Small GTPase Determinants for the Golgi Processing and Plasmalemmal Expression of Human Ether-a-go-go Related (hERG) K+ Channels

The pro-arrhythmic Long QT syndrome (LQT) is linked to 10 different genes (LQT1–10). Approximately 40% of genotype-positive LQT patients have LQT2, which is characterized by mutations in the human ether-a-go-go related gene (hERG). hERG encodes the voltage-gated K⁺ channel α-subunits that form the pore of the rapidly activating delayed rectifier K⁺ current in the heart. The purpose of this study was to elucidate the mechanisms that regulate the intracellular transport or trafficking of hERG, because trafficking is impaired for about 90% of LQT2 missense mutations. Protein trafficking is regulated by small GTPases. To identify the small GTPases that are critical for hERG trafficking, we coexpressed hERG and dominant negative (DN) GTPase mutations in HEK293 cells. The GTPases Sar1 and ARF1 regulate the endoplasmic reticulum (ER) export of proteins in COPII and COPI vesicles, respectively. Expression of DN Sar1 inhibited the Golgi processing of hERG, decreased hERG current (I_hERG) by 85% (n ≥ 8 cells per group, *, p < 0.01), and reduced the plasmalemmal staining of hERG. The coexpression of DN ARF1 had relatively small effects on hERG trafficking. Surprisingly, the coexpression of DN Rab11B, which regulates the endosomal recycling, inhibited the Golgi processing of hERG, decreased I_hERG by 79% (n ≥ 8 cells per group; *, p < 0.01), and reduced the plasmalemmal staining of hERG. These data suggest that hERG undergoes ER export in COPII vesicles and endosomal recycling prior to being processed in the Golgi. We conclude that hERG trafficking involves a pathway between the ER and endosomal compartments that influences expression in the plasmalemma.The human KCNH2 or ether-a-go-go related gene (hERG)³ encodes the voltage-gated K⁺ channel α-subunits that oligomerize to form the pore of the rapidly activating delayed rectifier K⁺ current (I_Kr) in cardiac myocytes (1–3). Hundreds of hERG mutations are linked to the congenital pro-arrhythmic Type 2 Long QT syndrome (LQT2) and functional studies suggest that these mutations result in a loss of normal hERG K⁺ channel (hERG) function (4, 5). In LQT2, missense mutations are the dominant abnormality and many LQT2 missense mutations reduce hERG K⁺ current (I_hERG) by decreasing the intracellular transport or trafficking of hERG to the Golgi apparatus (Golgi) and the cell surface membrane (plasmalemma) (6). Therefore, disruption of hERG K⁺ channel trafficking appears to be a principal mechanism for disease.Movement of proteins between membrane-bound intracellular compartments is mediated by small transport vesicles, which bud from a donor compartment to fuse with an appropriate acceptor compartment. The trafficking of many transmembrane and secretory proteins between the ER and Golgi compartments is dependent on the small GTPases ADP-ribosylation factor 1 (ARF1) and Sar1, which regulate the formation of coat-associated protein complex I (COPI) and II (COPII) vesicles, respectively (7–19). These small GTPases facilitate the polymerization of transport vesicle protein coats on the donor membrane. Vesicular cargo selection, docking, and fusion to the target membrane are regulated by adaptor proteins, SNARE proteins, and Rab GTPases. To rationally develop novel therapeutic targets that may increase the expression of trafficking-deficient LQT2 mutant channels, the molecular mechanisms that regulate the trafficking of hERG need to be explored. The purpose of this study is to identify transport proteins that regulate the trafficking of wild type (WT) hERG. We used a strategy of testing specific WT GTPases or ones containing dominant negative (DN) mutations to interfere with their function.     

Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome)

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.     

Predictive Models to Determine Imagery Strategies Employed by Children to Judge Hand Laterality

A commonly used paradigm to study motor imagery is the hand laterality judgment task. The present study aimed to determine which strategies young children employ to successfully perform this task. Children of 5 to 8 years old (N = 92) judged laterality of back and palm view hand pictures in different rotation angles. Response accuracy and response duration were registered. Response durations of the trials with a correct judgment were fitted to a-priori defined predictive sinusoid models, representing different strategies to successfully perform the hand laterality judgment task. The first model predicted systematic changes in response duration as a function of rotation angle of the displayed hand. The second model predicted that response durations are affected by biomechanical constraints of hand rotation. If observed data could be best described by the first model, this would argue for a mental imagery strategy that does not involve motor processes to solve the task. The second model reflects a motor imagery strategy to solve the task. In line with previous research, we showed an age-related increase in response accuracy and decrease in response duration in children. Observed data for both back and palm view showed that motor imagery strategies were used to perform hand laterality judgments, but that not all the children use these strategies (appropriately) at all times. A direct comparison of response duration patterns across age sheds new light on age-related differences in the strategies employed to solve the task. Importantly, the employment of the motor imagery strategy for successful task performance did not change with age.     

