Biomedical subjects

J Gitschier

Publications and source records attributed to J Gitschier.

79 records · Page 5Linked to original sources

PubMed1985

Genetic modifications of voltage-sensitive sodium channels in Drosophila: gene dosage studies of the seizure locus.

We have identified a genetic locus in Drosophila melanogaster whose product appears to have a structural role in the formation of functional voltage-sensitive sodium channels. This locus, designated seizure, is defined by two temperature-sensitive alleles (seits-1 and seits-2), each of which causes convulsive seizures followed by a rapid but reversible paralysis of adults at restrictive temperatures above 38 degrees C. Previous work had shown that seits-2 extracts display an altered pH dependence and an abnormally high Kd for [3H]-saxitoxin binding at high temperatures, suggesting that sodium channels in seits-2 mutants have an altered structure (Jackson F. R., S. D. Wilson, G. R. Strichartz, and L. M. Hall (1984) Nature 308: 189-191). These binding studies have now been extended to extracts of seits-1 which have a Kd not significantly different from wild-type at all assay temperatures. However, seits-1 extracts show a reduced number of saxitoxin binding sites (Bmax) relative to wild-type. This reduction is only 5 to 18% at 0 degree C but is 17 to 37% at 39 degrees C, suggesting that under certain conditions sodium channels in the seits-1 mutant are more labile than those of wild-type. Cytogenetic studies demonstrate that the seizure locus maps within region 60A to 60B8-10 on the second chromosome. Gene dosage analysis of approximately 99.7% of the genome, including this second chromosome region, failed to detect a wild-type locus whose dose affected saxitoxin-binding activity. Nevertheless, the mutant seits-2 allele has codominant and dose-dependent effects on paralytic behavior and saxitoxin-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1980

Saxitoxin binding to sodium channels in head extracts from wild-type and tetrodotoxin-sensitive strains of Drosophila melanogaster.

Extracts prepared from heads of Drosophila melanogaster show high-affinity binding (KD = 1.9 nM) of [3H]saxitonin, a compound known to bind to and block voltage-sensitive sodium channels in other organisms. The interaction between saxitoxin and the Drosophila saxitoxin receptor is non-cooperative and reversible with a half-life of 18.3 s for binding at 4 degrees C. The saturable binding is specifically inhibited by tetrodotoxin with a K1 = 0.30 nM. The number of saturable binding sites in the extract is 97 fmol/mg protein. Since approx. 50% of the binding activity is recovered in the extract, the number of binding sites in the head is estimated to be 6.4 fmol/mg head. Nerve conduction in Drosophila larvae is completely blocked after 20 min in a bathing solution containing 200 nM tetrodotoxin. A comparison between the binding and the electrophysiological studies in Drosophila and other organisms suggests that the Drosophila saxitoxin receptor is part of the voltage-sensitive sodium channel involved in the propagation of action potentials. A mutant (ttxs), which is abnormally sensitive to dietary tetrodotoxin, is shown to be indistinguishable from wild type with respect to [3H]saxitonin-binding properties and physiological sensitivity to tetrodotoxin. These studies provide techniques which can be used to identify mutants with defects in the saxitoxin-binding component of the sodium channel.

Animals↗

PubMedDate not supplied

Characterization of the human factor VIII gene.

The complete 186,000 base-pair (bp) human factor VIII gene has been isolated and consists of 26 exons ranging in size from 69 to 3,106 bp and introns as large as 32.4 kilobases (kb). Nine kb of mRNA and protein-coding DNA has been sequenced and the mRNA termini have been mapped. The relationship between internal duplications in factor VIII and evolution of the gene is discussed.

Base Sequence↗

PubMedDate not supplied

Expression of active human factor VIII from recombinant DNA clones.

DNA clones encoding the complete 2,351 amino acid sequence for human factor VIII have been isolated and used to produce biologically active factor VIII in cultured mammalian cells. The recombinant protein corrects the clotting time of plasma from haemophiliacs and has many of the biochemical and immunological characteristics of serum-derived factor VIII.

