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C F Huang

Publications and source records attributed to C F Huang.

At least 73 records · Page 4Linked to original sources
PubMed1994

Calcium influx blocks the skeletal muscle acetylcholine receptor alpha-subunit gene in vivo.

The transcriptional activity of the acetylcholine receptor alpha-subunit gene was measured in denervated chick skeletal muscle in response to calcium-active drugs, using a ribonuclease protection version of the conventional run-off assay. The L-channel agonist (-)Bay-K6844 and the calcium ionophore A23187 mimicked, and the intracellular chelator BAPTA and the calcium channel blockers D600 and nifedipine blocked, the effect of electrostimulation. These results suggest that influx of extracellular calcium is an integral component of the membrane depolarization-receptor gene inactivation cascade.

Animals↗
PubMed1994

Rapid inhibition of myogenin-driven acetylcholine receptor subunit gene transcription.

In investigating the coupling of depolarization and transcription in skeletal muscle we have focused on how protein kinase C suppresses acetylcholine receptor subunit genes. The activity of acetylcholine receptor subunit promoters in non-muscle cells co-transfected with myogenic factors and E proteins was measured, and their response to protein kinase C activation analyzed. To simplify interpretation of results, gene activities rather than levels of reporter enzymes were assayed, transcriptional effects of phorbol esters were determined, with drug exposures brief enough to preclude kinase depletion, and analysis was carried out with HeLa cells, which are not liable to myogenic conversion. Myogenin, which had been postulated previously to play a role in denervation supersensitivity (Neville et al., Mol. Cell. Neurobiol., 12, 511-527, 1992), was found to be the only myogenic factor whose inactivation kinetics can account for the plasma membrane-protein kinase C-receptor gene cascade observed in intact muscle (Huang et al., Neuron, 9, 671-678, 1992).

Gene Expression Regulation↗
PubMed1994

Depolarization-transcription signals in skeletal muscle use calcium flux through L channels, but bypass the sarcoplasmic reticulum.

Membrane depolarization inactivates acetylcholine receptor (AChR) genes in skeletal muscle. We have studied this process in C2C12 cells, focusing on the role of calcium. Cytoplasmic calcium was monitored with fluo-3, and the activity of receptor genes was measured with a sensitive transcript elongation assay. Removal of extracellular calcium or blockage of L-type calcium channels disrupts signaling, even when release of calcium from the sarcoplasmic reticulum (SR) is not impeded, whereas L channel agonists induce signaling without membrane depolarization or release of calcium from intracellular stores. Activators of calcium release from the SR do not inhibit AChR genes, either in C2C12 or in chicken skeletal muscle in vivo. It appears that calcium ions do not act as messengers between sarcolemma and nucleus but target a sensor near their port of entry where they initiate a signal that bypasses the SR.

Action Potentials↗
PubMed1994

Gene amplification and high-level expression of human pro-urokinase cDNA in Chinese hamster ovary cells.

Human Pro-Urokinase (Pro-UK) is expressed in CHO-DHFR- cells at high efficiency by co-transfecting the mouse dhfr gene and Pro-UK cDNA under the control of the SV40 late promoter. After gene co-amplification, the product level could reach 2-3 micrograms/10(6) cells/24 h in the presence of 5 x 10(-6) mol/L MTX, and the levels can be further raised to 3.5-4 micrograms/10(6) cells/24 h by PMA superinduction. The copy number of Pro-UK cDNA in the genomes of host cells is about 200-300 copies/cell. The Western blot analysis shows that the recombinant Pro-UK has similar molecular weight to its natural counterpart, and also the amidolytic activity of the product is determined by S-2444 assay.

Animals↗
PubMed1993

Glutathione as a cellular defence against arsenite toxicity in cultured Chinese hamster ovary cells.

