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Serine 129 (S129) phosphorylation of α-synuclein (αSyn) is a central feature of Lewy body (LB) disease pathology. Although the neighboring tyrosine residues Y125, Y133, and Y136 are also phosphorylation sites, little is known regarding potential roles of phosphorylation cross-talk between these sites and its involvement in the pathogenesis of LB disease. Here, we found that αSyn aggregates are predominantly phosphorylated at Y136 in the Lewy body dementia brain, which is mediated by unexpected kinase activity of Casein kinase 2 (CK2). Aggregate formation with S129 and Y136 phosphorylation of recombinant αSyn (r-αSyn) were induced by CK2 but abolished by replacement of S129 with alanine (S129A) in vitro. Mutation of Y136 to alanine (Y136A) promoted aggregate formation and S129 phosphorylation of r-αSyn by CK2 in vitro. Introduction of Y136A r-αSyn oligomers into cultured cells exhibited increased levels of aggregates with S129 phosphorylation compared to wild-type r-αSyn oligomers. In addition, aggregate formation with S129 phosphorylation induced by introduction of wild-type r-αSyn oligomers was significantly attenuated by CK2 inhibition, which resulted in an unexpected increase in Y136 phosphorylation in cultured cells. Our findings suggest the involvement of CK2-related αSyn Y136 phosphorylation in the pathogenesis of LB disease and its potential as a therapeutic target., (© 2021. The Author(s).)

Introduction: Prion diseases are fatal neurodegenerative disorders caused by the deposition of abnormal prion protein aggregates (PrP Sc ) in the central nervous system. This study aimed to evaluate the use of iodinated pyridyl benzofuran (IPBF) derivatives as single-photon emission computed tomography (SPECT) probes for the detection of cerebral PrP Sc deposits., Methods: In vitro binding assays of IPBF derivatives were carried out in the recombinant mouse prion protein (rMoPrP) and brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. SPECT imaging of 5-(5-[ 123 I]iodobenzofuran-2-yl)-N-methylpyridin-2-amine ([ 123 I]IPBF-NHMe) was performed on mBSE-infected and mock-infected mice., Results: Fluorescence microscopy results showed that fluorescence signals of IPBF derivatives corresponded to the thioflavin-T positive amyloid deposits of PrP Sc in the brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. Among the IPBF derivatives, 5-(5-iodobenzofuran-2-yl)-N-methylpyridin-2-amine (IPBF-NHMe) exhibited the highest binding affinity to the recombinant mouse prion protein (rMoPrP) aggregates with a K i of 14.3 nM. SPECT/computed tomography (CT) imaging and ex vivo autoradiography demonstrated that the [ 123 I]IPBF-NHMe distribution in brain tissues of mBSE-infected mice co-localized with PrP Sc deposits., Conclusion: [ 123 I]IPBF-NHMe appears to be a prospective SPECT tracer for monitoring prion deposits in living brain tissues., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Prion diseases are fatal neurodegenerative disorders associated with the deposition of abnormal prion protein aggregates (PrP Sc ) in the brain tissue. Here, we report the development of 125 I-labeled iodobenzofuran (IBF) derivatives as single photon emission computed tomography (SPECT) imaging probes to detect cerebral PrP Sc deposits. We synthesized and radioiodinated several 5-IBF and 6-IBF derivatives. The IBF derivatives were evaluated as prion imaging probes using recombinant mouse prion protein (rMoPrP) aggregates and brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. Although all the IBF derivatives were strongly adsorbed on the rMoPrP aggregates, [ 125 I]5-IBF-NHMe displayed the highest adsorption rate and potent binding affinity with an equilibrium dissociation constant ( K d -positive amyloid deposits in the mBSE-infected mouse brains. Biodistribution studies in normal mice demonstrated slow uptake and clearance from the brain of Sc -positive amyloid deposits in the mBSE-infected mouse brains. Biodistribution studies in normal mice demonstrated slow uptake and clearance from the brain of 125 I-IBF derivatives. Among the derivatives, [ 125 showed the highest peak brain uptake [2.59% injected dose (ID)/g at 10 min] and good clearance (0.51% ID/g at 180 min). Although the brain distribution of IBF derivatives should still be optimized for 2 showed the highest peak brain uptake [2.59% injected dose (ID)/g at 10 min] and good clearance (0.51% ID/g at 180 min). Although the brain distribution of IBF derivatives should still be optimized for in vivo imaging, these compounds showed prospective binding properties to PrP Sc . Further chemical modification of these IBF derivatives may contribute to the discovery of clinically applicable prion imaging probes.

Our previous study indicated that recombinant human soluble thrombomodulin (rhsTM) could attenuate brain damage when administered as a bolus in the cerebral ischaemic early phase. Then, we considered that treatment with rhsTM may show therapeutic effects even when administered in the ischaemic delayed phase, because rhsTM has an action of inhibiting high-mobility group box 1 (HMGB1) as a late mediator of lethal systemic inflammation. This study was performed to investigate the effects of delayed treatment with rhsTM on ischaemic brain damage induced by high HMGB1 level in mice subjected to 4-hour middle cerebral artery occlusion (MCAO). One day after MCAO, rhsTM was administered intraperitoneally at a dose of 1 or 5 mg/kg once a day for 7 days. Neurological score, motor coordination and HMGB1 levels were measured 1, 3 and 7 days after MCAO. The presence of activated microglia was evaluated 7 days after MCAO. Systemic HMGB1 levels increased 1 to 7 days after MCAO and were higher at 7 days compared with day 1. At the same time, survival rate decreased, and activated microglia increased in the infarct area. Treatment with rhsTM improved neurological score, motor coordination, survival and prevented brain damage. Moreover, rhsTM decreased both HMGB1 level and number of activated M1 microglia. The results of this study indicated that rhsTM improved functional outcomes via inhibition of HMGB1 up-regulation and M1 microglial activation in the cerebral ischaemic delayed phase. rhsTM may become a new therapeutic agent with a wide therapeutic time window in patients with cerebral ischaemia., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Pathological α-synuclein (αSyn) has been shown to retain the ability to propagate as prions in humans and animals. However, the molecular basis underlying the prion-like properties of αSyn remains poorly understood. We examined whether brain tissues from cases of dementia with Lewy bodies (DLB), which contain serine 129 (Ser129)-phosphorylated insoluble aggregates of αSyn, exhibit prion-like seeding activity in vitro using the real-time quaking-induced conversion (RT-QuIC) seeding assay. Brain tissues from cases of diffuse neocortical DLB yielded a 50% seeding dose of 10 7.3 -10 9.8 /g brain. The RT-QuIC assay could discriminate between DLB and other neurological and neurodegenerative disorders, suggesting its potential applicability for differential diagnosis. Insoluble aggregates of αSyn>250 kDa detected only in DLB brain tissues by Western blotting analysis were specifically phosphorylated at Ser129. Therefore, we postulated that Ser129-phosphorylated insoluble aggregates of αSyn have prion-like seeding activity. However, insoluble aggregates of recombinant human αSyn (rSyn) with increased β-sheet structures showed little seeding activity in either the phosphorylated or nonphosphorylated state. In contrast, prefibrillar oligomers of rSyn showed seeding activity both with and without phosphorylation. The findings of the present study suggested that soluble oligomeric αSyn, but not the fully fibrillary form, is a seeding species in vitro.

