Search PubMed⌕ Search

Biomedical subjects

C P Tu

Publications and source records attributed to C P Tu.

At least 37 records · Page 2Linked to original sources
PubMed1991

Antineoplastic drug sensitivity of human MCF-7 breast cancer cells stably transfected with a human alpha class glutathione S-transferase gene.

Studies have suggested that the alpha class glutathione S-transferase (GST) may protect cells from the chemotherapeutic drugs chlorambucil and melphalan. In order to further define the function of human alpha class GST, a complementary DNA which encodes it was ligated into an expression vector under the direction of the human metallothionein-IIA promoter and stably transfected into human MCF-7 breast cancer cells in conjunction with the G418-selectable plasmid pSV2neo. Clonal cell lines were identified which expressed increased levels of GST enzyme activity (2.2- to 5.6-fold). The transfected cell lines also had increased peroxidase activity using cumene hydroperoxide as the substrate (1.9- to 3.8-fold) which is consistent with the intrinsic peroxidase activity of alpha class GSTs. Southern blot analysis indicated that genomic DNA from these cells contained a fragment indistinguishable from the transfected alpha class GST complementary DNA (850 base pairs); Northern blot analysis of total cellular RNA indicated that these cells contained appropriately sized alpha class GST RNA (980 nucleotides); and Western blot analysis indicated that, while MCF-7 cells contained no detectable alpha class GST protein, the transfected cells contained markedly elevated levels of alpha class GST but no detectable mu or pi class GST. These alpha class GST transfected cells had increased resistance to ethacrynic acid (2.1- to 3.0-fold). However, the transfected cells failed to show any increased resistance measured at the drug dosage which inhibited 50% of the colony formation to the chemotherapeutic drugs chlorambucil, melphalan, Adriamycin, or cisplatin under conditions of either continuous or 1-h drug exposure. Neither was there any change in sensitivity to the cytotoxins benzo(a)pyrene, benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti), or 1-chloro-2,4-dinitrobenzene. These studies indicate that expression of this human alpha class GST by itself in MCF-7 human breast cancer cells does not confer resistance to the chemotherapeutic drugs tested under the conditions used in these studies.

Antineoplastic Agents↗
PubMed1991

High-dose-rate afterloading technique in the radiation treatment of uterine cervical cancer: 399 cases and 9 years experience in Taiwan.

Carcinoma of the uterine cervix has been the leading malignant neoplasm treated in our department. A comparative study was carried out using conventional low dose rate (LDR) with 137Cs sources (less than or equal to 100 cGy/hr) versus high dose rate (HDR) with 60Co sources (greater than 100 cGy/min) in the intracavitary (IC) application following external pelvic irradiation. A total of 399 patients were treated with external RT plus HDR radiation treatment alone from February 1980 through December 1985. Stage IIb and IIIb comprised 79.4% of cases (317 cases). The rate of initial complete response of the tumor, local control, and survival rate seemingly were better in the HDR group, but there was no significant difference statistically. The actuarial survival rates in all cases/definitive RT cases are 85%/85% for Stage 0-IIa, 53%/70% for IIb, 43%/49% for IIIa, 43%/53% for IIIb, 42%/47% for IVa, respectively. Complications were similar and the rectal complications were slightly higher in HDR group. The combination of pelvic irradiation with HDR intracavitary irradiation was more convenient for patients and also for personnel. The HDR technique may be a good substitution for IC treatment.

Adult↗
PubMed1990

Characterization of glutathione S-transferases from day-old chick livers.

Glutathione S-transferases (GSTs, EC 2.5.1.18) were isolated from the liver cytosolic fraction of 1 day old Leghorn chicks by S-hexylglutathione and glutathione affinity columns arranged in tandem. After sample loading, the affinity columns were detached from each other and developed separately. Four groups of GSTs (CL 1, 2, 3, and 4) were eluted from the hexylglutathione column, and an additional group of GSTs (CL 2 and 5) was eluted from the glutathione affinity column. CL 2, CL 3, and CL 5 were further purified to homogeneity by chromatofocusing, and the substrate specificities of each group were determined. Fractions from the chromatofocusing column were analyzed by native IEF electrophoresis. Protein bands were electroblotted onto PVDF membrane for N-terminal sequence analysis or extracted from IEF gel and rerun on SDS-PAGE to determine the subunit composition of each GST dimer. CL 2, CL 3, and CL 5 can form homodimers, whereas CL 1 and CL 4 exist only as CL 1-2 and CL 3-4 heterodimers. CL 2 and CL 5 have N-terminal amino acid sequences homologous to rat liver Yb and Ya GSTs, respectively. CL 1 has a unique N-terminal sequence that is not homologous to any known GSTs.

