Biomedical subjects

C F Wu

Publications and source records attributed to C F Wu.

At least 19 recordsLinked to original sources

PubMed2001

Enhancement of organophosphorus hydrolase yield in Escherichia coli using multiple gene fusions.

It was previously shown that organophosphorus hydrolase (OPH) expression and purification could be tracked by fluorescence of green fluorescent protein (GFP) when synthesized as an N-terminal fusion with GFP (Cha et al., 2000; Wu et al., 2000). In order to enhance OPH productivity while utilizing the advantage of the reporter protein (GFP), two copies of OPH were cloned in tandem following the gfp(uv) gene (e.g., GFP-OPH(n=2)). Both anti-GFP and anti-OPH Western blots demonstrated that a higher yield was achieved in comparison to the one copy fusion (GFP-OPH). Importantly, the fusion protein was still fluorescent as determined via microscopy. In contrast, a fusion containing two copies of OPH without GFP, and an operon fusion of two OPHs with two independent ribosomal binding sites, did not result in a higher yield than one OPH expressed alone.

Artificial Gene Fusion↗

PubMed2001

Pseudoginsenoside-F11 attenuates morphine-induced signalling in Chinese hamster ovary-mu cells.

Pseudoginsenoside-F11 (PF11), an ocotillol type saponin isolated from Panax quinquefolium L., has been shown to antagonize the behavioral actions of morphine. Biochemical experiments revealed that PF11 could inhibit diprenorphine (DIP) binding with an IC50 of approximately 6.1 microM and reduced the binding potency of morphine in Chinese hamster ovary (CHO)-mu cells. Furthermore, PF11 significantly attenuated morphine-stimulated [35S]GTPgammaS binding in a dose dependent manner, and strongly decreased the efficacy of morphine to inhibit intracellular cAMP production. In addition, PF11 pretreatment could also significantly inhibit naloxone induced cAMP overshoot in the morphine-pretreated cells. However, PF11 per se had no effect on either [35S]GTPgammaS binding or intracellular cAMP accumulation. These data suggested that PF11 antagonized the morphine stimulated opioid receptor signalling directly at the cellular level.

Adenylyl Cyclases↗

PubMed2001

Sacral parasite conjoined twin.

BACKGROUND: A variant of conjoined twins is one in which one twin is incomplete. CASE: A female infant was born vaginally at 40 weeks' gestation to a healthy primipara. No important abnormalities were noted during prenatal examinations. The infant was fully developed in all external aspects except for a parasitic body conjoined with her sacrococcygeal region. Separated by operation 2 weeks after birth, the parasite contained lower limbs, adipose tissue, muscles, and a bowel sac. Over 4 years of observation, no abnormalities have been found since the operation. CONCLUSION: Obstetricians should be aware of the existence of a parasite twin during prenatal examinations and of the importance of the differential diagnosis of parasite and teratoma, a neoplasm with malignant potential.

Diagnosis, Differential↗

PubMed2001

Reversal of morphine-induced memory impairment in mice by withdrawal in Morris water maze: possible involvement of cholinergic system.

The effects of morphine and morphine withdrawal on memory performance were examined in mice by using Morris water maze task. Morphine-induced memory impairment at the doses of 5 and 10 mg/kg recovered after repeated administration. Oxotremorine, a muscarinic receptor agonist, at the dose of 0.1 mg/kg ip, and physostigmine, a cholinesterase inhibitor, at the dose of 0.1 mg/kg ip, significantly antagonized morphine (10 mg/kg sc)-induced memory impairment in mice. Furthermore, repeated naloxone (0.5 mg/kg ip) attenuated scopolamine (0.2 mg/kg ip)-induced memory impairment. By using escalating doses of morphine for 13 days, morphine-induced memory impairment was continuously maintained. When withdrawal was precipitated by naloxone (5 mg/kg ip), or administration of oxotremorine (0.1 and 0.2 mg/kg ip) or physostigmine (0.05 and 0.1 mg/kg ip), the impairment was completely reversed. These results suggest that morphine-induced memory impairment could be partially due to the inhibition of the central cholinergic activity.

