Biomedical subjects

T S Su

Publications and source records attributed to T S Su.

At least 19 recordsLinked to original sources

PubMed2001

A silent mutation induces exon skipping in the phenylalanine hydroxylase gene in phenylketonuria.

An A-->T substitution in cDNA nucleotide 1197 (c.1197A/T) of the human phenylalanine hydroxylase (PAH) gene has been regarded as a silent mutation, because both the wild-type (GUA) and the mutant (GUU) alleles encode a valine residue at codon 399 (V399 V). The nucleotide c.1197 is located at the 3'-end of exon 11at position -3 of the exon-intron junction. To explore whether the substitution exerts any effects on the processing of the PAH mRNA, illegitimate PAH transcripts from lymphoblast cultures of a phenylketonuria (PKU) patient heterozygous for c.1197A/T were analyzed by the polymerase chain reaction following reverse-transcription (RT-PCR). mRNAs with an exon 11 deletion were revealed. Furthermore, by using an R408 W mutation in the paternal allele as a marker, sequence analysis of the RT-PCR products indicates that virtually all PAH transcripts from the maternal allele with the c. 1197A/T substitution do not contain exon 11. To address whether this substitution is the main determinant for exon skipping, PAH minigenes with or without the substitution were constructed and transfected to a human hepatoma cell line. Analysis of the transcription products by S1 nuclease mapping clearly indicated that such exon 11 skipping was directly associated with the c.1197A/T substitution. Thus, this study demonstrates that the c.1197A/T substitution in the PAH gene is not just a neutral polymorphism but a mutation that induces post-transcriptional skipping of exon 11 leading to a PKU phenotype.

Base Sequence↗

PubMed2001

A nonsense mutation is responsible for the RNA-negative phenotype in human citrullinaemia.

Citrullinaemia is an inborn error of metabolism resulting from a deficiency of argininosuccinate synthetase. Previous studies of RNA of argininosuccinate synthetase of citrullinaemia patients using S1 nuclease analysis have identified a class of so-called RNA-negative alleles in which no stable mRNA can be detected. To investigate the nature of mutation responsible for such a phenotype, a compound heterozygous citrullinaemia carrying an RNA-negative allele and an allele with a 3' splice site mutation in intron 6 (IVS6-2A>G) was analysed. Using sequences of a DNA polymorphism and the IVS6-2A>G mutation as markers, approximately equal amounts of pre-mRNAs from allelic genes were detected suggesting that RNA-negative phenotype could not be the result of defect in transcription initiation. A C-to-T transition converting the CGA arginine codon at residue 279 to a TGA termination codon (R279X) was identified by cDNA sequencing. No accumulation of partially spliced pre-mRNAs containing introns immediately upstream and downstream of the nonsense mutation was observed. In addition, no mRNA species of abnormal size was detected when cDNA from the RNA-negative allele was analysed. Hence, there is no indication of nonsense-associated altered splicing (NAS). The most likely event responsible for the RNA-negative phenotype appears to be nonsense-mediated mRNA decay (NMD).

Alleles↗

PubMed2000

[Study on the packings of affinity chromatography for the separation of urokinase].

Two kinds of affinity chromatographic packings for the separation of urokinase were synthesized by coupling of p-amino benzamidine (p-ABZ) to commercially available sepharose and polyepoxypropyl methacrylate (PEPMA). Then they were applied for separating crude urokinase. It was found that the average recovery of bioactivity on sepharose was higher than that on PEPMA, resulting in 108.3% and 43.4% respectively. The high rigidity of PEPMA permits fast flow of protein solutions and operation by higher-pressure affinity chromatography. The average purification times were 36.9 folds for PEPMA column and nine-folds for Sepharose column. The purification of crude urokinase described in this paper demonstrates that PEPMA column is effective for purifying biological products in a large scale.

Chromatography, Affinity↗

PubMed1999

Evidence that mutational activation of the ras genes may not be involved in aflatoxin B(1)-induced human hepatocarcinogenesis, based on sequence analysis of the ras and p53 genes.

