies that react with the human brain, rat brain, and rat thymus.

AMPA receptor protein in developing rat brain: glutamate receptor-1 expression and localization change at regional, cellular, and subcellular levels with maturation.

We tested the hypothesis that the regional, cellular, and synaptic localizations of the glutamate receptor 1 (GluR 1) subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor are regulated developmentally in rat brain. By immunoblotting, GluR1 was first detected in whole brain at embryonic day E15.5, and levels increased progressively during late embryonic (E20) and early postnatal (P2-P11) days. Regionally, GluR1 increased in cerebral cortex but decreased in striatum with postnatal maturation. These changes occurred in the presence of increased presynaptic maturation, as determined by synaptophysin detection. By immunocytochemistry, distinct cellular populations showed different temporal profiles of GluR1 expression during postnatal maturation. The neocortex and hippocampus showed a progressive maturation-related enrichment of GluR1, whereas the striatum showed a gradual reduction in GluR1 during maturation. In cerebellum, GluR1 protein was expressed transiently at restricted times postnatally by granule cells (P0-P11) and Purkinje cells (P13-P19), but by P21 and thereafter these neurons had sparse GluR1 immunoreactivity. By immunoelectron microscopy. GluR1 was found in neurites, specifically in both dendritic and axon terminal components of developing synapses. GluR1 was clustered at the plasma membrane of apparent growth cone appositions, neuronal cell bodies, and dendrites of developing neurons. The presence of GluR1 at presynaptic sites dissipated with synaptic maturation, as GluR1 became confined to the somatodendritic compartment as maturation progressed. We conclude that the regional expression as well as the cellular and synaptic localizations of the GluR1 are developmentally regulated and are different in immature and mature brain. Differences in glutamate receptor expression and synaptic localization in immature and mature brain may be relevant to the phenomenon that the perinatal and adult brain differ in their regional vulnerability to hypoxia-ischemia and excitotoxicity.

Cellular and synaptic localization of the neuronal glutamate transporters excitatory amino acid transporter 3 and 4.

Glutamate transport is a primary mechanism for the synaptic inactivation of glutamate. Excitatory amino acid transporter 4 (EAAT4) is a novel glutamate transporter with properties of a ligand-gated chloride channel that was recently cloned from human brain. The present study was an investigation of the protein expression and cellular localization of EAAT4 in human and rat brain, and comparison with another neuronal glutamate transporter, EAAT3 (rabbit excitatory amino acid carrier 1; EAAC1). Regional immunoblot analysis of EAAT4, using a monospecific oligopeptide (carboxy-terminal) affinity-purified polyclonal antibody, revealed that the protein was restricted to the central nervous system. The EAAT4 protein was largely expressed in cerebellum, with a much lower expression in hippocampus, neocortex, striatum, brain stem and thalamus. Immunohistochemical studies showed intense EAAT4 immunoreactivity in the human and rat cerebellar Purkinje cells with a somatodendritic localization. Other brain regions including neocortex, hippocampus, striatum showed faint neuropil staining of EAAT4. Immunogold localization identified EAAT4 protein at plasma membranes of Purkinje cell dendrites and spines. In the hippocampus and neocortex, EAAT4 immunoreactivity was found mainly at small calibre dendrites. Rarely, EAAT4 immunoreactivity was found in astrocytic cell processes of forebrain. In the cerebellum, EAAT4 localization partly overlapped with the neuronal localization of EAAT3 (EAAC1). Immunoreactivity for EAAT3 was enriched in the somatodendritic compartment of the Purkinje cells like EAAT4, but EAAT3 was also found in Purkinje cell axons and in boutons in deep cerebellar nuclei, as well as in granular cells and stellate cells. Our results indicate that EAAT4 protein is largely localized to cerebellar cortex and lower levels of EAAT4 protein are present in forebrain by immunoblot and immunohistochemistry. Both neuronal glutamate transporter EAAT3 (EAAC1) and EAAT4 are located at somatodendritic compartment of Purkinje cells, and probably contribute to glutamate re-uptake mechanisms at Purkinje cell synapses.

Molecular cloning and expression of the rat EAAT4 glutamate transporter subtype.