Reconstructed human epidermis for skin absorption testing: results of the German prevalidation study

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.     

Biosynthesis and maturation of alpha-N-acetylglucosaminidase in normal and Sanfilippo B-fibroblasts

The biosynthesis of alpha-N-acetylglucosaminidase in normal and Sanfilippo B fibroblasts was studied by labeling cells with [35S]methionine and isolation of the enzyme by immunoprecipitation. The immunoprecipitated polypeptides were separated by polyacrylamide gel electrophoresis and visualized by fluorography. alpha-N-acetylglucosaminidase is synthesized as a precursor of an apparent mol. wt. of 87,000. Intracellular processing of the precursor yields two polypeptides of apparent mol. wts. of 73,000 and 76,000 via several intermediates. It is accomplished within 3 days after synthesis. Less than 30% of the newly synthesized precursor is secreted. In the presence of 10 mM NH4Cl, secretion is enhanced to more than 80%. In our study, no alpha-N-acetylglucosaminidase polypeptides could be detected in fibroblasts from patients affected with either the severe or mild form of Sanfilippo disease, type B.     

Intercellular exchange of lysosomal enzymes: enzyme assays in single human fibroblasts after co-cultivation.

Intercellular exchange of N-acetyl-β-D-glucosaminidase (EC 3.2.1.30) β-galactosidase (EC 3.2.1.23) and acid α-glucosidase (EC 3.2.1.20) was studied after cocultivation of normal and enzyme deficient human fibroblasts in confluent cultures. Enzyme activities were measured in single cells using microchemical procedures. After co-cultivation of normal control fibroblasts and those from a patient with Sandhoff's disease an increase of activity of N-acetyl-β-D-glucosaminidase was found in Sandhoff cells, together with a decrease of activity in normal control cells. After co-cultivation of normal fibroblasts and those from patients with glycogenosis II and GM1-gangliosidosis, no indication was found for intercellular transfer of acid α-glucosidase and β-galactosidase respectively. The significance of the results is discussed in respect of the hypothesis of Hickman and Neufeld about secretion and uptake of lysosomal enzymes.     

Habitat Association in Two Genetic Groups of the Insect-Pathogenic Fungus Metarhizium anisopliae: Uncovering Cryptic Species?

Strains of insect-pathogenic fungi with high virulence toward certain pest insects have great potential for commercial biological control applications. Identifying such strains has been a central theme in using fungi for biological control. This theme is supported by a persistent paradigm in insect pathology which suggests that the host insect is the predominant influence on the population genetics of insect-pathogenic fungi. In this study, a population genetics analysis of the insect-pathogenic fungus Metarhizium anisopliae from forested and agricultural habitats in Ontario, Canada, showed a nonrandom association of alleles between two distinct, reproductively isolated groups (index of multilocus association = 1.2). Analyses of the mitochondrial DNA showed no differences between the groups. The two groups were associated with different habitat types, and associations with insect hosts were not found. The group from forested areas showed an ability for cold-active growth (i.e., 8°C), while the group from the agricultural area showed an ability for growth at high temperatures (i.e., 37°C) and resilience to UV exposure. These results represent a significant paradigm shift; habitat selection, not host insect selection, drives the population structure of these insect-pathogenic deuteromycetous fungi. With each group we observed recombining population structures as well as clonally reproducing lineages. We discuss whether these groups may represent cryptic species. Worldwide, M. anisopliae may be an assembly of cryptic species, each adapted to certain environmental conditions. The association of fungal genotypes with habitat but not with host insects has implications on the criteria for utility of this, and perhaps other, fungal biocontrol agents.     