The prion-like seeding of misfolded α-synuclein (αSyn) involved in the pathogenesis of Lewy body diseases (LBD) remains poorly understood at the molecular level. Using the real-time quaking-induced conversion (RT-QUIC) seeding assay, we investigated whether brain tissues from cases of dementia with Lewy bodies (DLB), which contain serine 129 (Ser129)-phosphorylated insoluble aggregates of αSyn, can convert Escherichia coli-derived recombinant αSyn (r-αSyn) to fibrils. Diffuse neocortical DLB yielded 50% seeding dose (SD 50 ) values of 10 7 ~10 10 /g brain. Limbic DLB was estimated to have an SD 50 value of ~10 5 /g brain. Furthermore, RT-QUIC assay discriminated DLB from other neurological and neurodegenerative disorders. Unexpectedly, the prion-like seeding was reconstructed in reactions seeded with oligomer-like species, but not with insoluble aggregates of r-αSyn, regardless of Ser129 phosphorylation status. Our findings suggest that RT-QUIC using r-αSyn can be applied to detect seeding activity in LBD, and the culprit that causes prion-like seeding may be oligomeric forms of αSyn.

Background: Aquaporin 4 (AQP4) is a water-selective transport protein expressed in astrocytes throughout the central nervous system. AQP4 level increases after cerebral ischemia and results in ischemic brain edema. Brain edema markedly influences mortality and motor function by elevating intracranial pressure that leads to secondary brain damage. Therefore, AQP4 is an important target to improve brain edema after cerebral ischemia. The Japanese herbal Kampo medicine, goreisan, is known to inhibit AQP4 activity. Here, we investigated whether goreisan prevents induction of brain edema by cerebral ischemia via AQP4 using 4-hour middle cerebral artery occlusion (4h MCAO) mice., Methods: Goreisan was orally administered at a dose of 500 mg/kg twice a day for 5 days before MCAO. AQP4 expression and motor coordination were measured by Western blotting and rotarod test, respectively., Results: Brain water content of 4h MCAO mice was significantly increased at 24 hours after MCAO. Treatment with goreisan significantly decreased both brain water content and AQP4 expression in the ischemic brain at 24 hours after MCAO. In addition, treatment with goreisan alleviated motor coordination deficits at 24 hours after MCAO., Conclusions: The results of this study suggested that goreisan may be a useful new therapeutic option for ischemic brain edema., (Copyright © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved.)

Prion diseases are caused by deposition of abnormal prion protein aggregates (PrP Sc ) in the central nervous system. This study aimed to develop in vivo imaging probes that can detect cerebral PrP Sc deposits. We synthesized several quinacrine-based acridine (AC) derivatives with 2,9-substitution and radioiodinated them. The AC derivatives were evaluated as prion-imaging probes using recombinant mouse prion protein (rMoPrP) aggregates and brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. The distribution of these compounds in mice was also evaluated. The 2-methoxy derivative [ 125 I]2 exhibited the highest binding affinity for rMoPrP aggregates with an equilibrium dissociation constant (K d ) value of 43.4nM. Fluorescence imaging with 2 showed clear signals at the thioflavin T (ThT)-positive amyloid deposits in the mBSE-infected mouse brain. Although a discrepancy was observed between the in vitro binding of AC derivatives to the aggregates and in vivo distribution of these compounds in the brain and we failed to identify prospective prion-imaging probes in this study, the AC derivatives may be considered a useful scaffold for the development of in vivo imaging probes. Further chemical modification of these AC derivatives may discover clinically applicable prion imaging probes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Prion diseases are fatal neurodegenerative diseases characterised by deposition of amyloid plaques containing abnormal prion protein aggregates (PrP(Sc)). This study aimed to evaluate the potential of radioiodinated flavonoid derivatives for single photon emission computed tomography (SPECT) imaging of PrP(Sc). In vitro binding assays using recombinant mouse PrP (rMoPrP) aggregates revealed that the 4-dimethylamino-substituted styrylchromone derivative (SC-NMe2) had higher in vitro binding affinity (Kd = 24.5 nM) and capacity (Bmax = 36.3 pmol/nmol protein) than three other flavonoid derivatives (flavone, chalcone, and aurone). Fluorescent imaging using brain sections from mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice demonstrated that SC-NMe2 clearly labelled PrP(Sc)-positive prion deposits in the mice brain. Two methoxy SC derivatives, SC-OMe and SC-(OMe)2, also showed high binding affinity for rMoPrP aggregates with Ki values of 20.8 and 26.6 nM, respectively. In vitro fluorescence and autoradiography experiments demonstrated high accumulation of [(125)I]SC-OMe and [(125)I]SC-(OMe)2 in prion deposit-rich regions of the mBSE-infected mouse brain. SPECT/computed tomography (CT) imaging and ex vivo autoradiography demonstrated that [(123)I]SC-OMe showed consistent brain distribution with the presence of PrP(Sc) deposits in the mBSE-infected mice brain. In conclusion, [(123)I]SC-OMe appears a promising SPECT radioligand for monitoring prion deposit levels in the living brain.