Glutamate transport is a primary mechanism for the synaptic inactivation of glutamate. Excitatory amino acid transporter 4 (EAAT4) is a novel glutamate transporter with properties of a ligand-gated chloride channel that was recently cloned from human brain. Here we report the cloning of rat EAAT4 (rEAAT4) cDNA from rat cerebellum. The nucleotide sequence of rEAAT4 was 88% identical to the human sequence, and the predicted peptide was 89% identical to the human protein. The transport activity encoded by rEAAT4 has high affinity for L-glutamate. In Xenopus laevis oocytes expressing rEAAT4, L-glutamate and other transporter substrates elicited a current predominantly carried by chloride ions. Like human EAAT4, the rEAAT4 mRNA was largely restricted to cerebellar Purkinje cells; the rEAAT4 protein was localized to Purkinje cell somas and dendrites.

Expression of hMTH1 in the hippocampi of control and Alzheimer's disease.

The oxidized purine nucleoside triphosphatase, hMTH1, has a critical role towards preventing errors caused by oxidized purine nucleoside triphosphates such as 8-oxo-dGTP and 2-hydroxy-dATP. We investigated the immunohistochemical expression of hMTH1 in human hippocampal postmortem tissues representing non-neurological disease and Alzheimer's disease (AD). In the non-neurological subjects the hMTH1 protein was enriched in the stratum lucidum at CA3 corresponding to mossy fiber synapses. In AD subjects, the synaptic immunoreactivities at CA3 were significantly decreased, whereas they tended to be increased at the entorhinal cortex. We suggest that the expression of hMTH1 indicates indirect evidence of oxidative stress and its regulation is regionally differentiated in AD.

Dorsal horn lesion resulting from spinal root avulsion leads to the accumulation of stress-responsive proteins.

The aim of this study was to demonstrate acute to subacute molecular episodes in the dorsal horn following root avulsion using immunohistochemical methods with the markers for synapses, astrocytes and such stress-responsive molecules as heat shock proteins (Hsps) and p38 MAP kinase (p38). Among them, Hsp27 was accumulated selectively in the injured substantia gelatinosa 24 h after avulsion injury. The localization of Hsp27 in astrocytes within the substantia gelatinosa was confirmed by the double immunofluorescence method using anti-Hsp27 antibody and either anti-synaptophysin antibody or anti-glutamine synthetase antibody and by immunoelectron microscopy for Hsp27. The pattern of Hsp27 expression subsequently changed from glial pattern to punctate pattern by 7 days. Immunoelectron microscopy revealed that the punctate pattern in the subacute stage corresponded to distal parts of the astrocytic processes. Hsp27 immunoreaction was decreased 21 days after root avulsion. In the distal axotomy model, Hsp27 was accumulated later in the ipsilateral dorsal horn in a punctate pattern from 7 days after the axotomy. Phosphorylation of p38 was detected in microglia in the dorsal horn following both avulsion and axotomy. Substance P was slightly decreased in the injured substantia gelatinosa in both the avulsion and axotomy models around 14-21 days. We conclude that Hsp27 is a useful marker for demonstrating dorsal horn lesions following avulsion injury and that avulsion injury may induce Hsp27 in the dorsal horn more rapidly than distal axotomy.

Altered expressions of glutamate transporter subtypes in rat model of neonatal cerebral hypoxia-ischemia.

Glutamate transporters are essential for maintaining the extracellular levels of glutamate at synaptic clefts and are regulated developmentally in a subtype-specific manner. We investigated chronological changes of immunoreactivities for glial glutamate transporters GLAST and GLT-1 and a neuronal glutamate transporter, EAAC1, in postnatal 7-day-old rat neocortices and hippocampi at 12, 24, 48 and 72 h after hypoxia-ischemia. Glutamate transporter subtypes are differentially expressed in the ischemic core and the boundary area of the neonatal rat brain with hypoxia-ischemia. Expressions of these glutamate transporters decreased in the ischemic core at 12 h, then immunoreactivities for GLAST and GLT-1 were recovered at the hippocampus. This was accompanied by a GFAP-positive gliosis at 72 h, whereas these immunoreactivities were reduced at the neocortex in the ischemic core. Glial glutamate transporters, especially GLAST, were noted in some astrocytes appearing as apoptosis as well as shrunken pyramidal neurons mainly in the boundary area of the neocortex. Increased perikaryal expression of EAAC1 was associated with that of MAP2 at the border of the boundary area. These temporal and regional expressions of glutamate transporters may contribute towards understanding the excitotoxic cell death mechanism in hypoxic-ischemic encephalopathy during the perinatal period.

Cost-sharing and pharmaceutical utilisation and expenditure in Russia.

