Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription.

At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration.

Cerebral blood flow and freezing of gait in Parkinson's disease.

OBJECTIVE: We investigated the relationship between freezing of gait (FOG) severity in Parkinson's disease (PD) and regional cerebral blood flow (rCBF) using single-photon emission computed tomography (SPECT) and evaluated the effect of selegiline therapy. METHOD: We evaluated 54 patients with PD (FOG positive: 21 patients, and FOG negative: 33 patients) with N-isopropyl-p-[I-123] iodoamphetamine ((123) I-IMP) SPECT and the Unified Parkinson's Disease Rating Scale (UPDRS) part III, Mini-Mental State Examination (MMSE), and Beck Depression Inventory. [Correction added on 18 April 2012, after online publication: In the preceding statement, 55 instead of 54 patients with PD were evaluated, and FOG negative consisted of 34 instead of 33 patients] Furthermore, we examined rCBF in FOG-negative patients treated with levodopa with or without selegiline. RESULTS: Z-values of bilateral Brodmann areas (BA) 10 and 11 and left BA32 showed significant increases in the FOG-positive group compared with the FOG-negative group. [Correction added on 18 April 2012, after online publication: In the preceding statement, Z-values was changed to Z-scores] There were significantly positive correlations between Z-values of these areas and FOG score, especially on both sides of BA11. [Correction added on 18 April 2012, after online publication: In the preceding statement, Z-values was changed to Z-scores] An increase in Z-values in bilateral BA10 and 11 and left BA32 in the levodopa-selegiline treatment group after 1 year was significantly inhibited compared with the levodopa treatment group. [Correction added on 18 April 2012, after online publication: In the preceding statement, left BA32 was changed to right BA32, and Z-values was changed to Z-scores] CONCLUSION: There was a close relationship between FOG severity in PD and an increase in rCBF in BA 10, 11 and 32. Furthermore, selegiline's FOG prevention effect may be related to maintaining rCBF in these same areas.

The relationship between depression and regional cerebral blood flow in Parkinson's disease and the effect of selegiline treatment.

OBJECTIVES: We examined the relationship between severity of depression in Parkinson's disease (PD) and regional cerebral blood flow (rCBF) using single photon emission computed tomography (SPECT) and the reaction to levodopa-selegiline combination therapy. MATERIALS AND METHODS: We evaluated 52 patients with PD and nine age-matched controls with SPECT and the Unified Parkinson's Disease Rating Scale (UPDRS) part III, Mini-Mental State Examination (MMSE), and Beck Depression Inventory (BDI) to evaluate depression severity and its connection with rCBF. Furthermore, we examined rCBF in patients with PD treated with levodopa with or without selegiline. RESULTS: A significant fall in rCBF was observed in the bilateral posterior cingulate, hippocampus, and cuneus and the superior parietal and primary visual areas in PD patients with minor depression and in all regions in those with major depression. Elevations in UPDRS part III and BDI scores and falls in MMSE scores were of significantly lower magnitude in the levodopa-selegiline group than in the levodopa group. Whole brain rCBF fell significantly less in the levodopa-selegiline group than in the levodopa group. CONCLUSIONS: These results indicate that selegiline controlled not only worsening of motor function and cognitive function in PD but also aggravation of minor depression, and restrained a fall in whole brain rCBF.

Vascular and brain dopamine beta-hydroxylase activity in young spontaneously hypertensive rats.

Dopamine beta-hydroxylase activity was higher in mesenteric vessels, adrenal glands, and serum of 3-week-old spontaneously hypertensive rats but lower in the locus coeruleus than it was in the control Wistar-Kyoto rats. The results support the concept that the nervous system is an important regulator of blood pressure.

Effects of tyrosine administration on serum biopterin in normal controls and patients with Parkinson's disease.

