Mineralization of the deep gray matter with age: a retrospective review with susceptibility-weighted MR imaging.

BACKGROUND AND PURPOSE: Susceptibility-weighted imaging (SWI) is an advanced MR imaging sequence that can be implemented at high resolution. This sequence can be performed on conventional MR imaging scanners and is very sensitive to mineralization. The purpose of this study was to establish the course of mineralization in the deep gray matter with age by using SWI. MATERIALS AND METHODS: We retrospectively reviewed susceptibility-weighted images of 134 patients (age range, 1 to 88 years). Inclusion criteria comprised a normal conventional MR imaging (T1, T2, and fluid-attenuated inversion recovery sequences). We statistically analyzed the relative signal intensities of the globus pallidus, putamen, substantia nigra, caudate nucleus, red nucleus, and thalamus for correlation with age. The putamen was graded according to a modified scale, based on previous work that described a systematic pattern of mineralization with age. Bands of hypointensity in the globus pallidus, dubbed "waves," were also evaluated. RESULTS: We documented decreasing intensity (ie, increasing mineralization) with age in all deep gray matter areas analyzed. We confirmed the age-related posterolateral to anteromedial progression of mineralization in the putamen. Characteristic medial and lateral bands of mineralization were exhibited in the globus pallidus in all children and young adults older than 3 years. Finally, an increase in the number of "waves" present in the globus pallidus was associated with increased age by category. CONCLUSION: This study documents the course and pattern of mineralization in the deep gray matter with age, as determined by SWI. These findings may play a role in evaluating diseased brains in the future.

Clinical, molecular, and PET study of a case of aceruloplasminaemia presenting with focal cranial dyskinesia.

Aceruloplasminaemia is a rare recessive disorder caused by mutations in the gene encoding the multicopper ferroxidase ceruloplasmin, thought to be involved in cellular iron export. Primary intracellular iron accumulation characterises this disorder. We investigated a case of aceruloplasminaemia early in the course of the disease by structural and functional neuroimaging and correlated the results with the clinical findings. The patient, a diabetic 59 year old lady, presented with perioral dyskinesia. Magnetic resonance imaging (MRI) revealed massive iron accumulation in the basal ganglia, notably sparing the pallidum, and along the cortical surface. However, most of these structures had preserved metabolic activity as evaluated by fluorodeoxyglucose positron emission tomography (FDG-PET). Voxel based analysis of FDG-PET data showed a significant hypometabolism only in the heads of the caudate nuclei. Molecular genetic analysis revealed compound heterozygosity for two null mutations in the ceruloplasmin gene, a rather surprising finding for a very rare recessive disease, suggesting that aceruloplasminaemia could be somewhat more frequent than is commonly thought and could therefore be underdiagnosed.

Identification of two distinct vasodilator pathways activated by ATP in the mesenteric bed of the rat.

Adenosine 5'-triphosphate (ATP) has important roles in the cardiovascular system, modulating vascular tone by acting as both a vasoconstrictor and a vasodilator. The dilator function of ATP is traditionally thought to be monophasic and mediated primarily by nitric oxide (NO). Here we have identified the endothelium-dependent biphasic nature of ATP-induced vasodilatation of the rat isolated mesenteric bed and investigated the two distinct pathways involved. ATP, at doses of 1x10(-11) to 1x10(-8) moles, induced transient relaxations that were inhibited by the NO synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME: 1x10(-4) M), the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ: 3x10(-6) M) and KCl (6x10(-2) - 1.2x10(-1) M). At doses upwards of 1x10(-8) moles (1x10(-8) - 3x10(-7) moles), ATP also induced prolonged vasodilatations which were unaltered by L-NAME, L-NAME (1x10(-3) M) and indomethacin (1x10(-5) M), or by ODQ, but were abolished in the presence of KCl. In addition, the cannabinoid CB(1) receptor antagonist SR141716A (1x10(-5) M) was found to inhibit the second prolonged phase of vasodilatation. However, at the concentration used SR141716A is reported to be non-selective. A second CB(1) receptor antagonist, AM251 (1x10(-6) M), had a small but significant inhibitory effect on the second phase of ATP-induced vasodilatation. SR141716A, AM251 and KCl (6x10(-2) - 1.2x10(-1) M) all inhibited anandamide-induced relaxation of the isolated mesenteric bed. These observations demonstrate that ATP stimulates vasodilatation of the mesenteric bed by two distinct mechanisms involving the release of NO and an EDHF. In the absence of better pharmacological tools we can only speculate as to the involvement of an endogenous CB(1) receptor ligand in these responses.

