Prevalence of Lawsonia intracellularis in swine herds in Taiwan.
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Animals↗
Biomedical subjects
C F Wu
Publications and source records attributed to C F Wu.
Atrial natriuretic peptide induces apoptosis in neonatal rat cardiac myocytes.
Early heart failure is characterized by elevated plasma atrial natriuretic peptide (ANP) levels, but little is known about the direct effects of ANP on cardiac myocytes. In neonatal rat cardiac myocytes, ANP induced apoptosis in a dose-dependent and cell type-specific manner. Maximum effects occurred at 1 microM ANP, with a 4-5-fold increase in apoptotic cells, reaching a maximum apoptotic index of 19%. In contrast, the maximum apoptotic index of ANP-treated non-myocytes was 1.1 +/- 0.2%, equivalent to control cultures. ANP treatment also sharply reduced levels of Mcl-1 mRNA, a Bcl-2 homologue, coincident with the increase in the incidence of apoptosis. ANP induction of apoptosis was receptor-dependent and mediated by cyclic GMP: the effect was mimicked by 8-bromo-cGMP, a membrane-permeable analog, and by sodium nitroprusside, an activator of soluble guanylyl cyclase, and was potentiated by a cGMP-specific phosphodiesterase inhibitor, zaprinast. Interestingly, norepinephrine, a myocyte growth factor, inhibited ANP-induced apoptosis via activation of the beta-adrenergic receptor and elevation of cyclic AMP. These results show that ANP is a specific effector of cardiac myocyte apoptosis in culture via receptor-mediated elevation of cGMP. Furthermore, at least in this model, ANP and norepinephrine may have opposing roles in the modulation of cardiac myocyte growth and survival.
3',5'-Cyclic-GMP Phosphodiesterases↗
Alterations in frequency coding and activity dependence of excitability in cultured neurons of Drosophila memory mutants.
Mutants of the Drosophila dunce (dnc) and rutabaga (rut) genes, which encode a cAMP-specific phosphodiesterase and a calcium/calmodulin-responsive adenylyl cyclase, respectively, are deficient in short-term memory. Altered synaptic plasticity has been demonstrated at neuromuscular junctions in these mutants, but little is known about how their central neurons are affected. We examined this problem by using the "giant" neuron culture, which offers a unique opportunity to analyze mutational effects on neuronal activity and the underlying ionic currents in Drosophila. On the basis of instantaneous frequency and first latency of spikes evoked by current steps, four categories of firing patterns (tonic, adaptive, delayed, and interrupted) were identified in wild-type neurons, revealing interesting parallels to those commonly observed in vertebrate CNS neurons. The distinct firing patterns were correlated with expression of different ratios of 4-aminopyridine- and tetraethylammonium-sensitive K+ currents. Subsets of dnc and rut neurons displayed abnormal spontaneous spikes and altered firing patterns. Altered frequency coding in mutant neurons was demonstrated further by using stimulation protocols involving conditioning with previous activity. Abnormal spike activity and reduced K+ current remained in double-mutant neurons, suggesting that the opposite effects on cAMP metabolism by dnc and rut do not counterbalance the mutual functional defects. The aberrant spontaneous activity and altered frequency coding in different stimulus paradigms may present problems in the stability and reliability of neural circuits for information processing during certain behavioral tasks, raising the possibility of modulation in neuronal excitability as a cellular mechanism underlying learning and memory.
Action Potentials↗
Characterization and function of Xnf7 during early development of Xenopus.
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Animals↗
Mechanisms of natriuretic-peptide-induced growth inhibition of vascular smooth muscle cells.