The cytotoxic effect of arsenite seems to be inversely related to the intracellular glutathione (GSH) content, and GSH seems to facilitate the metabolism of arsenic in cell. Arsenite is also known to induce chromosome aberration, to enhance the cytotoxicity and clastogenicity of ultraviolet (UV) light, and to inhibit UV-induced DNA repair. We have investigated whether these toxic effects of arsenite and the cellular arsenic content are also modulated by the intracellular GSH. A 2-h pretreatment of the cultured ovary (CHO) cells with GSH reduced the clastogenicity and cytotoxicity of arsenite. The enhancing effects of arsenite on chromosome aberrations and cell destruction induced by UV were also reduced by a 2-h pretreatment with GSH. The inhibitory effect of arsenite on the strand-break rejoining during UV-induced DNA repair was reduced by GSH pretreatment and was enhanced by pretreatment with buthionine sulfoximine, which is known to deplete the cellular GSH. The cellular arsenic content was reduced by GSH pretreatment and increased by buthionine sulfoximine pretreatment. GSH given before or simultaneously with arsenite, effectively reduced the clastogenicity and coclastogenicity of arsenite. GSH given after treatment with arsenite decreased the cellular arsenic content, and increased the cell survival, but did not reduce the clastogenicity or the coclastogenicity of arsenite.

Animals↗
PubMed1993

Control of myogenic factor genes by the membrane depolarization/protein kinase C cascade in chick skeletal muscle.

Myogenic factor genes were found to respond differentially to electrical stimulation of denervated chick skeletal muscle. Myogenin gene activity declined rapidly (t1/2: approximately 2 min), comparable to the rate of acetylcholine receptor (AChR) gene inactivation, while other myogenic bHLH genes either lost activity more slowly (MyoD) or not at all (myf5, herculin). Protein kinase C (PKC) is known to couple membrane activity to AChR gene inactivation; myogenin gene transcription was also rapidly blocked by the PKC activator PMA, whereas electrostimulation remained without effect on myogenin gene activity in muscle that was either exposed to the kinase inhibitor staurosporine or chronically treated with PMA to deplete PKC. These results attest to a special role for myogenin in the activation of AChR genes in denervation supersensitivity.

Alkaloids↗
PubMed1993

[Effect of implantation of an Ommaya reservoir on prognosis for cryptococcal meningitis].

Cryptococcal meningitis has a high mortality rate of central nervous infection. The patients usually die of the disease itself, or complications from increased intracranial pressure. Early diagnosis and treatment, including surgical drainage, will improve the results. In this series, twenty-one patients with high intracranial pressure (ICP > 300 mmH2 O) are presented. Fourteen received implantation of Ommaya reservoir to aspirate cerebrospinal fluid (CSF) for relief of symptoms of ICP. Meanwhile 4 of these 14 patients also received intraventricular injection of amphotericin B because of poor response to systemic drugs. Another seven patient received systemic drug therapy only. Survival during therapy occurred in 11 of 14 patients in the surgical group, compared with only 1 of 7 patients treated by drug therapy alone (P = 0.019). In the 14 patients who received implantation of an Ommaya reservoir, there was one complication of CSF leakage when the reservoir ruptured because of repeated aspiration. For patients with cryptococcal meningitis with high ICP, early implantation of an Ommaya reservoir will improve the survival rate.

Adolescent↗
PubMed1992

Protein kinase C couples membrane excitation to acetylcholine receptor gene inactivation in chick skeletal muscle.

The signaling pathway connecting membrane depolarization and gene activity in skeletal muscle remains largely unknown. Using transcription elongation (run-on) analysis we have found that electrical stimulation of denervated chick skeletal muscle in vivo rapidly and selectively results in inactivation of acetylcholine receptor (AChR) subunit genes. We have studied the possible involvement of protein kinase C (PKC) in this response and have observed that electrical stimulation increases the activity of PKC in the nucleus by over two orders of magnitude within 10 min; phorbol esters, within minutes after intramuscular application, block AChR subunit genes in the absence of electrical activity; and the activity-triggered gene inactivation is blocked by the protein kinase inhibitor staurosporine or by enzyme depletion resulting from chronic pretreatment of muscle with phorbol esters. We conclude that PKC is an integral component of the pathway coupling membrane excitation and AChR gene control.