Antiproliferative B cell translocation gene 2 protein is down-regulated post-transcriptionally as an early event in prostate carcinogenesis.

B cell translocation gene 2 (BTG2) is a p53 target that negatively regulates cell cycle progression in response to DNA damage and other stress. The objective of this study was to examine the expression, regulation and tumor suppressor properties of BTG2 in prostate cells. By immunohistochemistry BTG2 protein was detected in approximately 50% of basal cells in benign glands from the peripheral zone of the human prostate. BTG2 was expressed in all hyperproliferative atrophic peripheral zone lesions examined (simple atrophy, post-atrophic hyperplasia and proliferative inflammatory atrophy), but was undetectable or detectable at very low levels in the hyperproliferative epithelial cells of HGPIN and prostate cancer. BTG2 mRNA was detected in non-malignant prostate epithelial (PE) cells and in LNCaP cells, but not in PC-3 cells, consistent with p53-dependent regulation. In PE cells BTG2 protein was detected in areas of cell confluence by immunohistochemistry. BTG2 protein in LNCaP cells was undetectable by immunohistochemistry but was detected by immunoblotting at 8- to 9-fold lower levels than in PE cells. BTG2 protein levels were shown to be regulated by the ubiquitin-proteosome system. Forced expression of BTG2 in PC-3 cells was accompanied by a decreased rate of cell proliferation and decreased tumorigenicity of these cells in vivo. Taken together, these findings suggest that BTG2 functions as a tumor suppressor in prostate cells that is activated by cell quiescence, cell growth stimuli as part of a positive feedback mechanism and in response to DNA damage or other cell stress. The low steady-state levels of BTG2 protein in HGPIN and prostate cancer, a potential consequence of increased proteosomal degradation, may have important implications in the initiation and progression of malignant prostate lesions. Furthermore, these findings suggest that a significant component of the p53 G(1) arrest pathway might be inactivated in prostate cancer even in the absence of genetic mutations in p53.

The metallochaperone Atox1 plays a critical role in perinatal copper homeostasis.

Copper plays a fundamental role in the biochemistry of all aerobic organisms. The delivery of this metal to specific intracellular targets is mediated by metallochaperones. To elucidate the role of the metallochaperone Atox1, we analyzed mice with a disruption of the Atox1 locus. Atox1(-/-) mice failed to thrive immediately after birth, with 45% of pups dying before weaning. Surviving animals exhibited growth failure, skin laxity, hypopigmentation, and seizures because of perinatal copper deficiency. Maternal Atox1 deficiency markedly increased the severity of Atox1(-/-) phenotype, resulting in increased perinatal mortality as well as severe growth retardation and congenital malformations among surviving Atox1(-/-) progeny. Furthermore, Atox1-deficient cells accumulated high levels of intracellular copper, and metabolic studies indicated that this defect was because of impaired cellular copper efflux. Taken together, these data reveal a direct role for Atox1 in trafficking of intracellular copper to the secretory pathway of mammalian cells and demonstrate that this metallochaperone plays a critical role in perinatal copper homeostasis.

The neuronal adaptor protein X11alpha interacts with the copper chaperone for SOD1 and regulates SOD1 activity.

The neuronal adaptor protein X11alpha participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human X11alpha, and we isolated a clone encoding domains II and III of the copper chaperone for Cu,Zn-superoxide dismutase-1 (CCS). The X11alpha/CCS interaction was confirmed in coimmunoprecipitation studies plus glutathione S-transferase fusion protein pull-down assays and was shown to be mediated via PDZ2 of X11alpha and a sequence within the carboxyl terminus of domain III of CCS. CCS delivers the copper cofactor to the antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for its activity. Overexpression of X11alpha inhibited SOD1 activity in transfected Chinese hamster ovary cells which suggests that X11alpha binding to CCS is inhibitory to SOD1 activation. X11alpha also interacts with another copper-binding protein found in neurons, the Alzheimer's disease amyloid precursor protein. Thus, X11alpha may participate in copper homeostasis within neurons.

Wilson's disease.