OBJECTIVE: While natriuretic peptides can inhibit growth of vascular muscle cells (VSMC), controversy exists as to whether this effect is mediated via the guanylate cyclase-coupled receptors, NPR-A and NPR-B, or the clearance receptor, NPR-C. The original aim of this study was to examine the mechanism by which the NPR-C receptor regulates growth. METHODS: Rat VSMC were characterized with regard to natriuretic peptide receptor expression by RT/PCR and radioligand binding studies. The effect on growth following addition of the peptides and the ligands for NPR-C was measured by [3H]thymidine incorporation. Cyclic guanosine monophosphate (cGMP) levels were determined by radioimmunoassay and mitogen activating protein kinase activity was based on the phosphorylation of myelin basic protein. RESULTS: In rat VSMC, passages 4-12, both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) dose-dependently inhibited serum and PDGF-induced VSMC growth. In contrast, NPR-C specific ligands alone had no effect on cell growth but enhanced growth inhibition when co-administered with ANP and CNP. ANP and CNP also decreased PDGF-BB-stimulated MAP kinase activity. Once again, NPR-C specific ligands alone had no effect but enhanced the effects of ANP. Furthermore, a cGMP specific phosphodiesterase inhibitor dose-dependently inhibited VSMC growth and markedly enhanced natriuretic-peptide-induced inhibition at low peptide concentrations. To examine a potential mechanism for the controversy concerning the NPR-C, we investigated the autocrine expression of ANP and CNP by VSMC and found that mRNA encoding both peptides could be detected by RT/PCR. CONCLUSION: Our findings indicate that the guanyl-cyclase-linked receptors mediate the antiproliferative actions of the natriuretic peptides on vascular smooth muscle cell growth. Moreover, we hypothesize that the apparent inhibition of growth by NPR-C specific ligands reported by others may be due to stabilization of natriuretic peptides produced by the cultured VSMC and subsequent action of these peptides at guanyl-cyclase-linked receptors.
Animals↗
Nonradioactive method for the determination of internucleosomal cleavage associated with apoptosis.
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Animals↗
Morphometric description of the wandering behavior in Drosophila larvae: aberrant locomotion in Na+ and K+ channel mutants revealed by computer-assisted motion analysis.
Wandering is a simple behavior in Drosophila larvae prior to metamorphosis. Using the Dynamic Image Analysis System (DIAS) initially developed for analyzing amoeboic movements of single cells, we have analyzed videotaped behaviors of Drosophila larvae at the wandering stage. Previous studies show that mutations in the Na+ channel gene paralytic (para) cause paralysis at 29 degrees C, and mutations in the K+ channel beta subunit gene Hyperkinetic (Hk) lead to leg-shaking under ether anesthesia. The application of DIAS revealed quantifiable abnormalities in the larval locomotion of both ion channel mutants even under "permissive" conditions. Analysis of centroid movement indicates that, compared to wild type, both Hk and para larvae crawled at a slower average speed, but a similar peak instantaneous speed during a contraction cycle. Nevertheless, contraction in the body length was greater in mutants, implying a lower efficiency in conversion of muscular contraction to distance translocation. In addition, each mutant produced a characteristic crawling pattern distinct from the wild-type control. The larval crawling pattern was determined by periods of linear locomotion interposed by non-locomotive, "searching and decision-making" episodes, after which the crawling was resumed in a new direction. Our results demonstrate that mutations in single ion channel subunits resulted in stereotypic modifications in locomotion control and crawling patterns, and that DIAS is a powerful tool in revealing subtle differences in animal behavior and quantifying mutational effects on the interplay of discrete behavioral components.
Animals↗
Reduced growth cone motility in cultured neurons from Drosophila memory mutants with a defective cAMP cascade.
Drosophila memory mutants dunce (dnc) and rutabaga (rut) are known to have altered intracellular cAMP levels, nerve terminal growth, and plasticity of synaptic transmission. Because the growth cone is responsible for neurite outgrowth and synaptogenesis, video microscopy was used to examine growth cone morphology and behavior of mutant neurons in larval CNS cultures. We found that growth cone exploratory movement was nearly arrested by both mutations, even though they change cAMP levels in opposite directions. The dnc phenotype could be mimicked by normal neurons when perfused with dibutyryl cAMP (db-cAMP) or forskolin. In contrast, rut growth cones became active when perfused with db-cAMP. Furthermore, motility was also restored by counterbalancing the effects of the two genes in double mutants, indicating that dynamic control of growth cone motility in developing Drosophila neurons requires optimal cAMP levels within an operational range. These findings represent the first demonstration of altered growth cone properties in learning and memory mutants and establish in a natural setting the role of cAMP in growth cone motility and neuronal plasticity.
Animals↗
Altered habituation of an identified escape circuit in Drosophila memory mutants.