After administration of tyrosine, total concentration of biopterin, the cofactor for tyrosine hydroxylase, was increased in the striatum, adrenal glands, and serum of rats, and in the serum of humans. Serum biopterin is lower in patients with Parkinson's disease than in normal controls. After oral administration of tyrosine, the increase in serum biopterin concentration was smaller in patients with Parkinson's disease (less than twofold) than in healthy controls (three-to sevenfold). These results suggest that tyrosine may have a regulatory role in biopterin biosynthesis and that patients with Parkinson's disease may have some abnormality in the regulation of biopterin biosynthesis.

Synaptic integration mediated by striatal cholinergic interneurons in basal ganglia function.

The physiological role of striatal cholinergic interneurons was investigated with immunotoxin-mediated cell targeting (IMCT). Unilateral cholinergic cell ablation caused an acute abnormal turning behavior. These mice showed gradual recovery but displayed abnormal turning by both excess stimulation and inhibition of dopamine actions. In the acute phase, basal ganglia function was shifted to a hyperactive state by stimulation and suppression of striatonigral and striatopallidal neurons, respectively. D1 and D2 dopamine receptors were then down-regulated, relieving dopamine-predominant synaptic perturbation but leaving a defect in controlling dopamine responses. The acetylcholine-dopamine interaction is concertedly and adaptively regulated for basal ganglia synaptic integration.

Biochemistry of postmortem brains in Parkinson's disease: historical overview and future prospects.

Biochemical studies on postmortem brains of patients with Parkinson's disease (PD) have greatly contributed to our understanding of the molecular pathogenesis of this disease. The discovery by 1960 of a dopamine deficiency in the nigro-striatal dopamine region of the PD brain was a landmark in research on PD. At that time we collaborated with Hirotaro Narabayashi and his colleagues in Japan and with Peter Riederer in Germany on the biochemistry of PD by using postmortem brain samples in their brain banks. We found that the activity, mRNA level, and protein content of tyrosine hydroxylase (TH), as well as the levels of the tetrahydrobiopterin (BH4) cofactor of TH and the activity of the BH4-synthesizing enzyme, GTP cyclohydrolase I (GCHI), were markedly decreased in the substantia nigra and striatum in the PD brain. In contrast, the molecular activity (enzyme activity/enzyme protein) of TH was increased, suggesting a compensatory increase in the enzyme activity. The mRNA levels of all four isoforms of human TH (hTH1-hTH4), produced by alternative mRNA splicing, were also markedly decreased. This finding is in contrast to a completely parallel decrease in the activity and protein content of dopamine beta-hydroxylase (DBH) without changes in its molecular activity in cerebrospinal fluid (CSF) in PD. We also found that the activities and/or the levels of the mRNA and protein of aromatic L-amino acid decarboxylase (AADC, DOPA decarboxylase), DBH, phenylethanolamine N-methyltransferase (PNMT), which synthesize dopamine, noradrenaline, and adrenaline, respectively, were also decreased in PD brains, indicating that all catecholamine systems were widely impaired in PD brains. Programmed cell death of the nigro-striatal dopamine neurons in PD has been suggested from the following findings on postmortem brains: (1) increased levels of pro-inflammatory cytokines such as TNF-alpha and IL-6; (2) increased levels of apoptosis-related factors such as TNF-alpha receptor R1 (p 55), soluble Fas and bcl-2, and increased activities of caspases 1 and 3; and (3) decreased levels of neurotrophins such as brain-derived nerve growth factor (BDNF). Immunohistochemical data and the mRNA levels of the above molecules in PD brains supported these biochemical data. We confirmed by double immunofluorescence staining the production of TNF-alpha and IL-6 in activated microglia in the putamen of PD patients. Owing to the recent development of highly sensitive and wide-range analytical methods for quantifying mRNAs and proteins, future assays of the levels of various mRNAs and proteins not only in micro-dissected brain tissues containing neurons and glial cells, but also in single cells from frozen brain slices isolated by laser capture micro-dissection, coupled with toluidine blue, Nissl staining or immunohistochemical staining, should further contribute to the elucidation of the molecular pathogenesis of PD and other neurodegenerative or neuropsychiatric diseases.

