Biomedical subjects

Y C Liu

Publications and source records attributed to Y C Liu.

At least 217 records · Page 12Linked to original sources

PubMed1993

Characterization of a fusion protein composed of the extracellular domain of c-kit and the Fc region of human IgG expressed in a baculovirus system.

The extracellular domain of c-kit, which is the receptor for stem cell factor (SCF), was fused genetically to the Fc portion of human immunoglobulin G1. This chimeric protein, c-kitFc, was then expressed in the baculovirus system. The fusion product was secreted into the serum-free culture medium as a soluble dimeric form of approximately 210 Kda. The recombinant protein was easily purified by protein A affinity chromatography at approximately 25 micrograms protein per ml of medium. Binding assay and cross-linking assay showed that the fusion protein retained high affinity for binding SCF (Kd = 0.3 nM). Addition of the chimeric protein into the culture medium of SCF-dependent cells inhibited cell proliferation in a dose-dependent manner. These results suggest that the dimeric c-kitFc protein can be used as an antagonist of SCF for the study of hematopoietic progenitor cells.

Animals↗

PubMed1993

Dependence of trp repressor-operator affinity, stoichiometry, and apparent cooperativity on DNA sequence and size.

A series of chemically synthesized trp and mutant operator DNAs was employed to examine trp repressor binding. Although only a single repressor-operator complex was observed for most DNAs as reported previously, varying DNA sequence revealed two retarded complexes with an additional band of faster mobility. The relative intensity of the two retarded bands with varying repressor concentrations suggests that cooperative interactions between dimers may occur in the formation of the predominant repressor-operator complex. Direct stoichiometry measurements demonstrated that a 2:1 stoichiometry (two dimers per operator) is found in the primary repressor-operator complex band and that a 1:1 stoichiometry is observed, when present, for the minor repressor-operator complex band of faster mobility. Similar retardation patterns with a single complex of 2:1 stoichiometry were observed for 40-base pair (bp) trp operators corresponding to TrpEDCBA, aroH, and Trp-PL (a derivative of TrpEDCBA with increased symmetry) operator sequences as well as to hybrid operators containing a half-binding site from TrpEDCBA in conjunction with lac operator sequences, although the apparent affinity for the half-site DNAs was diminished by 10-fold. In contrast, the prominence of the 1:1 dimer-operator complex for Trp-PR, a different derivative of TrpEDCBA with increased symmetry, suggests that sequence context may diminish cooperativity between dimers. The stoichiometry observed was also dependent on the length of TrpEDCBA operator DNA used, shifting from primarily 2:1 for 40- and 33-bp TrpEDCBA DNA to primarily 1:1 for 29-, 26-, and 20-bp TrpEDCBA DNAs. In addition, the stability of the repressor-operator complex to electrophoresis is reduced for DNA lengths of 33, 29, and 26 bp. Based on the binding data and footprinting patterns for two hybrid 40-bp TrpEDCBA/lac half-binding site DNAs, it appears that repressor associates tightly to the specific trp half-site, whereas the nonspecific half of the DNA is more loosely bound. These results suggest that repressor dimer-dimer interaction may be an important feature in the trp repressor-operator interaction.

Allosteric Regulation↗

PubMed1993

Molecular cloning and functional expression of a cDNA encoding glycosylation-inhibiting factor.

By using probes based on partial amino acid sequence of glycosylation-inhibiting factor (GIF) from a mouse T-cell hybridoma, a full-length cDNA encoding mouse GIF was isolated. A cDNA clone encoding human GIF was isolated from cDNA libraries of a GIF-producing human T-cell hybridoma by using mouse GIF cDNA as a probe. The cDNAs encode a putative 12.5-kDa peptide of 115 amino acids. Northern blot analysis demonstrated a single, 0.6-kb transcript. Polyclonal rabbit antibodies against the Escherichia coli-derived recombinant 13-kDa peptide bound hybridoma-derived GIF. Although the peptide did not contain a signal peptide sequence, transfection of the cDNA into COS-1 cells resulted in secretion of 13-kDa peptide, but the peptide had substantially less bioactivity than the hybridoma-derived GIF. However, expression of a chimeric cDNA encoding a fusion protein consisting of the N-terminal pro region of calcitonin precursor and human GIF and cotransfection with furin cDNA to allow intracellular cleavage of the fusion protein resulted in secretion of 13-kDa peptide that was comparable to hybridoma-derived GIF in its bioactivity. Both the 13-kDa peptide and GIF bioactivity in the transfected COS-1 supernatant bound to a monoclonal antibody against hybridoma-derived human GIF. These results indicate that the 13-kDa peptide represents recombinant GIF, but posttranslational modification of the peptide is important for generation of the bioactivity. The GIF cDNA had high homology with the cDNA encoding macrophage migration inhibitory factor. However, the recombinant GIF failed to inhibit migration of human monocytes, and recombinant human macrophage migration inhibitory factor did not have GIF bioactivity.

