Biomedical subjects

R O Brady

Publications and source records attributed to R O Brady.

At least 19 recordsLinked to original sources

PubMed1997

Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

Amino Acid Sequence↗

PubMed1997

alpha-Galactosidase A deficient mice: a model of Fabry disease.

Fabry disease is an X-linked inherited metabolic disorder that is caused by a deficiency of alpha-galactosidase A (alpha-Gal A). Progressive deposition of neutral glycosphingolipids that have terminal a-linked galactosyl moieties in vascular endothelial cells causes renal failure along with premature myocardial infarctions and strokes in patients with this condition. No specific treatment is available for patients with this disorder at this time. An animal model of this condition would be valuable for exploring therapeutic strategies for patients with Fabry disease. We report here the generation of alpha-Gal A deficient mice by gene targeting and an analysis of the resulting phenotype. The knockout mice display a complete lack of alpha-Gal A activity. The mice, however, appeared clinically normal at 10 weeks of age. Ultrastructural analysis revealed concentric lamellar inclusions in the kidneys, and confocal microscopy using a fluorescent-labeled lectin specific for alpha-D-galactosyl residues showed accumulation of substrate in the kidneys as well as in cultured fibroblasts. Lipid analysis revealed a marked accumulation of ceramidetrihexoside in the liver and the kidneys. These findings indicate the similarity of the pathophysiological process in the mutant mice and in patients with Fabry disease. The deficiency of alpha-Gal A activity and the accumulation of material containing terminal alpha-galactosyl residues in cultured embryonic fibroblasts derived from alpha-Gal A(-/0) mice were corrected by transducing these cells with bicistronic multidrug resistance retroviruses containing human alpha-Gal A cDNA.

Animals↗

PubMed1997

D-serine as a neuromodulator: regional and developmental localizations in rat brain glia resemble NMDA receptors.

D-Serine is localized in mammalian brain to a discrete population of glial cells near NMDA receptors, suggesting that D-serine is an endogenous agonist of the receptor-associated glycine site. To explore this possibility, we have compared the immunohistochemical localizations of D-serine, glycine, and NMDA receptors in rat brain. In the telencephalon, D-serine is concentrated in protoplasmic astrocytes, which are abundant in neuropil in close vicinity to NMDA receptor 2A/B subunits. Ultrastructural examination of the CA1 region of hippocampus reveals D-serine in the cytosolic matrix of astrocytes that ensheath neurons and blood vessels, whereas NR2A/B is concentrated in dendritic spines. By contrast, glycine immunoreactivity in telencephalon is the lowest in brain. During postnatal week 2, D-serine levels in cerebellum are comparable to those in adult cerebral cortex but fall to undetectable levels by day 26. During week 2, we observe parallel ontogeny of D-serine in Bergmann glia and NR2A/B in Purkinje cells, suggesting a role for astrocytic D-serine in NMDA receptor-mediated synaptogenesis. D-Serine in the radial processes of Bergmann glia is also well positioned to regulate NMDA receptor-dependent granule cell migration. In the inner granule layer, D-serine is found transiently in protoplasmic astrocytes surrounding glomeruli, where it could regulate development of the mossy fiber/granule cell synapse. D-Serine seems to be the endogenous ligand of glycine sites in the telencephalon and developing cerebellum, whereas glycine predominates in the adult cerebellum, olfactory bulb, and hindbrain.

Animals↗

PubMed1997

Leukodystrophy in patients with ovarian dysgenesis.

We describe clinical, biochemical, pathological, and spectroscopic findings in 4 women, aged 15 to 29 years, from three unrelated families who had a unique combination of a central nervous system white matter disease and primary ovarian failure. All had normal initial development but 3 had borderline low IQ and academic difficulties in primary school. Puberty did not develop in 2 patients and was arrested in a third patient. The fourth patient had premature ovarian failure at the age of 13 years. Head magnetic resonance imaging showed diffuse white matter disease, with frontal cortical atrophy in the most clinically advanced patient. All patients had normal karyotype and normal findings on extensive evaluations for known leukodystrophies, for other metabolic diseases, and for causes of ovarian failure. Proton magnetic resonance spectroscopic imaging showed reduction of choline-containing compounds in the affected white matter in all patients and reduction of N-acetylaspartate in the unaffected frontal white matter of 2 patients. All patients had evidence of primary gonadal insufficiency with a normal hypothalamic-hypophyseal axis. Pathological analysis showed streak ovaries in 1 patient and signs of hypomyelination, and gliosis on brain biopsy in another patient. In conclusion, we present a novel group of patients who have in common leukodystrophy, primary ovarian dysfunction, and magnetic resonance spectroscopic abnormalities.