The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrPSc. Real-time quaking-induced conversion can amplify very small amounts of PrPSc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt-Jakob disease patients demonstrated that 50% seeding dose (SD50) is reached approximately 10(10)/g brain (values varies 10(8.79-10.63)/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrPSc concentrations in the samples, determined by dot-blot assay, was 0.6-5.4 μg/g brain; therefore, we estimated that 1 SD50 unit was equivalent to 0.06-0.27 fg of PrPSc. The SD50 values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission.

Oestrogen receptor (ER)α plays important roles in the development and function of various neuronal systems through activation by its ligands, oestrogens. To visualise ERα-positive neurons, we generated transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the ERα promoter. In three independent tg lines, GFP-positive neurons were observed in areas previously reported to express ERα mRNA, including the lateral septum, bed nucleus of the stria terminalis, medial preoptic nucleus (MPO), hypothalamus, and amygdala. In these areas, GFP signals mostly overlapped with ERα immunoreactivity. GFP fluorescence was seen in neurites and cell bodies of neurons. In addition, the network and detailed structure of neurites were visible in dissociated and slice cultures of hypothalamic neurons. We examined the effect of oestrogen deprivation by ovariectomy on the structure of the GFP-positive neurons. The area of ERα-positive cell bodies in the bed nucleus of the stria terminalis and MPO was measured by capturing the GFP signal and was found to be significantly smaller in ovariectomy mice than in control mice. When neurons in the MPO were infected with an adeno-associated virus that expressed small hairpin RNA targeting the ERα gene, an apparent induction of GFP was observed in this area, suggesting a negative feedback mechanism in which ERα controls expression of the ERα gene itself. Thus, the ERα promoter-GFP tg mice will be useful to analyse the development and plastic changes of the structure of ERα-expressing neurons and oestrogen and its receptor-mediated neuronal responses., (© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Testosterone is known to play an important role in the regulation of male-type sexual and aggressive behavior. As an aromatised metabolite of testosterone, estradiol-induced activation of estrogen receptor α (ERα) may be crucial for the induction of these behaviors in male mice. However, the importance of ERα expressed in different nuclei for this facilitatory action of testosterone has not been determined. To investigate this issue, we generated an adeno-associated virus vector expressing a small hairpin RNA targeting ERα to site-specifically knockdown ERα expression. We stereotaxically injected either a control or ERα targeting vector into the medial amygdala, medial pre-optic area (MPOA), or ventromedial nucleus of the hypothalamus (VMN) in gonadally intact male mice. Two weeks after injection, all mice were tested biweekly for sexual and aggressive behavior, alternating between behavior tests each week. We found that suppressing ERα in the MPOA reduced sexual but not aggressive behavior, whereas in the VMN it reduced both behaviors. Knockdown of ERα in the medial amygdala did not alter either behavior. Additionally, it was found that ERα knockdown in the MPOA caused a parallel reduction in the number of neuronal nitric oxide synthase-expressing cells. Taken together, these results indicate that the testosterone facilitatory action on male sexual behavior requires the expression of ERα in both the MPOA and VMN, whereas the testosterone facilitatory action on aggression requires the expression of ERα in only the VMN., (© 2013 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Amyloid β (Aβ) oligomers are presumed to be one of the causes of Alzheimer's disease (AD). Previously, we identified the E693Δ mutation in amyloid precursor protein (APP) in patients with AD who displayed almost no signals of amyloid plaques in amyloid imaging. We generated APP-transgenic mice expressing the E693Δ mutation and found that they possessed abundant Aβ oligomers from 8months of age but no amyloid plaques even at 24months of age, indicating that these mice are a good model to study pathological effects of Aβ oligomers. To elucidate whether Aβ oligomers affect proteome levels in the brain, we examined the proteins and phosphoproteins for which levels were altered in 12-month-old APP(E693Δ)-transgenic mice compared with age-matched non-transgenic littermates. By two-dimensional gel electrophoresis (2DE) followed by staining with SYPRO Ruby and Pro-Q Diamond and subsequent mass spectrometry techniques, we identified 17 proteins and 3 phosphoproteins to be significantly changed in the hippocampus and cerebral cortex of APP(E693Δ)-transgenic mice. Coactosin like-protein, SH3 domain-bind glutamic acid-rich-like protein 3 and astrocytic phosphoprotein PEA-15 isoform 2 were decreased to levels less than 0.6 times those of non-transgenic littermates, whereas dynamin, profilin-2, vacuolar adenosine triphosphatase and creatine kinase B were increased to levels more than 1.5 times those of non-transgenic littermates. Furthermore, 2DE Western Blotting validated the changed levels of dynamin, dihydropyrimidinase-related protein 2 (Dpysl2), and coactosin in APP(E693Δ)-transgenic mice. Glyoxalase and isocitrate dehydrogenase were increased to levels more than 1.5 times those of non-transgenic littermates. The identified proteins could be classified into several groups that are involved in regulation of different cellular functions, such as cytoskeletal and their interacting proteins, energy metabolism, synaptic component, and vesicle transport and recycling. These findings indicate that Aβ oligomers altered the levels of some proteins and phosphoproteins in the hippocampus and cerebral cortex, which could illuminate novel therapeutic avenues for the treatment of AD., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

There is great potential in the use of positron emission tomography (PET) and suitable radiotracers for the study of cyclooxygenase type 2 (COX-2) enzyme in living subjects. In the present study, we prepared and evaluated five ¹¹C-labeled ester and amide analogs derived from indomethacin as potential PET imaging agents for the in vivo visualization of the brain COX-2 enzyme. Five ¹¹C-labeled COX-2 inhibitors, with different lipophilicities and moderate COX-2 inhibitory activity, were prepared by treatment of the corresponding O-desmethyl precursors with [¹¹C]methyl triflate and purified by HPLC (radiochemical yields of 55-71%, radiochemical purity of >93%, and the specific activities of 22-331 GBq/µmol). In mice, radioactivity in the brain for all radiotracers was low, with very low brain-to-blood ratios. A clear inverse relationship was observed between brain uptake at 1 min postinjection and the lipophilicity (experimental log P₇.₄) of the studied ¹¹C-radiotracers. Pretreatment of mice with cyclosporine A to block P-glycoproteins caused a significant increase in brain uptake of radioactivity following injection of the ¹¹C-radiotracer compared to control. HPLC analysis showed that each radiotracer was rapidly metabolized, and a few metabolites, which were more polar than the original radiotracers, were found in both plasma and brain. No specific binding of the tracers towards the COX-2 enzyme in the brain was clearly revealed by in vivo blocking study. Further structural refinement of the tracer agent is necessary for better enhancement of brain uptake and for sufficient metabolic stability.