Genetic approaches in Drosophila have advanced our understanding of the molecular mechanisms of different forms of learning, including habituation, but relevant neural components have not been explored. We show that a well defined neural circuit that underlies an escape response can be habituated, providing for the first time excellent opportunities for studying physiological parameters of learning in a functional circuit in the fly. Compared with other forms of conditioning, relatively little is known of the physiological mechanisms of habituation. The giant fiber pathway mediates a jump-and-flight escape response to visual stimuli. The jump may also be triggered electrically at multiple sites in the tethered fly. This response shows parameters of habituation, including frequency-dependent decline in responsiveness, spontaneous recovery, and dishabituation by a novel stimulus, attributable to plasticity in the brain. Mutations of rutabaga that diminish cAMP synthesis reduced the rate of habituation, whereas dunce mutations that increase cAMP levels led to a detectable but moderate increase in habituation rates. Surprisingly, habituation was extremely rapid in dunce rutabaga double mutants. This corresponds to the extreme defects seen in double mutants in other learning tasks, and demonstrates that defects of the rutabaga and dunce products interact synergistically in ways that could not have been predicted on the basis of simple counterbalancing biochemical effects. Although habituation is localized to afferents to the giant fiber, cAMP mutations also affected performance of thoracic portions of the pathway on a millisecond time scale that did not account for behavioral plasticity. More significantly, spontaneous recovery and dishabituation were not as clearly affected as habituation in mutants, indicating that these processes may not overlap entirely in terms of cAMP-regulating mechanisms. The analysis of habituation of the giant fiber response in available learning and memory mutants could be a crucial step toward realizing the promise of memory mutations to elucidate mechanisms in neural circuits that underlie behavioral plasticity.
Animals↗
In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels.
The physiological roles of the beta, or auxiliary, subunits of voltage-gated ion channels, including Na+, Ca2+, and K+ channels, have not been demonstrated directly in vivo. Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel beta subunit, providing a unique opportunity to delineate the in vivo function of auxiliary subunits in K+ channels. We found that the Hk beta subunit modulates a wide range of the Shaker (Sh) K+ current properties, including its amplitude, activation and inactivation, temperature dependence, and drug sensitivity. Characterizations of the existing mutants in identified muscle cells enabled an analysis of potential mechanisms of subunit interactions and their functional consequences. The results are consistent with the idea that via hydrophobic interaction, Hk beta subunits modulate Sh channel conformation in the cytoplasmic pore region. The modulatory effects of the Hk beta subunit appeared to be specific to the Sh alpha subunit because other voltage- and Ca(2+)-activated K+ currents were not affected by Hk mutations. The mutant effects were especially pronounced near the voltage threshold of IA activation, which can disrupt the maintenance of the quiescent state and lead to the striking neuromuscular and behavioral hyperexcitability previously reported.
4-Aminopyridine↗
Heteromultimeric interactions among K+ channel subunits from Shaker and eag families in Xenopus oocytes.
Heteromultimeric interactions of K+ channel subunits across different families have been thought to contribute to the functional diversity of ionic currents, as suggested by previous genetic evidence. We present here direct demonstration in Xenopus oocytes that subunits from distinct eag and Shaker families functionally interact, most likely as heteromultimeric channels. Coexpression with eag accelerates the inactivation and slows the recovery from inactivation of the transient Shaker current. Site-directed mutagenesis indicates that the eag carboxyl terminus is crucial for this interaction, exerting effects preferentially on N-type inactivation. Many members of the eag and Shaker families have now been identified and their human homologs implicated in cardiac and neurological disorders. Studies on channel subunit interactions may prove important in understanding the disease pattern and the complex functions of the brain.
Alternative Splicing↗
K252a-potentiation of EGF-induced neurite outgrowth from PC12 cells is not mimicked or blocked by other protein kinase activators or inhibitors.
Epidermal growth factor (EGF) has recently been shown to cause certain strains of PC12 cells to extend short neurites. This EGF-induced differentiation of PC12 was found to be potentiated by the protein kinase inhibitor, K252a, in that PC12 cells treated with both EGF and K252a extended long branched neurites similar to those induced by nerve growth factor (NGF). As reported here no other protein kinase inhibitor or activator mimicked or blocked the effect of K252a on EGF-induced PC12 differentiation. Cyclic adenosine 3',5'-monophosphate (cAMP) also potentiated EGF-induced neurite outgrowth from PC12 cells, but the mechanism of this potentiation was different from that of K252a. Cells that had been exposed to EGF and then stripped of their neurons extended neurites again when retreated with EGF in the absence of RNA synthesis or when treated with NGF in the absence of RNA synthesis. Thus EGF can prime PC12 cells for either EGF or for NGF, a finding that further suggests that EGF and NGF use similar signaling pathways to induced neuronal differentiation of PC12.