Adolescent↗

PubMed1997

Cocaine detoxification by human plasma butyrylcholinesterase.

The ability of human plasma butyrylcholinesterase (BChE) to detoxify cocaine in vivo was evaluated. Intravenous administration of BChE, at doses sufficient to increase the plasma levels of the enzyme as much as 800-fold, produced no adverse effects on the cardiovascular, autonomic, or central nervous systems of rats. Most of the enzyme could be recovered in the plasma immediately after administration and remained active with a beta-t(1/2) of 21.6 +/- 2.4 hr. Pretreatment of chloralose-urethane anesthetized rats with BChE, 0.1-7.8 mg/kg, decreased the hypertensive and arrhythmogenic effects produced by cocaine and increased the lethal dose of cocaine by three- to fourfold. Treatment of conscious rats with 1 and 10 mg/kg BChE decreased the incidence of seizures and deaths produced by a prior dose of cocaine (80 mg/kg, i.p.). These results suggest that BChE would provide a safe and highly efficacious treatment for cocaine intoxication.

Animals↗

PubMed1997

Therapeutic use of butyrylcholinesterase for cocaine intoxication.

The most common complications of cocaine ingestion are on the cardiovascular and central nervous systems and produce chest pain and generalized seizures. In humans, decreased levels of butyrylcholinesterase (BChE) (EC 3.1.1.8) have been associated with sustained effects of cocaine and life-threatening complications. Administration of purified human BChE has previously been demonstrated to protect against cocaine-associated cardiovascular toxicity in rats. A shift in the metabolism of cocaine as well as enhanced metabolism may be the underlying mechanism of the enzyme. Therefore, levels of the parent drug and four metabolites were determined in rat plasma after i.p. administration of a lethal cocaine dose, followed by i.v. administration of BChE. Plasma and brain concentrations of cocaine were lowered by 80% after BChE administration. Furthermore, the metabolic profile of cocaine in the plasma was altered. The concentration of ecgonine methylester was doubled although the concentration of ecgonine, a secondary metabolite of cocaine, was reduced. The level of benzoylecgonine was reduced by one-half while norcocaine was absent. Cocaine-associated effects upon the central nervous system were also shown to be reduced by administration of BChE to conscious rats. Furthermore, our studies in the cat have also shown that purified BChE shifts the metabolic profile of cocaine (1 mg/kg) to the pharmacologically inactive products ecgonine methylester and ecgonine. Pretreatment with BChE (0.27, 1.0, and 10.0 mg/kg) ameliorated the hypertensive effects of cocaine (1 mg/kg) by reducing the duration and the extent of BP elevation by 66%. Administration of the enzyme, 1 min after cessation of cocaine infusion, resulted in an immediate attenuation in the cocaine-induced broadening of the QRS complex. These results suggest that BChE could be an effective and rapid therapy for the treatment of life-threatening cocaine-induced cardiovascular effects in human while clearing the total body burden of cocaine.

Animals↗

PubMed1996

Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles.

The use of Moloney murine leukemia virus (Mo-MLV)-based vectors to deliver therapeutic genes into target cells is limited by their inability to transduce nondividing cells. To test the capacity of HIV-based vectors to deliver genes into nondividing cells, we have generated replication-defective HIV type 1 (HIV-1) reporter vectors carrying neomycin phosphotransferase or mouse heat stable antigen, replacing the HIV-1 sequences encoding gp160. These vectors also harbor inactive vpr, vpu, and nef coding regions. Pseudotyped HIV-1 particles carrying either the ecotropic or the amphotropic Mo-MLV envelope proteins or the vesicular stomatitis virus G protein were released after single or double transfections of either human 293T or monkey COS-7 cells with titers of up to 10(7) colony-forming units per milliliter. A simple ultrafiltration procedure resulted in an additional 10- to 20-fold concentration of the pseudotyped particles. These vectors along with Mo-MLV-based vectors were used to transduce primary human skin fibroblasts and human peripheral blood CD34+ cells. The HIV-1 vector system was significantly more efficient than its Mo-MLV-based counterpart in transducing human skin fibroblasts arrested at the G0/G1 stage of the cell cycle by density-dependent inhibition of growth. Human CD34+ cells were transduced efficiently using HIV-1 pseudotype particles without prior stimulation with cytokines.

Animals↗

PubMed1996

Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death.