Alkaloids↗
PubMed1992

The transition from late G1 to early S phase is most vulnerable to the coclastogenic effect of ultraviolet radiation plus arsenite.

It has previously been reported that chromosome aberrations induced by ultraviolet (UV) radiation can be enhanced by treatment with sodium arsenite for 24 h post-irradiation. Using synchronized CHO-K1 cells, it has now been established that cells in the transitional stage from late G1 to early S phase are most vulnerable to the coclastogenic effect of treatment with UV radiation and arsenite. This result cannot be explained by the special vulnerability of cells in the late G1 to early S transition to UV clastogenicity, as the coclastogenic effects of UV and caffeine or UV and arabinofuranosylcytosine were detected when treating the mid-S but not late-G1 or G2 phase cells.

Animals↗
PubMed1992

Traumatic tension pneumocephalus--intracerebral pneumatocele: a case report.

An 18-year-old Chinese man, suffering from head injury in a motorcycle accident, with right traumatic frontal intracerebral hemorrhage and frontal bone fractures, underwent emergency craniotomy with evacuation of hematoma at a local out-patient clinic and three months later, he suffered from CSF rhinorrhea. Headache, nausea and vomiting developed a week prior to admission. Tension pneumocephalus was diagnosed by computed tomography (CT) scan and plain skull X-ray. The fistula tract of the ethmoid sinus was investigated by radioisotope albumin (RISA) cisternography. The patient subsequently underwent emergency craniotomy for decompression. During the operation, we found that the intracerebral pneumatocele in the right frontal lobe communicated with the ipsilateral ethmoidal sinus, through which extracranial air ingressed and CSF egressed. This pneumatocele was unroofed and the fistula was temponaded by pericranial muscles and the Gelfomas sealed by tissue glue. Finally duraplasty was performed. Follow-up CT scan revealed that pneumocephalus subsided and the patient was cured of CSF rhinorrhea on discharge. As a surgical emergency, tension pneumocephalus can be successfully treated only by early diagnosis and early treatment.

Adolescent↗
PubMed1991

Expression of pro-urokinase cDNA in Chinese hamster ovary cell line.

Expression vectors containing the pro-urokinase (pro-UK) cDNA (pSV2-proUK) and a dihydrofolate reductase cDNA (pSV2-dhfr or MMTV-dhfr) were cotransfected into CHO-dhfr- cells by the calcium phosphate precipitation technique. The dhfr+ transformants were selected by fibrinolytic agarose plate assay. Two colonies, named CLF-14 and CLF-8, exhibited significantly high expression levels of the biological activity of urokinase-type plasminogen activator (mu-Pa). They reached more than 24 IU/10(6) cells/48 h and 16 IU/10(6) cells/48 h, respectively. Examination of the cell supernatants for mu-Pa antigenicity using ELISA method also showed strong positive results, and the quantities of expression were about 0.14-0.22 micrograms/10(6) cells/48 h and 0.08-0.14 micrograms/10(6) cells/48 h, respectively. The mu-Pa secreted by stable transformed cells could be completely inhibited by UK anti-serum, but not by tissue-type plasminogen activator (t-PA) antiserum nor by normal rabbit serum.

Animals↗
PubMed1990

Vasopressin-induced polyphosphoinositide and phosphatidylcholine degradation in fibroblasts. Temporal relationship for formation of phospholipase C and phospholipase D hydrolysis products.