Animals↗

PubMed2001

Combinatorial screening for enzyme-mediated coupling. Tyrosinase-catalyzed coupling to create protein--chitosan conjugates.

In nature, tyrosinase-generated o-quinones are commonly involved in processes that lead to functional biomaterials. These biomaterials are chemically complex and have been difficult to analyze. Furthermore, the cascade of reactions involving o-quinones is poorly understood, and it has been difficult to mimic ex vivo for materials processing. We report the use of a combinatorial approach to learn how tyrosinase and low molecular weight phenolic precursors can be used to generate biologically active protein-polysaccharide conjugates. Specifically, we screened various phenolic coupling precursors and various reaction conditions for the coupling of proteins onto the polysaccharide chitosan. Several natural phenols were identified as appropriate precursors for the coupling of polyhistidine tagged organophosphorus hydrolase (His-OPH) onto chitosan films. OPH activity was retained upon coupling and subsequent studies indicated that the histidine tag was not necessary for coupling. Using conditions identified for His-OPH coupling, we observed that various biologically active proteins (cytochrome c, OPH, and His-CAT) could be coupled onto chitosan films. The glycosylated protein horseradish peroxidase was not effectively coupled onto chitosan under the conditions studied. In all cases studied, we observed that coupling required a phenolic precursor, suggesting that tyrosinase is unable to couple by reaction with surface tyrosyl residues of the target protein. In conclusion, this study illustrates a combinatorial approach for the "discovery" of conditions to couple biologically active proteins onto chitosan through natural, quinone-based processes.

Biocompatible Materials↗

PubMed2001

DNA microarray-based identification of genes controlled by autoinducer 2-stimulated quorum sensing in Escherichia coli.

Bacterial cell-to-cell communication facilitates coordinated expression of specific genes in a growth rate-II and cell density-dependent manner, a process known as quorum sensing. While the discovery of a diffusible Escherichia coli signaling pheromone, termed autoinducer 2 (AI-2), has been made along with several quorum sensing genes, the overall number and coordination of genes controlled by quorum sensing through the AI-2 signal has not been studied systematically. We investigated global changes in mRNA abundance elicited by the AI-2 signaling molecule through the use of a luxS mutant that was unable to synthesize AI-2. Remarkably, 242 genes, comprising ca. 5.6% of the E. coli genome, exhibited significant transcriptional changes (either induction or repression) in response to a 300-fold AI-2 signaling differential, with many of the identified genes displaying high induction levels (more than fivefold). Significant induction of ygeV, a putative sigma(54)-dependent transcriptional activator, and yhbH, a sigma(54) modulating protein, suggests sigma(54) may be involved in E. coli quorum sensing.

Amino Acid Sequence↗

PubMed2001

Distinct roles of CaMKII and PKA in regulation of firing patterns and K(+) currents in Drosophila neurons.

The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the cAMP-dependent protein kinase A (PKA) cascades have been implicated in neural mechanisms underlying learning and memory as supported by mutational analyses of the two enzymes in Drosophila. While there is mounting evidence for their roles in synaptic plasticity, less attention has been directed toward their regulation of neuronal membrane excitability and spike information coding. Here we report genetic and pharmacological analyses of the roles of PKA and CaMKII in the firing patterns and underlying K(+) currents in cultured Drosophila central neurons. Genetic perturbation of the catalytic subunit of PKA (DC0) did not alter the action potential duration but disrupted the frequency coding of spike-train responses to constant current injection in a subpopulation of neurons. In contrast, selective inhibition of CaMKII by the expression of an inhibitory peptide in ala transformants prolonged the spike duration but did not affect the spike frequency coding. Enhanced membrane excitability, indicated by spontaneous bursts of spikes, was observed in CaMKII-inhibited but not in PKA-diminished neurons. In wild-type neurons, the spike train firing patterns were highly reproducible under consistent stimulus conditions. However, disruption of either of these kinase pathways led to variable firing patterns in response to identical current stimuli delivered at a low frequency. Such variability in spike duration and frequency coding may impose problems for precision in signal processing in these protein kinase learning mutants. Pharmacological analyses of mutations that affect specific K(+) channel subunits demonstrated distinct effects of PKA and CaMKII in modulation of the kinetics and amplitude of different K(+) currents. The results suggest that PKA modulates Shaker A-type currents, whereas CaMKII modulates Shal-A type currents plus delayed rectifier Shab currents. Thus differential regulation of K(+) channels may influence the signal handling capability of neurons. This study provides support for the notion that, in addition to synaptic mechanisms, modulations in spike activity patterns may represent an important mechanism for learning and memory that should be explored more fully.