Cannabidiol, a non-psychoactive component of cannabis, has been reported to have interactions with Delta(9)-tetrahydrocannabinol (Delta(9)-THC). However, such interactions have not sufficiently been clear and may have important implications for understanding the pharmacological effects of marijuana. In the present study, we investigated whether cannabidiol modulates the pharmacological effects of Delta(9)-THC on locomotor activity, catalepsy-like immobilisation, rectal temperature and spatial memory in the eight-arm radial maze task in mice. In addition, we measured expression level of cannabinoid CB(1) receptor at striatum, cortex, hippocampus and hypothalamus. Delta(9)-THC (1, 3, 6 and 10 mg/kg) induced hypoactivity, catalepsy-like immobilisation and hypothermia in a dose-dependent manner. In addition, Delta(9)-THC (1, 3 and 6 mg/kg) dose-dependently impaired spatial memory in eight-arm radial maze. On the other hand, cannabidiol (1, 3, 10, 25 and 50 mg/kg) did not affect locomotor activity, catalepsy-like immobilisation, rectal temperature and spatial memory on its own. However, higher dose of cannabidiol (10 or 50 mg/kg) exacerbated pharmacological effects of lower dose of Delta(9)-THC, such as hypoactivity, hypothermia and impairment of spatial memory. Moreover, cannabidiol (50 mg/kg) with Delta(9)-THC (1 mg/kg) enhanced the expression level of CB(1) receptor expression in hippocampus and hypothalamus. Cannabidiol potentiated pharmacological effects of Delta(9)-THC via CB(1) receptor-dependent mechanism. These findings may contribute in setting the basis for interaction of cannabinoids and to find a cannabinoid mechanism in central nervous system.

In addition to their well-known role in neural development, the neurotrophins BDNF and NGF help mediate the plasticity that occurs in the brain to promote learning. Exposure to learning procedures often leads to increases in neurotrophins, while exposure to stress often results in decreases. It is unclear how the neurotrophins would respond to an aversive learning task. Therefore, BDNF and NGF content in the dorsal striatum, hippocampus, and basal forebrain was measured following discrete trial lever-press escape/avoidance conditioning. Conditioning significantly increased levels of both neurotrophins in hippocampus and basal forebrain, relative to home cage controls (HCC). Contrary to expectations, the dorsal striatum did not show any significant changes. However, significant correlations were observed between dorsal striatal neurotrophins and aspects of avoidance performance. This may indicate that the dorsal striatum is involved in the performance aspects of the task. Results are discussed in terms of the role of neurotrophins in the acquisition of new information, and the neural structures involved in different types of memory.

The mechanism of radiation-induced diffuse brain injury was investigated using a model of delayed myelination in the irradiated neonatal rat brain in which the number of oligodendrocytes decreases without associated necrosis of the cerebral white matter. Immunohistochemical analysis using antibody against the large myelin-associated glycoprotein, a specific marker of oligodendrocytes at an early stage of development, showed that the number of the oligodendrocytes associated with myelination decreased in the irradiated hemisphere 1 day after irradiation and remained low until 5 days after irradiation. In situ terminal deoxynucleotidyl transferase-mediated nick end-labeling assay revealed that apoptosis mainly occurred in the cerebral white matter of the irradiated hemisphere. Three hours after irradiation, apoptotic cells were found in the subcortical white matter and the periventricular white matter. Six hours after irradiation, apoptotic cells were found in the internal capsule, and the numbers of apoptotic cells in the periventricular white matter and subcortical white matter increased. One day after irradiation, the number of apoptotic cells in the periventricular white matter decreased. Three days after irradiation, apoptotic cells were not observed in the cerebral white matter. These results suggest that the oligodendrocytes associated with myelination may be damaged via radiation-induced apoptosis, and depletion of the oligodendrocytes may cause delay of myelination.

The amino acid sequence predicted from a rat liver cDNA library indicated that the precursor of beta-AlaAT I (4-aminobutyrate aminotransferase, beta-alanine-oxoglutarate aminotransferase) consists of a mature enzyme of 466 amino acid residues and a 34-amino acid terminal segment, with amino acids attributed to the leader peptide. However, the mass of beta-AlaAT I from rat brain was larger than that from rat liver and kidney, as assessed by Western-blot analysis, mass spectroscopy and N-terminal sequencing. The mature form of beta-AlaAT I from the brain had an ISQAAAK- peptide on the N-terminus of the liver mature beta-AlaAT I. Brain beta-AlaAT I was cleaved to liver beta-AlaAT I when incubated with fresh mitochondrial extract from rat liver. These results imply that mature rat liver beta-AlaAT I is proteolytically cleaved in two steps. The first cleavage of the motif XRX( downward arrow)XS is performed by a mitochondrial processing peptidase, yielding an intermediate-sized protein which is the mature brain beta-AlaAT I. The second cleavage, which generates the mature liver beta-AlaAT I, is also carried out by a mitochondrial endopeptidase. The second peptidase is active in liver but lacking in brain.

