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- ENaC (9)
- apoptosis (6)
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- 5-Methylcytosine (3)
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2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency is caused by mutations in the HADH2 gene
(2003)
Striated muscle contraction is regulated by the translocation of troponin-tropomyosin strands over the thin filament surface. Relaxation relies partly on highly-favorable, conformation-dependent electrostatic contacts between actin and tropomyosin, which position tropomyosin such that it impedes actomyosin associations. Impaired relaxation and hypercontractile properties are hallmarks of various muscle disorders. The α-cardiac actin M305L hypertrophic cardiomyopathy-causing mutation lies near residues that help confine tropomyosin to an inhibitory position along thin filaments. Here, we investigate M305L actin in vivo, in vitro, and in silico to resolve emergent pathological properties and disease mechanisms. Our data suggest the mutation reduces actin flexibility and distorts the actin-tropomyosin electrostatic energy landscape that, in muscle, result in aberrant contractile inhibition and excessive force. Thus, actin flexibility may be required to establish and maintain interfacial contacts with tropomyosin as well as facilitate its movement over distinct actin surface features and is, therefore, likely necessary for proper regulation of contraction.
A structural mapping of mutations causing succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency
(2013)
Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare inherited metabolic disorder of ketone metabolism, characterized by ketoacidotic episodes and often permanent ketosis. To date there are ~20 disease-associated alleles on the OXCT1 gene that encodes the mitochondrial enzyme SCOT. SCOT catalyzes the first, rate-limiting step of ketone body utilization in peripheral tissues, by transferring a CoA moiety from succinyl-CoA to form acetoacetyl-CoA, for entry into the tricarboxylic acid cycle for energy production. We have determined the crystal structure of human SCOT, providing a molecular understanding of the reported mutations based on their potential structural effects. An interactive version of this manuscript (which may contain additional mutations appended after acceptance of this manuscript) may be found on the web address:
http://www.thesgc.org/jimd/SCOT
AAV-encoded expression of TRAIL in experimental human colorectal cancer leads to tumor regression
(2004)
Hydrogen sulfide (H2S) is well known as a highly toxic environmental chemical threat. Prolonged exposure to H2S can lead to the formation of pulmonary edema. However, the mechanisms of how H2S facilitates edema formation are poorly understood. Since edema formation can be enhanced by an impaired clearance of electrolytes and, consequently, fluid across the alveolar epithelium, it was questioned whether H2S may interfere with transepithelial electrolyte absorption. Electrolyte absorption was electrophysiologically measured across native distal lung preparations (Xenopus laevis) in Ussing chambers. The exposure of lung epithelia to H2S decreased net transepithelial electrolyte absorption. This was due to an impairment of amiloride-sensitive sodium transport. H2S inhibited the activity of the Na+/K+-ATPase as well as lidocaine-sensitive potassium channels located in the basolateral membrane of the epithelium. Inhibition of these transport molecules diminishes the electrochemical gradient which is necessary for transepithelial sodium absorption. Since sodium absorption osmotically facilitates alveolar fluid clearance, interference of H2S with the epithelial transport machinery provides a mechanism which enhances edema formation in H2S-exposed lungs.
The development of pulmonary edema can be considered as a combination of alveolar flooding via increased fluid filtration, impaired alveolar-capillary barrier integrity, and disturbed resolution due to decreased alveolar fluid clearance. An important mechanism regulating alveolar fluid clearance is sodium transport across the alveolar epithelium. Transepithelial sodium transport is largely dependent on the activity of sodium channels in alveolar epithelial cells. This paper describes how sodium channels contribute to alveolar fluid clearance under physiological conditions and how deregulation of sodium channel activity might contribute to the pathogenesis of lung diseases associated with pulmonary edema. Furthermore, sodium channels as putative molecular targets for the treatment of pulmonary edema are discussed.
Gene expression and cell growth rely on the intracellular concentration of amino acids, which in metazoans depends on extracellular amino acid availability and transmembrane transport. To investigate the impact of extracellular amino acid concentrations on the expression of a concentrative amino acid transporter, we overexpressed the main kidney proximal tubule luminal neutral amino acid transporter B0AT1-collectrin (SLC6A19-TMEM27) in MDCK cell epithelia. Exogenously expressed proteins co-localized at the luminal membrane and mediated neutral amino acid uptake. However, the transgenes were lost over few cell culture passages. In contrast, the expression of a control transgene remained stable. To test whether this loss was due to inappropriately high amino acid uptake, freshly transduced MDCK cell lines were cultivated either with physiological amounts of amino acids or with the high concentration found in standard cell culture media. Expression of exogenous transporters was unaffected by physiological amino acid concentration in the media. Interestingly, mycoplasma infection resulted in a significant increase in transgene expression and correlated with the rapid metabolism of L-arginine. However, L-arginine metabolites were shown to play no role in transgene expression. In contrast, activation of the GCN2 pathway revealed by an increase in eIF2α phosphorylation may trigger transgene derepression. Taken together, high extracellular amino acid concentration provided by cell culture media appears to inhibit the constitutive expression of concentrative amino acid transporters whereas L-arginine depletion by mycoplasma induces the expression of transgenes possibly via stimulation of the GCN2 pathway.