Action Potentials↗

PubMed2001

Tumorhead, a Xenopus gene product that inhibits neural differentiation through regulation of proliferation.

Tumorhead (TH) is a novel maternal gene product from Xenopus laevis containing several basic domains and a weak coiled-coil. Overexpression of wild-type TH resulted in increased proliferation of neural plate cells, causing expansion of the neural field followed by neural tube and craniofacial abnormalities. Overexpressed TH protein repressed neural differentiation and neural crest markers, but did not inhibit the neural inducers, pan-neural markers or mesodermal markers. Loss of function by injection of anti-TH antibody inhibited cell proliferation. Our data are consistent with a model in which tumorhead functions in regulating differentiation of the neural tissues but not neural induction or determination through its effect on cell proliferation.

Amino Acid Sequence↗

PubMed2001

Minilaparotomy for internal drainage of a symptomatic lymphocele after renal transplantation.

Development of a lymphocele after renal transplantation is a well-described complication that occurs with relative frequency. Management options include simple aspiration with sclerotherapy and operative marsupialization of the lymphoceles into the peritoneal cavity. Laparoscopic internal drainage has been favored in recent reports. Still, the laparoscopic procedure is associated with a number of potential problems, including difficulty in localizing lymphoceles if laparoscopic ultrasound is unavailable. In addition, lobulated lymphoceles are more difficult to completely dissect with laparoscopy, and lymphoceles always occur on the anterior and medial aspects of the graft which are easily approached directly from the anterior skin. We tried to resolve these complications by modifying traditional laparotomy and proved that laparoscopy is a progressive, popular procedure, and that fenestration with finger dissection of the lobulated lymphoceles through minilaparotomy is still a reliable, effective procedure.

Adult↗

PubMed2000

A novel leg-shaking Drosophila mutant defective in a voltage-gated K(+)current and hypersensitive to reactive oxygen species.

1,1'-Dimethyl-4,4'-bipyridinium dichloride (methyl viologen; paraquat), an herbicide that causes depletion of NADPH and generates excessive reactive oxygen species (ROS) in vivo, has been used to screen for ROS-sensitive Drosophila mutants. One mutant so isolated, named quiver(1) (qvr(1)), has a leg-shaking phenotype. Mutants of the Shaker (Sh), Hyperkinetic (Hk), and ether a go-go (eag) genes, which encode different K(+) channel subunits that regulate the A-type K(+) current (I(A)) in different ways, exhibit leg shaking under ether anesthesia and have heightened metabolic rates and shortened life spans. We found that Sh, Hk, and eag mutant flies were all hypersensitive to paraquat. Double-mutant combinations among the three channel mutations and qvr(1) had drastically enhanced sensitivity to paraquat. Synaptic transmission at the larval neuromuscular junction was increased in the qvr(1) mutant to the level of Sh mutants. Similar to eag Sh double mutants, double mutants of eag and qvr(1) showed striking enhancement in synaptic transmission and a wings-down phenotype, the hallmarks of extreme hyperexcitability. Voltage-clamp experiments demonstrated that the qvr(1) mutation specifically disrupted the Sh-dependent I(A) current without altering the other currents [I(K), Ca(2+)-activated fast (I(CF)) and slow (I(CS)) currents, and I(Ca)] in larval muscles. Several deficiency strains of the qvr locus failed to complement qvr(1) and confirmed that ether-induced leg shaking, reduced I(A) current, and paraquat hypersensitivity map to the same locus. Our results suggest that the qvr gene may encode a novel K(+) channel-related polypeptide and indicate a strong link between a voltage-activated K(+) current and vulnerability to ROS.