Glycogenolysis during recovery from muscular work. The time course of phosphorylase activity is dependent on Pi concentration in the abdominal muscle of the shrimp Crangon crangon

1) Glycogen is degraded in the abdominal muscle of the shrimp Crangon crangon (Decapoda, Crustacea) during the recovery period following work. The regulation of post-exercise glycogen breakdown and the properties of glycogen phosphorylase (EC 2.4.1.1) have been studied: 2) Glycogen phosphorylase exists as unphosphorylated b-form and phosphorylated a-form, the latter contains 1 molecule phosphate/subunit. Both forms of phosphorylase are dimers, isoenzymes have not been detected. 3) The purified b-form is inactive in absence of AMP and has very low affinities for AMP and Pi. For half-maximum activation 0.33 +/- 0.04 mM AMP is necessary, and the Km-value for Pi at 1 mM AMP is 48 +/- 5 mM. IMP does not affect the activity of the b-form. 4) The a-form is active without effectors, its Km-value for Pi is 5.3 +/- 1.5 mM. The proportion of phosphorylase a increases in vivo, from about 25% at rest, to approximately 90% upon work and remains at this high level during the first minutes of recovery. 5) It is concluded that the glycogenolytic flux in the abdominal muscle of the shrimp even during post-exercise periods depends on the level of the a-form the activity of which is restricted in time and extent by the cytoplasmic Pi concentration (Kamp, G. & Juretschke, H. P. (1987) Biochim. Biophys. Acta 929, 121-127).     

The rate of uptake and efflux of phosphatidylcholine from human erythrocytes depends on the fatty acyl composition of the exchanging species

The rate of uptake of radioactive phosphatidylcholine molecules of different fatty acid composition in intact erythrocytes as facilitated by a phosphatidylcholine-specific transfer protein has been studied. When trace amounts of radiolabeled phosphatidylcholine molecules are present in donor vesicles consisting of egg phosphatidylcholine and cholesterol, the transfer of the radiolabeled species depends strongly on their fatty acyl composition: dipalmitoylphosphatidylcholine is transferred at the lowest rate, 1-saturated-2-unsaturated species are transferred faster and the highest rate is observed for dioleoyl phosphatidylcholine. Transfer of the various phosphatidylcholine molecules was measured furthermore using donor systems in which the bulk phosphatidylcholine was varied in its fatty acyl composition. Also in this type of experiment, the transfer protein preferentially stimulated transfer of unsaturated phosphatidylcholine molecules, especially from an environment containing more saturated molecules. Finally, the efflux of labeled phosphatidylcholine from intact erythrocytes to plasma in the absence of the phosphatidylcholine-specific transfer protein was studied and it became clear that in this case the nature of the effused molecules itself, rather than the composition of the bulk lipids, determined the effuse rates. An important conclusion to be drawn from these experiments is that radiolabeled phosphatidylcholine molecules, when used as markers for phospholipid exchange or transfer, should resemble in their fatty acid composition the composition of the bulk lipid in order to provide reliable data on rates and extents of the process studied.     

Molecular mechanisms of asbestos-induced lung epithelial cell apoptosis

Asbestos causes pulmonary fibrosis (asbestosis) and malignancies (bronchogenic lung cancer and mesothelioma) by mechanisms that are not fully elucidated. Accumulating evidence show that alveolar epithelial cell (AEC) apoptosis is a crucial initiating and perpetuating event in the development of pulmonary fibrosis following exposure to a wide variety of noxious stimuli, including asbestos. We review the important molecular mechanisms underlying asbestos-induced AEC apoptosis. Specifically, we focus on the role of asbestos in augmenting AEC apoptosis by the mitochondria- and p53-regulated death pathways that result from the production of iron-derived reactive oxygen species (ROS) and DNA damage. We summarize emerging evidence implicating the endoplasmic reticulum (ER) stress response in AEC apoptosis in patients with idiopathic pulmonary fibrosis (IPF), a disease with similarities to asbestosis. Finally, we discuss a recent finding that a mitochondrial oxidative DNA repair enzyme (8-oxoguanine DNA glycosylase; Ogg1) acts as a mitochondrial aconitase chaperone protein to prevent oxidant (asbestos and H₂O₂)-induced AEC mitochondrial dysfunction and intrinsic apoptosis. The coupling of mitochondrial Ogg1 to mitochondrial aconitase is a novel mechanism linking metabolism to mitochondrial DNA that may be important in the pathophysiologic events resulting in oxidant-induced toxicity as seen in tumors, aging, and respiratory disorders (e.g. asbestosis, IPF). Collectively, these studies are illuminating the molecular basis of AEC apoptosis following asbestos exposure that may prove useful for developing novel therapeutic strategies. Importantly, the asbestos paradigm is elucidating pathophysiologic insights into other more common pulmonary diseases, such as IPF and lung cancer, for which better therapy is required.