Wilson's disease is an autosomal recessive disorder of copper metabolism resulting from the absence or dysfunction of a copper transporting P-type ATPase encoded on chromosome 13. This ATPase is expressed in hepatocytes where it is localized to the trans-Golgi network and transports copper into the secretory pathway for incorporation into ceruloplasmin and excretion into the bile. Under physiologic circumstances, biliary excretion represents the sole mechanism for copper excretion, and thus affected individuals have progressive copper accumulation in the liver. When the capacity for hepatic storage is exceeded, cell death ensues with copper release into the plasma, hemolysis, and tissue deposition. Presentation in childhood may include chronic hepatitis, asymptomatic cirrhosis, or acute liver failure. In young adults, neuropsychiatric symptoms predominate and include dystonia, tremor, personality changes, and cognitive impairments secondary to copper accumulation in the central nervous system. The laboratory diagnosis of Wilson's disease is confirmed by decreased serum ceruloplasmin, increased urinary copper content, and elevated hepatic copper concentration. Molecular genetic analysis is complex as more than 100 unique mutations have been identified and most individuals are compound heterozygotes. Copper chelation with penicillamine is an effective therapy in most patients and hepatic transplantation is curative in individuals presenting with irreversible liver failure. Elucidation of the molecular genetic basis of Wilson's disease has permitted new insights into the mechanisms of cellular copper homeostasis.

Hepatic iron overload in aceruloplasminaemia.

We report the case of a 52 year old male with diabetes mellitus and long standing evidence of hepatic iron excess. Initially considered to have haemochromatosis, this patient was reevaluated when hepatic iron stores were found to be unaffected by a prolonged course of weekly phlebotomy. The development of neurological disease prompted diagnostic consideration of aceruloplasminaemia, which we confirmed by demonstration of a novel frameshift mutation in the ceruloplasmin gene. Our inability to resolve the patient's iron overload by regular phlebotomy is consistent with recent animal studies indicating an essential role for ceruloplasmin in cellular iron efflux. Evaluation of this case underscores the clinical relevance of aceruloplasminaemia in the differential diagnosis of hepatic iron overload and provides insight into the pathogenetic mechanisms of hepatocellular iron storage and efflux.

Interpretation of Emergency Department radiographs: a comparison of emergency medicine physicians with radiologists, residents with faculty, and film with digital display.

OBJECTIVE: We determined the relative value of teleradiology and radiology resident coverage of the emergency department by measuring and comparing the effects of physician specialty, training level, and image display method on accuracy of radiograph interpretation. MATERIALS AND METHODS: A sample of four faculty emergency medicine physicians, four emergency medicine residents, four faculty radiologists, and four radiology residents participated in our study. Each physician interpreted 120 radiographs, approximately half containing a clinically important index finding. Radiographs were interpreted using the original films and high-resolution digital monitors. Accuracy of radiograph interpretation was measured as the area under the physicians' receiver operating characteristic (ROC) curves. RESULTS: The area under the ROC curve was 0.15 (95% confidence interval [CI], 0.10-0.20) greater for radiologists than for emergency medicine physicians, 0.07 (95% CI, 0.02-0.12) greater for faculty than for residents, and 0.07 (95% CI, 0.02-0.12) greater for films than for video monitors. Using these results, we estimated that teleradiology coverage by faculty radiologists would add 0.09 (95% CI, 0.03-0.15) to the area under the ROC curve for radiograph interpretation by emergency medicine faculty alone, and radiology resident coverage would add 0.08 (95% CI, 0.02-0.14) to this area. CONCLUSION: We observed significant differences between the interpretation of radiographs on film and on digital monitors. However, we observed differences of equal or greater magnitude associated with the training level and physician specialty of each observer. In evaluating teleradiology services, observer characteristics must be considered in addition to the quality of image display.

Copper chaperone for superoxide dismutase is essential to activate mammalian Cu/Zn superoxide dismutase.

Recent studies in Saccharomyces cerevisiae suggest that the delivery of copper to Cu/Zn superoxide dismutase (SOD1) is mediated by a cytosolic protein termed the copper chaperone for superoxide dismutase (CCS). To determine the role of CCS in mammalian copper homeostasis, we generated mice with targeted disruption of CCS alleles (CCS(-/-) mice). Although CCS(-/-) mice are viable and possess normal levels of SOD1 protein, they reveal marked reductions in SOD1 activity when compared with control littermates. Metabolic labeling with (64)Cu demonstrated that the reduction of SOD1 activity in CCS(-/-) mice is the direct result of impaired Cu incorporation into SOD1 and that this effect was specific because no abnormalities were observed in Cu uptake, distribution, or incorporation into other cuproenzymes. Consistent with this loss of SOD1 activity, CCS(-/-) mice showed increased sensitivity to paraquat and reduced female fertility, phenotypes that are characteristic of SOD1-deficient mice. These results demonstrate the essential role of any mammalian copper chaperone and have important implications for the development of novel therapeutic strategies in familial amyotrophic lateral sclerosis.