Neuropeptide Y (NPY), one of the most abundant peptide transmitters in the mammalian brain, is assumed to play an important role in behavior and its disorders. To understand the long-term modulation of neuronal functions by NPY, we raised transgenic mice created with a novel central nervous system (CNS) neuron-specific expression vector of human Thy- gene fragment linked to mouse NPY cDNA. In situ hybridization analysis demonstrated transgene-derived NPY expression in neurons (e.g., in the hippocampus, cerebral cortex, and the arcuate nucleus of the hypothalamus) in the transgenic mice. The modest increase of NPY protein in the brain was demonstrated by semiquantitative immunohistochemical analysis and by radioreceptor assay (115% in transgenic mice compared to control littermates). Double-staining experiments indicated colocalization of the transgene-derived NPY message and NPY protein in the same neurons, such as in the arcuate nucleus. The transgenic mice displayed behavioral signs of anxiety and hypertrophy of adrenal zona fasciculata cells, but no change in food intake was observed. The anxiety-like behavior of transgenic mice was reversed, at least in part, by administration of corticotropin-releasing factor (CRF) antagonists, alpha-helical CRF9-41, into the third cerebral ventricle. These results suggest that NPY has a role in anxiety and behavioral responses to stress partly via the CRF neuronal system. This genetic model may provide a unique opportunity to study human anxiety and emotional disorders.

Phosphodiesterase I/nucleotide pyrophosphatase is a widely expressed membrane-bound enzyme that cleaves diester bonds of a variety of substrates. We have cloned brain-type cDNA for this enzyme from rat brain and designated it PD-I alpha (M. Narita, J. Goji, H. Nakamura, and K. Sano, 1994, J. Biol. Chem. 269: 28235-28242). In this study we have isolated cDNA and genomic DNA encoding human PD-I alpha. Human PD-I alpha cDNA, designated PDNP2 in HGMW nomenclature, has a 2589-nucleotide open reading frame encoding a polypeptide of 863 amino acids with a calculated M(r) of 99,034. Northern blot analysis revealed that human PD-I alpha transcript was present in brain, lung, placenta, and kidney. The database analysis showed that human PD-I alpha was identical with human autotaxin (ATX), a novel tumor motility-stimulating factor, except that human PD-I alpha lacks 156 nucleotides and 52 amino acids of human ATX. Human PD-I alpha and human ATX are likely to be alternative splicing products from the same gene. The 5' region of the human PDNP2 gene contains four putative binding sites of transcription factor Sp1 without typical TATA or CAAT boxes, and there is a potential octamer binding motif in intron 2. From the results of fluorescence in situ hybridization, the human PDNP2 gene is located at chromosome 8q24.1.

We have isolated cDNA clones encoding the rat brain phosphodiesterase I/nucleotide pyrophosphatase (PD-I alpha), a novel member of the membrane phosphodiesterase I gene family. PD-I alpha cDNA has a 2,655-nucleotide open reading frame encoding a polypeptide of 885 amino acids with a calculated M(r) of 101,302. Northern blot analysis revealed that PD-I alpha transcript was abundantly present in cerebrum and cerebellum while its level was quite low in other tissues. In situ hybridization analysis revealed that PD-I alpha mRNA is localized in secretory epithelial cells in the brain and the eye including choroid plexus epithelial cells, ciliary epithelial cells, iris pigment epithelial cells, and retinal pigment cells. Localization of PD-I alpha mRNA was also observed in glial cells in the molecular layer of the cerebellum. These results indicate that this protein might be involved in the synthesis of adenosine as well as in the regulation of the secretion/transport of epithelial cells and glial cells in the central nervous system.

Rab GDP dissociation inhibitor (GDI) is a regulatory protein for Rab proteins that controls not only the GDP/GTP exchange reaction of Rab proteins but also their translocation between the cytosol and cell membranes. Recently, rab GDI cDNAs have been isolated from human, bovine, rat, and Drosophila, and these Rab GDI proteins indicated an important role in the regulation of intracellular vesicle traffic. In this study, we have isolated two different rab GDI cDNAs, designated rat rab GDI alpha and beta, from a rat brain cDNA library using bovine rab GDI cDNA as a probe. Rat rab GDI alpha and beta show 99 and 86% amino acid identity with bovine rab GDI, respectively, indicating that rat rab GDI alpha is a rat counterpart of bovine rab GDI. Both rat Rab GDI alpha and beta proteins expressed in Escherichia coli showed a similar degree of activity of regulating the GDP/GTP exchange reaction to that of bovine Rab GDI using Rab3A and Rab11 as substrates. Northern blot analysis revealed that rab GDI alpha mRNA was expressed abundantly in brain but much less in other tissues, whereas rat rab GDI beta mRNA was ubiquitously expressed. Reverse transcription-polymerase chain reaction analysis revealed that astrocytes expressed rat rab GDI beta gene but not rat rab GDI alpha gene. These results indicate that Rab GDI alpha is involved in sorting of highly specialized vesicles in brain such as neurosecretory vesicles, whereas Rab GDI beta plays a general role in vesicular trafficking in diverse types of cells.

The entire coding sequences for five possible human cadherins, named cadherin-4, -8, -11, -12 and -13, were determined. The deduced amino acid sequences of cadherin-4 and cadherin-13 showed high homology with those of chicken R-cadherin or chicken T-cadherin, suggesting that cadherin-4 and cadherin-13 are mammalian homologues of the chicken R-cadherin or T-cadherin. Comparison of the extracellular domain of these proteins with those of other cadherins and cadherin-related proteins clarifies characteristic structural features of this domain. The domain is subdivided into five subdomains, each of which contains a cadherin-specific motif characterized by well-conserved amino acid residues and short amino acid sequences. Moreover, each subdomain has unique features of its own. The comparison also provides additional evidence for two structurally different types of cadherins: the first type includes B-, E-, EP-, M, N-, P- and R-cadherins and cadherin-4; the second type includes cadherin-5 through cadherin-12. Cadherin-13 lacks the sequence corresponding to the cytoplasmic domain of typical cadherins, but the extracellular domain shares most of the features common to the extracellular domain of cadherins, especially those of the first type of cadherins, suggesting that cadherin-13 is a special type of cadherin. These results, and those of other recent cloning studies, indicate that many cadherins with different properties are expressed in various tissues of different organisms.

smg p25A/rab 3A is a member of the rab family of small guanosine 5'-trisphosphate (GTP)-binding proteins, which are postulated to act as specific regulators of membrane trafficking in exocytosis and endocytosis. In neural tissues, smg p25A/rab 3A is expressed only in neurons but not in glia. In this study, expression of a regulatory protein for smg p25A/rab 3A, guanine nucleotide dissociation inhibitor (smg p25A/rab 3A GDI), in neurons and glia isolated from rat brain was examined. The smg p25A/rab 3A GDI mRNa was detected as early as on gestational day 14 in the fetal brain, and its level reached a maximum 5 days after birth. By immunoblot analysis, smg p25A/rab 3A GDI was detected in the homogenate of rat hippocampus as well as in that of astrocytes in primary culture. By immunocytochemical analysis, the smg p25A/rab 3A GDI immunoreactivity was detected in hippocampal neurons and oligodendrocytes. The immunoreactivity of astrocytes was much lower than that of neurons and oligodendrocytes. These results indicate that smg p25A/rab 3A GDI may play a fundamental role in membrane trafficking both neurons and glia by interacting with multiple rab family proteins.

