Parkinson's Disease Master Regulators on Substantia Nigra and Frontal Cortex and Their Use for Drug Repositioning.

Parkinson's disease (PD) is among the most prevalent neurodegenerative diseases. Available evidences support the view of PD as a complex disease, being the outcome of interactions between genetic and environmental factors. In face of diagnosis and therapy challenges, and the elusive PD etiology, the use of alternative methodological approaches for the elucidation of the disease pathophysiological mechanisms and proposal of novel potential therapeutic interventions has become increasingly necessary. In the present study, we first reconstructed the transcriptional regulatory networks (TN), centered on transcription factors (TF), of two brain regions affected in PD, the substantia nigra pars compacta (SNc) and the frontal cortex (FCtx). Then, we used case-control studies data from these regions to identify TFs working as master regulators (MR) of the disease, based on region-specific TNs. Twenty-nine regulatory units enriched with differentially expressed genes were identified for the SNc, and twenty for the FCtx, all of which were considered MR candidates for PD. Three consensus MR candidates were found for SNc and FCtx, namely ATF2, SLC30A9, and ZFP69B. In order to search for novel potential therapeutic interventions, we used these consensus MR candidate signatures as input to the Connectivity Map (CMap), a computational drug repositioning webtool. This analysis resulted in the identification of four drugs that reverse the expression pattern of all three MR consensus simultaneously, benperidol, harmaline, tubocurarine chloride, and vorinostat, thus suggested as novel potential PD therapeutic interventions.

Nicotinamide N-methyltransferase expression in SH-SY5Y neuroblastoma and N27 mesencephalic neurones induces changes in cell morphology via ephrin-B2 and Akt signalling.

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to produce 1-methylnicotinamide (MeN). We have previously shown that NNMT expression protected against neurotoxin-mediated cell death by increasing Complex I (CxI) activity, resulting in increased ATP synthesis. This was mediated via protection of the NDUFS3 subunit of CxI from degradation by increased MeN production. In the present study, we have investigated the effects of NNMT expression on neurone morphology and differentiation. Expression of NNMT in SH-SY5Y human neuroblastoma and N27 rat mesencephalic dopaminergic neurones increased neurite branching, synaptophysin expression and dopamine accumulation and release. siRNA gene silencing of ephrin B2 (EFNB2), and inhibition of Akt phosphorylation using LY294002, demonstrated that their sequential activation was responsible for the increases observed. Incubation of SH-SY5Y with increasing concentrations of MeN also increased neurite branching, suggesting that the effects of NNMT may be mediated by MeN. NNMT had no significant effect on the expression of phenotypic and post-mitotic markers, suggesting that NNMT is not involved in determining phenotypic fate or differentiation status. These results demonstrate that NNMT expression regulates neurone morphology in vitro via the sequential activation of the EFNB2 and Akt cellular signalling pathways.

Protein-protein interactions in the assembly and subcellular trafficking of the BACE (beta-site amyloid precursor protein-cleaving enzyme) complex of Alzheimer's disease.

The correct assembly of the BACE (beta-site amyloid precursor protein-cleaving enzyme or beta-secretase) complex and its subsequent trafficking to cellular compartments where it associates with the APP (amyloid precursor protein) is essential for the production of Abeta (amyloid beta-peptide), the protein whose aggregation into senile plaques is thought to be responsible for the pathogenesis of AD (Alzheimer's disease). These processes rely upon both transient and permanent BACE-protein interactions. This review will discuss what is currently known about these BACE-protein interactions and how they may reveal novel therapeutic targets for the treatment of AD.

Regulation of the lipidation of beta-secretase by statins.

Statins inhibit the dimerization of beta-secretase [BACE (beta-site amyloid precursor protein-cleaving enzyme)] by inhibiting the lipidation of BACE and associated proteins. Our studies have demonstrated a clearly defined temporal sequence for these reactions in the assembly of the BACE complex, which may provide targets for the treatment of Alzheimer's disease.