Animals↗

PubMed2000

Opposite effects of sulpiride and SCH 23390 on ethanol-induced striatal ascorbic acid release in intact and 6-hydroxydopamine lesioned rats.

The effects of L-sulpiride and SCH 23390 on ethanol-induced striatal ascorbic acid (AA) release in normal and 6-hydroxydopamine-lesioned rats were studied by using microdialysis coupled to high performance liquid chromatography with electrochemical detection. Ethanol (3.0 g/kg i.p.) significantly stimulated striatal AA release by 200% above the baseline in normal, 6-hydroxydopamine-lesioned, and reserpine-treated rats. L-Sulpiride, a dopamine D(2) antagonist, at the dose of 100 mg/kg i.p., decreased basal ascorbic acid release and showed an inhibitory tendency on ethanol-induced ascorbic acid release. However, at the higher dose of 200 mg/kg i.p., L-sulpiride significantly inhibited ethanol-induced ascorbic acid release in both normal and 6-hydroxydopamine-treated rats. SCH 23390, a dopamine D(1) antagonist, at the doses of 0.5 and 1.0 mg/kg i.p., potentiated ethanol-induced ascorbic acid release in normal rats. However, the potentiation of SCH 23390 on ethanol effect was not significant in 6-hydroxydopamine-treated rats at the dose of 1.0 mg/kg i.p. The present study demonstrates that opposite actions exist in the regulation of ethanol-induced ascorbic acid release in the striatum by dopamine D(1) and D(2) receptor blockade. It also suggests that the postsynaptic dopamine receptors are involved in mediation of ethanol-induced ascorbic acid release in rat striatum.

Animals↗

PubMed2000

Differential effects of acute administration of haloperidol and clozapine on ethanol-induced ascorbic acid release in rat striatum.

Antipsychotic drugs were initially considered to act predominantly through their antagonism at dopamine D(2)-like receptors. However, reports have demonstrated that the typical neuroleptic drug haloperidol and the atypical neuroleptic drug clozapine showed differential actions in clinical, behavioral and biochemical studies. Since ascorbic acid has a potential usefulness in psychological therapeutics, the present study investigates the actions of these two drugs on ethanol-induced ascorbic acid release in the striatum in order to help explain the different mechanisms of these drugs. The results showed that clozapine, at the doses of 15 and 30 mg/kg, i.p., had no effect on basal ascorbic acid release. However, a synergistic tendency at a dose of 15 mg/kg and a significant synergism at a dose of 30 mg/kg were observed on ascorbic acid release when clozapine was used with ethanol. In contrast, haloperidol, at the doses of 0.5, 1.0 and 2.0 mg/kg, i.p., administered alone did not affect the basal release of striatal ascorbic acid, and when used together with ethanol had neither a potentiating nor an antagonizing effect on ethanol-induced ascorbic acid release. Chlorpromazine, a nonselective dopamine receptor antagonist, at the dose of 5 mg/kg, i.p., affected neither the basal nor the ethanol-induced ascorbic acid release. Ritanserin, a 5-HT(2) receptor antagonist, at the dose of 1 mg/kg, s.c., significantly antagonized ethanol-induced ascorbic acid release. These results demonstrate that clozapine dose-dependently potentiates the stimulatory effect of ethanol on striatal ascorbic acid release and this effect of clozapine may not be related to its dopamine D(2) receptor antagonism.

Animals↗

PubMed2000

Role of cAMP cascade in synaptic stability and plasticity: ultrastructural and physiological analyses of individual synaptic boutons in Drosophila memory mutants.