Although cyclin-dependent kinase 5 (Cdk5) is closely related to other cyclin-dependent kinases, its kinase activity is detected only in the postmitotic neurons. Cdk5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in developing brain. Cdk5 purified from nervous tissue phosphorylates neuronal cytoskeletal proteins including neurofilament proteins and microtubule-associated protein tau in vitro. These findings indicate that Cdk5 may have unique functions in neuronal cells, especially in the regulation of phosphorylation of cytoskeletal molecules. We report here generation of Cdk5(-/-) mice through gene targeting and their phenotypic analysis. Cdk5(-/-) mice exhibit unique lesions in the central nervous system associated with perinatal mortality. The brains of Cdk5(-/-) mice lack cortical laminar structure and cerebellar foliation. In addition, the large neurons in the brain stem and in the spinal cord show chromatolytic changes with accumulation of neurofilament immunoreactivity. These findings indicate that Cdk5 is an important molecule for brain development and neuronal differentiation and also suggest that Cdk5 may play critical roles in neuronal cytoskeleton structure and organization.

Animals↗

PubMed1996

Correction in trans for Fabry disease: expression, secretion and uptake of alpha-galactosidase A in patient-derived cells driven by a high-titer recombinant retroviral vector.

Fabry disease is an X-linked metabolic disorder due to a deficiency of alpha-galactosidase A (alpha-gal A; EC 3.2.1.22). Patients accumulate glycosphingolipids with terminal alpha-galactosyl residues that come from intracellular synthesis, circulating metabolites, or from the biodegradation Of senescent cells. Patients eventually succumb to renal, cardio-, or cerebrovascular disease. No specific therapy exists. One possible approach to ameliorating this disorder is to target corrective gene transfer therapy to circulating hematopoietic cells. Toward this end, an amphotropic virus-producer cell line has been developed that produces a high titer (>10(6) i.p. per ml) recombinant retrovirus constructed to transduce and correct target cells. Virus-producer cells also demonstrate expression of large amounts of both intracellular and secreted alpha-gal A. To examine the utility of this therapeutic vector, skin fibroblasts from Fabry patients were corrected for the metabolic defect by infection with this recombinant virus and secreted enzyme was observed. Furthermore, the secreted enzyme was found to be taken up by uncorrected cells in a mannose-6-phosphate receptor-dependent manner. In related experiments, immortalized B cell lines from Fabry patients, created as a hematologic delivery test system, were transduced. As with the fibroblasts, transduced patient B cell lines demonstrated both endogenous enzyme correction and a small amount of secretion together with uptake by uncorrected cells. These studies demonstrate that endogenous metabolic correction in transduced cells, combined with secretion, may provide a continuous source of corrective material in trans to unmodified patient bystander cells (metabolic cooperativity).

3T3 Cells↗

PubMed1996

Molecular cloning and chromosomal mapping of the mouse gene encoding cyclin-dependent kinase 5 regulatory subunit p35.

A neural-specific activating subunit, p35, of cyclin-dependent kinase 5 (Cdk5) was recently reported to differ from other mammalian cyclins, suggesting a new type of regulatory subunit for Cdk activity. The mouse gene encoding p35, Cdk5r, was isolated from a mouse 129/SvJ genomic library, and the genomic structure of Cdk5r was characterized. The most notable features of Cdk5r are the absence of introns in the amino acid coding region and the high homology of amino acid sequence among species. The 5'-flanking region of Cdk5r contained no canonical TATA or CAAT box but had several putative promoter elements, including Sp1, AP2, MRE, and NGFIA. The mouse Cdk5r transcript was detected only in the brain by Northern blot analysis. Mouse Cdk5r was mapped to a position on mouse chromosome 11.

Amino Acid Sequence↗

PubMed1996

A bicistronic therapeutic retroviral vector enables sorting of transduced CD34+ cells and corrects the enzyme deficiency in cells from Gaucher patients.