Protein lipidation of BACE.

Our research has concentrated upon the protein lipid modification of BACE [beta-site amyloid precursor protein cleaving enzyme (beta-secretase)], of which very little is currently known. Lipidation influences the production of Abeta (amyloid beta-protein) by promoting the dimerization of BACE.

Expression of cysteine dioxygenase (EC 1.13.11.20) and sulfite oxidase in the human lung: a potential role for sulfate production in the protection from airborne xenobiotica.

AIMS: The lung is one of the major sites of phase I cytochrome P450 enzyme and phase II sulfotransferase expression, which together are thought to act as an enzymic barrier against the unimpeded transfer of airborne xenobiotics into the lung parenchyma and systemic circulation. Sulfate for conjugation is produced primarily from the oxidation of cysteine, begun by cysteine dioxygenase (CDO), and completed with the conversion of sulfite to sulfate via sulfite oxidase (SO). Little is known about the site of expression of these two enzymes in the alveoli of the human lung. METHODS: Antibodies and oligonucleotide probes raised against both CDO and SO were used for immunohistochemistry and in situ hybridisation, respectively, to investigate the expression of CDO and SO in human lung alveoli. RESULTS: CDO and SO were expressed in alveolar epithelial cells, which is also the site of expression of cytochrome P450 1B1. CONCLUSIONS: These results demonstrate that the two key enzymes in sulfate production are expressed in the same locale as phase I and phase II enzymes, and that these two enzymes may be involved in the production of sulfate for the maintenance of a metabolic barrier against the entry of airborne xenobiotics and the synthesis of important structural proteins and proteoglycans.

The aetiology of idiopathic Parkinson's disease.

Agents potentially involved in the aetiology of idiopathic Parkinson's disease are discussed. These include factors regulating dopaminergic neurogenesis (Nurr 1, Ptx-3, and Lmx1b) and related proteins, together with genes involved in familial Parkinson's disease (alpha synuclein, parkin, and ubiquitin carboxy terminal hydroxylase L1), and endogenous and environmental agents.

Renal localisation of rat cysteine dioxygenase.

BACKGROUND/AIMS: Cysteine dioxygenase (CDO, EC 1.13.11.20) catalyses the conversion of cysteine to cysteine sulphinic acid and controls the rate-limiting step of sulphate production. Many neurological and non-neurological diseases are associated with abnormalities in CDO activity, giving rise to reduced availability of sulphate. The importance of the kidney in the sulphation of xenobiotics has long been recognised, but little is known about the renal expression of key enzymes in this pathway. In order to address this, this report demonstrates the expression of CDO in the kidney. METHODS: Two previously characterised antibodies were used to investigate the localisation and expression of CDO using immunohistochemistry, in-situ hybridisation and Western blotting. RESULTS: Renal CDO was shown to exist as a 68-kDa protein, which was unaffected by levels of cysteine and methionine that had been previously shown to induce hepatic CDO. CDO protein expression was present in the proximal convoluted tubules of the cortex and the collecting ducts of both the medulla and papilla. DISCUSSION: These results suggest that renal CDO is immunologically identical to that of the liver. Its expression in the kidney tubules, the major site of sulphation in the kidney, suggests that CDO in the kidney may play a role in both xenobiotic metabolism and sodium and water homeostasis.

Cysteine dioxygenase: regional localisation of protein and mRNA in rat brain.