Amino Acid Sequence↗

PubMed1993

Trp repressor interaction with bromodeoxyuridine-substituted operators alters UV-induced perturbation pattern in a sequence-dependent manner.

In order to explore DNA sites influenced by the trp repressor-operator interaction, bromodeoxyuridine (BrdU) was chemically incorporated into the TrpEDCBA, TrpR, and aroH operators at selected thymidine positions. Different patterns of repressor protection from strand scission in the two halves of complexes with the TrpEDCBA, TrpR, and aroH operators suggest different local environments despite the highly symmetric sequences. Although protection was observed at multiple sites in the operators in the presence of repressor, UV irradiation did not lead to a cross-linked repressor-operator complex. This result indicates the absence of close contacts in the major groove between suitable repressor residues and the 5-methyl of thymidines. Upon trp repressor binding and UV irradiation, in addition to protection from strand scission, multiplets were observed at some sites, notably within CTAG sequences in the BrdU-substituted operators. This phenomenon (termed band migration) may result from distortion by the trp repressor of the BrdU-substituted operator DNA and consequent exposure of different sites along the backbone to strand scission. Interestingly, UV footprinting of two BrdU-substituted TrpEDCBA variant operators showed different patterns when base pair symmetry was matched to each side of the symmetry axis. These observations suggest that alterations in the UV photolysis pattern in response to protein binding result from DNA structural alterations that are sequence dependent.

Bacterial Proteins↗

PubMed1993

Processing of a fusion protein by endoprotease in COS-1 cells for secretion of mature peptide by using a chimeric expression vector.

The subtilisin-related proprotein convertase furin is expressed in various mammalian tissues. Expecting that COS-1 cells have a furin-like endoprotease, we constructed a fusion expression vector for production of a recombinant foreign protein having no signal peptide or a protein in truncated form into secreted mature protein. A cDNA fragment encoding N-terminal procalcitonin (pro-CT) of human calcitonin precursor was inserted into the mammalian expression vector pME18S. We used PCR techniques to generate four kinds of cDNAs encoding the C terminus of the pro-CT with Arg residues at P4 (Arg-Xaa-Lys-Arg), P6 (Arg-Xaa-Xaa-Xaa-Lys-Arg), or both (Arg-Xaa-Arg-Xaa-Lys-Arg), in addition to the Lys-Arg motif at the cleavage site, in order to determine the conditions for efficient processing in nonendocrine cells, such as COS-1 cells. The cDNA coding for the Fc fragment of human immunoglobulin G1 was fused in-frame to the cDNA encoding pro-CT at its C terminus. Upon transfection of the chimeric plasmids into COS-1 cells, almost all of the fusion protein with the Arg residues at both P4 and P6 were processed into secreted Fc product, even without cotransfection of furin. These results indicate that COS-1 cells have a furin-like endoprotease and suggest that pro-CT, with the Arg residues at both P4 and P6, can be used as a carrier peptide for expression of a foreign protein having no signal peptide or a protein in truncated form in COS-1 cells.

Amino Acid Sequence↗

PubMed1993

DNA hypomethylation of proliferating cell nuclear antigen gene in human hepatocellular carcinoma is not due to cell proliferation.

Proliferating cell nuclear antigen (PCNA) gene expresses preferentially in proliferative cells or tissues. The levels of PCNA mRNA are very low in livers of adult mammals. Expression of PCNA gene in hepatocellular carcinoma tissues was, however, elevated; and 5'-CCGG-3' sequences of the gene in neoplastic tissue were less methylated. Such DNA hypomethylation was concluded, on the basis of two observations, not to be due to the cell proliferation in hepatoma tissues. First, while the expression of PCNA was increased during serum-stimulation of quiescent Hep G2 cells, the DNA methylation pattern of PCNA gene remained unchanged. Second, in rat liver regeneration, the PCNA mRNA level rose and declined, but the DNA methylation status of PCNA gene was unaltered. Therefore, the DNA hypomethylation of the PCNA gene found in hepatocellular carcinoma was not due to cell proliferation, but a possible consequence of cell transformation.

Animals↗

PubMed1993

A 13C NMR study on ibuprofen metabolism in isolated rat liver mitochondria.