Molecular mechanism of the relation of monoamine oxidase B and its inhibitors to Parkinson's disease: possible implications of glial cells.

Monoamine oxidases A and B (MAO A and MAO B) are the major enzymes that catalyze the oxidative deamination of monoamine neurotaransmitters such as dopamine (DA), noradrenaline, and serotonin in the central and peripheral nervous systems. MAO B is mainly localized in glial cells. MAO B also oxidizes the xenobiotic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to a parkinsonism-producing neurotoxin, 1-methyl-4-phenyl-pyridinium (MPP+). MAO B may be closely related to the pathogenesis of Parkinson's disease (PD), in which neuromelanin-containing DA neurons in the substantia nigra projecting to the striatum in the brain selectively degenerate. MAO B degrades the neurotransmitter DA that is deficient in the nigro-striatal region in PD, and forms H2O2 and toxic aldehyde metabolites of DA. H2O2 produces highly toxic reactive oxygen species (ROS) by Fenton reaction that is catalyzed by iron and neuromelanin. MAO B inhibitors such as L-(-)-deprenyl (selegiline) and rasagiline are effective for the treatment of PD. Concerning the mechanism of the clinical efficacy of MAO B inhibitors in PD, the inhibition of DA degradation (a symptomatic effect) and also the prevention of the formation of neurotoxic DA metabolites, i.e., ROS and dopamine derived aldehydes have been speculated. As another mechanism of clinical efficacy, MAO B inhibitors such as selegiline are speculated to have neuroprotective effects to prevent progress of PD. The possible mechanism of neuroprotection of MAO B inhibitors may be related not only to MAO B inhibition but also to induction and activation of multiple factors for anti-oxidative stress and anti-apoptosis: i.e., catalase, superoxide dismutase 1 and 2, thioredoxin, Bcl-2, the cellular poly(ADP-ribosyl)ation, and binding to glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Furthermore, it should be noted that selegiline increases production of neurotrophins such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrphic factor (GDNF), possibly from glial cells, to protect neurons from inflammatory process.

Role of cytokines in inflammatory process in Parkinson's disease.

We investigated whether the cytokines produced in activated microglia in the substantia nigra (SN) and putamen in sporadic Parkinson's disease (PD) are neuroprotective or neurotoxic. In autopsy brains of PD, the number of MHC class II (CR3/43)-positive activated microglia, which were also ICAM-1 (CD 54)-, LFA-1 (CD 11a)-, TNF-alpha-, and IL-6-positive, increased in the SN and putamen during progress of PD. At the early stage activated microglia were mainly associated with tyrosine hydroxylase (TH)-positive neurites in the putamen, and at the advanced stage with damaged TH-positive neurons in the SN. The activated microglia in PD were observed not only in the nigro-striatal region, but also in various brain regions such as the hippocampus and cerebral cortex. We examined the distribution of activated microglia and the expression of cytokines and neurotrophins in the hippocampus of PD and Lewy body disease (LBD). The levels of IL-6 and TNF-alpha mRNAs increased both in PD and LBD, but those of BDNF mRNA and protein drastically decreased specifically in LBD, in which neuronal loss was observed not only in the nigro-striatum but also in the hippocampus. The results suggest activated microglia in the hippocampus to be probably neuroprotective in PD, but those to be neurotoxic in LBD. As an evidence supporting this hypothesis, two subsets of microglia were isolated from mouse brain by cell sorting: one subset with high production of reactive oxygen species (ROS) and the other with no production of ROS. When co-cultured with neuronal cells, one microglia clone with high ROS production was neurotoxic, but another clone with no ROS production neuroprotective. On the other hand, Sawada with coworkers found that a neuroprotective microglial clone in a culture experiment converted to a toxic microglial clone by transduction of the HIV-1 Nef protein with increasing NADPH oxidase activity. Taken together, all these results suggest that activated microglia may change in vivo from neuroprotective to neurotoxic subtsets as degeneration of dopamine neurons in the SN progresses in PD. We conclude that the cytokines from activated microglia in the SN and putamen may be initially neuroprotective, but may later become neurotoxic during the progress of PD. Toxic change of activated microglia may also occur in Alzheimer's disease and other neurodegenerative diseases in which inflammatory process is found.