Amino Acid Sequence↗
PubMed1990

Elevation of rat liver mRNA for selenium-dependent glutathione peroxidase by selenium deficiency.

Selenium-dependent glutathione peroxidase (Se-GSH-Px, GSH-H2O2 oxidoreductase EC 1.11.1.9) is the best characterized selenoprotein in higher animals, but the mechanism whereby selenium becomes incorporated into the enzyme protein remains under investigation. To elucidate the mechanism of insertion of selenium into Ge-GSH-Px further, we have systematically analyzed and compared the results of Western blot, in vitro translation immunoprecipitation, and Northern blot experiments conducted with liver proteins and RNAs obtained from rats fed on selenium-deficient and selenium-supplemented diets. The anti-serum employed in this study was raised against an electrophoretically pure Se-GSH-Px preparation obtained from rat livers by a simplified purification procedure involving separation by high performance liquid chromatography on a hydrophobic interaction column. Different forms of Se-GSH-Px, including apo-protein, cross-reacted with this antiserum and Western blot analysis found no Se-GSH-Px protein present in livers from rats fed on selenium-deficient diets. By contrast, a distinct protein band corresponding to purified Se-GSH-Px was detected in livers from selenium-supplemented animals, a result consistent with the finding that the Se-GSH-Px activity was reduced to undetectable levels in livers of selenium-deficient rats. The in vitro translation experiments, however, indicated not only that mRNA for Se-GSH-Px was present during selenium deficiency but also that its translation products contained 2-3-fold as much immunoprecipitable protein as the products of poly(A) RNA from livers of selenium-supplemented rats. This result suggests that the Se-GSH-Px mRNA may be increased in the selenium-deficient state. Elevated levels of Se-GSH-Px mRNA were directly demonstrated in Northern blot experiments employing cDNA clone pGPX1211 as a probe. A similar increase in Se-GSH-Px mRNA was observed in such other tissues as kidney, testis, brain, and lung tissue, in selenium-deficient states. The present data support the co-translational mechanism for the incorporation of selenium into Se-GSH-Px in rat liver.

Animals↗
PubMed1990

The gene for the microsomal glutathione S-transferase is on human chromosome 12.

The microsomal glutathione S-transferase (GST) is a unique membrane-bound GST structurally distinct from the cytosolic GSTs. A cDNA encoding this 154 amino acid protein has recently been isolated and characterized. Using the cDNA as the hybridization probe, we now report the assignment of the human microsomal GST gene to chromosome 12 through the use of a panel of mouse-human somatic cell hybrid lines. This locus has recently been designated as GST 12. In addition, genomic Southern blotting data suggest that the human microsomal GST is encoded by a single- or very-low-copy gene. Therefore, the human GST gene superfamily resides on at least four separate chromosomes: 1 (GST 1), 6 (GST 2), 11 (GST 3), and 12 (GST 12).

Animals↗
PubMed1990

Drosophila glutathione S-transferase 1-1 shares a region of sequence homology with the maize glutathione S-transferase III.

We have characterized a Drosophila glutathione S-transferase (RX:glutathione R-transferase, EC 2.5.1.18) cDNA encoding a protein of 209 amino acids. The cDNA was expressed in Escherichia coli harboring the expression plasmid construct pGTDml-KK. The active enzyme, designated as Drosophila glutathione S-transferase 1-1, had a specific activity toward 1-chloro-2,4-dinitrobenzene comparable to that for the mammalian glutathione S-transferases but did not have as broad a substrate specificity pattern. There is a region of 44 amino acids in this enzyme that shares 66% identity with an analogous region of maize glutathione S-transferase III. Drosophila glutathione S-transferase 1-1 had no obvious homology to any mammalian or parasitic glutathione S-transferases. The gene was found to be a member of a multigene family.

Amino Acid Sequence↗
PubMed1990

[Evaluation of quality assurance program in high dose rate remote after-loading brachytherapy].