Animals↗
Two isoforms of Drosophila dynamin in wild-type and shibire(ts) neural tissue: different subcellular localization and association mechanisms.
The temperature-sensitive mutations of the shibire (shi) gene in Drosophila cause endocytic arrest, resulting in neurotransmission block and paralysis at high temperatures. However, underlying mechanism for the defects is not yet known. We examined the subcellular distribution of dynamin, a product of the shi gene, by immunoblotting and immunocytochemical assays. Two isoforms of dynamin with apparent M(r) of 92 kD and 94 kD have been detected in wild-type and shi(n) adult neural tissue. The two isoforms were reproducibly associated with different subcellular fractions of head homogenates. The 94kD isoform is fractionated in the low speed (2.000 x g) pellet containing plasma membrane fragments, and the 92kD isoform in the high speed (130,000 x g) pellet. In this procedure, very little dynamin remained in the high speed supernatant fraction. The 94 kD isoform represents the majority (65-75%) of total dynamin and appears to be a peripheral membrane protein. It can be extracted from the low speed membrane pellet by high salt, Na2CO3 (pH 11) or Triton X-100 treatments. Extracted 94kD dynamin from both wild-type and mutant homogenates is able to reassociate with artificial phospholipid vesicles at both permissive and restrictive temperatures. Binding of the 94 kD dynamin to liposomes appears to be pH-dependent, varying most significantly within the physiological pH range, which may be functionally important. The 92 kD isoform cannot be released by high salt or Na2CO3 treatments and only a small fraction is released by Triton X-100, suggesting a different mechanism of association with cell structures. The distribution of the two isoforms is not altered by the presence of stabilized microtubules in homogenates. No apparent degradation or subcellular redistribution of mutant dynamin was detected in two shi(n) alleles after heat shock or block of the dynamin GTPase activity, suggesting that intracellular redistribution or degradation of mutant dynamin are not involved in the endocytosis arrest in these mutants. These observations resemble the effect of endocytosis arrest by GTP-gamma-S in rat brain synaptosomes (Takei et al., 1995), in which dynamin is trapped at the neck of invaginated pits but is absent in the clathrin-coated distal end undergoing internalization. Our finding that endocytosis arrest by shi(n) mutations and GTP-gamma-S do not lead to cumulation of dynamin in the low speed pellet fraction further suggests that the 94 kD isoform remains associated with the plasma membrane during coated vesicle pinch-off and that the two isoforms do not appear to correspond to different functional states of dynamin but are likely to be involved in separate cellular compartments within the membrane cycling pathway (e.g., the plasma membrane, endosomes, and endoplasmic reticulum).
Amino Acid Sequence↗
Functional expression of Shaker K+ channels in cultured Drosophila "giant" neurons derived from Sh cDNA transformants: distinct properties, distribution, and turnover.
Expression of transgenic Shaker (Sh) channels has not previously been examined in Drosophila neurons. We studied K+ current by whole-cell recording in cultured "giant" neurons derived from germline transformants. Independent lines were generated by using a P-element vector, in which transcription of the 29-4 cDNA, one of the Sh splicing variants (Iverson and Rudy, 1990), was under the control of a heat shock (HS)-inducible promoter. Transformants in wild-type and two different Sh mutant backgrounds all exhibited an HS-inducible, A-type K+ current that was characterized by a much slower recovery from inactivation and a higher sensitivity to 4-aminopyridine than native K+ currents of Sh 29-4 currents expressed in Xenopus oocytes. Despite similarities in the kinetic and pharmacological properties of the HS-induced current in all backgrounds examined, host-dependent differences in the peak current amplitude have been consistently observed between multiple lines of 29-4 ShM and 29-4 Sh120 that might reflect differential channel subunit assembly in different hosts. Isolation of the novel 29-4 currents allowed determination of the channel turnover rate in cultured neurons. These currents persisted for up to 3 d or more, comparable with the durations previously reported for Na+ and Ca2+ channels. Surprisingly, the percentage of cells expressing inactivating K+ currents remained approximately the same with or without HS induction, suggesting that some mechanisms exist to restrict functional expression of inactivating K+ channels, including transgenic Sh channels and those not encoded by the Sh locus, to certain types of neurons.