A 13C NMR approach was taken to study the biotransformation of (R)- and (S)-[2-13C,2-2H]ibuprofen-CoA in isolated rat liver mitochondria. The 13C chemical shift induced by 2H substitution for the C-2 of [2-13C,2-2H]-ibuprofen-CoA is about 0.5 ppm relative to the corresponding 1H-bearing thioester. The C-2 resonances for [2-13C]- and [2-13C,2-2H]ibuprofen-CoA are 5 ppm downfield with respect to their acid counterparts. Consequently, this technique permits real time analyses of the metabolic fate of the doubly labeled substrate in a quantitative manner. The initial rate of the epimerization of (S)-[2-13C,2-2H]ibuprofen-CoA relative to the (R)-counterpart by intact mitochondria was estimated to be 1.5, which is in line with the equilibrium constant determined with the purified epimerase. Contrary to the reports by other groups, this 13C NMR study did not find spontaneous hydrolysis of the CoA-thioester upon being exposed to intact mitochondria. Current focus of this investigation is to extend this NMR methodology to the levels of whole cells and live animals to gain a better understanding of the enantioselective metabolism of ibuprofen.

Animals↗

PubMed1993

DNA methylation is not involved in growth regulation of gene expression of proliferating cell nuclear antigen.

Expression of proliferating cell nuclear antigen (PCNA) gene is growth regulated. By Southern blot analysis with restriction enzymes such as HpaII and MspI, no change of DNA methylation state in this gene was detected during rat liver regeneration. There is only one HpaII site located in the promoter region of rat PCNA gene and this HpaII site was found to be demethylated in both normal and regenerating livers. Human hepatoma cell lines Hep G2 and Hep 3B were used to study the relation of PCNA gene expression and the DNA methylation. There are 16 HpaII sites in human PCNA gene, and according to the HpaII and MspI restriction patterns, many of the HpaII sites were methylated in vivo. Upon serum stimulation of quiescent cells, no change of DNA methylation state in the HpaII and HhaI sites was found. Demethylation by the methylation inhibitor, 5-azacytidine, did not affect the expression of PCNA gene in the hepatoma cells. With the human primary fibroblasts, Y2, the demethylation by 5-azacytidine did not seem to change the growth dependence of PCNA gene expression. This is consistent with the observation that the expression of PCNA gene of some cultured cell lines such as CHO.K1, in which the PCNA gene was unmethylated, showed growth dependence. Also, no variation in methylation pattern of PCNA gene was found among the different rat tissues in which the expression of PCNA varies. Therefore, we conclude that DNA methylation is not involved in growth regulation of the PCNA gene expression.

Animals↗

PubMed1993

Identification of a disease resistance locus in Arabidopsis that is functionally homologous to the RPG1 locus of soybean.

A new disease resistance locus in Arabidopsis, RPS3, was identified using a previously cloned avirulence gene from a non-Arabidopsis pathogen. The avrB avirulence gene from the soybean pathogen Pseudomonas syringae pv. glycinea was transferred into a P. syringae pv. tomato strain that is virulent on Arabidopsis, and conversion to avirulence was assayed on Arabidopsis plants. The avrB gene had avirulence activity on most, but not all, Arabidopsis ecotypes. Of 53 ecotypes examined, 45 were resistant to a P. syringae pv. tomato strain carrying avrB, and eight were susceptible. The inheritance of this resistance was examined using crosses between the resistant ecotype Col-0 and the susceptible ecotype Bla-2. In F2 plants from this cross, the ratio of resistant:susceptible plants was approximately 3:1, indicating that resistance to P. syringae expressing avrB is determined by a single dominant locus in ecotype Col-0, which we have designated RPS3. Using RFLP analysis, RPS3 was mapped to chromosome 3, adjacent to markers M583 and G4523, and < or = 1 cM from another disease resistance locus, RPM1. In soybean, resistance to P. syringae strains that carry avrB is controlled by the locus RPG1. Thus, RPG1 and RPS3 both confer avrB-specific disease resistance, suggesting that these genes may be homologs.

Arabidopsis↗

PubMed1993

An EGF-pseudomonas exotoxin A recombinant protein with a deletion in toxin binding domain specifically kills EGF receptor bearing cells.

