High-throughput mutational analysis of TOR1A in primary dystonia

<p>Abstract</p> <p>Background</p> <p>Although the c.904_906delGAG mutation in Exon 5 of <it>TOR1A </it>typically manifests as early-onset generalized dystonia, DYT1 dystonia is genetically and clinically heterogeneous. Recently, another Exon 5 mutation (c.863G>A) has been associated with early-onset generalized dystonia and some ΔGAG mutation carriers present with late-onset focal dystonia. The aim of this study was to identify <it>TOR1A </it>Exon 5 mutations in a large cohort of subjects with mainly non-generalized primary dystonia.</p> <p>Methods</p> <p>High resolution melting (HRM) was used to examine the entire <it>TOR1A </it>Exon 5 coding sequence in 1014 subjects with primary dystonia (422 spasmodic dysphonia, 285 cervical dystonia, 67 blepharospasm, 41 writer's cramp, 16 oromandibular dystonia, 38 other primary focal dystonia, 112 segmental dystonia, 16 multifocal dystonia, and 17 generalized dystonia) and 250 controls (150 neurologically normal and 100 with other movement disorders). Diagnostic sensitivity and specificity were evaluated in an additional 8 subjects with known ΔGAG DYT1 dystonia and 88 subjects with ΔGAG-negative dystonia.</p> <p>Results</p> <p>HRM of <it>TOR1A </it>Exon 5 showed high (100%) diagnostic sensitivity and specificity. HRM was rapid and economical. HRM reliably differentiated the <it>TOR1A </it>ΔGAG and c.863G>A mutations. Melting curves were normal in 250/250 controls and 1012/1014 subjects with primary dystonia. The two subjects with shifted melting curves were found to harbor the classic ΔGAG deletion: 1) a non-Jewish Caucasian female with childhood-onset multifocal dystonia and 2) an Ashkenazi Jewish female with adolescent-onset spasmodic dysphonia.</p> <p>Conclusion</p> <p>First, HRM is an inexpensive, diagnostically sensitive and specific, high-throughput method for mutation discovery. Second, Exon 5 mutations in <it>TOR1A </it>are rarely associated with non-generalized primary dystonia.</p

Neurosteroidogenesis in oligodendrocytes and Purkinje neurones of cerebellar cortex of dogs

/0000-0002-0636-4214WOS: 000221913400006PubMed: 15144282The cerebellum is a steroidogenic organ that expresses steroidogenic enzymes and produces neurosteroids. Purkinje neurones appear to be the most active steroidogenic cells in the cerebellar cortex. These neurones express 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 side-chain cleavage (P450scc), 17alpha-hydroxylase/c17, 201yase (P450c17), P450 aromatase (P450arom) and produce pregnenolone, progesterone, dehydroepiandrosterone, androstenedion, oestradion and oestrone. Oligodendrocytes are predominantly the producer of myeline protein. The oligodendrocytes were identified by immunohistochemistry using a monoclonal antibody against myeline 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a myeline specific enzyme. In this study we have examined the distribution of 3beta-HSD and CNPase by immunohistochemistry using monoclonal antibody in canine cerebellar cortex. The localization of oligodendrocytes within the cerebellar cortex was determined to be close to Purkinje neurones. This result suggests that endogenous progesterone synthesized de novo in the Purkinje neurone can promote myeline protein synthesis in oligodentrocytes

Distribution of 3 beta-hydroxysteroid dehydrogenase in the cerebellum in canine distemper virus infection

/0000-0002-0636-4214WOS: 000179500000008PubMed: 12443737The cerebella of eight dogs naturally infected with canine distemper virus (CDV) and two normal dogs were examined immunohistochemically for glial fibrillary acidic protein (GFAP) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). The clinical diagnosis of canine distemper was Confirmed histopathologically and by the immunohistochemical demonstration of CDV antigen. In all dogs (healthy and infected), the Purkinje cells of the cerebellum were immunolabelled for 3beta-HSD activity. In infected dogs, 3beta-HSD labelling was prominent in astrocytes (particularly in areas of astrocytosis) whereas in healthy dogs such immunolabelling was weak. Double immunolabelling demonstrated that all GFAP-positive cells (especially in demyelinating areas) were also positive for 3beta-HSD. The results suggest that 3beta-HSD expression by astrocytes is associated with demyelination in CDV infection. (C) 2002 Elsevier Science Ltd. All rights reserved

The expression of CD14 antigen in experimental encephalitic listeriosis in rabbits

/0000-0002-0636-4214WOS: 000221704400004The present study examined the distribution of Listeria monocytogenes 1/2a (L.m. 1/2a) and the antigen CD14 by using immunohistochemical methods in the brain of healthy and experimentally infected rabbits with L.m. 1/2a. The ten New Zealand White rabbits were inoculated by intracerebral route, and 4 rabbits were used for control. Only the brains of the animals were removed during necropsy, and they were analysed by conventional histological techniques. By Haematoxylin and Eosin staining all L.m. 1/2a inoculated rabbits presented meningitis or meningoencephalitis. In healthy rabbits, the immunohistochemical staining of brains for CD14 was very slight and seen only in the lumen of the vessels, whereas, in infected rabbits the CD14 immunoreactivity was more prominent especially in inflammation areas of parenchyma and meninges. Moreover, the CD14 positive immunostainings were seen massively in L.m. positive inflamed areas. The present study provides the immunohistochemical expression of CD14 and L.monocytogenes antigen in the brain and a positive correlation was obtained between CD14 and this infectious agent