Brain copper content and cuproenzyme activity do not vary with prion protein expression level.

Prion diseases are neurodegenerative disorders that result from conformational transformation of a normal cell surface glycoprotein, PrP(C), into a pathogenic isoform, PrP(Sc). Although the normal physiological function of PrP(C) has remained enigmatic, the recent observation that the protein binds copper ions with micromolar affinity suggests a possible role in brain copper metabolism. In this study, we have used mice that express 0, 1, and 10 times the normal level of PrP to assess the effect of PrP expression level on the amount of brain copper and on the properties of two brain cuproenzymes. Using mass spectrometry, we find that the amount of ionic copper in subcellular fractions from brain is similar in all three lines of mice. In addition, the enzymatic activities of Cu-Zn superoxide dismutase and cytochrome c oxidase in brain extracts are similar in these groups of animals, as is the incorporation of (64)Cu into Cu-Zn superoxide dismutase both in cultured cerebellar neurons and in vivo. Our results differ from those of another set of published studies, and they require a re-evaluation of the role of PrP(C) in copper metabolism.

Structure, expression, and chromosomal localization of the mouse Atox1 gene.

Copper trafficking in eukaryotes involves small proteins termed metallochaperones, which mediate copper delivery to specific intracellular sites. Previous studies in yeast and human cell lines have suggested that Atox1 plays a critical role in copper delivery to the secretory pathway. In the present study, a mouse Atox1 (mAtox1) cDNA was cloned and shown to encode an open reading frame with 85% amino acid identity to human Atox1. RNA blot analysis revealed that mAtox1 was expressed as a single transcript in multiple tissues, and immunoblotting indicated that the relative abundance of mAtox1 mRNA directly correlated with mAtox1 protein. Analysis of the mAtox1 gene locus revealed a genomic structure with four exons encompassing a total of 14.5 kb. RFLP and haplotype analyses indicated that the mAtox1 locus was tightly linked to the Trhr and D15Bir7 loci on mouse chromosome 15. Taken together, these data reveal marked evolutionary conservation of Atox1 structure and provide a genomic organization and localization that will aid in the genetic deciphering of the molecular role of this protein in copper homeostasis.

Interaction of the copper chaperone HAH1 with the Wilson disease protein is essential for copper homeostasis.

The delivery of copper to specific sites within the cell is mediated by distinct intracellular carrier proteins termed copper chaperones. Previous studies in Saccharomyces cerevisiae suggested that the human copper chaperone HAH1 may play a role in copper trafficking to the secretory pathway of the cell. In this current study, HAH1 was detected in lysates from multiple human cell lines and tissues as a single-chain protein distributed throughout the cytoplasm and nucleus. Studies with a glutathione S-transferase-HAH1 fusion protein demonstrated direct protein-protein interaction between HAH1 and the Wilson disease protein, which required the cysteine copper ligands in the amino terminus of HAH1. Consistent with these in vitro observations, coimmunoprecipitation experiments revealed that HAH1 interacts with both the Wilson and Menkes proteins in vivo and that this interaction depends on available copper. When these studies were repeated utilizing three disease-associated mutations in the amino terminus of the Wilson protein, a marked diminution in HAH1 interaction was observed, suggesting that impaired copper delivery by HAH1 constitutes the molecular basis of Wilson disease in patients harboring these mutations. Taken together, these data provide a mechanism for the function of HAH1 as a copper chaperone in mammalian cells and demonstrate that this protein is essential for copper homeostasis.

Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux.

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism. Affected individuals evidence iron accumulation in tissue parenchyma in association with absent serum ceruloplasmin. Genetic studies of such patients reveal inherited mutations in the ceruloplasmin gene. To elucidate the role of ceruloplasmin in iron homeostasis, we created an animal model of aceruloplasminemia by disrupting the murine ceruloplasmin (Cp) gene. Although normal at birth, Cp(-/-) mice demonstrate progressive accumulation of iron such that by one year of age all animals have a prominent elevation in serum ferritin and a 3- to 6-fold increase in the iron content of the liver and spleen. Histological analysis of affected tissues in these mice shows abundant iron stores within reticuloendothelial cells and hepatocytes. Ferrokinetic studies in Cp(+/+) and Cp(-/-) mice reveal equivalent rates of iron absorption and plasma iron turnover, suggesting that iron accumulation results from altered compartmentalization within the iron cycle. Consistent with this concept, Cp(-/-) mice showed no abnormalities in cellular iron uptake but a striking impairment in the movement of iron out of reticuloendothelial cells and hepatocytes. Our findings reveal an essential physiologic role for ceruloplasmin in determining the rate of iron efflux from cells with mobilizable iron stores.