We constructed two chimeric toxins; one composed of epidermal growth factor (EGF) and pseudomonas exotoxin A (PE), designated EGF-PE and the other composed of EGF and PE with a deletion of the Ia domain (cell-binding domain), designated EGF-PE (delta Ia). Both chimeric toxins reacted with anti-EGF and anti-PE antibodies. The cell-killing experiments showed that EGF-PE, but not EGF-PE (delta Ia), was cytotoxic to the murine fibroblast cell line NR6, which carried the PE receptor, but not the EGF receptor. However, after NR6 was transfected with DNA for the expression of human EGF receptor, the transfected cell line, designated NRHER5, over-expressed human EGF receptors and became sensitive to EGF-PE(delta IA). The cytotoxicity of EGF-PE(delta Ia), but not EGF-PE, to NRHER5 can be completely blocked by an excess amount of EGF. To completely reverse the cytotoxicity of EGF-PE on NRHER5, both the EGF receptor pathway and the PE receptor pathway need to be blocked. These results suggest that EGF-PE exhibits both EGF and PE binding activities, while EGF-PE(delta IA) possesses only EGF binding activity. Thus, EGF-PE(delta Ia) may be a better chimeric toxin than EGF-PE in terms of target specificity to EGF receptor bearing cells. We, therefore, examined the cytotoxicity of EGF-PE(delta Ia) to various human cancer cell lines. We find that human cancer cells containing more EGF receptors are more sensitive to EGF-PE(delta Ia).

ADP Ribose Transferases↗

PubMed1993

Seroprevalence of Chlamydia pneumoniae in Taiwan.

Chlamydia pneumoniae (TWAR) is a relatively newly discovered respiratory tract pathogen which was first isolated in Taiwan. In order to describe the seroepidemiology of C. pneumoniae in Taiwan, we evaluated 1,085 stored serum samples: 904 from patients, 97 from umbilical cord blood samples, and 84 from medical personnel at the Veterans General Hospital-Kaohsiung, between January 1 and April 30, 1991. Antibodies were determined by the use of a microimmunofluorescence test using elementary bodies of C. pneumoniae AR-39 as antigen. Sera were tested with screening titres of 16 and 512 for immunoglobulin G antibody. The antibody prevalence was found to be 23.1% in young children (6 months to 10 years old), rising to 66.7% in teenagers, and to 96.2% in older age groups. These rates were higher than any reported earlier. The progressively increasing rates of seropositivity found in older individuals indicated a surge of reinfection in these age groups. Only 5 cases were found with micro-IF IgG titres equal to or greater than 512. All were asymptomatic according to the hospital records. In addition to a high prevalence rate in Taiwan, the data also showed high infection rates in teenagers and elderly people.

Adolescent↗

PubMed1993

[A study of cerebral vasospasm after subarachnoid hemorrhage with transcranial Doppler].

Transcranial Doppler (TCD) was performed in 64 patients with subarachnoid hemorrhage (SAH) to detect the blood velocity in middle cerebral artery (MCA). Based on Poiseuille's principle, the increase of blood velocity was used as an indicator of cerebral vasospasm (CVS). It was found that 61.67% of the patients with SAH developed CVS, especially in the second week after SAH. The incidence of CVS in patients below 50 years old was higher than that in patients above 51 years old. The results showed that TCD is valuable in evaluation of CVS after SAH.

Adult↗

PubMed1993

Acute Q fever: first case report in Taiwan.

A 70-year-old retired fisherman presented with fever, chills and headache for four days. The clinical presentation was mild, including a self-limiting fever, pneumonia and elevated liver enzyme levels. Acute Q fever was proved by a four-fold rise of phase II IgG antibodies. Interestingly, the man denied any animal contact or travelling over the past year. This case may suggest that Q fever should be added to the list of differential diagnoses for acute febrile disease in Taiwan.

Acute Disease↗

PubMed1993

The influence of position and PEEP on arterial blood gas during operation.

From December, 1990 to December, 1991, we studied the influence of PEEP and positional change to arterial blood gas in 60 ASA class II or III, aged 20 to 65 years, non-obese patients. The patients were assigned randomly into six groups. Group 1: head down position without PEEP. Group 2: head down position with PEEP 5 cmH2O. Group 3: lithotomy position without PEEP. Group 4: lithotomy position with PEEP 5 cmH2O. Group 5: supine position without PEEP. Group 6: supine position with PEEP 5 cmH2O. Blood gas analysis were performed at 5, 15, 30, 60, 120, and 180 minutes after positional change in group 1-4 and after anesthesia in group 5 and 6. The ventilator settings were: tidal volume -10 ml x 25 x (height in meters)2, rate 8/min. The results of significant difference inter-grouply were: PaO2 at 15 minutes; PaCO2 at 5 minutes; pH at 60, 120, and 180 minutes; base excess (BE) at 120 and 180 minutes. As the time progressed, PaO2 decreased in group 1, 2, and 3; PaCO2 decreased in group 3 and 5; pH decreased in group 3; BE decreased in all groups. PaCO2 were between 30-40 mmHg and no hypocarbia produced in all groups. In conclusion, ventilation of the non-obese patient based on 10 ml x 25 x (height in meters)2 x 8/min produce normocarbia and PaO2 more than 80 mmHg with 50% oxygen. No significant difference of PaO2 were found whether there were position change or PEEP 5 cmH2O.