Urinary putrescine, spermidine, and spermine in human blood and solid cancers and in an experimental gastric tumor of rats.

An improved method of assay of urinary polyamines (putrescine, spermidine, and spermine) was applied to the study of cancer patients and an experimental gastric tumor of rats. Although total polyamines (putrescine, spermidine, and spermine) in urine of patients with blood and solid cancers were significantly high, putrescine concentrations also increased significantly and were shown to be of diagnostic aid even in solid cancers. A significant increase in putrescine was also noted in the urine of rats with experimental stomach tumors induced by N-methyl-N-nitro-N'-nitrosoguanidine.

Increase of urinary putrescine in 3,4-benzopyrene carcinogenesis and its inhibition by putrescine.

A significant increase in putrescine was noted in the urine of mice with experimental s.c. tumors induced by a single injection of 3,4-benzopyrene solution (2.52 mg of 3,4-benzopyrene in 0.5 ml of tricaprylin). When 10 mg of putrescine were added to the 3,4-benzopyrene solution, the development of tumors was completely inhibited and the increase of urinary putrescine in mice was suppressed simultaneously. Animal weight data of a control group receiving only putrescine indicated that the inhibitory effect of putrescine is not due to its toxicity.

Reversible squamous cell characteristics induced by vitamin A deficiency in a small cell lung cancer cell line.

A cultured small cell lung cancer cell line (Lu-134-B-S) established from a xenotransplanted tumor in a nude mouse, which had originated from a primary focus of small cell lung cancer, showed morphological changes when the medium was changed from RPMI 1640 supplemented with 10% fetal calf serum to RPMI 1640 supplemented with 10% delipidized fetal calf serum. That is, it consisted of "classic" small cells in the former medium, but after eight passages in the latter medium many cells became squamous cells, possessing abundant eosinophilic cytoplasm and intercellular bridges. Immunohistochemically, they reacted to antikeratin and antiinvolucrin antibodies. Electron microscopically, well developed desmosomes and associated tonofibrils were noted, and electrophoretically, the amount of medium (Mr 57,000 and 59,000) and large-sized (Mr 67,000) keratins were found to increase with the change of the medium. These changes reversed to the original small cell morphology within 4 weeks after addition of vitamin A (retinoic acid) to the medium. These findings suggested that deficiency of vitamin A caused the change of the cell from small to squamous cell and vice versa.

Selective toxicity of 5-S-cysteinyldopa, a melanin precursor, to tumor cells in vitro and in vivo.

The effect of 5-S-cysteinyl-L-3,4-dihydroxyphenylalanine (cys-dopa), an intermediate in the pathway from L-3,4-dihydroxyphenylalanine (L-dopa) to pheomelanin, on the growth of eight human tumor cell lines in culture was compared to that of L-dopa. The tumor cell lines tested comprise two neuroblastomas (NB-1 and YT-nu), two amelanotic melanomas (HMV and SEKI), a gastric carcinoma (MKN-28), and three squamous cell carcinomas (HeLa-S3, KB, and a salivary gland carcinoma). Cys-dopa at a concentration of 1 mM inhibited growth of NB-1 (66%), YT-nu (67%), HMV (44%), SEKI (60%), MKN-28 (47%), HeLa-S3 (24%), KB (64%), and salivary gland carcinoma (33%), while L-dopa exhibited similar or even lower degree of inhibition at a concentration of 6 mM. On the other hand, both catechols had little effect on the growth of two fibroblasts derived originally from normal tissues (mouse fibroblast L929 and Chinese hamster fibroblast Don-6). Cys-dopa and L-dopa inhibited DNA and protein synthesis in YT-nu cells, but RNA synthesis was less affected. Treatment with cys-dopa at a dose of 1000 mg/kg i.p. for 7 days prolonged by 50% the life span of mice inoculated with L1210 leukemia. Normal mice given cys-dopa at a dose of 1000 mg/kg for 12 days showed no signs of toxicity. These results suggest the potential of cys-dopa as an antitumor agent.

