Clinical significance of ERG rearrangement subtype and its association with increased p53 expression in Japanese and German prostate cancer.

UNLABELLED: This study investigated differences in prevalence of the androgen-regulated transmembrane protease serine 2 (TMPRSS2) and ETS transcription factor family member, v-ets erythroblastosis virus E26 oncogene homolog (ERG) fusion gene (TMPRSS2-ERG fusions) in clinically localized prostate cancer Japanese and German patients. A total of 105 specimens, including 69 Japanese and 36 German patients, were collected. The status of TMPRSS2-ERG fusion was determined by fluorescence in situ hybridization, and correlations of the TMPRSS2-ERG fusion with clinicopathological characteristics and immunohistochemistry were studied. Gene fusions were identified in 20% (14/69) of Japanese and 53% (19/36) of German patients (P < 0.001). The difference in the type of gene fusion between the two ethnic groups was statistically significant (P=0.024). Overexpression of ERG protein was significantly associated with gene fusion. Biochemical recurrence was significantly higher in patients with ERG overexpression than in those without, and not related to TMPRSS2-ERG fusion status. Interestingly, two types of gene fusions (deletion and increase of copy number) were significantly associated with increased p53 expression (P = 0.005). Association of specific gene fusions harboring higher genomic alterations with p53 expression levels suggests that p53 mutation might drive more aggressive arrangements of TMPRSS2-ERG fusion in prostate cancer. KEYWORDS: ERG, p53, prostate cancer, TMPRSS2-ERG fusion.

The effects of Patent Blue dye on peripheral and cerebral oxyhaemoglobin saturations.

We measured the effect of Patent Blue dye on oxyhaemoglobin saturations after injection into breast tissue: 40 women had anaesthesia for breast surgery maintained with sevoflurane or propofol (20 randomly allocated to each). Saturations were recorded with a digital pulse oximeter, in arterial blood samples and with a cerebral tissue oximeter before dye injection and 10, 20, 30, 40, 50, 60, 75, 90, 105 and 120 min afterwards. Patent Blue did not decrease arterial blood oxyhaemoglobin saturation, but it did reduce mean (SD) digital and cerebral oxyhaemoglobin saturations by 1.1 (1.1) % and 6.8 (7.0) %, p < 0.0001 for both. The falsely reduced oximeter readings persisted for at least 2 h. The mean (SD) intra-operative digital pulse oxyhaemoglobin readings were lower with sevoflurane than propofol, 97.8 (1.2) % and 98.8 (1.0) %, respectively, p < 0.0001.

A simple method for enhancing hybridization efficiency in chromosome and array comparative genomic hybridization.

The accuracy of comparative genomic hybridization (CGH) analysis is affected by hybridization efficiency. We describe here a simple method for enhancing hybridization efficiency. The hybridization procedure is essentially the same as that of conventional methods. Hybridization solution containing denatured DNA probe mixture was applied to a metaphase chromosome slide or DNA chip slide and covered with a coverslip. In the new method, however, the slide was inverted by turning the coverslip downward prior to hybridization. We termed this method the inverted slide method. To estimate the efficiency of the new method, metaphase chromosome slides and DNA chip slides were treated by both the conventional and inverted slide methods and incubated in a moist chamber at 37°C for 12, 24, 48, and 72 h. Hybridization signals were approximately 1.5 to 2 times brighter on the slides using the inverted slide method than those using the conventional method after 48 and 72 h of incubation. Furthermore, topographical differences in fluorescence intensity were smaller in slides using the inverted-slide method than in those prepared by the conventional method. The inverted slide method is methodologically very simple and improves the resolution of CGH.

Establishment of the transformants expressing human cytochrome P450 subtypes in HepG2, and their applications on drug metabolism and toxicology.