Cultured fibroblasts (REF52 cells) were employed to investigate phospholipid degradation in response to vasopressin (VP) treatment. There have been few studies in fibroblasts which characterize the pattern and relationship of phosphatidylinositol 4,5-bisphosphate (PIP2) and non-phosphoinositide hydrolysis elicited by VP. Here we demonstrate that VP-induced PIP2 hydrolysis is closely accompanied by phosphatidylcholine (PC) degradation by phospholipase D. Cells prelabeled with [3H]arachidonic acid showed rapid formation and diminution of [3H]diacylglycerol (DG) (5-15s) when treated with VP; this was accompanied by a reduction in polyphosphoinositide radioactivity. Radiolabeled inositol trisphosphate was generated with a similar time frame. In cells prelabeled with [3H]myristic acid, which is predominantly incorporated into cellular PC, VP elicited the generation of [3H]myristoyl phosphatidate (PA) as early as 15 s, in the absence of an increase in labeled DG. In the presence of ethanol the pattern of [3H]myristoyl phosphatidylethanol (PEt) formation coincided with [3H]myristoyl-PA formation in the absence of ethanol. PEt was similarly formed, in response to VP treatment, in cells prelabeled with 1-O-[3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine. The formation of PC-derived [3H]myristoyl-DG was characterized by a lag period of approximately 1 min, after which DG increased steadily over a 10-min period. Biphasic formation of DG was observed in cells prelabeled with [3H]arachidonic acid, and the formation of [3H]PA occurred in an uninterrupted fashion. Two protein kinase C agonists, phorbol diester and dioctanoylglycerol, elicited the formation of [3H]myristoyl-PEt. The inclusion of staurosporine, a protein kinase C inhibitor, blocked VP-induced [3H]myristoyl-PEt formation by 88%. These data demonstrate that VP elicits the coordinated hydrolysis of PIP2 by phospholipase C and PC hydrolysis by phospholipase D. This event results in the prolonged generation of PA and biphasic formation of DG. From the time courses shown, we hypothesize that the early generation of PA, heretofore ascribed to products of the polyphosphoinositide cycle, are in part derived from PC by phospholipase D.

Animals↗
PubMed1990

Phorbol diesters stimulate the accumulation of phosphatidate, phosphatidylethanol, and diacylglycerol in three cell types. Evidence for the indirect formation of phosphatidylcholine-derived diacylglycerol by a phospholipase D pathway and direct formation of diacylglycerol by a phospholipase C pathway.

The effect of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), on phospholipid degradation was investigated in three cell lines of dissimilar origin, Madin-Darby canine kidney cells (MDCK), rat aorta smooth muscle cells (RASM), and bovine pulmonary artery endothelial cells (BPAE). In cells prelabeled with [3H]myristic acid, which is predominantly incorporated into phosphatidylcholine (PC), TPA treatment (80 nM) in the absence or presence of ethanol (2%) in the culture medium resulted in either the rapid generation of [3H]phosphatidate (PA) or the sustained accumulation of [3H]phosphatidylethanol (PEt), respectively. Increases in [3H]PA and [3H]PEt were paralleled by quantitative decreases in cellular [3H]PC radioactivity. TPA-induced [3H]PEt formation occurred in a similar fashion, irrespective of the presence of Ca2+ in the culture medium. The experiments demonstrate that TPA elicits PC degradation by phospholipase D (PLD) in cells of diverse origin. Data from further experiments revealed a complex relationship between TPA-induced [3H]PA and [3H]diacylglycerol (DG) generation in the three cell lines that was suggestive of dual pathways for the generation of [3H]DG. Experiments to discern the pathways for TPA-induced, PC-derived DG were conducted by comparing the variation of [3H]PA and [3H]DG formation in the absence and in the presence of increasing ethanol concentrations in the culture medium. With increasing amounts of ethanol, the formation of [3H]PA decreased at the expense of [3H]PEt formation, and depending upon the pathway operable, the amount of [3H]DG formed was either decreased, indicative of indirect formation of DG via PA phosphohydrolase, or not modified, indicative of DG formation by a direct phospholipase C (PLC) pathway. Increasing the concentration of ethanol in the medium blocked TPA-induced [3H]DG generation in MDCK cells in a concentration-dependent manner, while the formation of [3H]PEt increased at the expense of [3H]PA formation. In BPAE cells the presence of ethanol likewise reduced TPA-elicited formation of DG. Conversely, in two smooth muscle cell lines, RASM and A-10, ethanol was without influence on TPA-induced formation of [3H]DG, although [3H]PEt was generated at the expense of [3H]PA. In RASM cells prelabeled with [3H]choline, TPA induced the release to the medium of [3H]choline and [3H]phosphocholine, indicative of both PLD and PLC activation. These results show that TPA elicits DG formation from PC in MDCK cells predominantly by an indirect pathway, whereas in arterial smooth muscle cells DG is formed in part by the direct action of PLC.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Recombinant bivalent live vaccines against neonatal colibacillosis in piglets.