Exposure to aflatoxin B(1) (AFB(1)) is one of the risk factors for developing hepatoma. In rats, activation of the ras gene is a prevalent event in AFB(1)-induced hepatocarcinogenesis. It is not clear whether a similar event occurs in humans. By analysis of codon 249 of the p53 gene, six of 36 human hepatoma samples were found to show a G-->T transversion, suggesting that AFB(1) may be a risk factor for hepatocarcinogenesis. However, analysis at codons 12, 13, and 61 in the ras family genes revealed a A-->T transversion at codon 61 of the N-ras gene in a single tumor. Apparently, ras activation is rare in human hepatoma, and the mutation detected might not be induced by AFB(1). This suggests that activation of the ras gene may not be a major event in AFB(1)-related human hepatocarcinogenesis.

Aflatoxin B1↗

PubMed1999

The encapsidation signal of hepatitis B virus facilitates preC AUG recognition resulting in inefficient translation of the downstream genes.

Hepatitis B virus (HBV) DNA polymerase (P) is translated from a bicistronic pregenomic RNA via a ribosomal leaky scanning mechanism. Another viral transcript, the preC RNA, differs from pregenomic RNA by the presence of some 30 nt at the 5' end that encompass the preC initiation codon. This RNA is used exclusively for expression of the precore protein which is a precursor of secreted HBeAg. Factors leading to inefficient translation of the P and C proteins from the preC RNA were explored using a genetic approach in transient transfection assays. Our data indicate that when translating the precore protein, the elongation arrest that occurs during targeting of nascent polypeptide chains to the endoplasmic reticulum interferes with the scanning of the 40S ribosomal subunits. Such interference seems to hinder initiation of the ribosomes at the downstream genes. Furthermore, the presence of the preC initiator codon in the preC mRNA has resulted in a reduction in the number of scanning ribosomes reaching the C and P initiator codons compared with the case of pregenomic RNA. Finally, although the preC initiator codon is in a suboptimal context for translation initiation, an RNA secondary structure, the encapsidation signal, located downstream to the initiator codon is shown to enhance codon recognition, resulting in a depletion of the number of 40S ribosomal subunits available for scanning of the downstream AUG codons. This study demonstrates that the HBV encapsidation signal plays an additional role in facilitating recognition of the preC initiator codon.

Amino Acid Sequence↗

PubMed1998

Characterization of hepatitis B virus integrant that results in chromosomal rearrangement.

A hepatitis B virus (HBV) integrant was cloned from the genomic DNA library of human hepatocellular carcinoma cell line, Hep3B. Sequence analysis of the restriction fragment bearing the virus-host junction revealed that its integration pattern was the common type, with the right junction located at the cohesive region. The open reading frame of the major viral surface antigen was intact with rearranged preS1 and core sequences. The X protein, although truncated, maintained the trans-activating activity to simian virus 40 enhancer/promoter. S1 nuclease mapping showed that 4.0-, 2.9-, and 2.2-kb HBV RNAs detected in Hep3B cells were transcribed from this integrant using preS2/S promoter. By somatic-cell hybrid mapping, the left and right cellular flanking sequences were assigned to chromosomes 13 and 4, respectively. The results of this study support the notion that integrated hepatitis B virus, resulting in chromosomal rearrangement as well as the production of the carboxy-terminal truncated X protein with trans-activating activity, is important for viral hepatocarcinogenesis.

Chromosomes, Human, Pair 13↗

PubMed1998

Usage of cryptic splice sites in citrullinemia fibroblasts suggests role of polyadenylation in splice-site selection during terminal exon definition.