Transformants with stable expression of a series of human cytochrome P450 (CYP) subtypes in the human hepatic cell line, HepG2, were established. These transformants are designated Hepc/1A1.4, Hepc/1A2.9, Hepc/2A6L.14, Hepc/2B6.68, Hepc/2C8.46, Hepc/2C9.1, Hepc/2C19.12, Hepc/2D6.39, Hepc/2E1.3-8 and Hepc/3A4.2-30, which stably expressed human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, respectively. The expression of the CYP subtypes in the transformants was confirmed by both determination of enzyme activities and the reverse transcriptase polymerase chain reaction (RT-PCR) procedure. The apparent K(m) values of the expressed CYP subtypes for their specific substrates were close to those of human liver microsomes. In addition to their CYP activities, these transformants retained glucuronide- and sulfate-conjugating activities. Furthermore, the activities of CYP2C9, CYP2D6 and CYP3A4 were inhibited by their specific inhibitors. The cytotoxicity of acetaminophen (APAP), cyclophosphamide (CPA) and benz[a]anthracene (BA) were analyzed by CYP-expressing transformants. The cytotoxicity depended on the expression of CYP subtypes and increased in a dose-dependent manner. These results show the metabolic activation of APAP, CPA and BA by the specific CYP subtypes expressed in the transformants and demonstrate the usefulness of these transformants for in vitro metabolic and toxicological studies in human liver.

Exon 3 of tyrosine hydroxylase gene: lack of association with Japanese schizophrenic patients.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine (DA) biosynthesis. Exon 3 of the human TH gene encodes the sequence from Ser31 to Glu104 of type 1 enzyme, which contains the critical parts for regulation of the catalytic activity. The amino acid residues Gly36-Arg37-Arg38 were identified as a key sequence for DA to exert its inhibitory effect on catalytic activity. Therefore, we screened the nucleotide sequences of exon 3 from 201 Japanese patients with schizophrenia to explain the elevation in the synaptic or presynaptic DA concentrations in the schizophrenic brain, based on the hypothesis that any mutation changing the amino acid sequence Gly36-Arg37-Arg38 would result in the elevation of DA synthesis, due to a reduced inhibitory effect of DA on the catalytic activity. However, no mutated sequences of exon 3 and both exon-intron boundaries were detected in any of the patients examined. Polymorphisms generating Val81 and Met81 were compared of the distributions of genotype and allele between the patients and 175 Japanese healthy controls, which did not suggest an association between the polymorphism and schizophrenia. These results indicate that exon 3 of the human TH gene lacks association with schizophrenia in Japanese patients.

Expression of GTP cyclohydrolase I in murine locus ceruleus is enhanced by peripheral administration of lipopolysaccharide.

Among the enzymes involved in the system for catecholamine biosynthesis, GTP cyclohydrolase I (GCH) contributes to the system as the first and rate-limiting enzyme for the de novo biosynthesis of tetrahydrobiopterin (BH4), which is the cofactor for tyrosine hydroxylase (TH). Therefore, we investigated whether the endotoxemia caused by an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) can modulate BH4 production in the norepinephrine nuclei, i.e. the locus ceruleus (LC; A6) and central caudal pons (A5), in C3H/HeN mice and whether such a change in BH4, if any, can result in the modification of norepinephrine production in these nuclei. After a 5-microg i.p. injection of LPS, the protein expression of GCH and TH in both nuclei was examined by immunohistochemistry. The staining intensity of GCH-positive cells increased at 6 h, whereas no significant change in the staining intensity of TH-positive cells was detected. Next, we measured the contents of BH4, norepinephrine, and its metabolites 4-hydroxy-3-methoxyphenylglycol (MHPG) and DL-4-hydroxy-3-methoxymandelic acid (VMA) in these nuclei after LPS i.p. injection. The BH4 content increased to a statistically significant level at 2 and 4 h after the injection. The contents of MHPG and VMA also showed a time-course similar to that of BH4. These data can be rationalized to indicate that an increased supply of BH4 in the LC increased TH activity and resulted in an increase in norepinephrine production rate at the site. This is the first report that sheds light on BH4 as a molecule that intervenes during endotoxemia to increase norepinephrine production rate in the LC.

Diagnostic significance of tau protein in cerebrospinal fluid from patients with corticobasal degeneration or progressive supranuclear palsy.