Corrective gene transfer for therapeutic intervention in metabolic and hematopoietic disorders has been hampered by the relatively inefficient transduction of human hematopoietic stem cells. To overcome this, a bicistronic recombinant retrovirus has been generated that delivers both a therapeutic glucocerebrosidase (GC) cDNA for the treatment of Gaucher disease, and a small murine cell surface antigen (heat-stable antigen [HSA]) as a selectable marker. An amphotropic retroviral-producing cell clone was created, and filtered supernatant was used to transduce NIH 3T3 cells. Sorting of transduced cells by flow cytometry enabled separation into populations based on cell surface fluorescence intensity derived from the expressed HSA. Significant increases in GC enzyme activity were seen for the transduced and especially the transduced and sorted cells. Similarly, increases in GC specific activity were seen in transduced and sorted skin fibroblasts from a patient with Gaucher disease. To streamline future transfer and sorting protocols for hematopoietic cells, transformed B-cell lines from Gaucher patients were created. Type I B cells were transduced and sorted, and large increases in GC specific activity occurred with concomitant increases in integrated retroviral copy numbers. In addition, toward the goal of using this selectable approach for corrective gene transfer to bone marrow stem cells, CD34+ cells were isolated from normal BM donors, transduced, and sorted based on cell surface expression of HSA. Proviral DNA was detected in approximately 40% of clonogenic progenitor colonies derived from unsorted, transduced CD34+ cells, demonstrating the high titer of the vector. However, after sorting, 100% of the colonies had the corrective GC cDNA, demonstrating the efficiency of this selective system for human hematopoietic progenitors. It is expected that strategies based on this approach will allow sorting of transduced cells of many types before implantation of transduced cells to animals or patients. This vector system may also be used to simplify manipulations and studies on retroviral-mediated gene delivery in vitro and for in vivo models.

3T3 Cells↗

PubMed1996

High-level production of recombinant human lysosomal acid alpha-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fibroblasts from patients with Pompe disease.

Infantile Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. We constructed a plasmid containing a 5'-shortened human acid alpha-glucosidase cDNA driven by the cytomegalovirus promoter, as well as the aminoglycoside phosphotransferase and dihydrofolate reductase genes. Following transfection in dihydrofolate reductase-deficient Chinese hamster ovary cells, selection with Geneticin, and amplification with methotrexate, a cell line producing high levels of the alpha-glucosidase was established. In 48 hr, the cells cultured in Iscove's medium with 5 mM butyrate secreted 110-kDa precursor enzyme that accumulated to 91 micrograms.ml-1 in the medium (activity, > 22.6 mumol.hr-1.ml-1). This enzyme has a pH optimum similar to that of the mature form, but a lower Vmax and Km for 4-methylumbelliferyl-alpha-D-glucoside. It is efficiently taken up by fibroblasts from Pompe patients, restoring normal levels of acid alpha-glucosidase and glycogen. The uptake is blocked by mannose 6-phosphate. Following intravenous injection, high enzyme levels are seen in heart and liver. An efficient production system now exists for recombinant human acid alpha-glucosidase targeted to heart and capable of correcting fibroblasts from patients with Pompe disease.

Animals↗

PubMed1996

Enzyme replacement and gene therapy for Gaucher's disease.

The lipid storage disorders have long been considered primary candidates for enzyme replacement therapy. This goal has been achieved with a remarkable degree of success in Gaucher's disease. Among the accomplishments that were important to obtain clinical benefit were the development of a large-scale procedure to purify human placental glucocerebrosidase and a method to target this enzyme to lipid-storing macrophages through glycoform modification. In addition, the effectiveness of recombinantly produced macrophage-targeted glucocerebrosidase has recently been demonstrated. Because macrophages originate from stem cells in the bone marrow, ex vivo transduction of these cells with retroviral vectors containing the cDNA for human glucocerebrosidase is being explored for the genetic therapy of Gaucher's disease.

Carbohydrate Sequence↗

PubMed1996

Radiographic findings in type 3b Gaucher disease.

The purpose of this paper is to describe the radiographic findings in type 3 b Gaucher disease, a chronic neuronopathic form of the illness with severe systemic manifestations. Between 1980 and 1985 17 consecutive patients were evaluated with radiography of the chest, long bones and spine, CT of the head and chest, abdominal sonography, and MRI of the head, abdomen and spine. Clinical manifestations were severe, and led to death from hepatic, pulmonary or cardiac failure in nine patients. Type 3 b Gaucher disease shares the same spectrum of radiographic findings observed in type 1 disease, but the systemic manifestations are more severe. Pulmonary infiltrates, thoracic lymph node enlargement, vertebral compression fractures and osteonecrosis of the long bones occur much more frequently in patients with type 3 b disease.

Adolescent↗

PubMed1996

Analysis of the lipids of normal and Gaucher bone marrow.