Two novel cell surface antigens on small cell lung carcinoma defined by mouse monoclonal antibodies NE-25 and PE-35.

Two mouse monoclonal antibodies, NE-25 and PE-35, defining novel cell surface antigens of small cell lung carcinoma (SCLC) were produced. The molecular weight of NE-25 and PE-35 antigens estimated by radioimmunoprecipitation was 25,000 and 35,000, respectively. NE-25 antigen was expressed on the majority of cell lines and tumor specimens of SCLC among lung carcinoma. These NE-25-positive cell lines showed typical growth morphology as SCLC classic lines and expressed high levels of neuroendocrine biomarkers, such as aromatic L-amino acid decarboxylase, while NE-25 antigen-negative lines lacked apparent neuroendocrine properties. This antigen was expressed also on a subset of neoplastic cells with (neuro)endocrine properties, including pulmonary carcinoid, and on various tumors of nervous tissues, such as neuroblastoma. Among the normal cells, Kulchitski cells of lung, thyroid gland, adrenal gland, Langerhans islet, and nervous tissues were positive. Thus, the expression of NE-25 antigen is closely associated with the neural and/or (neuro)endocrine differentiation state. On the contrary, PE-35 antigen was present on four major types of lung carcinomas as well as on squamous cell carcinoma and adenocarcinomas of various tissues, but it was absent from nervous tissue tumors. Thus, PE-35 antibody showed a "pan-epithelial" reactivity. Analysis by NE-25 and PE-35 antibodies provided evidence for the heterogeneities of SCLC by demonstrating four surface phenotypes, with the NE-25+/PE-35+ phenotype being most common. In addition, the results supported the current understanding that various histological types of lung carcinoma, including SCLC, are derived from a stem cell of the bronchial epithelium.

Reduction of adriamycin toxicity by ascorbate in mice and guinea pigs.

The effect of ascorbate in reducing Adriamycin toxicity has been examined in mice and guinea pigs. Ascorbate had no effect on the antitumor activity of Adriamycin in mice inoculated with leukemia L1210, but it significantly prolonged the life of mice and guinea pigs treated with Adriamycin. Adriamycin elevated lipid peroxide levels in serum and liver, and ascorbate prevented the elevation. The significant prevention of Adriamycin-induced cardiomyopathy by ascorbate was proved by means of electron microscopy. The earliest alterations of dilation of the sarcoplasmic reticulum and transverse tubular system and the appearance of a large number of cytoplasmic fat droplets, which were seen in cardiac tissue from guinea pigs receiving Adriamycin, were apparently reduced in animals that were treated with ascorbate.

Modest neuropsychological deficits caused by reduced noradrenaline metabolism in mice heterozygous for a mutated tyrosine hydroxylase gene.

Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme for the biosynthesis of catecholamines that are considered to be involved in a variety of neuropsychiatric functions. Here, we report behavioral and neuropsychological deficits in mice carrying a single mutated allele of the TH gene in which TH activity in tissues is reduced to approximately 40% of the wild-type activity. In the mice heterozygous for the TH mutation, noradrenaline accumulation in brain regions was moderately decreased to 73-80% of the wild-type value. Measurement of extracellular noradrenaline level in the frontal cortex by the microdialysis technique showed a reduction in high K(+)-evoked noradrenaline release in the mutants. The mutant mice displayed impairment in the water-finding task associated with latent learning performance. They also exhibited mild impairment in long-term memory formation in three distinct forms of associative learning, including active avoidance, cued fear conditioning, and conditioned taste aversion. These deficits were restored by the drug-induced stimulation of noradrenergic activity. In contrast, the spatial learning and hippocampal long-term potentiation were normal in the mutants. These results provide genetic evidence that the central noradrenaline system plays an important role in memory formation, particularly in the long-term memory of conditioned learning.