The genes of colonization factor K88 and avirulent heat-labile enterotoxin LT A-B+ of enterotoxigenic E. coli (ETEC) have been ligated, and a vaccine strain containing recombinant plasmid that efficiently expresses two antigens has been obtained. Using the activity of beta-galactosidase as a selection marker instead of drug resistance, another bivalent vaccine strain with the same expression level has also been constructed. The vaccine strains have no toxicity and do not cause any adverse reactions. In challenge study and field trials, a high degree of protection from colibacillosis was afforded to piglets when their dams were immunized orally or parenterally. Practice of bivalent live vaccines including colonization factor and enterotoxin antigens and without antibiotic resistance gene shows effective.

Adhesins, Escherichia coli↗
PubMed1990

The construction of a non-antibiotic resistant ETEC vaccine strain containing K88ac and LT-B antigens.

By inserting the LacZ gene into the plasmid containing ETEC LT-B antigen gene we obtained a recombinant plasmid which could express the beta-galactosidase and LT-B antigen, but has the Tc resistant gene. The K88ac antigen gene was then inserted into the BamHI site of the Tc resistant region in the above plasmid. The final recombinant plasmid has no antibiotic resistance gene but can stably express both LT-B and K88ac antigens, as well as the beta-galactosidase as a tracking marker.

Antigens, Bacterial↗
PubMed1990

Construction of a bivalent oral vaccine for prevention of typhoid fever and cholera diarrhea.

A recombinant plasmid pMM-CTB containing the gene for production of the nontoxic B subunit of Vibrio cholera was transferred into a safe, effective and attenuated oral vaccine Ty21a strain of Salmonella typhi. The resulting Ty21a (pMM-CTB) could steadily produce CT-B subunit that was secreted extracellularly and had the same antigenicity as CT-B produced by V. cholera. Furthermore, the characteristics of the antigenicity, the persistance in mice and the galactose sensitivity possessed in the strain of Ty21a were also retained in Ty21a (pMM-CTB). A bivalent vaccine containing Ty21a (pMM-CTB) and the killed whole cell of V. cholera was then constructed which had good immunogenecity for typhoid fever and cholera diarrhea.

Animals↗
PubMed1989

[Cloning of the gene relative to invasive ability of Shigella flexneri 5].

A genomic library of S. flexneri 5 large plasmid (140 Md) was constructed using the cosmid pJB8 as vector. There were more than 4,000 transformants in the library. 66 clones were picked out from the library by hybridization with 17kb gene probe relative to invasive ability. A few clones were analyzed. The results indicated that all these clones tested contained recombinant plasmids, they could hybridize with 17 kb gene probe. When these recombinant plasmids were digested with EcoR1, 17 kb fragments which hybridized with 17 kb gene probe always existed in these recombinant plasmids. It showed that all these recombinant plasmids contained the DNA fragments relative to invasive ability. It provides the possibility to construct an oral living vaccine against S. flexneri.

Cloning, Molecular↗