The role of copper in neurodegenerative disease.

Copper is an essential trace metal which plays a fundamental role in the biochemistry of the human nervous system. Menkes disease and Wilson disease are inherited disorders of copper metabolism and the dramatic neurodegenerative phenotypes of these two diseases underscore the essential nature of copper in nervous system development as well as the toxicity of this metal when neuronal copper homeostasis is perturbed. Ceruloplasmin contains 95% of the copper found in human plasma and inherited loss of this essential ferroxidase is associated with progressive neurodegeneration of the retina and basal ganglia. Gain-of-function mutations in the cytosolic copper enzyme superoxide dismutase result in the motor neuron degeneration of amyotrophic lateral sclerosis and current evidence suggests a direct pathogenic role for copper in this process. Recent studies have also implicated copper in the pathogenesis of neuronal injury in Alzheimer's disease and the prion-mediated encephalopathies, suggesting that further elucidation of the mechanisms of copper trafficking and metabolism within the nervous system will be of direct relevance to our understanding of the pathophysiology and treatment of neurodegenerative disease.

Intracellular localization of the Menkes and Wilson's disease proteins and their role in intracellular copper transport.

Copper is a heavy metal ion essential for the activity of a variety of enzymes in the body. In excess, copper is a very toxic ion and therefore efficient regulation of its metabolism is required. This is dramatically illustrated by the genetic disorders X-linked Menkes disease and autosomal recessive Wilson's disease. In 1993, both the Menkes and Wilson's genes were isolated and it was found that these genes encode homologous cation copper transporting P-type ATPase proteins. The Menkes protein (ATP7A) is expressed in most tissues, except liver. In contrast, the Wilson's protein (ATP7B) is abundantly expressed in liver. Intracellular localization of those proteins was investigated. Both ATP7A and ATP7B are localized in the trans-Golgi network and post-Golgi vesicular compartment (PGVC) in the cell. This intracellular localization was altered by the copper content present in the cell. This result may support the hypothesis that ATP7A and ATP7B are involved in cellular copper transport and those proteins could be suitable models for elucidating intracellular copper metabolism.

New concepts of histological changes in experimental augmentation cystoplasty: insights into the development of neoplastic transformation at the enterovesical and gastrovesical anastomosis.

PURPOSE: To our knowledge the pathogenesis of malignancy associated with ileal cystoplasty, ureterosigmoidostomy and ileal conduits is currently unknown. To gain further insights into the mechanism of neoplastic transformation we studied histological changes in a canine augmentation cystoplasty model. MATERIALS AND METHODS: Enterocystoplasty and gastrocystoplasty were performed using a 5 to 7 cm. patch of ileum in 8 dogs and gastric antrum in 6. Specimens were harvested 4 months postoperatively. Representative 3 microm sections of the enterovesical and gastrovesical junctions were stained with hematoxylin and eosin. Uroplakin expression was assessed using an indirect peroxidase method subjected to double staining with alcian blue and periodic acid-Schiffreagent. RESULTS: The bladder portion of the augmentation cystoplasty had 3 to 4 stratified cell layers covered with a distinctive umbrella cell layer. Strong uroplakin staining was visible in all cell layers except the basal layer. At the enterovesical and gastrovesical junctions 6 to 10 layers of hyperplastic, urothelial appearing cells covered the glandular epithelium of the ileal and gastric segments. These cells expressed uroplakins. At this junction zone there was a marked decrease of underlying enteric glands, which had atrophied in proportion to the degree of urothelial hyperplasia. Double staining of uroplakin stained sections with alcian blue and periodic acid-Schiff reagent revealed mucosubstances in hyperplastic urothelial cells covering the enteral segments, indicating that the cells co-expressed uroplakins and mucins. CONCLUSIONS: Histological changes in this experimental canine model of augmentation cystoplasty indicated that the overgrowth of hyperplastic transitional epithelium develops at the enterovesical and gastrovesical junctions. These cells express not only uroplakins, but also mucosubstances. Our results suggest that the migrated hyperplastic urothelial cells have undergone changes characteristic of the enteric and gastric epithelium, which may have important implications in the pathogenesis of malignancy in bladder augmentations.