Citrullinemia is a human genetic disease caused by a deficient argininosuccinate synthetase. In fibroblasts established from a citrullinemia patient with a mutation at the 3' splice site of the terminal intron of the gene, three cryptic 3' splice sites; i.e., SA1275, SA1636, and SA1663, residing on the terminal exon were activated. The usage of the cryptic sites showed a gradient, with the most downstream site having the highest usage; i.e., SA1663 > SA1636 > SA1275. However, when these cryptic sites were relocated to the internal exon, SA1636 was used the most. The splice-site strength of SA1636 was at least 10-fold higher than that of SA1663 in this situation. The results suggest that the preferential usage of SA1663 residing on the terminal exon may depend on its proximity to the poly(A) signal rather than on the strength of the splice site. Furthermore, when the strength of the downstream-most splice site increased, almost all the RNAs spliced to this site. However, in the presence of the wild-type splice site, all the RNAs were processed to the authentic site. Apparently, the selection of splice site can be revealed only when the sites being selected do not differ too much in their strength. By using a naturally occurring human mutant gene as a model, this study reveals that polyadenylation may play an important role in the selection of splice site during terminal exon definition.

Amino Acid Metabolism, Inborn Errors↗

PubMed1998

Translational regulation of hepatitis B virus polymerase gene by termination-reinitiation of an upstream minicistron in a length-dependent manner.

Hepatitis B virus (HBV) polymerase (P) gene is translated from the bicistronic pregenomic RNA with the core (C) gene in the first cistron. The P ORF is preceded by the C AUG and three AUG codons within the C region, where a minicistron of 7 amino acids can potentially be translated. Our results indicate that the efficiency of the P gene translation initiation was about 10% of that of the C gene when both genes were fused in-frame to a lacZ reporter in an mRNA similar in structure to the pregenomic RNA. By mutational analysis, about 74% of the translation initiation of HBV P gene was shown to be by ribosomes that reinitiated after terminating translation of this minicistron, while the rest was by two mechanisms: one by ribosomes leaky scanning through every upstream AUG and the other by ribosomal backwards scanning to the P AUG after finishing the translation of the C gene. The efficiency of termination-reinitiation depended on the size of the minicistron, i.e. the reinitiation efficiency decreased about 50% when the size increased from 24 nt to 57 nt. When a 44 nt HBV sequence comprising the minicistron was inserted at the 5' untranslated region of the cat gene, CAT expression was regulated in a similar way to that of the HBV P gene. Moreover, when transfection occurred with an HBV expression plasmid containing an inactivated minicistron, production of virus-like particles dropped to about one-third of the wild-type level, suggesting that the termination-reinitiation mechanism is indeed important for HBV P gene expression.

Amino Acid Sequence↗

PubMed1998

[Ischemic preconditioning of the heart can be simulated by pharmacologic hibernation enkephalins].

The protective effects of ischemic preconditioning on the myocardium can be abolished by pretreatment with opioid receptor antagonists. We investigated, if--vice versa--the protective effect of preconditioning may be induced by systemic pretreatment with the delta-opioid receptor agonist D-Ala2-DLeu5-enkephalin (DADLE). DADLE is known to be very similar to the trigger substance that induces natural hibernation. Isolated working rat hearts of male Wistar rats (n = 32) were subjected to 45 minutes of global hypothermic ischemia at 30 degrees C followed by 25 minutes of normothermic reperfusion. Hearts from rats injected with 1 mg/kg DADLE intravenously 1 hour before isolated perfusion were either preconditioned by one cycle of 5 minutes of normothermic ischemia followed by 5 minutes of normothermic reperfusion or hearts were not preconditioned during preischemic perfusion. Untreated preconditioned and non-preconditioned hearts served as controls. Ischemic preconditioning alone and DADLE alone improved the recovery of aortic flow and reduced the creatine kinase leakage significantly during postischemic reperfusion. Over and above that, pretreatment with DADLE prior to ischemic preconditioning significantly further enhanced functional recovery and reduced the creatine kinase leakage, when compared to DADLE alone and preconditioning alone. Therefore we conclude, that DADLE attenuates ischemic injury in isolated rat hearts and enhances the protective effects of ischemic preconditioning. The data suggest that ischemic preconditioning and DADLE act via a similar pathway.

Animals↗

PubMed1996

Constitutive production of colony-stimulating factors by human hepatoma cell lines: possible correlation with cell differentiation.