Distinguishing corticobasal degeneration (CBD) from progressive supranuclear palsy (PSP) is clinically and pathologically difficult, and a useful biological marker to discriminative these two diseases has been a subject of clinical interest. In the present study, we assessed tau protein levels in cerebrospinal fluids by sandwich ELISA to distinguish CBD from PSP. The subjects consisted of 27 cases of CBD, 30 cases of PSP, and 36 healthy controls (CTL). The tau values in CBD were significantly higher than those in PSP (P<0.001) and those in CTL (P<0.001). The assay of CSF tau provided diagnostic sensitivity of 81.5% and specificity of 80.0% between CBD and PSP according to receiver-operating characteristic (ROC) curve analysis. When values were compared separately with respect to stage of the disease, differences in the values for moderate CBD vs. moderate PSP had the greatest significance (P<0.001 sensitivity 92.3%, specificity 100.0%), followed by cases of mild CBD and PSP (P<0.005, sensitivity 100.0%, specificity 87.5%). The values in severe CBD and PSP were not significantly different (P=0.07, sensitivity 100%, specificity 75.0%). Using data obtained from a larger number of disease cases, we confirmed our previous findings that tau protein levels in cerebrospinal fluids in patients with CBD are significantly higher than those in patients with PSP. Because tau protein levels in cerebrospinal fluids are significantly higher in early CBD cases than in early PSP cases, measurement of tau protein levels in cerberospinal fluids may be useful for the differential diagnosis of early CBD from early PSP.

Human midbrain dopamine neurons express serotonin 2A receptor: an immunohistochemical demonstration.

We demonstrated intense serotonin (5-HT) 2A receptor immunoreactivity in the human ventral tegmental area (VTA) using by a recently raised antibody against 5-HT2A receptor. The substantia nigra (SN) neurons also showed 5-HT2A receptor immunoreactivity. Double immunohistochemistry of 5-HT2A receptor and tyrosine hydroxylase (TH) revealed many neurons doubly labeled by 5-HT2A receptor and TH in the VTA and SN. It is suggested that activity of human midbrain dopaminergic neurons might be strongly regulated via 5-HT2A receptors at the level of their originating nuclei.

The carbohydrate deposits detected by histochemical methods in the molecular layer of the dentate gyrus in the hippocampal formation of patients with schizophrenia, Down's syndrome and dementia, and aged person.

Post-mortem brain tissue was obtained from 28 patients with brain disorders, of which 15 had clinically diagnosed schizophrenia, 6 Alzheimer type dementia, 5 dementia with tangles and 2 cases of Down's syndrome. The controls were 22 cases from autopsies without brain disorders or with no known episodes of brain disorder. The tissues were stained for the detection of carbohydrate deposits in the hippocampal formation, using lectin, immunohistochemical and conventional staining methods. The staining revealed the existence of spherical deposits in the inner and middle molecular layers of the dentate gyrus in the hippocampal formation which contained fucose, galactose, N-acetyl galactosamine, N-acetyl glucosamine, sialic acid, mannose and chondroitin sulfate. The number of the deposits was higher in patients with brain disorder such as schizophrenia, Alzheimer type dementia, dementia with tangles or Down's syndrome, and in some aged individuals, in comparison to those in younger individuals. No deposits were detected in a few younger or aged individuals. Spherical deposits 3-10 microm in diameter may be an immature form of the corpora amylacea, since they were similar in the histochemical characteristics with lectin, immunohistochemical and conventional staining methods. However, differing staining ability by hematoxylin, periodic acid Schiff's reagent and antibodies against the intracellular degraded proteins such as ubiquitin and tau-protein was observed. The antibodies against ubiquitin and tau-protein showed clear reactivity with the corpora amylacea and no reactivity with spherical deposits, indicating that the corpora amylacea has an intracellular origin and spherical deposits an extracellular matrix origin. The results obtained in this study indicate that not only neuronal degeneration but also unusual glycometabolism in neurons may disturb the neuronal function and cause brain disorders, and that spherical deposits may cause dysfunction of the neuronal network in the dentate gyrus of the hippocampus which is closely linked with recognition and memory functions.

Cholinergic neurons with monoamine oxidase type B (MAOB)-activity in the laterodorsal tegmental nucleus of the mouse.