Cysteine dioxygenase (CDO) converts cysteine to cysteinesulphinic acid and is the rate-limiting step in sulphate production. Most studies have centred upon the hepatic form of the enzyme, but several studies have investigated brain CDO using activity assays and western blotting. The aim of this study was to investigate the expression of CDO in the rat brain using a combination of immunohistochemistry and in situ hybridisation. Affinity-purified anti-R and anti-H CDO antibodies were immunoprecipitated using rat brain homogenate to determine whether the antibodies could remove enzyme activity. Immunohistochemistry and in situ hybridisation were then used to determine the cellular and regional expression of both CDO protein and mRNA. Immunoprecipitation of rat brain homogenate removed up to 98% and 70% (anti-R and anti-H, respectively) of enzyme activity. Nonimmune sheep serum had no effect upon enzyme activity. CDO protein and mRNA was localised solely to the neurones of the brain, including the pyramidal cells of the hippocampus and the Purkinje cells of the cerebellum. Regional localisation varied, with high levels of expression in the hippocampus, the dentate gyrus, the outer cortices of the brain, and the substantia nigra. The relative expression of CDO activity and protein in these regions is most probably a result of the relative abundance of neurones in these regions. CDO expression in the brain may have several possibilities functions, the most likely being the prevention of free radical production by the autoxidation of cysteine and dopamine.

Development of an in vitro model for cysteine dioxygenase expression in the brain.

The development of an in vitro model for cysteine dioxygenase (CDO) expression in the brain would provide a useful model for determining the mechanisms for the regulation of CDO expression that does not involve the use of animals. Here we demonstrate the screening and characterization of a cell line that expresses CDO, the primary metabolizing enzyme of cysteine and the regulatory point of sulfate production. A panel of four commercially available tumor-derived human brain cell lines, each representing one major class of brain cell, were screened using western blotting and activity assay for cysteine dioxygenase expression. One cell line, TE 671 (human medulloblastoma) was found to express both a protein of approximately 70 kDa and CDO activity. Nuclease protection assay (NPA) of mRNA isolated from TE 671 showed the expression of a CDO mRNA. Reverse transcription-polymerase chain reaction of this mRNA and sequencing of the cDNA obtained showed that this was indeed CDO. Treatment of TE 671 cells with cysteine resulted in the upregulation of CDO mRNA, whereas treatment with tumor necrosis factor alpha resulted in the downregulation of CDO mRNA, as evidenced using NPA. The characterization of an in vitro model for CDO expression provides a useful tool for the investigation of this important enzyme, which may have an etiological role in the pathogenesis of Parkinson's disease.

Human brain cytochrome P450 1B1: immunohistochemical localization in human temporal lobe and induction by dimethylbenz(a)anthracene in astrocytoma cell line (MOG-G-CCM).

CYP1B1, a new member of human cytochrome P450 family 1, is involved in the xenobiotic detoxification metabolism and possibly activation of numerous procarcinogens and promutagens. Localization of CYP1B1 in human temporal lobe and its induction in astrocytoma cell line (MOG-G-CCM) by 7,12-dimethylbenz(a)anthracene (DMBA) was investigated using antibodies against human CYP1B1. A single band of approximately 58 kDa size in both human temporal lobe and in MOG-G-CCM was detected by Western blot analysis. Treatment of MOG-G-CCM cells with DMBA resulted in approximately 2.8-fold induction of CYP1B1. CYP1B1 immunoreactivity was detected at the blood-brain interface areas of the temporal lobe as evidenced by co-localization with CD34 antigen. These results suggest that this enzyme may be important in brain xenobiotic metabolism acting as an enzymatic barrier.

Midterm results of endovascular stented grafts for the treatment of isolated iliac artery aneurysms.