In the past 9 years, we have routinely used high-dose-rate remote afterloading systems, a Toshiba RAL-302-2 remote afterloading system and a self-fabricated Ir-192 system, for radiation therapy of various cancers, particularly cancers of the uterine cervix and nasopharynx. The frequency of using these two systems is more than 30 times per week. These machines can deliver high-dose radiation (300-1000 cGy) to a specifically planned target on the patient in a few minutes, therefore, the performance of the systems requires well planned dosimetry and quality control assurance in order to achieve effective therapeutic results and ensure safety of the patient under treatment. Any malfunction of the machines may cause over-radiation that exceeds a planned dose to the patient, and any distortion in the distribution of a planned dose may give an unexpected high dose of radiation to the therapist operating the machines. Hence, a quality assurance program must be enforced regularly to avoid or minimize any possible accidents. Based on information and recommendations reported in the literature, as well as from our own experience, we set up a program of routine checks to correct and adjust the machinery conditions and to ensure the optimum operational status of the equipment on a weekly, monthly, semiannual and annual basis. In checking the Co-60 machine, we found that the rate of occurrence for abnormal source position was 3.9% and that for the timer was 2.8%. In the Ir-192 machine checks, abnormal conditions were found only in the source position at the rate of 7.8%.

Brachytherapy↗
PubMed1989

[Radiation safety program at the Chang Gung Memorial Hospital].

There are many areas in several departments where radiation-producing facilities are routinely used for diagnosis and therapy in Chang Gung Memorial Hospital. We used RD-8 Wallac survey meters to survey the surroundings of controlled and noncontrolled areas. The results of the survey showed background level in the noncontrolled areas and under 0.2 mR/hr in the controlled areas. In diagnostic fluoroscopy, the worker wore 0.5mm lead apron and received an exposure under 0.5 mR/hr. The operator of the portable X-ray machine was exposed to an exposure of background level when he was 4.5 meter from target and wore a 0.5mm lead apron. The patients in the nuclear medicine department were given 15m Ci Tc-99m for bone scan and the exposure rate was under 0.5 mR/hr at 1.0 meter from the patient. The patient loaded with 55 mCi of Cs-137 for brachytherapy produced exposure rate of 1.5 mR/hr behind lead shielding placed beside the patient bed, and the surroundings outside the room was at background level. We also checked the execution and enforcement of the radiation safety regulations which was found to have met the requirements of the Atomic Energy Council.

Hospital Departments↗
PubMed1989

Amino acid substitutions in the human glutathione S-transferases confer different specificities in the prostaglandin endoperoxide conversion pathway.

The human glutathione S-transferases 1-1 and 2-2, which differ from each other by 11 amino acids, have different catalytic activities against cumene hydroperoxide and t-butyl hydroperoxide. Using prostaglandin H2 as the peroxide substrate, we found that GSH S-transferase 1-1 catalyzed the transformation of prostaglandin H2 to prostaglandin F2 alpha and E2 at a 4:1 ratio whereas GSH S-transferase 2-2 produced primarily prostaglandin D2 and F2 alpha at a 4:1 ratio. Our results indicate that GSH S-transferases catalyze the reduction and isomerization of prostaglandin H2 endoperoxide in vitro. We suggest that the amino acid substitutions between these two isozymes may be responsible for the difference in catalytic specificities. We propose that these isozymes are important reagents for the biosynthesis of various prostaglandins.

Dinoprost↗
PubMed1989

Characterization of a variant rat glutathione S-transferase by cDNA expression in Escherichia coli.

We have isolated a glutathione S-transferase Yb1 subunit cDNA from a lambda gt11 cDNA collection constructed from rat testis poly(A) RNA enriched for glutathione S-transferase mRNA activities. This Yb1 cDNA, designated pGTR201, is identical to our liver Yb1 cDNA clone pGTR200 except for a shorter 5'-untranslated sequence. Active glutathione S-transferase is expressed from this Yb1 cDNA driven by the tac promoter on the plasmid construct pGTR201-KK. The expressed glutathione S-transferase protein begins with the third codon (Met) of the cDNA, and is missing the N-terminal proline of rat liver glutathione S-transferase 3-3. Therefore, our Escherichia coli expressed glutathione S-transferase protein represents a variant form of glutathione S-transferase 3-3 (Yb1Yb1), designated GST 3-3(-1). The expressed Yb1 subunits are assembled into a dimer as purified from sonicated E. coli crude extracts. In the absence of dithiothreitol three active isomers can be resolved by ion-exchange chromatography. The pure protein has an extinction coefficient of 9.21 x 10(4) M-1 cm-1 at 280 nm or E0.1% 280 = 1.78 and a pI at 8.65. It has a substrate specificity pattern similar to that of the authentic glutathione S-transferase 3-3. The GST 3-3(-1) has a KM of 202 microM for reduced GSH and of 36 microM for 1-chloro-2,4-dinitrobenzene. The turnover number for this conjugation reaction is 57 s-1. Results of kinetic studies of this reaction with GST 3-3(-1) are consistent with a sequential substrate binding mechanism. We conclude that the first amino acid proline of glutathione S-transferase 3-3 is not essential for enzyme activities.