Separation of two PZ-peptidases from bovine dental follicle.

Two PZ-peptidases (EC 3.4.-) (A and B) cleaving a synthetic substrate for collagenase, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (PZ-peptide) have been separated from the particulate fraction of bovine dental follicle. PZ-peptidase A had a molecular weight of 220 000, an optimum pH at 8.0-8.5, and a Km value of 67 muM toward PZ-peptide at pH 7.1, whereas PZ-peptidase B had a molecular weight of 20 000, an optimum pH at 6.5-6.7, and a Km value of 400 muM toward PZ-peptide at pH 7.1. Two similar enzymes were also isolated from the soluble fraction. Since the pH-activity curve of the crude tissue preparations such as homogenate, microsomes and soluble supernatant had two peaks at 6.5-6.7 and 8.0-8.5, both PZ-peptidase A and B may exist in situ as two independent active enzymes.

Mouse sepiapterin reductase: an enzyme involved in the final step of tetrahydrobiopterin biosynthesis. Primary structure deduced from the cDNA sequence.

We carried out the cloning of a mouse cDNA encoding a sepiapterin reductase which is involved in the final step of tetrahydrobiopterin biosynthesis as a first step toward gene-targeting technique in mice. The sequence contained 1245 nucleotides consisting of an open reading frame of 783 nucleotides encoding a protein of 261 amino acid residues whose molecular weight was 27,851, a 5'-untranslated region of 21 nucleotides and a 3'-untranslated region of 441 nucleotides containing poly(A) tail. The amino acid sequence of mouse sepiapterin reductase revealed the identity of 88% with rat and 74% with human sequence.

Comparison of characteristics of bovine aromatic L-amino acid decarboxylase with human enzyme.

Aromatic L-amino acid decarboxylase (AADC) was purified from bovine adrenal medulla and properties of this enzyme were compared with those of AADC from human pheochromocytoma. The molecular weights of the subunits were identical between human and bovine enzymes and estimated to be 50,000 by SDS-polyacrylamide gel electrophoresis. An isoelectric point of the human enzyme was 5.7, while the bovine enzyme showed several distinct bands at the region of pH 4.9-5.3 in the absence of urea. Multiplicity of the isoelectric point of bovine AADC disappeared in the presence of urea. These results showed that there were some differences between the properties of human and bovine AADC in spite of the high homology (88%) in their primary structures.

Characterization of protein kinases from bovine parotid glands. The effect of tolbutamide and its derivative on these partially purified enzymes.

1. Four fractions of protein kinase (EC 2.7.1.37) activity (Peak IH, IIH, IIIC and IVC) have been resolved and partially purified from the 100 000 X g supernatant fraction of bovine parotid glands by DEAE-cellulose and phosphocellulose chromatographies. 2. The protein kinases of Peak IH and IIH were adenosine 3',5'-monophosphate (cyclic AMP) -dependent and had similar enzymic properties. The enzyme activities of Peak IIIC and IVC were cyclic-AMP independent, but there were some distinct differences between their properties. The protein kinase in Peak IIIC was activated by 0.2 M NaCl or KCl and phosphorylated casein preferentially as the substrate, utilizing only ATP as a phosphate donor. On the other hand, the protein kinase in Peak IVC was inhibited by univalent salts and preferred phosvitin to casein, utilizing either ATP or GTP as a phosphate donor. 3. Tolbutamide increased the Km value for ATP and the dissociation constant for cyclic AMP, resulting in the inhibition of cyclic-AMP dependent protein kinase activity in the presence of cyclic AMP. Tolbtamide and its carboxy derivative, 1-butyl-3-p-carboxyphenylsulfonylurea, exerted almost no inhibitory effect on either the cyclic-AMP dependent protein kinase activities in the absence of cyclic AMP or on the cyclic-AMP independent protein kinase activities.