4-Aminopyridine↗
Co-assembly of potassium channel subunits in Drosophila: the combinatorial hypothesis revisited.
A diversity of voltage-gated K+ channels has been identified which participates in the regulation of membrane excitability and neural plasticity. After decades of intensive molecular, genetic, pharmacological and electrophysiological studies, the structure and function of K+ channels have been elucidated in great detail. In particular, impressive progress has been made in recent years from studies in Drosophila and the Xenopus oocyte expression system. This review will focus on the variety and physiological functions of K+ channels found in Drosophila. Also discussed is the possible role of coassembly of different combinations of channel subunits in the generation of K+ channel diversity. Various lines of evidence from in situ experiments in Drosophila and heterologous channel expression in Xenopus oocytes will be reviewed.
Animals↗
Differential physiology and morphology of motor axons to ventral longitudinal muscles in larval Drosophila.
Morphological and physiological characteristics of the two major motor axons supplying the commonly studied ventral longitudinal muscle fibers (6 and 7) of third-instar Drosophila melanogaster larvae were investigated. The innervating terminals of the two motor axons differ in the size of their synapse-bearing varicosities. The terminal with the larger varicosities also fluoresces more brightly when stained with the vital fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-Asp) and occupies a larger total contact area on the muscle fiber. Through selective simultaneous recording of synaptic currents from identified boutons in living preparations during elicitation of synaptic potentials, it was shown that the axon with the smaller varicosities generates a large excitatory junction potential (EJP) in muscle 6 and that the axon with the larger varicosities generates a smaller EJP. Short-term facilitation is more pronounced for the smaller EJP. In preparations treated with 4-Di-2-Asp, the fluorescence of smaller varicosities increases with stimulation that elicits the large EJPs, indicating an activity-dependent entry of calcium that enhances mitochondrial fluorescence. The differences in morphology and physiology of the two axons are similar to, though less pronounced than, those observed in "phasic" and "tonic" motor axons of crustaceans.
Animals↗
Calcium/calmodulin-dependent protein kinase II and potassium channel subunit eag similarly affect plasticity in Drosophila.
Similar defects in both synaptic transmission and associative learning are produced in Drosophila melanogaster by inhibition of calcium/calmodulin-dependent protein kinase II and mutations in the potassium channel subunit gene eag. These behavioral and synaptic defects are not simply additive in animals carrying both an eag mutation and a transgene for a protein kinase inhibitor, raising the possibility that the phenotypes share a common pathway. At the molecular level, a portion of the putative cytoplasmic domain of Eag is a substrate of calcium/calmodulin-dependent protein kinase II. These similarities in behavior and synaptic physiology, the genetic interaction, and the in vitro biochemical interaction of the two molecules suggest that an important component of neural and behavioral plasticity may be mediated by modulation of Eag function by calcium/calmodulin-dependent protein kinase II.
Amino Acid Sequence↗
Altered mechanoreceptor response in Drosophila bang-sensitive mutants.
Bang-sensitive mutants of Drosophila melanogaster (bas1, bssMW1, eas2, tko25t) display seizure followed by paralysis when subjected to mechanical shock. However, no physiological or biochemical defect has been found to be common to all of these mutants. In order to observe the effects of bang-sensitive mutations upon an identified neuron, and to study the nature of mechanically induced paralysis, we examined the response of a mechanosensory neuron in these mutants. In each single mutant and the double mutant bas1 bssMW1, the frequency of action potentials in response to a bristle displacement was reduced. This is the first demonstration of a physiological defect common to several of the bang-sensitive mutations. Adaptation of spike frequency, cumulative adaptation to repeated stimulation (fatigue) and the time course of recovery from adaptation were also examined. Recovery from adaptation to a conditioning stimulus was examined in two mutants (bas1 and bssMW1), and initial recovery from adaptation was greater in both mutants. Quantification of receptor potentials was complicated by variability inherent in extracellular recording conditions, but examination of the waveform and range of amplitudes did not indicate clear mutant defects. Therefore the differences observed in the spike response may be due to an alteration of the transfer from receptor potentials to action potential production. DNA sequence analysis of tko and eas has indicated that they encode apparently unrelated biochemical products. Our results suggest that these biochemical lesions lead to a common physiological defect in mechanoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Adaptation, Physiological↗