A panel of two poorly differentiated (HA22T/VGH and SK-Hep-1) and six well-differentiated (HuH-6-cl 5, HuH-7, PLC/PRF/5, Hep G2, Hep 3B, and Tong) human hepatocellular carcinoma (HCC) cell lines were studied for the production of colony-stimulating factors (CSFs) using the granulocyte and macrophage colony formation (CFU-GM) assay, immunocytochemical staining, and Northern blotting. Medium conditioned by untreated HA22T/VGH cells contained a high level of CSFs that could stimulate the in vitro colony formation of human myeloid progenitor cells. The HA22T/VGH cell-derived CSF had an apparent molecular weight of 23 kD. Its activity could be effectively neutralized by antiserum against granulocyte-macrophage CSF (GM-CSF) but not by antibodies to other hematopoietic growth factors, including G-CSF, M-CSF, interleukin-3 (IL-3), and IL-6. Correspondingly, immunocytochemical studies using monoclonal anti-GM-CSF showed a strong positive reaction in the cytoplasm of the HA22T/VGH cells. Northern blot analysis revealed that untreated HA22T/VGH cells expressed a considerable amount of GM-CSF mRNA, confirming that GM-CSF production was constitutive. At optimal concentrations, lipopolysaccharide (LPS), IL-1beta, interferon-gamma (IFN-gamma), and tumor-promoting phorbol diester (TPA) could all stimulate HA22T/VGH cells to secrete GM-CSF. In addition to HA22T/VGH, SK-Hep-1 cells could also produce GM-CSF, although less effectively, whereas all the well-differentiated HCC cell lines tested were negative for CSF production. Morphologic, cytochemical, and immunocytochemical examinations demonstrated that both poorly differentiated CSF-producing HCC cell lines (HA22T/VGH and SK-Hep-1) were macrophage-like in morphology, possessed nonspecific esterase (NSE) activity, and expressed CD14, CD68, and HLA-DR on their surface, while all the well-differentiated HCC cell lines were epithelioid and lacked myeloid differentiation antigens. These results suggest that monocytoid features and CSF production may be differentiation markers of hepatocytes at the immature stages, amd that the HA22T/VGH and SK-Hep-1 cell lines may be valuable tools for the study of hepatic function and differentiation.

Blotting, Northern↗

PubMed1995

A nuclear post-transcriptional event responsible for overproduction of argininosuccinate synthetase in a canavanine-resistant variant of a human epithelial cell line.

The Canr1 cell line, a canavanine-resistant variant of the cultured human epithelial cell line, RPMI 2650, overproduces argininosuccinate synthetase more than 200-fold. Run-on transcription assays showed no significant difference in transcription initiation of the argininosuccinate synthetase gene between Canr1 and RPMI 2650 cells. Furthermore, no difference in the relative transcription rate was seen along this 63-kb gene, suggesting that neither transcription initiation nor elongation is responsible for differential expression of argininosuccinate synthetase in these two cell lines. However, when isolated nuclei were labeled for a longer period of time in the transcription assay, precursor RNA of argininosuccinate synthetase in RPMI 2650 cells was found to be very labile. Apparently, a nuclear event affecting precursor RNA stability is responsible for the dramatic difference in argininosuccinate synthetase levels in these two cell lines. Using a microsatellite polymorphic marker, it was demonstrated that argininosuccinate synthetase from both alleles of the gene in Canr1 cells was overexpressed. This suggests that a trans-acting mechanism may be responsible for regulation of overproduction in this cell line. Furthermore, when protein synthesis was blocked by cycloheximide, less precursor RNA was observed in Canr1 cells. These data suggest that a labile protein factor(s) participates in the regulation.

Argininosuccinate Synthase↗

PubMed1995

Demonstration of the presence of protease-cutting site in the spacer of hepatitis B viral Pol protein.