Mutations of the genes rutabaga (rut) and dunce (dnc) affect the synthesis and degradation of cAMP, respectively, and disrupt learning in Drosophila. Combined ultrastructural analysis and focal electrophysiological recording in the larval neuromuscular junction revealed a loss of stability and fine tuning of synaptic structure and function in both mutants. Increased ratios of docked/undocked vesicles and poorly defined synaptic specializations characterized dnc synapses. In contrast, rut boutons possessed fewer, although larger, synapses with lower proportions of docked vesicles. At reduced Ca(2+) levels, decreased quantal content coupled with an increase in failure rate was seen in rut boutons and reduced pair-pulse facilitation were found in both rut and dnc mutants. At physiological Ca(2+) levels, strong enhancement, instead of depression, in evoked release was observed in some dnc and rut boutons during 10 Hz tetanus. Furthermore, increased variability of synaptic transmission, including fluctuation and asynchronicity of evoked release, paralleled an increase in synapse size variation in both dnc and rut boutons, which might impose problems for effective signal processing in the nervous system. Pharmacological and genetic studies indicated broader ranges of physiological alteration by dnc and rut mutations than either the acute effects of cAMP analogs or the available mutations that affect cAMP-dependent protein kinase (PKA) activity. This is consistent with previous reports of more severe learning defects in dnc and rut mutations than these PKA mutants and allows identification of the phenotypes involving long-term developmental regulation and those conferred by PKA.

Alleles↗

PubMed2000

Spontaneous acetylcholine secretion from developing growth cones of Drosophila central neurons in culture: effects of cAMP-pathway mutations.

We describe a novel bioassay system that uses Xenopus embryonic myocytes (myoballs) to detect the release of acetylcholine from Drosophila CNS neurons. When a voltage-clamped Xenopus myoball was manipulated into contact with cultured Drosophila "giant" neurons, spontaneous synaptic current-like events were registered. These events were observed within seconds after contact and were blocked by curare and alpha-bungarotoxin, but not by TTX and Cd(2+), suggesting that they are caused by the spontaneous quantal release of acetylcholine (ACh). The secretion occurred not only at the growth cone, but also along the neurite and at the soma, with significantly different release parameters among various regions. The amplitude of these currents displayed a skewed distribution. These features are distinct from synaptic transmission at more mature synapses or autapses formed in this culture system and are reminiscent of the transmitter release process during early development in other preparations. The usefulness of this coculture system in studying presynaptic secretion mechanisms is illustrated by a series of studies on the cAMP pathway mutations, dunce (dnc) and PKA-RI, which disrupt a cAMP-specific phosphodiesterase and the regulatory subunit of cAMP-dependent protein kinase A, respectively. We found that these mutations affected the ACh current kinetics, but not the quantal ACh packet, and that the release frequency was greatly enhanced by repetitive neuronal activity in dnc, but not wild-type, growth cones. These results suggest that the cAMP pathway plays an important role in the activity-dependent regulation of transmitter release not only in mature synapses as previously shown, but also in developing nerve terminals before synaptogenesis.

Acetylcholine↗

PubMed2000

Observations of green fluorescent protein as a fusion partner in genetically engineered Escherichia coli: monitoring protein expression and solubility.

We have constructed three plasmid vectors for the expression of green fluorescent protein (GFP) fusion proteins using the following motif: (His)(6)-GFP-EK-X, where X represents chloramphenicol acetyl-transferase (CAT), human interleukin-2 (hIL-2), and organophosphorous hydrolase (OPH), respectively, (His)(6) represents a histidine affinity ligand for purification, and EK represents an enterokinase cleavage site for recovering the protein-of-interest from the fusion. The CAT and OPH fusion products ( approximately 63 kDa GFP/CAT and approximately 70 kDa GFP/OPH) were expressed at 4.85 microg/mL (19.9 microg/mg-total protein) and 1.42 microg/mL (4.2 microg/mg-total protein) in the cell lysis supernatant, and, in both cases, enzymatic activity was retained while coupled to GFP. In the case of hIL-2 fusion ( approximately 52 kDa), however, the GFP fluorescence was significantly reduced and most of the fusion was retained in the cell pellet. Linear relationships between GFP fluorescence and CAT or OPH concentration, and with enzymatic activity of CAT or OPH, indicated, for the first time, that in vivo noninvasive quantification of proteins-of-interest, was made possible by simple measurement of GFP fluorescence intensity. The utility of GFP as a reporter was not realized without disadvantages however, in particular, an incremental metabolic cost of GFP was found. This could be offset by many benefits foreseen in expression and purification efficiencies.