This paper presents estimates of the impact of exemption status, and other socio-economic variables, on pharmaceutical use in Russia. Estimates are derived from a newly collected household survey covering around four thousand households. Separate results for a zero-inflated negbin model of utilisation of prescriptions and for a two-part model of the overall level of household expenditure on pharmaceuticals are presented. Full exemption from prescription charges is shown to increase the utilisation of prescription items and reduce the probability of the households incurring drug expenditure.

Reconstitution of shunted mantle in experimental hydrocephalus.

The morphological mechanism of the reconstitution of shunted mantle was studied histopathologically in 22 kaolin-treated hydrocephalic puppies. A remarkable attenuation of cerebral mantle to less than 1 cm in thickness was seen on computerized tomography (CT) scans of four animals sacrificed 1 to 2 months after kaolin treatment (preshunt group). Ventricular shunting resulted in successful recovery of the mantle on repeated CT scans obtained 1 to 2 months after shunting in seven animals (postshunt group). In the remaining 11 animals the cerebral mantle, which had been reduced to 4 mm in thickness prior to shunting, failed to recover even 2 months after the procedure (shunt-refractory group). On gross inspection, the preshunt specimens showed marked thinning of the white matter, with the cortical ribbon well preserved, while the postshunt specimens consisted predominantly of thickened white matter. Histopathological examination of the attenuated white matter of the preshunt specimens showed decreased nerve-fiber density, myelin destruction with myelin regeneration and/or repair of myelin sheaths, and reactive astrocytosis, which were prominent especially in the periventricular white matter. The main findings in the reconstituted white matter of the postshunt specimens were extensive myelin regeneration of residual axons and remarkable astroglial proliferation with mesenchymal reaction, particularly at capillaries. No clear evidence of increased numbers of nerve fibers or axonal regeneration was observed. The shunt-refractory specimens showed remarkable attenuation of cortex, in which reduced numbers of neurons and loss of cortical lamination were noted, with vestigial white matter. The results indicate that astroglial proliferation with mesenchymal reaction and myelin regeneration contribute to the reconstitution of the cerebral mantle volume following ventricular shunting in this model. It is suggested that the critical factor for mantle reconstitution in chronic hydrocephalus is whether cortex is preserved.

Temporal lobe tumor demonstrating ganglioglioma and pleomorphic xanthoastrocytoma components. Case report.

The case is reported of a 16-year-old boy with a left temporal lobe tumor composed of a ganglioglioma and a pleomorphic xanthoastrocytoma. Histologically, the tumor had two different components. One component involved the cortex of the left posterior temporal lobe and showed an aggregation of neuronal cells with an astroglial stroma. Ultrastructurally, numerous dense-cored vesicles, diagnosed as ganglioglioma, were found in the neuronal cells. The other component involved the adjacent cortex and white matter of the left anterior temporal lobe and the surrounding subarachnoid space. This was composed of pleomorphic cells with many multinucleated giant cells and occasional foamy cells. Most of the tumor cells were positive for glial fibrillary acidic protein. These features correspond well to earlier descriptions of pleomorphic xanthoastrocytoma. At 24 months following total tumor extirpation, the patient is alive and has had no evidence of tumor recurrence.

High-performance liquid chromatography-fluorescence determination of dinitropyrenes in soil after column chromatographic clean-up and on-line reduction.

In order to quantify 1,3-dinitropyrene (DNP), 1,6-DNP and 1,8-DNP in soil, we developed an efficient clean-up procedure and a sensitive determination method using fluorescence detection. DNP isomers were efficiently cleaned by three stages of fractionation, i.e., a silica gel open column chromatography using stepwise elution and two further purification steps by high-performance liquid chromatography (PPLC) using a monomeric-type octadecylsilyl (ODS) column and a polymeric-type ODS column. The recoveries of DNPs during the whole clean-up process were 94% or more. The fraction corresponding to DNPs was injected into an analytical polymeric-type ODS column for HPLC to separate DNP isomers. The effluent from the analytical ODS column was directly introduced to a catalyst column, which was packed with 5 microns alumina coated with platinum and rhodium (Pt-Rh), in order to reduce DNPs to diamino compounds, and then the fluorescence of diaminopyrenes was detected. The immediate detection of diaminopyrene isomers after on-line reduction afforded a sensitive detection of DNP isomers. The detection limits for DNPs were in the range of 0.7 to 4 pg. These developed methods were applied to four soil samples collected at parks in residential areas.

Resveratrol, a naturally occurring polyphenol, induces sister chromatid exchanges in a Chinese hamster lung (CHL) cell line.