PURPOSE: Isolated aneurysms of the iliac arteries are uncommon lesions that require surgical repair to prevent rupture. METHODS: During a 4-year period, we used endovascular stented grafts (EGs) to treat 28 iliac artery aneurysms that were not associated with aortic aneurysms. Twenty-five patients, with a total of 24 common iliac (15 right, nine left) and four internal iliac (two right, two left) artery aneurysms, underwent endovascular grafting. There were 24 men and 1 woman, with a mean age of 74 years (range, 51 to 88 years). Combined common and internal iliac artery aneurysms were present in three patients. Nineteen patients who underwent treatment with EGs were administered epidural anesthesia (22 epidural, two local, one general). Before surgery, one patient had lower extremity embolization and ischemia from the aneurysm, three had abdominal or back pain, and the remaining were asymptomatic. The EGs were constructed of polytetrafluoroethylene grafts and balloon expandable stents. RESULTS: Four procedure-related complications (12%) occurred (distal extremity embolization, n = 1; wound complications, n = 2; colonic mucosal ischemia, n = 1). Only a minimal reduction in the aneurysmal diameter was seen in 90% of the iliac artery aneurysms treated. The remaining lesions showed no change in size, and no aneurysm had an increase in cross-sectional diameter on computed tomographic images enduring a follow-up period up to 4 years (mean, 24 months). One aneurysm ruptured after successful endovascular exclusion, and the patient underwent treatment with open repair. The 3-year primary patency rate of iliac EGs was 86%. CONCLUSION: EGs appear to show satisfactory safety and efficacy for the repair of isolated aneurysms of the iliac arteries.

Tissue harmonic imaging sonography: evaluation of image quality compared with conventional sonography.

OBJECTIVE: The purpose of this study was to determine if tissue harmonic imaging (THI) sonography produced higher quality images than did conventional sonography. SUBJECTS AND METHODS: A prospective study was performed on 89 patients to compare the image quality of THI sonography with that of conventional sonography. Each examination was performed using THI sonography (transmitted frequency, 2.0 MHz; received frequency, 4.0 MHz) and conventional sonography at 2.5 and 4.0 MHz. The pancreatic area was studied in 60 patients, and other anatomic areas were studied in 68 patients. The images were then graded for penetration, detail, and total image quality. Graders were unaware of the sonographic technique. RESULTS: Of the 60 pancreatic examinations, THI sonography was the best technique for penetration in 45, detail in 54, and total image quality in 50. For the pancreas, THI sonography was significantly better than 2.5-MHz conventional sonography for penetration (p = .0002), detail (p < .0001), and total image quality (p < .0001). THI sonography was significantly better than 4.0-MHz conventional sonography for penetration (p < .0001), detail (p < .0001), and total image quality (p < .0001). Of the 68 examinations of other anatomic areas, THI sonography was the best technique for penetration in 42, detail in 57, and total image quality in 58. For other anatomic areas, THI sonography was significantly better than 2.5-MHz conventional sonography for penetration (p = .05), detail (p < .0001), and total image quality (p < .0001). THI sonography was significantly better than 4.0-MHz conventional sonography for penetration (p < .0001), detail (p < .0001), and total image quality (p < .0001). CONCLUSION: The THI technique improved sonographic image quality.

Hepatic localisation of rat cysteine dioxygenase.

BACKGROUND/AIM: Cysteine dioxygenase (CDO, E.C. 1.13.11.20) is the main catabolic enzyme of cysteine, metabolising cysteine to cysteinesulphinic acid. CDO abnormality has been implicated in a number of neurological and non-neurological diseases, with CDO deficiency possibly leading to excitotoxic damage to the brain and impaired Phase II metabolism in the liver. METHODS: Two novel anti-CDO antibodies raised against linear synthetic peptides corresponding to two distinct epitopes on the 22 kDa gene product of the CDO-I gene were used for immunohistochemistry and Western blotting. These antibodies were characterised by their ability to both block and precipitate CDO enzyme activity as well as the ability of the respective antigenic peptides to absorb the antibodies and prevent the immunodetection of CDO. RESULTS: The antibodies were found to detect the presence of a 68 kDa protein, which was subsequently shown to be CDO. Distribution was found to be centrilobular and did not alter when CDO was induced with cysteine or methionine; however, the intensity of staining increased, indicating an increase in the levels of CDO in that region. CONCLUSIONS: These results suggest that the 68 kDa Type II is the predominant isoform in vitro and in vivo and that its centrilobular localisation may allow CDO to initiate the production of sulphate and taurine for Phase II conjugation in the liver.