Effects of dexamethasone (DEX) on the relative abundance of myelin basic protein (MBP), proteolipid protein (PLP) and glial fibrillary acidic protein (GFAP) mRNAs in the developing rat brain were examined. After DEX (1.0 mg/kg body weight) or saline was administered intraperitoneally to 3-day-old rats for 7 consecutive days, wet weight, DNA content and the relative abundance of the glia-specific mRNAs in cerebrum and cerebellum were analyzed at postnatal days (P) 10, 20 and 30. DEX decreased both wet weight and DNA content in cerebellum more profoundly than in cerebrum. The appearance of MBP, PLP and GFAP mRNAs in cerebellum preceded that in cerebrum in the control group. In cerebrum, the relative abundance of MBP and PLP mRNAs was significantly less in the DEX group than that in the control group at P20 and P30. The relative abundance of the GFAP mRNA was significantly less in the DEX group than in the control group at P10 and P20, but there was no significant difference at P30. In cerebellum, a significant decrease in the abundance of MBP, PLP and GFAP mRNAs in the DEX group was observed only at P10 but not at P20 and P30. Our findings indicate that DEX suppresses expression of genes related to glial functions, especially myelination when administered in the early postnatal period.

It has been proposed that lithium ion desensitizes neuronal receptors that function via the inositol phospholipid signaling mechanism. We examined the effects of lithium chloride on the morphologic outcome after 5 minutes of cerebral ischemia induced in gerbils by occluding both common carotid arteries under brief halothane anesthesia. In three treated groups of 10 gerbils each, 5 meq/kg i.p. lithium chloride was given 2 days, 1 day, and 2 hours before ischemia; 2 hours before ischemia; or immediately after the end of ischemia. Corresponding control groups of nine or 10 gerbils each received equivalent volumes of saline injected at comparable times. All gerbils were perfusion-fixed 1 week later, and neuronal density of the hippocampal CA1 pyramidal cells was determined. Lithium induced very mild intraischemic systemic hypothermia, but postischemic hyperthermia developed in both treated and control groups. Neuronal densities were equal in corresponding groups. The results indicate that our regimen of lithium administration provides no benefit in survival of hippocampal neurons, and intraischemic hypothermia of less than 0.8 degrees C is not protective. Other strategies to inactivate the signal transduction system that is specific for excitatory neurotransmission should be evaluated.

We have recently reported that middle cerebral artery (MCA) occlusion in the rat produces a uniform pattern of cerebral ischemia in an acute phase. This study was done to determine if this model is also useful for quantitative evaluation of infarction size in a chronic phase. [Methods] Sprague-Dawley rats were anesthetized with halothane and left MCA was occluded via transretro-orbital approach. The following studies were done. Neuropathological study was done one week after MCA occlusion. After perfusion fixation, the brain was cut into 6 coronal slices and stained sections were examined. Local cerebral blood flow patterns were observed by 14C-iodoantipyrine autoradiographic technique 1, 2, and 5 days after the occlusion. [Results] Neuropathological studies invariably showed infarct in the cortex and the lateral part of the basal ganlia. The ratio of the infarct to the total areas of both hemispheres in 6 coronal sections was 14.05 +/- 2.66% (Mean +/- SD) in MCA occluded animals (N = 14) and 0.59 +/- 0.46% in sham operated animals (N = 12). Relative to the contralateral hemisphere, marked reduction in CBF was seen in the territory of the MCA and moderate reductions were also seen in the surrounding areas. The same pattern of increased CBF as previously reported was also seen in the ipsilateral substantia nigra and globus pallidus 1, 2, and 5 days after the occlusion. These results indicate the usefulness of this chronic focal cerebral infarction model in the evaluation of infarction.

The correlation between the changes of regional cerebral blood flow (rCBF) and the histological changes were examined using the middle cerebral arterial (MCA) occlusion model in cats. A total of 24 adult cats were tracheostomized and anesthetized by inhalation of halothane. The right MCA was clipped by the transorbital approach. Two hours after the application, the clip was removed and the brain was recirculated for two hours. Then, the brain was perfusion-fixated and the histological studies were carried out. The animals were separated into two groups according to the severity of histological damages using light and electron microscope. Severe cortical damage was present in 8 cats (Group A). In the remaining 16 cats, little or no cortical damage was found (Group B). The averaged rCBF values before occlusion were 45.4 +/- 2.3 ml/100 gm/min in group A and 46.5 +/- 1.6 in group B, showing no statistically significant difference. Between these two groups, however, there was a statistically significant difference in the averaged rCBF values during the ischemic period. In group A, the averaged rCBF values during MCA occlusion was only 6.8 +/- 0.9 and in group B, it was 25.3 +/- 0.8. In the recirculation period, there was a prompt and uniform recovery of rCBF in group B. Whereas in group A, a marked diversity of rCBF ranging from oligemia to hyperemia ensued. This is presumably a reflection of inhomogeneous blood flow, or patchy non-filling of the cerebral cortex. The critical values of rCBF as to the occurrence of severe cortical damage in two-hours MCA occlusion is considered to lie between the lowest value of group B and the highest value of group A, i.e., around 12--15 ml/100 gm/min.