Immunohistochemical investigation of CD14 in experimental rabbit pneumonic pasteurellosis

/0000-0002-0636-4214WOS: 000227165300003The present study investigated the immunolocalization of CD 14 and the bacterial antigen of Pasteurella multocida serotype A :3 in normal and pneumonic rabbit lungs. To induce pneumonia, P. multocida serotype A :3 (4 x 10(6) CFU/ml) was inoculated intra-tracheally to twelve New Zealand White rabbits (Oryctolagus cuniculus) at age of 8-10 weeks while four rabbits received physiologic saline with the same route. Nine infected rabbits died with clinical sings of the disease between the 3(rd) and 13(th) days post inoculation. On day 14, the remaining three infected and control rabbits were euthanatized. The histopathological evaluation of the lung samples collected from nine infected rabbits with clinical symptoms revealed severe cases of fibrinopurulent and necrotic pneumonia and fibrinopurulent pleuritis. The bacterial antigen was immunolocalized in macrophages and degenerating leukocytes around the necrotic tissue and in exudates occupying the lumens of bronchia, bronchioles, and alveoli. CD14 immunoreactivity was present in alveolar macrophages, neutrophiles, alveolar epithelial cell surfaces, and lumens of the capillaries of the pneumonic lungs. In three rabbits, inoculated but presenting no clinical symptoms and no histopathological lesions, the bacterial antigen was found in the alveolar wall; however, like in the controls, CD14 immunoreactivity was present only in the lumen of capillaries. Double labelling immunohistochemistry indicated that CD14 was closely associated with the bacterial antigen in inflamed regions of the lung. Results suggested that interaction of the bacterial antigens and CD14 may have a critical role in lung pathologies resulting from P. multocida serotype A :3 infection

Immunolocalization of 3 beta-hydroxysteroid dehydrogenase in normal and hyperplastic ram prostates

/0000-0002-0636-4214; ATMACA, HASAN TARIK/0000-0001-8379-4114WOS: 000233827400002PubMed: 16009527The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) is essential in the synthesis of all steroids by cleaving dehydroepiandrosterone to androstenedione. In the present study, 3 beta-HSD immunoreactivity was investigated in the prostate of Akkaraman breed rams aged older than 3 years. Five normal and five hyperplastic ram prostates were processed for immunohistochernistry. Prostate hyperplasia was determined by histopathological evaluation of 375 ram prostate and confirmed with significantly (P 0.05), suggesting that locally produced steroids have little or no effect onthe pathogenesis of the ram prostate hyperplasia which affects a very small proportion of the ram population (5 out of 375). (c) 2005 Elsevier Inc. All rights reserved

Immunohistochemical localisation of 3 beta-hydroxysteroid dehydrogenase in Sarcocystis spp.

YILDIZ, Kader/0000-0001-5802-6156; /0000-0002-0636-4214WOS: 000223295000005PubMed: 152437983beta-Hydroxysteroid-dehydrogenase (3beta-HSD) is an isoenzyme that catalyses an essential step in the synthesis of all classes of active steroid hormones. The presence of steroid hormones of the vertebrate type in invertebrates is acknowledged in addition to a group of steroid-like hormones called ecdysteroids that were present in arthropods and helminths. In the present study, 3beta-HSD was detected in the bradyzoites enclosed in sarcocysts of Sarcocystis spp. with immunohistochemistry. The results suggest that self-originating steroid hormones may play important roles in the development of Sarcocystis spp., and possibly in the regulation of the reciprocal immune interaction between the host and these parasites

Polioencephalomalacia in cattle: A consequence of prolonged feeding barley malt sprouts

WOS: 000235893300003PubMed: 16533327Polioencephalomalacia (PEM) in ruminants has been recognized as a consequence of excess sulphur intake. The present study describes clinical, gross and histopathological findings of PEM following an abrupt change of diet in two ranches housing 2750 dairy and 2300 beef cattle. As a result of severe PEM, 256 cattle died or were slaughtered. Clinical findings included circling, hypersensitivity, excessive salivation, hypermetria, incoordination, blindness and death. The first clinical signs occurred in beef calves (6-8 months old) at a holding facility. Clinical signs of the disorder continued intermittently during the 5-month period in both ranches and were more evident in calves and lactating dairy cows. The affected cattle did not respond to thiamine injections. Clinical signs disappeared gradually following removal of barley malt sprouts from the diet. Although macroscopic lesions were not apparent in the brain tissues of some animals, histopathology typical of PEM was found in most cases: spongiosis in the neuropil and neuronal necrosis, haemorrhage, capillary hyperplasia, fibrinoid degeneration in arterioles, multifocal liquefaction necroses in the grey matter and abundance of gitter cells with vacuolar large cytoplasm. Sulphide in rumen fluid of a clinically affected animal was measured as 1.55 mg/dl, which is considerably higher than that collected from two control cows (mean 0.21 mg/dl). The total sulphur content of the diet containing barley malt sprouts was estimated to be 0.45%, which is also higher than the National Research Council (NRC) maximum tolerable levels. In conclusion, PEM can result from excess barley malt sprout intake because of its higher sulphur content. Clinical signs may occur shortly after the intake of barley malt sprout as outbreaks with a higher number of deaths or as an ongoing periodic condition