Molecular genetic studies have revealed that the human hepatitis B viral (HBV) Pol protein, a polypeptide of about 94 kDa, contains four domains. These are the 5'-terminal protein, spacer, RNA reverse transcriptase/DNA polymerase, and RNase H, respectively, from the amino (N) to carboxy (C) terminus. No evidence indicates as yet the involvement of a specific protease in cleaving the Pol protein or the presence of protease-cutting sites in the Pol protein. An in vitro-translated Pol protein was shown to be cleaved by purified thrombin but not in the presence of its inhibitor, hirudin. Two thrombin-cutting sites, spanning 194 amino acids, were then deduced by thrombin digestion of Pol protein with various lengths of C-terminal deletion. These two putative cutting sites, one located in the spacer region and the other in the beginning of the polymerase region, were found to be conserved at similar positions in the Pol protein of all hepadnaviruses. By using a novel method called the LacZ localization assay (LLA), it was demonstrated that a tripartite fusion protein containing the nucleus localization sequence (NLS) of SV40 large T Ag, the putative thrombin cutting sequence (Ile-Arg-Ile-Pro-Arg320-Thr) of HBV Pol protein and the full length beta-galactosidase of E. coli, exhibited a lower percentage (approximately 53%) of targeting into the nucleus of transfected hepatoma cells when compared with a similar tripartite protein containing a single mutation (Arg320 residue into Trp320) of HBV Pol protein (approximately 78%) or with a bipartite protein of SV40 NLS-beta-galactosidase (approximately 90%). These results indicate that the putative thrombin-cutting site in the spacer region of HBV Pol protein could be cleaved by a cellular protease resulting in the separation of NLS sequence from the beta-galactosidase and rendering a lower frequency of X-gal staining in the nucleus.

Amino Acid Sequence↗

PubMed1993

Analysis of glucocorticoid receptors in human hepatocellular carcinoma and HepG2 cells.

Hepatocellular carcinoma is the leading cause of male cancer death in Taiwan. We have found that the level of glucocorticoid receptor in hepatocellular carcinoma is significantly higher than that in the peritumoral tissue. In this study, we used a rat liver glucocorticoid receptor complementary DNA probe to examine the expression of glucocorticoid receptor gene in 15 paired samples of hepatocellular carcinoma and their peritumoral tissues. No differences in genomic DNA patterns of the glucocorticoid receptor gene were found between the tumor and peritumoral tissues. The amount of glucocorticoid receptor was found to be significantly higher in hepatoma samples than in peritumoral liver samples. The levels of glucocorticoid receptor messenger RNAs were increased in most tumors compared with their peritumoral samples. To examine the function of glucocorticoid receptors in hepatoma, we examined the expression of glucocorticoid receptor and its relation to cell-cycle progression in human HepG2 cells. Using specific monoclonal antibodies and flow cytometric study, we found glucocorticoid receptor to be expressed constitutively in all cell-cycle phases. In addition, hydrocortisone treatment of HepG2 cells resulted in increased expression of glucocorticoid receptors and increased secretion of alpha-fetoprotein. RU-486, a glucocorticoid antagonist, blocked the hydrocortisone effect, indicating that glucocorticoid receptors are functional in HepG2 cells. Taken together, our data suggest that glucocorticoids and their receptors play an important role in the growth of hepatoma.

Adult↗

PubMed1992

Identification of a missense phenylketonuria mutation at codon 408 in Chinese.

A single base transition of G to A at codon 408 of the phenylalanine hydroxylase gene is identified. This missense mutation results in the substitution of Arg408 for Gln408 (R408Q) and accounts for about 5% of phenylketonuria (PKU) chromosomes among Chinese. This mutation is in linkage disequilibrium with restriction fragment length polymorphism haplotype 4. In addition, another mutation (R408W), at the same codon and prevalent on haplotype 2 PKU chromosomes in Caucasians, is identified in a PKU allele of haplotype 41. Previously, this mutation has been observed on a haplotype 44 background in Chinese PKU patients.

Alleles↗

PubMed1991

DNA polymorphisms and deletion analysis of the Duchenne-Becker muscular dystrophy gene in the Chinese.