Animals↗

PubMedDate not supplied

Genetic mapping and diagnosis of haemophilia A achieved through a BclI polymorphism in the factor VIII gene.

Haemophilia A is the most common inherited bleeding disorder in man, affecting approximately 1 male in 10,000. The disease is caused by a deficiency in the gene for factor VIII, a component of the intrinsic coagulation pathway. Due to the broad range of clotting activity in normal and heterozygous females, it is often difficult to confirm the status of women at risk for carrying the disease. A genetic marker in the form of a restriction fragment length polymorphism (RFLP) within or tightly linked to the factor VIII gene would serve as a tag for the haemophilia gene, thus allowing both accurate carrier detection and improved, earlier prenatal diagnosis by chorionic villi sampling. The recent isolation of the factor VIII gene has allowed a search for RFLPs within the gene, and we report here the identification of a common polymorphism within the factor VIII gene, revealed by the restriction enzyme BclI, which can be used diagnostically in about 42% of all families. Although the disease haemophilia A has been mapped to the distal portion of Xq, the BclI RFLP makes possible higher-resolution genetic linkage mapping with respect to other polymorphic markers on this portion of the X chromosome. We have established close linkage of the factor VIII gene to several useful RFLP markers, including the highly informative marker St14. These markers should also be useful for prenatal diagnosis of haemophilia A and for detection of its carriers.

Chromosome Mapping↗

PubMedDate not supplied

Detection and sequence of mutations in the factor VIII gene of haemophiliacs.

The most common inherited bleeding disorder in man, haemophilia A, is caused by defect in factor VIII, a component in the blood coagulation pathway. The X-chromosome-linked disease almost certainly stems from a heterogeneous collection of genetic lesions. Because, without proper treatment, haemophilia can be a fatal disease, new mutations are necessary to account for its constant frequency in the population. In addition, haemophilia A displays a wide range of severity, and some 15% of haemophiliacs generate high levels of antibodies against factor VIII ('inhibitor patients'). The present work elucidates the molecular genetic basis of haemophilia in some individuals. Using the recently cloned factor VIII gene as a probe, we have identified two different nonsense point mutations in the factor VIII gene of haemophiliacs, as well as two different partial deletions of the gene. Our survey of 92 haemophiliacs indicates no firm correlation between antibody (inhibitor) production and gross gene defects.

Autoantibodies↗

PubMedDate not supplied

Brain MRI in neurodegeneration with brain iron accumulation with and without PANK2 mutations.

BACKGROUND AND OBJECTIVE: Patients with a clinical diagnosis of neurodegeneration with brain iron accumulation (NBIA, formerly called Hallervorden-Spatz syndrome) often have mutations in PANK2, the gene encoding pantothenate kinase 2. We investigated correlations between brain MR imaging changes, mutation status, and clinical disease features. METHODS: Brain MRIs from patients with NBIA were reviewed by 2 neuroradiologists for technical factors, including signal intensity abnormalities in specific brain regions, presence and location of atrophy, presence of white matter abnormality, contrast enhancement, and other comments. PANK2 genotyping was performed by polymerase chain reaction amplification of patient genomic DNA followed by automated nucleotide sequencing. RESULTS: Sixty-six MR imaging examinations from 49 NBIA patients were analyzed, including those from 29 patients with mutations in PANK2. All patients with mutations had the specific pattern of globus pallidus central hyperintensity with surrounding hypointensity on T2-weighted images, known as the eye-of-the-tiger sign. This sign was not seen in any studies from patients without mutations. Even before the globus pallidus hypointensity developed, patients with mutations could be distinguished by the presence of isolated globus pallidus hyperintensity on T2-weighted images. Radiographic evidence for iron deposition in the substantia nigra was absent early in disease associated with PANK2 mutations. MR imaging abnormalities outside the globus pallidus, including cerebral or cerebellar atrophy, were more common and more severe in mutation-negative patients. No specific MR imaging changes could be distinguished among the mutation-negative patients. CONCLUSION: MR imaging signal intensity abnormalities in the globus pallidus can distinguish patients with mutations in PANK2 from those lacking a mutation, even in the early stages of disease.

Adolescent↗