In vitro effect of the cysteine metabolites homocysteic acid, homocysteine and cysteic acid upon human neuronal cell lines.

Cysteine (CYS) is a non-essential amino acid which elicits excitotoxic properties via the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor. CYS levels are known to be elevated in association with neurological disease such as Alzheimers Disease (AD) and Parkinsons Disease (PD). We have previously reported studies investigating the toxicity of CYS and its major metabolite cysteinesulfinic acid (CSA) to human neuronal cell lines in vitro and in continuation of this we now report the toxicity of other compounds (Homocysteic Acid, HCA; Homocysteine, HCYS; and Cysteic Acid, CA) in the CYS metabolic pathway. Three cell lines, all of human origin and derived from separate discrete areas of the brain were used in the neurotoxicity assays. Lactate dehydrogenase (LDH) release was assayed as a measure of cell death. The cell lines investigated showed varying degrees of toxic responses which were the reverse of those seen when they were exposed to CYS or CSA. The SK.N.SH (Neuroblastoma) cell line, which exhibits a high toxic response to CYS and CSA, gave a low toxic response to HCA and CA while the TE 671 (Medulloblastoma) cell line, which exhibits a low toxic response to CYS and CSA, showed a high toxic response to HCYS, HCA and CA. However, the U-87 MG (Glioblastoma) cell line, which has a median toxic response to CYS and CSA, also has median response to HCYS, HCA and CA. These results show that toxic responses are cell-type specific for CYS and its metabolites and this may be reflected in the patterns of neurodegeneration observed in such diseases as AD and PD. HCYS is selectively toxic to medulloblastoma cells; this may explain why high HCYS levels result in neural tube defects in prenatal humans, where the same cell-type is involved.

Torsion of the fallopian tube: progression of sonographic features.

Isolated torsion of the fallopian tube is a rare gynecologic condition that is difficult to diagnose preoperatively. We present the sonographic and CT findings over a 48-hour period in a case of isolated torsion of the fallopian tube. The radiologic features of isolated torsion have been described previously; however, to our knowledge, the progressive findings have not been previously reported.

Cysteine dioxygenase: regional expression of activity in rat brain.

The levels of expression of cysteine dioxygenase (CDO) protein and activity were investigated in nine functionally distinct regions of the rat brain before and after induction with methionine by Western analysis and an activity assay. Activity expression ranged from no activity in the brain-stem to high activity expression in the olfactory bulb and basal ganglia. Upon exposure to 400 mg/l methionine for 5 days, significant induction of expression was observed in the basal ganglia, brain-stem, cerebellum, hippocampus, midbrain and olfactory bulb. Protein expression appeared to correlate with activity expression when levels before and after induction were compared. This non-uniformity of expression may reflect different physiological functions of CDO in these areas.

MR imaging in male infertility.

In patients with male infertility, endorectal magnetic resonance (MR) imaging provides high-resolution images of the prostate gland and ejaculatory apparatus. The multiplanar capability of MR imaging allows production of a detailed map of the reproductive tract for guiding treatment. Causes of male infertility can be classified as congenital, acquired, infectious, or hormonal. Wolffian duct abnormalities include agenesis of the kidney, vas deferens, or seminal vesicle and cysts of the vas deferens, seminal vesicle, or urogenital sinus-ejaculatory duct. Müllerian duct abnormalities are less common and consist of müllerian duct cysts and utricle cysts. Cowper duct cysts and peripheral-zone prostatic cysts are acquired causes of male infertility. Prostatitis, an infectious cause of male infertility, may mimic carcinoma on long repetition time/echo time images. A low testosterone levels is one of the hormonal causes of male infertility. Pitfalls in the interpretation of MR images can be avoided by familiarity with normal and abnormal findings in patients with male infertility.