Quantitative chemical shift magnetic resonance imaging (QCSI) is currently being utilized for measuring the extent of bone marrow involvement and its response to enzyme replacement therapy in patients with Gaucher's disease. Quantitation of the major lipid species in human bone marrow is required to accurately interpret QCSI data on bone marrow composition. The major lipid species in bone marrow specimens from normal individuals and from patients with type 1 Gaucher's disease were analyzed by thin-layer and high-pressure liquid chromatography. In normal marrow (N = 5), triglycerides were by far the most abundant lipid (278 +/- 70 mg/gm wet wt), with other non-polar lipids and phospholipids totaling less than 20 mg/gm wet weight. The concentration of glucocerebroside in normal marrow was 0.061 +/- 0.06 mg/gm wet weight. Gaucher marrow (N = 9) had dramatically lower triglyceride levels (51 +/- 53 mg/gm wet wt), and as expected, it had markedly elevated levels of glucocerebroside (7.1 +/- 3.4 mg/gm wet wt). The other major non-polar lipids and phospholipids were measured in selected specimens, but none were found that differed so profoundly between normal and Gaucher's disease. These data support a model of bone marrow alteration in Gaucher's disease in which triglyceride-rich adipocytes are progressively replaced by storage cells, leading to an overall reduction in total lipid content. This phenomenon provides an explanation for the changes in proton signal intensity observed in QCSI studies of Gaucher bone marrow.

Adult↗

PubMed1996

Molecular cloning of the mouse apolipoprotein D gene and its upregulated expression in Niemann-Pick disease type C mouse model.

Apolipoprotein D (ApoD) is a member of the lipocalin superfamily. The primary structure and diverse expression of ApoD suggest that this protein is a multiligand, multifunctional glycoprotein. Here we report the structure of the mouse ApoD gene, which is composed of six exons spanning approximately 20 kb in length. All the exon-intron splice junctions follow the consensus GT/AG sequence. The 5'-flanking region of the mouse ApoD gene contains several putative regulatory elements, including FSE-2, GRE, SDR, MRE, IL-6RE, and TATA box. Northern blot analysis revealed that ApoD was highly expressed in the brain and adipose tissue in mouse. Lower levels of expression were observed in the heart, lung, thymus, testis, and salivary glands. In situ hybridization for the brain showed that ApoD mRNA was mainly localized in the subarachnoid space including the pia. In the Niemann-Pick disease type C mouse model, ApoD expression was upregulated in many organs such as brain, adipose tissue, heart, and thymus, presumably due to enhanced ApoD synthesis in perivascular fibroblasts.

Animals↗

PubMed1995

Structural organization and expression of the mouse gene encoding alpha-galactosidase A.

alpha-Galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22; alpha GalA) is a lysosomal enzyme that hydrolyses the alpha-D-galactosyl residues from glycosphingolipids. Fabry disease, an inhibited X-linked recessive human metabolic disorder, results from a mutation in the alpha GalA gene at Xq22. As a prerequisite for generating a mouse model of Fabry disease by gene targeting, we have isolated and characterized the mouse alpha GalA gene and cDNA. A cloned mouse alpha GalA cDNA encoded a putative precursor protein of 419 amino acids (aa), including a 31-aa signal peptide (SP). The deduced aa sequence showed high homology (79%) with the human alpha GalA protein. Nucleotide sequence analysis of genomic clones revealed that the overall structure and organization of the gene was very similar to that of human alpha GalA. All exon-intron splice junctions conformed to the GT/AG consensus sequence. Comparison of genomic and cDNA sequences revealed the occurrence of two putative polyadenylation signals whose alternative use results in the two mouse alpha GalA transcripts of 1.4 and 3.6 kb. The 5'-flanking region of mouse alpha GalA had no typical TATA box. Several putative promoter-associated elements including Sp1, AP1 and a potential cAMP-responsive element (CRE) were identified. Northern blot analysis revealed the widespread tissue distribution of mouse alpha GalA transcripts. Lower expression levels, however, were observed in some tissues, implying tissue-specific differences in alpha GalA promoter function.

Amino Acid Sequence↗

PubMed1995

Molecular cloning and chromosomal mapping of the mouse cyclin-dependent kinase 5 gene.

Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in neurons. In vitro, Cdk5 purified from the nervous tissue phosphorylates both high-molecular-weight neurofilament and microtubule-associated tau. The mouse gene encoding Cdk5 (Cdk5) was found to be 5 kb in length and divided into 12 exons. All of the exon-intron junctions matched the expected consensus sequence with the exception of the splice junction for intron 9, which has AT and AC dinucleotides instead of the usual GT and AG bordering sequence. In the 5'-flanking region of mouse Cdk5, several putative promoter elements were present, including AP1, Sp1, PuF, and TATA motifs. A metal regulatory element was also identified at position -207 to -201. Nucleotide sequence analysis of mouse Cdk5 showed high identity to the homologues of other vertebrate species, indicating that this kinase is highly conserved during evolution. Mouse Cdk5 was mapped to the centromeric region of mouse chromosome 5.

Amino Acid Sequence↗