Animals↗
PubMed1989

Dosimetry of high dose rate intracavitary radiation therapy using Co-60 and Ir-192 sources.

For decades, the high dose rate sources of Co-60 and Ir-192 have been used in remote controlled afterloading brachytherapy. A small volume ion chamber was used to measure the dose rate in varying distances from the sources. The entire dose distribution around the source was tabulated and the data were used to calculate the treatment time for individualized patient setting. A solid diode detector was used to plot the relative distance dose and the symmetrical dose distribution of the sources. Thermoluminescence dosimetry (TLD) was used for spot-checking of the dose. The dose distribution in the clinically useful range of 2-5 cm was carefully estimated. When normalized at 2.0 cm from the source, the dose rate at 5.0 cm was 17.1% for the Co-60 source and 18.4% for the Ir-192 source. It is clearly demonstrated that Ir-192 offers no dosimetric advantages, which is contrary to what used to be believed by some clinicians. The parameters of the computer treatment planning system have to be corrected and then adopted into clinical application.

Brachytherapy↗
PubMed1988

Human glutathione S-transferases. The Ha multigene family encodes products of different but overlapping substrate specificities.

The human glutathione S-transferase cDNAs encoding subunits 1 and 2 contain intrinsic ribosome-binding sites in their 5'-untranslated regions for direct expression in Escherichia coli. We show that functional human GSH S-transferases 1-1 and 2-2 are synthesized from lambda gt11 cDNA clones lambda GTH1 and lambda GTH2 in phage lysates of E. coli Y1090, in lysogens of E. coli Y1089, and from the plasmid expression constructs in pKK223-3. The E. coli-expressed human GHS S-transferases 1-1 and 2-2 do not have blocked N termini in contrast to those directly purified from human livers. These two isozymes, with 11 amino acid substitutions between them, are similar in their Km values for GSH and 1-chloro-2,4-dinitrobenzene and Kcat values for this conjugation reaction. The human GSH S-transferase 2-2, however, is a more active GSH peroxidase than transferase 1-1 toward cumene hydroperoxide and t-butyl hydroperoxide. Our results indicate that different members of a GSH S-transferase gene family with limited amino acid substitutions have different with limited amino acid substitutions have different but overlapping substrate specificities. We propose that accumulation of single amino acid replacements may be an important mechanism for generating diversity in GSH S-transferases with various xenobiotic substrates. In situ chromosomal hybridization results show that the GSH transferase Ha genes are located in the region of 6p12.

Base Sequence↗
PubMed1988

The human liver glutathione S-transferase gene superfamily: expression and chromosome mapping of an Hb subunit cDNA.

We have isolated from a lambda gt10 cDNA library a clone lambda GTH4 which encodes a human liver glutathione S-transferase Hb subunit, designated as subunit 4. Expression of this cDNA in E. coli and subsequent purification and immunoblotting analysis provided a definitive assignment of a structure and function relationship. RNA blot hybridization with human liver poly(A) RNA revealed a single band of approximately 1200 nucleotides, comparable in size to the rat brain Yb3 mRNA. Divergence analysis of amino acid replacement sites in subunit 4 relative to the four rat Yb subunits revealed that it is most closely related to the brain-specific Yb3 subunit. This conclusion is further substantiated by the nucleotide sequence homology between lambda GTH4 and the Yb3 cDNA in their 3' untranslated region. In situ chromosome mapping has located this glutathione S-transferase gene in the region of p31 on chromosome 1. Results from many laboratories, including ours, indicate that the human glutathione S-transferases are encoded by a gene superfamily which is located on at least two different chromosomes.

Amino Acid Sequence↗
PubMed1988

Gene expression of rat glutathione S-transferases. Evidence for gene conversion in the evolution of the Yb multigene family.