We tested the genotoxicity of 3,5,4'-trihydroxystilbene (resveratrol), a polyphenolic phytoalexin found in grapes, in a bacterial reverse mutation assay, in vitro chromosome aberration (CA) test, in vitro micronucleus (MN) test, and sister chromatid exchange (SCE) test. Resveratrol was negative in the strains we used in the bacterial reverse mutation assay (S. typhimurium TA98 and TA100 and E. coli WP2uvrA) in the absence and presence of a microsomal metabolizing system. It induced structural CAs at 2.5-20 microg/ml and showed weak aneuploidy induction in a Chinese hamster lung (CHL) cell line. It induced MN cells and polynuclear and karyorrhectic cells after 48h treatments in the in vitro MN test. In the SCE test, resveratrol caused a clear cell-cycle delay; at 10 microg/ml, the cell cycle took twice as long as it did in the control. Resveratrol induced SCEs dose-dependently at up to 10 microg/ml, at which it increased SCE six-fold, and the number was almost as large as mitomycin C, a strong SCE inducer. No second mitoses were observed at 20 microg/ml even after 54h. Cell cycle analysis by FACScan indicated that resveratrol caused S phase arrest, and 48h treatment induced apoptosis. Our results suggest that resveratrol may preferentially induce SCE but not CA, that is, it may cause S phase arrest only when SCEs are induced.

Changes in concentrations of serum amyloid A protein, alpha 1-acid glycoprotein, haptoglobin, and C-reactive protein in feline sera due to induced inflammation and surgery.

To identify candidates for feline acute phase proteins, the concentrations of serum amyloid A protein (SAA), alpha 1-acid glycoprotein (alpha 1-AG), C-reactive protein (CRP), and haptoglobin (Hp) were measured in sera isolated from clinically normal and hospitalized (or diseased) cats, from cats with experimentally induced inflammation, and cats subjected to surgery for urinary diversion. Measurements were made by sandwich enzyme-linked immunosorbent assay and single radial immunodiffusion. The concentrations of SAA, alpha 1-AG, and Hp in sera from hospitalized cats were 7-11 times higher than in clinically normal cats. Similar results were obtained for the concentrations of SAA, alpha 1-AG, and Hp in cats with induced inflammation and cats subjected to surgery. By contrast, the serum concentration of feline CRP did not change significantly between clinically normal cats and hospitalized cats or inflammation-induced or post-surgery cats. Feline SAA concentration was found to increase earliest, with alpha 1-AG and Hp beginning to increase thereafter. From these results, feline SAA is concluded to be an acute phase reactant at the early stage of inflammation.

Immunohistochemical analysis in a case of idiopathic Lennox-Gastaut syndrome.

We herein report a neuropathological and immunohistochemical analysis of a brain from a 25-year-old male with idiopathic type of Lennox-Gastaut syndrome (LGS). The clinical pictures, such as seizure type and progressive mental deterioration with an initial normal psychomotor and mental development in a man were typical of LGS. A routine neuropathological examination showed no pronounced changes, such as neuronal loss, morphologically abnormal neurons, inflammation, vascular changes, Lafora bodies and tumor cells, except that mild gliosis was seen only in CA4 of the hippocampus. Numerous corpora amylacea were observed throughout the cerebral cortices subjacent to the pia mater. An immunohistochemical analysis showed no marked findings for such proteins as glutamate transporters, glutamate decarboxylase, glutamine synthetase, neuronal cytoskeleton proteins and heat-shock proteins. However, intense ubiquitin-immunostained neurons were only found in CA4 of the hippocampus, whereas numerous astrocytes showed a strong immunoreaction for glial fibrillary acidic protein, but showed an exclusively reduced immunoreactivity for metallothionein-I/II, zinc-chelating protein. Our findings thus suggest that the pathology in the hippocampus is either causally or consequentially associated with the seizures occurring in LGS.

MR imaging of multiple sclerosis simulating brain tumor.

Multiple sclerosis may sometimes present as a mass lesion that is indistinguishable from brain tumor both clinically and radiologically. We describe two cases of multiple sclerosis simulating brain tumor on computed tomography (CT) scans and magnetic resonance (MR) images, one of which was proved and another was suggestive to be demyelinating disease by biopsy. Steroid therapy produced regression of the lesions of MR images and CT scans. Our cases and others in the literature suggest strategies for detecting multiple sclerosis presenting as a mass lesion.