Chloramphenicol O-Acetyltransferase↗

PubMed2000

Isolation and characterization of mutants for the vesicular acetylcholine transporter gene in Drosophila melanogaster.

The Drosophila vesicular acetylcholine transporter gene (Vacht) is nested within the first intron of the choline acetyltransferase gene (Cha). To isolate Vacht mutants, we performed an F(2) genetic screen and identified mutations that failed to complement Df(3R)Cha(5), a deletion lacking Cha and the surrounding genes. Of these mutations, three mapped to a small genomic region where Cha resides. Complementation tests with a Cha mutant allele and rescue experiments using a transgenic Vacht minigene have revealed that two of these three mutations are nonconditional lethal alleles of Vacht (Vacht(1) and Vacht(2) ). The other is a new temperature-sensitive allele of Cha (Cha(ts3) ). Newly isolated Vacht mutants were used to reexamine the existing Cha mutations. We found that all deficiencies uncovering Cha also lack Vacht function, reflecting the nested organization of the two genes. The effective lethal phase for Vacht(1) is the embryonic stage, whereas that for Vacht(2) is the larval stage. Viable first-instar larvae homozygous for Vacht(2) showed reduced motility. Adult flies heterozygous for Vacht mutations were found to have defective responses in the dorsal longitudinal muscles following high-frequency brain stimulation. Since cholinergic synapses have been shown to be involved in the giant fiber pathway that mediates this response, the result suggested that reduction in the Vacht activity to 50% causes an abnormality in cholinergic transmission when stressed by a high-frequency stimulus.

Alleles↗

PubMed2000

A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase.

Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of the gene for green fluorescent protein (GFP) to the 5' end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP. Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations with inexpensive instrumentation based on detecting green fluorescence.

Aryldialkylphosphatase↗

PubMed2000

Sh and eag K(+) channel subunit interaction in frog oocytes depends on level and time of expression.

Subcellular clustering of ion channels critically affects neuronal function. Coexpression of Eag and Sh channel subunits in Xenopus oocytes leads to accelerated decay of the Sh-like transient K(+) current (Chen, M.-L., T. Hoshi, and C.-F. Wu. 1996. Neuron. 17:535-542). We report that such interaction depends critically on functional expression level (controlled by RNA injection quantities and indicated by current amplitudes) and developmental time after RNA injection. The accelerated decay became apparent 3 days after coinjection and increased thereafter. This was observed in different ionic conditions and at different voltage steps. However, decay was not accelerated at low expression levels, either within 1-2 days after injection or with reduced amounts of RNA. With sequential RNA injection, preformation of either Eag or Sh channels prevented interactions with the other subunit. The carboxyl terminus of Eag was found to be involved in accelerating, and in retarding recovery from, N-type inactivation. The interaction was reduced upon patch excision in macropatch measurements, suggesting involvement of cytosolic factors. We have reproduced the absence of interaction between Eag and Sh reported previously within 2 days after RNA injection and with low levels of current expression (Tang, C.-Y., C. T. Schulteis, R. M. Jiménez, and D. M. Papazian. 1998. Biophys. J. 75:1263-1270). Our findings demonstrate that heterologous expression of channels in Xenopus oocytes is a dynamic process influenced by cell physiology and development. These factors must be considered in interpreting the functional properties of heterologously expressed channels.

Animals↗