Adult↗

PubMed1993

Effects of bretylium tosylate on electrophysiologic properties of normal and digitalized papillary muscles of guinea pigs.

The effects of bretylium tosylate (BT) on the electrophysiologic properties of normal and digitalized papillary muscles isolated from guinea pigs were studied with regular glass microelectrode. BT prolonged effective refractory period (ERP) and action potential duration (APD) of normal papillary muscles. The ERP and APD of papillary muscles were shortened by perfusion with ouabain (Oua) 0.2 mumol.L-1. No recovery was seen in perfusion without drug for 30 min. In digitalized papillary muscles with Oua, ERP, APD90, and APD50 were prolonged by BT 120 mumol.L-1 form 175 +/- 20, 187 +/- 20, and 146 +/- 21 ms to 222 +/- 21, 220 +/- 19, and 190 +/- 19 ms, respectively. The results demonstrated that BT can prolong ERP and APD of papillary muscles digitalized with Oua.

Action Potentials↗

PubMed1993

Five-year experience of human immunodeficiency virus type 1 national screening program implemented at Veterans General Hospital-Taipei.

From July 1986 through June 1990, 33,199 sera from various risk groups were collected in Veterans General Hospital-Taipei for detection of antibody against human immunodeficiency virus, type 1 (HIV-1). Sixty-five samples were proved positive by Western blot analysis. Among individual high risk groups, hemophiliacs had the highest positive rate of 20/60 (29.41%), followed by homosexual/bisexual males (41/1,264, 3.24%). The overall positive rate was 65/33,199 (0.19%). Ten cases were recognized as acquired immunodeficiency syndrome (AIDS), 1 case had AIDS-related complex (ARC) and 4 case had other apparently symptomatic infections. Among these 15 cases, 7 expired, 1 lost of follow-up and 7 surviving cases are being treated with zidovudine (AZT). Most of symptomatic HIV-1 antibody positive cases had abnormal T4/T8 ratio of 0.39 +/- 0.54 as compared with the asymptomatic HIV-1 carriers at a ratio of 0.81 +/- 0.69. The opportunistic infections included Pneumocystis carinii pneumonia (PCP) in 6 case, disseminated cytomegalovirus infection in 6 cases, herpes zoster virus infection in 3 case, candidiasis in 4 cases, syphilis in 3 cases, pulmonary tuberculosis in 2 cases, and others with cryptococcosis, salmonellosis, Mycobacterium avium-intracellulare infection, gonorrhea, Staphylococcus aureus endocarditis and bacterial sepsis, etc. The natural history of HIV-1 infection to AIDS involved acute and persistent multiple infections. Although prevalence of HIV-1 infection was low in Taiwan, nationwide surveillance of HIV-1 infection in various risk groups is still needed.

AIDS-Related Opportunistic Infections↗

PubMed1992

A new procedure for management of extrahepatic portal obstruction. Proximal splenic-left intrahepatic portal shunt.

For management of obstruction of the extrahepatic portal trunk in patients with healthy livers, we designed an end-to-side anastomosis between the proximal splenic vein and the umbilical portion of the left intrahepatic portal vein and performed a splenectomy to relieve portal hypertension, treat hypersplenism, and restore hepatic portal flow. To our knowledge, no other procedure more adeptly restores original hepatic blood flow. Creation of an anastomosis between the coronary vein and umbilical portion of the left intrahepatic portal vein is an alternative method.

Adult↗

PubMed1992

Deletion studies to reveal the basis for size discrepancy in proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA), an essential component for DNA replication in eukaryotes, is a highly conserved nonhistone nuclear protein of 261 amino acids. The molecular weight of mammalian PCNA, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), differs notably from that predicted by the cDNA sequences, that is, 36,000 in comparison with 29,261 and 28,748 for human and rat PCNA, respectively. To investigate if this discrepancy is due to posttranslational modifications, we studied the PCNA protein synthesized by an in vitro transcription/translation system as well as the protein overproduced in bacteria. We found that both PCNA protein samples were indistinguishable from the authentic protein from the protein mobility in SDS-PAGE. The finding indicates that the size discrepancy is not due to the posttranslational modifications. Hence, the size discrepancy may be due to the protein sequence per se, namely a sequence-related anomaly in SDS-PAGE. Results from the analyses of a series of PCNA derivatives with various lengths of C- or N-terminal deletion indicate that the putative sequence is in the region of residues 128-150.

Amino Acid Sequence↗