No neurons in the laterodorsal tegmental nucleus (LDTg) show monoamine oxidase (MAO) activity in the rat or monkey. However, in our recent study, many LDTg neurons with MAO type B (MAOB)-activity were found in MAOA-deficient mice that were derived from C3H mouse line. In the present study, LDTg neurons with MAOB-activity were found not only in normal C3H mouse but also in BALB/C and C57BL/6 mouse lines: MAO histochemistry revealed LDTg neurons with MAO-activity even after pharmacological suppression of MAOA-activity with clorgyline, a specific MAOA inhibitor, but not after pharmacological suppression of MAOB-activity with deprenyl, a specific MAOB inhibitor. LDTg neurons with MAOB-activity also showed NADPH-diaphorase-activity, a marker of cholinergic neurons.

Effects of NTE-122, a novel acyl-CoA:cholesterol acyltransferase inhibitor, on cholesterol esterification and high-density lipoprotein-induced cholesterol efflux in macrophages.

We investigated the effects of a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, NTE-122 (trans-1,4-bis[[1-cyclohexyl-3-(4-dimethylamino phenyl)ureido]methyl]cyclohexane), on ACAT activities in macrophages originating from several species and high-density lipoprotein (HDL)-induced cholesterol efflux in phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells. NTE-122 inhibited cell-free ACAT activities in human PMA-treated THP-1 cells and mouse J774.1 cells with IC50 values of 0.88 and 360 nM, respectively. NTE-122 competively inhibited the ACAT activity in PMA-treated THP-1 cells. NTE-122 also inhibited cellular ACAT activities in PMA-treated THP-1 cells, rat peritoneal macrophages and J774.1 cells with IC50 values of 3.5, 84 and 6800 nM, respectively. Furthermore, NTE-122 prevented cholesterol accumulation in PMA-treated THP-1 cells incubated with acetylated low density lipoprotein, simultaneously with HDL, while it caused accumulation of a significant amount of free cholesterol in the absence and even in the presence of HDL. NTE-122 also enhanced HDL-induced cholesterol efflux from established foam cells converted from PMA-treated THP-1 cells. These results suggest that NTE-122, capable of inhibiting macrophage ACAT activity in humans more strongly than those in the other species, exhibits anti-atherogenic effects by preventing the foam cell formation and enhancing the foam cell regression in humans.

A comparison of tau protein in cerebrospinal fluid between corticobasal degeneration and progressive supranuclear palsy.

Many clinical and pathological discussions have been focused on the difficulty of differential diagnosis between corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) in recent years. This study was conducted to evaluate the usefulness of tau proteins in cerebrospinal fluid (CSF) for the differentiation of these two diseases. Subjects consisted of 10 patients with CBD (four males and six females with a mean age of 67.9+/-5.8 years), 12 patients with PSP (eight males and four females with a mean age of 62.6+/-5.8 years) and 36 control subjects (CTL) (16 males and 20 females with a mean age of 65.8+/-9.9 years). The CBD group included patients with probable CBD, while all the patients in the PSP group satisfied the diagnostic criteria developed by the National Institute of Neurological Disorders and Stroke and Society for PSP (NINDS-SPSP). CSF tau proteins were measured with the sandwich ELISA method (Innogenetics, Belgium). The CSF tau protein level was 320.1+/-86.5 pg/ml in the CBD group, 151.5+/-52.7 pg/ml in the PSP group and 128.7+/-91.7 pg/ml in the CTL group. Significant differences were noted in tau protein levels between the CBD group and both the PSP group (P<0.001) and the CTL group (P<0.005). We suggested that the measurement of CSF tau proteins may be useful for the differentiation between CBD and PSP.

Tyrosine hydroxylase and aromatic L-amino acid decarboxylase do not coexist in neurons in the human anterior cingulate cortex.

Immunoreactivity for aromatic L-amino acid decarboxylase (AADC), the second step dopamine-synthesizing enzyme, was found immunohistochemically in neurons of the human anterior cingulate cortex (ACC). Most of these neurons were located in layers V and VI and subcortical white matter; a small number were occasionally found in layer III. Double immunohistochemistry for tyrosine hydroxylase (TH: the first step dopamine-synthesizing enzyme) and AADC revealed that no neuronal cell bodies in the ACC were doubly immunostained for TH and AADC, suggesting that these TH-only- or AADC-only-immunoreactive neurons were not dopaminergic. AADC neurons in the human ACC might transform L-DOPA to dopamine, droxidopa to noradrenaline, and/or 5-hydroxytryptophan to serotonin.