Xanthine and uric acid, products of purine metabolism, were measured by reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection in rat forebrain following focal cerebral ischemia. Focal cerebral ischemia was induced in the rat by permanent occlusion of the left middle cerebral artery (MCA). Sprague-Dawley rats were anesthetized with halothane inhalation and left MCA was occluded via trans-retro-orbital approach. Normal and sham-operated rats were used as control animals. The animals were decapitated 2 (MCA = 5, Sham = 5), 4 (MCA = 7, Sham = 6), 8 (MCA = 5, Sham = 5), and 16 (MCA = 6, Sham = 6) hours or 1 (MCA = 5, Sham = 5), 2 (MCA = 6, Sham = 6), 7 (MCA = 7, Sham = 6), 14 (MCA = 6, Sham = 5), and 28 (MCA = 7, Sham = 5) days after the operation. The brains were removed and divided into right and left hemisphere. Each hemisphere was homogenized and centrifuged. The supernates were filtered with membrane filter. An aliquot of the filtrate was used for measurement of xanthine and uric acid in both of the ischemic and contralateral hemisphere by a HPLC system. In the normal group, xanthine and uric acid in the brain was 12.4 +/- 0.4 and 2.2 +/- 0.1 nmol/g tissue (mean +/- SEM), respectively. In the ischemic hemisphere, xanthine increased up to 57.7 +/- 5.2 nmol/g tissue 2 hours after MCA occlusion and reached a maximum value of 59.42 +/- 4.91 nmol/g tissue 4 hours following the induction of ischemia. Xanthine level was still high 8 hours after ischemia and then rapidly decreased to the normal value at day 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes of uric acid level in rat cerebral hemisphere after left middle cerebral artery (MCA) occlusion were studied by reversed-phase HPLC with electrochemical detection. Uric acid level in the normal group was 2.98 nmol/g tissue. Uric acid concentration of the left hemisphere in the left MCA-occluded group progressively increased after occlusion, and reached a maximum value of 67.26 nmol/g tissue 24 h after ischemia. Uric acid levels in the right hemisphere remained unchanged. Uric acid concentration of the left hemisphere in sham-operated group was 9.29 nmol/g tissue 24 h after the operation.

The effect of transient bilateral carotid occulsion on levels of free fatty acids, phospholipids, and lipid peroxides in the brain was studied in gerbils. During occulsion, both saturated and polyunsaturated free fatty acids increased strikingly to approximately 11-fold in total by 30 minutes. During recirculation, however, a selectively rapid decrement occurred in arachidonic acid, while saturated fatty acids gradually decreased to their basal levels in 180 minutes. The peroxide level, estimated by a thiobarbituric acid test, did not change during occlusion, but was elevated on reperfusion. Phosphatidylethanolamine content decreased throughout the periods examined. These results do not support a hypothesis that lipid peroxidation is initated during ischemia by the lack of oxygen at the terminus of the mitochondrial respiratory chain. Instead, it is suggested that severe cerebral ischemia disintegrates membrane phospholipids, probably through activation of hydrolytic enzymes, and that overt peroxidative processes take place during reflow by means of restoration of oxygen supply. The peroxidative reactions may indeed, cause additional damage during the postischemic phase.

Uric acid (UA) in the rat brain was measured by HPLC with an electrochemical detector following focal ischemia. At 24 h after the operation, the UA level in the ischemic center was 105.47 +/- 8.39 nmol/g tissue, whereas it was 8.36 +/- 1.86 in the sham-operated group. Allopurinol, xanthine oxidase inhibitor, almost completely inhibited this UA accumulation. These data demonstrate that the UA increase in the ischemic brain is due to the xanthine oxidase reaction.

It is important to have information about physical property of the brain in order to elucidate both the physical changes of the morbid brain and the physical mechanism of the traumatic brain injury. Under the hypothesis that reaction of the active brain to the dynamic load can be compared to the Maxwell-Voigt three dimensional model, elastic property of the brain was obtained as the Young's modulus (E: 10(-2) Kgf/cm2) of which error was less than 10%, and viscous property of the brain as the Viscous modulus (eta: 10(-2) Kgf.sec/cm2). And it was confirmed that the reactive pressure of the brain to dynamic load came from the surface to about 15 mm depth of the brain. In this report, experiments were done on the alive normal brains, the edematous ones nd necrotic ones which were produced by the cold injury (dry ice-aceton) in dogs (9.0--16.0 Kg). In the normal brain, E = 3.24 +/- 0.25, eta = 1.10 +/- 0.37 and these moduli were also stable when the physical conditions of the brain were stable. Under the dehydration by 20% mannitol, E increased in its value (p less than 0.01). But under the hydration by 5% glucose, E did not change at all. In the edematous brain, E = 3.28 +/- 0.44, eta = 1.74 +/- 0.06 and E of the edematous brain was almost same as that of normal ones, but under the dehydration, E of the edematous brain decreased (p less than 0.10), on the other hand it increased in its value under the hydration (p less than 0.05). In the necrotic brain, E = 1.60 +/- 0.14, eta = 0.82 +/- 0.28. Both moduli were of lower values and moreover they did not change its values at all under dehydration and hydration. As Young's modulus is the elastic index of the brain, the converse (1/E) should be compliance of the brain, that is to say, buffer effect of the brain. As for the compliance, the necrotic brain has maximum buffer effect and the over-hydrated edematous brain and the dehydrated normal ones have minimum buffer effect. From analysing the changes of the viscous moduli, it became clear that the viscous moduli took quite different functions in alive brains and in fatal ones, and it was suspected that the alive brain might not be so simple in its viscoelastic property.

Tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) directly activate in vitro Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), which normally requires unsaturated diacylglycerol. Kinetic analysis indicates that TPA can substitute for diacylglycerol and greatly increases the affinity of the enzyme for Ca2+ as well as for phospholipid. Under physiological conditions, the activation of this enzyme appears to be linked to the receptor-mediated phosphatidylinositol breakdown which may be provoked by a wide variety of extracellular messengers, eventually leading to the activation of specific cellular functions or proliferation. Using human platelets as a model system, TPA is shown to enhance the protein kinase C-specific phosphorylation associated with the release reaction in the total absence of phosphatidylinositol breakdown. Various phorbol derivatives which have been shown to be active in tumor promotion are also capable of activating this protein kinase in in vitro systems.