In our investigation of Duchenne muscular dystrophy (DMD)-Becker muscular dystrophy (BMD) gene in the Chinese, the analysis of relevant restriction fragment length polymorphisms (RFLPs) was first made in 30 normal female volunteers to determine their allele and genotype frequencies, and then in 29 DMD-BMD families for informativeness of different combinations of RFLPs in making carrier detection and prenatal diagnosis. We further screened the mutant gene, first with four 5' end intronic, genomic probes (pERT87-1, pERT87-8, pERT87-15, and XJ1.1) which did not show any deletions, and then with all dystrophin cDNA probes which disclosed 13 partial gene deletions out of 29 patients studied (45%). The deletions were nonrandomly distributed, clustering primarily near the central region of the gene. Fifty percent of the deletions involved single exon-containing HindIII restriction fragments, and again most were located near the center of the gene, emphasizing the importance of this area. Some exceptions were found against the previous suggestion that intactness of translational open reading frame resulted in a BMD phenotype. Neither the location of the breakpoints nor the length of the deletions was useful in predicting a certain phenotype. One of our patients had an intriguing pattern of partial gene deletion that lost part of the gene at the 3' end. Carrier determination was attempted by use of dosage analyses or identification of junction fragments which greatly improved accuracy and reliability.

Alleles↗

PubMed1991

Measurement of serum TSH level by ultrasensitive method in inhabitants of endemic goiter area supplied with iodized salt for 25 years.

Recently, we surveyed thyroid function and TSH concentration of villagers in an endemic goiter area where iodized salt had been supplied for 25 years; it was found that the serum FT3 and TSH levels determined with immunoradiometric analysis (IRMA) were higher and the FT4 level was lower than that of the controls. It was shown that the inhabitants of the endemic goiter area had subclinical hypothyroidism based on the "ultrasensitive" method for TSH assay. We suggest that the best biochemical techniques for monitoring the iodized salt prophylaxis program and the physiological response of the villagers to iodine should be the periodical measurement of serum TSH level using ultrasensitive assay and determination of FT4 level.

Antibodies, Monoclonal↗

PubMed1991

[Immunoradiometric measurement of serum thyrotropin levels in inhabitants who used iodized salt for 25 years in an endemic goiter area].

Recently we surveyed the thyroid function and TSH concentration of villagers in an endemic goiter area where iodized salt had been supplied for 25 years. We found that the serum FT3 and TSH (IRMA) level of villagers were higher and the FT4 level was lower than those of the controls, comparing with the RIA, which suggested that the inhabitants of the endemic goiter area had subclinical hypothyroidism based on the IRMA method for TSH assay. Therefore, we suggest that the best biochemical technique for monitoring the iodized salt prophylaxis program and the physiological response of villagers to iodine is measurement of serum TSH level with the ultrasensitive assay and FT4 level periodically.

Adolescent↗

PubMed1990

Analysis of a splice acceptor site mutation which produces multiple splicing abnormalities in the human argininosuccinate synthetase locus.

The cloned argininosuccinate synthetase gene from a citrullinemia patient's fibroblast cell line revealed a single base substitution (G to C) within the splice acceptor site of the last intron. The mutation abolished normal RNA splicing, and, by cDNA analysis, three abnormal splicing pathways were demonstrated. The major pathway involved the activation of a cryptic acceptor site in the last exon that resulted in a deletion of seven nucleotides in the mature RNA. Another pathway involved a downstream cryptic acceptor site, that is 388 nucleotides downstream from the first cryptic site. Northern blot analysis showed that this second cryptic site is present on the minor 2.7-kilobase mRNA, but not on the major species of argininosuccinate synthetase mRNA, which is 1.7-kilobases in length. Using this aberrant cDNA as a probe, the cDNA of the 2.7-kilobase mRNA was isolated and studied. Sequence analysis suggests that this species of RNA is the one that bypasses the polyadenylation signal employed by the 1.7-kilobase RNA. Since both transcripts encounter the same translation termination codon, both RNAs should encode identical protein. Furthermore, a tract of 22 repeats of d(CA).(GT) is found at the 3' end of the gene and this repeat sequence is present on the 2.7-kilobase RNA. The third pathway of the abnormal splicing revealed a rare class of transcript that has the last intron retained in the mature RNA. This study shows that in human the intron inclusion can occur through a naturally occurring point mutation. All these abnormally spliced RNAs resulted in a protein reading frame shift.

Amino Acid Metabolism, Inborn Errors↗