Specific localization of the guanosine triphosphate (GTP) cyclohydrolase I-immunoreactivity in the human brain.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH) is the first and rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, the cofactor of tyrosine hydroxylase (TH). Our previous study reported the presence of GCH in several neuronal groups in animal brains using a newly raised anti-GCH antibody. The present study aims at elucidating whether GCH and TH coexist in the same neurons of the human brain with the aid of immunohistochemical dual labeling. GCH-immunoreactivity was observed in the cell bodies and fibers of monoaminergic neurons of the human brain. Neurons which contain both enzymes are seen in the human substantia nigra, ventral tegmental area, locus coeruleus, dorsal raphe, and zona incerta. In these regions, almost all the cells also show immunoreactivity for aromatic L-amino acid decarboxylase (AADC), the second step enzyme for catecholamine synthesis, indicating that these neurons are catecholaminergic. However, some neurons in the dorsal and dorsomedial hypothalamic nuclei are stained only for GCH or TH. They appear to constitute an independent cell group in the human brain. The present observation suggests that L-dopa is not produced in the cells immunoreactive for TH but not for GCH, and that TH in these cells which lack GCH may have an unidentified role other than dopa synthesis.

Injection of 6-hydroxydopamine into the ventral tegmental area suppresses the increase in arterial pressure during REM sleep in the rat.

We have examined the effect of injection of 6-hydroxydopamine (6-OHDA) into the ventral tegmental area (VTA) on the changes in arterial blood pressure (AP) and heart rate (HR) during the transition from non-rapid eye movement (NREM) sleep to REM sleep. The 6-OHDA-treated rats showed suppression of the increase of AP and HR during REM sleep and of theta frequency in the cortical electroencephalogram (EEG) during wakefulness (W) and REM sleep. It is suggested that midbrain dopaminergic neurons are involved in the control of AP and HR during REM sleep and in the EEG theta activity.

Cholesterol-lowering effects of NTE-122, a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, on cholesterol diet-fed rats and rabbits.

Pharmacological characterization of NTE-122 (trans-1,4-bis[[1-cyclohexyl-3-(4-dimethylamino phenyl)ureido]methyl]cyclohexane), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, was performed with both in vitro and in vivo assay systems. NTE-122 inhibited microsomal ACAT activities of various tissues (liver of rabbit and rat, small intestine of rabbit and rat, and aorta of rabbit) and cultured cells (HepG2 and CaCo-2), with IC50 values from 1.2 to 9.6 nM. The inhibition mode of NTE-122 was competitive for HepG2 ACAT. NTE-122 had no effect on other lipid metabolizing enzymes, such as 3-hydroxy-3-methylglutaryl-CoA reductase, acyl-CoA synthetase, cholesterol esterase, lecithin:cholesterol acyltransferase, acyl-CoA:sn-glycerol-3-phosphate acyltransferase and cholesterol 7alpha-hydroxylase up to 10 microM. When NTE-122 was administered to the cholesterol diet-fed rats, serum and liver cholesterol levels were markedly reduced with an ED50 of 0.12 and 0.44 mg/kg/day, respectively. In the cholesterol diet-fed rabbits, NTE-122 significantly lowered plasma and liver cholesterol levels at more than 2 mg/kg/day. These results indicate that NTE-122 is a potent, selective and competitive inhibitor of ACAT, making it a worth while therapeutic agent for hypercholesterolemia and atherosclerosis.

Does tyrosinase exist in neuromelanin-pigmented neurons in the human substantia nigra?

It is controversial whether tyrosinase is involved in the neuromelanin-biosynthetic pathway. We examined tyrosinase-immunoreactivity in human substantia nigra neurons which contain neuromelanin pigments, using antibodies against human tyrosinase and human tyrosine hydroxylase. In human melanoma, the antibody to tyrosinase showed intense immunoreactivity while there was no immunoreactivity with antibody to tyrosine hydroxylase. In the human midbrain pigmented neurons, however, we could detect no tyrosinase-immunoreactivity while the neurons were strongly immunoreactive to tyrosine hydroxylase. The present results suggest that tyrosinase is not involved in the main pathway of neuromelanin biosynthesis.