The limited sodium availability of freshwater and terrestrial environments was a major physiological challenge during vertebrate evolution. The epithelial sodium channel (ENaC) is present in the apical membrane of sodium-absorbing vertebrate epithelia and evolved as part of a machinery for efficient sodium conservation. ENaC belongs to the degenerin/ENaC protein family and is the only member that opens without an external stimulus. We hypothesized that ENaC evolved from a proton-activated sodium channel present in ionocytes of freshwater vertebrates and therefore investigated whether such ancestral traits are present in ENaC isoforms of the aquatic pipid frog Xenopus laevis. Using whole-cell and single-channel electrophysiology of Xenopus oocytes expressing ENaC isoforms assembled from alpha beta gamma- or delta beta gamma-subunit combinations, we demonstrate that Xenopus delta beta gamma-ENaC is profoundly activated by extracellular acidification within biologically relevant ranges (pH 8.0-6.0). This effect was not observed in Xenopus alpha beta gamma-ENaC or human ENaC orthologs. We show that protons interfere with allosteric ENaC inhibition by extracellular sodium ions, thereby increasing the probability of channel opening. Using homology modeling of ENaC structure and site-directed mutagenesis, we identified a cleft region within the extracellular loop of the delta-subunit that contains several acidic amino acid residues that confer proton-sensitivity and enable allosteric inhibition by extracellular sodium ions. We propose that Xenopus delta beta gamma-ENaC can serve as a model for investigating ENaC transformation from a proton-activated toward a constitutively-active ion channel. Such transformation might have occurred during the evolution of tetrapod vertebrates to enable bulk sodium absorption during the water-to-land transition.
Dihydropyrimidine dehydrogenase (DPD) deficiency is an infrequently described autosomal recessive disorder of the pyrimidine degradation pathway and can lead to mental and motor retardation and convulsions. DPD deficiency is also known to cause a potentially lethal toxicity following administration of the antineoplastic agent 5-fluorouracil. In an ongoing study of 72 DPD deficient patients, we analysed the molecular background of 5 patients in more detail in whom initial sequence analysis did not reveal pathogenic mutations. In three patients, a 13.8 kb deletion of exon 12 was found and in one patient a 122 kb deletion of exon 14–16 of DPYD. In the fifth patient, a c.299_302delTCAT mutation in exon 4 was found and also loss of heterozygosity of the entire DPD gene. Further analysis demonstrated a de novo deletion of approximately 14 Mb of chromosome 1p13.3–1p21.3, which includes DPYD. Haploinsufficiency of NTNG1, LPPR4, GPSM2, COL11A1 and VAV3 might have contributed to the severe psychomotor retardation and unusual craniofacial features in this patient. Our study showed for the first time the presence of genomic deletions affecting DPYD in 7% (5/72) of all DPD deficient patients. Therefore, screening of DPD deficient patients for genomic deletions should be considered.
BACKGROUND
Neuronal migration is a crucial process that allows neurons to reach their correct target location to allow the nervous system to function properly. AP-2alpha is a transcription factor essential for neural crest cell migration and its mutation results in apoptosis within this cell population, as demonstrated by genetic models.
RESULTS
We down-modulated AP-2alpha expression in GN-11 neurons by RNA interference and observe reduced neuron migration following the activation of a specific genetic programme including the Adhesion Related Kinase (Axl) gene. We prove that Axl is able to coordinate migration per se and by ChIP and promoter analysis we observe that its transcription is directly driven by AP-2alpha via the binding to one or more functional AP-2alpha binding sites present in its regulatory region. Analysis of migration in AP-2alpha null mouse embryo fibroblasts also reveals an essential role for AP-2alpha in cell movement via the activation of a distinct genetic programme.
CONCLUSION
We show that AP-2alpha plays an essential role in cell movement via the activation of cell-specific genetic programmes. Moreover, we demonstrate that the AP-2alpha regulated gene Axl is an essential player in GN-11 neuron migration.
Apoptosis in the terminal endbud of the murine mammary gland: a mechanism of ductal morphogenesis
(1996)
ATM virtual studio services
(1996)
The term "virtual studio" refers to real-time 3D graphics systems used to render a virtual set in sync with live camera motion. As the camera pans and zooms, the virtual set is redrawn from the correct perspective. Using blue room techniques, actors in front of the real camera are then “placed in” the virtual set. Current virtual studio systems are centralized – the blue room, cameras, renderers etc. are located at a single site. However distributed configurations offer significant economies such as the sharing of expensive rendering equipment among many sites. This paper describes early expe- riences of the DVP1 project in the realization of a distributed virtual studio. In particular we de- scribe the first video production using a distributed virtual studio over ATM and make observations concerning network QOS requirements.
Autoantikörper und das Komplementsystem - Bedeutung für die Pathogenese der Rheumatoiden Arthritis?
(2002)
Bcl-2 is known to have dual antiproliferative and antiapoptotic roles. Overexpression of Bcl-2 in the mammary gland using a whey acidic protein (WAP) promoter-driven Bcl-2 transgene inhibits apoptosis in the mammary gland during pregnancy, lactation, and involution, and also counteracts apoptosis induced by overexpression of a mutant p53 transgene (WAP-p53 172 R-L). WAP-Bcl-2 mice and nontransgenic controls were treated with the carcinogen dimethylbenz(a)anthracene (DMBA). Surprisingly, the nontransgenic mice developed mammary tumors with decreased latency. Tumors arising in WAP-Bcl-2 mice displayed substantially reduced levels of proliferation relative to those seen in nontransgenic mice (P < 0.015), perhaps resulting in the observed increase in tumor latency following carcinogen treatment. This WAP-Bcl-2 mouse tumor model reflects the situation seen in some human breast cancers overexpressing Bcl-2, where expression of Bcl-2 has been shown to correlate with a lower proliferative index in tumors.
Benches and CAVEs
(1997)
Benches and Caves
(1998)
Benches and Caves
(1998)