Changes of the xanthine and uric acid (UA) levels in rat forebrain following focal cerebral ischemia were studied by reversed-phase HPLC with electrochemical detection. Focal ischemia was induced by occluding the left middle cerebral artery in the rat. The xanthine level in the normal group was 11.50 nmol/g tissue. In the ischemic group, the xanthine concentration in the ischemic hemisphere progressively increased after occlusion and reached a maximum value of 59.42 nmol/g tissue 4 h after operation. The UA level in the normal group was 2.20 nmol/g tissue, whereas in the ischemic group the UA concentration in the ischemic hemisphere gradually increased after occlusion, reaching a value of 38.53 nmol/g tissue 24 h after ischemia. The concentration of UA remained elevated in the ischemic hemisphere until 48 h after occlusion, and reached a maximum value of 38.98 nmol/g tissue. The xanthine and UA levels in the contralateral hemisphere remained unchanged. The xanthine and UA concentrations in the sham-operated group did not show a significant increase after operation. The time course of xanthine and UA levels suggests that in ischemic forebrain UA is formed from xanthine as a product of purine metabolism.

Brain free fatty acids (FFAs) and brain water content were measured in gerbils subjected to transient, bilateral cerebral ischemia under brief halothane anesthesia (nontreated group) and pentobarbital anesthesia (treated group). Mortality in the two groups was also evaluated. In nontreated animals, both saturated and mono- and polyunsaturated FFAs increased approximately 12-fold in total at the end of a 30-min period of ischemia; during recirculation, the level of free arachidonic acid dropped rapidly, while other FFAs gradually decreased to their preischemic levels in 90 min. In treated animals, the levels of total FFAs were lower than the nontreated group during ischemia, but higher at 90 min of reflow, and the decrease in the rate of free arachidonic acid was slower in the early period of reflow. Water content increased progressively during ischemia and recirculation with no extravasation of serum protein, but the values were consistently lower in the treated group. None of the nontreated animals survived for 2 weeks; in contrast, survival was 37.5% in the treated group. It is suggested that barbiturate protection from transient cerebral ischemia may be mediated by the attenuation of both membrane phospholipid hydrolysis during ischemia and postischemic peroxidation of accumulated free arachidonic acid.

The present study was conducted to overcome some of disadvantages of cerebral blood flow study by radionuclide, such as poor regionality of flow values and errors involved in pathological brains. Serial CT scanning was carried out during and after inhalation of 50 to 70% non-radioactive xenon in humans. Diffusible gas with high atomic number enhanced gray matter first 19 +/- 4 HU (mean +/- SD) on an average in Hounsfield unit (HU) and later white matter 24 +/- 4 HU. In seven normal subjects, blood flow in gray matter was 82 +/- 11 and that in white matter 24 +/- 5 ml/100 gm/min. Partition coefficient which is not readily obtainable by radionuclide study was 0.9 +/- 0.1 in normal gray matter and 1.4 +/- 0.2 in normal white matter. Two cases were presented to show k-map (clearance rate), lambda-map (partition coefficient) and CBF map, which displayed images of values calculated automatically, in black and white or color on CRT. The first case was a patient with metastatic brain tumor from lung in the left parietal region. The blood flow of the tumor was close to that of gray matter, whereas blood flow of edematous white matter surrounding the tumor was decreased to below 10 ml/100 g/min with partition coefficient ranged from 0 to 0.9. The second case was presented to demonstrate the resolution of the blood flow map obtained by this method. Multiple lacunar infarcts of the basal ganglia and white matter in the size of 1 to 3 mm, which were hardly identified on regular CT picture, were well visualized on CBF map. This method appeared to have several advantages over conventional isotope method and to provide useful clinical and research informations.

The present study was carried out in order to clarify the mechanism of calcium prolongation of ethanol-induced sleep. p-Chlorophenylalanine (PCPA, 300 mg/kg), alpha-methyltyrosine (alpha MPT, 100 mg/kg) and diethyldithiocarbamate (DDC, 250 mg/kg) were administered intraperitoneally (IP) to mice to reduce the levels of serotonin (5-HT), dopamine (DA) and norepinephrine (NE) respectively in the brain. Sleeping time was then measured following the administration of ethanol (4.5 g/kg, IP) both with and without CaCl2 (20 mumol/kg, intravenous (IV)). When saline (IP) plus CaCl2 (IV) was administered, the duration of ethanol-induced sleep was prolonged by 100% as compared with saline (IP) plus saline (IV). Duration of ethanol-induced sleep was not changed by PCPA, alpha MPT and DDC. On the other hand, the prolongation of ethanol-induced sleep by CaCl2 was antagonized by PCPA, alpha MPT and DDC. Also, only the DA level in the cerebrum was increased by 25% by administration of CaCl2. We suggest that the increase in ethanol-induced sleeping time due to CaCl2 results from the increase in biogenic amines in the brain.

In order to establish more strict and objective indication for EC-IC bypass, a retrospective study on the surgical effect on cerebral blood flow (CBF) and neurological function was carried out in 37 cases. STA-MCA bypass was carried out on 20 cases with IC occlusion and 17 cases with MCA occlusion. Twenty-two cases with completed stroke, 9 cases with TIA and 6 cases with RIND. All patients were able to ambulate before surgery, and none of them were hemiplegic. CBF study by 133Xe inhalation method was carried out before and 2 to 4 weeks after surgery. Bypass was patent in all cases. Thirty percent of all cases showed more than 16% increase in CBF (++ group) on the side of bypass surgery and another 30% demonstrated 6 to 15% increase (+). On the other hand 30% of cases appeared to be unchanged (+/-), and 10% showed more than 6% decrease (-). The less the pre-operative CBF value was, the more the post-operative CBF was expected to be increased. Patients with pre-operative CBF less than 60% of normal value (ISI = 63 +/- 4) showed remarkable increase (++), and those with 60 to 70% showed moderate increase (+). Contrarily, cases with more than 70% of normal value showed either no change or decrease. Neurological change was evaluated as to disappearance of TIA attack and improvement of hemiparesis or mental dysfunction. Seventy-seven percent of patients in groups with (++) and (+) effect on CBF showed improvement in neurological function, whereas only 27% of patients in groups with (+/-) and (-) effect demonstrated improvement, which was statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)