We have characterized a cDNA with complete coding sequence for the rat liver glutathione S-transferase subunit 4 (Yb2) isolated from a constructed lambda gt10 cDNA library. Functional expression of the cDNA sequence has resulted in the purification to homogeneity of an enzymatically active anionic glutathione S-transferase. In addition to three previously described Yb-type subunits (Yb1, Yb2, Yb3), we now report characterization of a fourth Yb subunit sequence in the form of a genomic DNA clone lambda GTR15-2. The Yb4 gene has no apparent defect, and the deduced Yb4 polypeptide sequence differs from the other three Ybs by 40 to 53 amino acids. The Yb4 gene organization is similar to that of the Yb2 gene in having a minimum of eight exons. Three out of the seven introns between the two genes are conserved to the extent of more than 88% nucleotide identity. We propose that gene conversion may have played a role in the evolution of these Yb genes.

Amino Acid Sequence↗
PubMed1988

Expression of glutathione peroxidase I gene in selenium-deficient rats.

We have characterized a cDNA pGPX1211 encoding rat glutathione peroxidase I. The selenocysteine in the protein corresponded to a TGA codon in the coding region of the cDNA, similar to earlier findings in mouse and human genes, and a gene encoding the formate dehydrogenase from E. coli, another selenoenzyme. The rat GSH peroxidase I has a calculated subunit molecular weight of 22,155 daltons and shares 95% and 86% sequence homology with the mouse and human subunits, respectively. The 3'-noncoding sequence (greater than 930 bp) in pGPX1211 is much longer than that of the human sequences. We found that glutathione peroxidase I mRNA, but not the polypeptide, was expressed under nutritional stress of selenium deficiency where no glutathione peroxidase I activity can be detected. The failure of detecting any apoprotein for the glutathione peroxidase I under selenium deficiency and results published from other laboratories supports the proposal that selenium may be incorporated into the glutathione peroxidase I co-translationally.

Amino Acid Sequence↗
PubMed1988

Gene expression of rat and human microsomal glutathione S-transferases.

We have isolated and characterized cDNA clones for both the rat and human liver microsomal GSH S-transferase (mGST) mRNAs. The rat sequence is 883 nucleotides long with 5'- and 3'-untranslated regions of 55 and 360 nucleotides, respectively, and contains two polyadenylation signals. The highest level of mRNA expression occurs in the rat liver, with widely varied levels of expression in extrahepatic tissues. Primer extension of rat liver poly(A) RNAs showed that only 12 nucleotides exist beyond the isolated cDNA. Southern blotting analysis of rat genomic DNA showed that this gene resides in a single EcoRV band of approximately 23 (kilobases) kb. Digestions with other restriction enzymes were consistent with the microsomal GST gene being a single copy gene with at least 3 exons spanning less than 12 kb. Human microsomal GST cDNAs were also characterized, and a 909 nucleotide sequence was determined with 5'- and 3'-untranslated regions of 74 and 368 nucleotides, respectively. RNA blot hybridization of human liver mRNA demonstrated that the human mGST is very similar in size to the rat mGST poly(A) RNA. Both the rat and human sequences contain a 154-amino acid reading frame and both show the same seven amino acid changes compared to the previously published protein sequence which upon reevaluation reveals the cDNA-deduced sequence to be correct. These two transcripts share 77% nucleotide similarity in the coding region, 47% in the 5'-untranslated regions and 61% in the 3'-untranslated regions. The rat and human microsomal GSTs show 95% conservation in amino acids. Several lines of evidence suggest that neither transcript contains a membrane-directing cleavable signal sequence.

Amino Acid Sequence↗
PubMed1988

Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family.

We have isolated from a constructed lambda gt11 expression library two classes of cDNA clones encoding the entire sequence of the maize GSH S-transferases GST I and GST III. Expression of a full-length GST I cDNA in E. coli resulted in the synthesis of enzymatically active maize GST I that is immunologically indistinguishable from the native GST I. Another GST I cDNA with a truncated N-terminal sequence is also active in heterospecific expression. Our GST III cDNA sequence differs from the version reported by Moore et al. [Moore, R. E., Davies, M. S., O'Connell, K. M., Harding, E. I., Wiegand, R. C., and Tiemeier, D. C. (1986) Nucleic Acids Res. 14:7227-7235] in eight reading frame shifts which result in partial amino acid sequence conservation with the rat GSH S-transferase sequences. The GST I and GST III sequences share approximately 45% amino acid sequence homology. Both the GST I and the GST III mRNAs contain different repeating motifs in front of the initiation codon ATG. Multiple poly(A) addition sites have been identified for these two classes of maize GSH S-transferase messages. Genomic Southern blotting results suggest that both GST I and GST III are present in single or low copies in the maize (GT112 RfRf) genome.

Amino Acid Sequence↗