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Biomedical subjects

J G Chang

Publications and source records attributed to J G Chang.

At least 127 records · Page 7Linked to original sources
PubMed1993

Cloning of a portion of the chromosomal gene and cDNA for human beta-fodrin, the nonerythroid form of beta-spectrin.

A 96-bp synthetic oligonucleotide corresponding to an amino acid sequence near the N-terminus of erythroid beta-spectrin was used to screen a human genomic library, and two overlapping recombinants were isolated. DNA sequence analysis established that the genomic fragment encoded beta-fodrin, the nonerythroid form of beta-spectrin, by correlation to a known amino acid sequence of human brain beta-fodrin. The genomic DNA contained regions that cross-hybridized with an erythroid beta-spectrin cDNA probe, and the DNA sequence of these regions revealed a high degree of identity with that of erythroid beta-spectrin and a similar exon/intron organization. A single-copy DNA fragment of the beta-fodrin genomic clone was used to screen a lymphoid cell cDNA library and two recombinants were isolated. The composite DNA sequence of these various genomic and cDNA clones encoded almost all of the first twelve 106 amino acid repeat segments of beta-fodrin that shared 58% identity and 75.5% similarity with the amino acid sequence of beta-spectrin and 66% identity with the nucleotide sequence of beta-spectrin cDNA. The chromosomal localization of the gene was determined to be chromosome 2 by hybridization of a single-copy probe derived from the cloned genomic DNA to DNA of a panel of somatic hybrid cell lines, and in situ hybridization localized the gene to band 2p21. beta-Fodrin was assigned the gene symbol SPTBN1.

Amino Acid Sequence↗
PubMed1993

Mutation analysis of K-ras oncogenes in gastroenterologic cancers by the amplified created restriction sites method.

A rapid, simple, and nonradioactive method for diagnosing point mutations of c-K-ras oncogenes in gastroenterologic cancers is described. This method involved the selective amplification of DNA fragments from cancer tissues of surgical specimens with specific oligonucleotide primers, followed by digestion with restriction enzymes that recognized artificially created or naturally occurring restriction sites. To detect codon 12 mutations, an artificial Msp I site was created by introducing a single nucleotide mismatch into the 5' mutagenesis primer. Using a similar approach, an Hae III site was created to detect codon 13 mutations. Bal I and MBo II sites were used to detect codon 61 mutations. A total of 61 gastroenterologic cancer cases were studied. Of 35 cases of colorectal cancer, 7 showed mutations: 6 at codon 12 and 1 at codon 13. In 1 of 2 cases of cholangiocellular carcinoma, point mutation at codon 12 was found. One case of duodenal cancer showed point mutation at codon 12. No mutations were found in the cases of hepatocellular carcinoma (4), gastric cancer (12), esophageal cancer (3), or pancreatic cancer (2).

Amino Acid Sequence↗
PubMed1993

Molecular basis and haematological characterization of beta-thalassaemia major in Taiwan, with a mutation of IVS-1 3' end TAG-->GAG in a Chinese patient.

We studied 41 patients with beta-thalassaemia major and their parents by using a combination of polymerase chain reaction (PCR) amplification, slot-blot hybridization of allele-specific oligonucleotide (ASO), and direct genomic sequencing. Eight different point mutations were characterized. C to T substitution at nucleotide (nt) 654 of intervening sequences (IVS) 2, accounting for 46.3% of mutant beta-globin genes, is the most common mutation in Taiwan, followed by frameshift codons 41/42 with four nucleotides (TCTT) deletion for 31.7%, A to G substitution at position -28 of promotor area for 8.5%, A to T substitution at codon 17 for 6.1%, frameshift codons 27/28 (insertion of C) for 2.4%, G to T substitution at nucleotide 1 of IVS-1 for 2.4%, frameshift codons 71/72 (insertion of A) and IVS-1 3 end TAG-->GAG for 1.2%. The former four mutations showed no obvious difference between two major ethnic groups in Taiwan. As to mutations in each individual of beta-thalassaemia major, the incidence of compound heterozygotes of two different mutations is much higher than homozygotes of single mutation, 78.0% v 22.0%. Compound heterozygotes of C to T substitution at nt 654 of IVS-2 and frameshift codons 41/42 with four nucleotides deletion is the most common pattern of beta-thalassaemia mutations in each individual (41.5%). The results are somewhat different from other documented reports concerning the mutations of beta-thalassaemia in southern China. This is the first report of mutation of IVS-1 3' end TAG-->GAG which causes consensus change in Chinese people. Patients with heterozygotes of beta zero and -28 beta(+)-thalassaemia mutations would have a greater delay in initial transfusion in comparison to patients with homozygotes of both beta zero-thalassaemia mutation, but their initial clinical manifestation might be aggravated when combined with a glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and an insult such as exposure to infection and certain drugs.

Adolescent↗
PubMed1993

Molecular characterization of Hb H disease by polymerase chain reaction.

We utilized the PCR method to amplify the alpha-thalassemia-1 breakpoint area of the Southeast Asia type and several regions of the alpha-globin gene cluster to diagnose rightward deletion (-alpha 3.7), leftward deletion (-alpha 4.2) or nondeletion forms of the Hb H disease. For the nondeletion form, a natural restriction site of MseI was used to detect the Hb Constant Spring (Hb CS) or other termination codon mutations. Another naturally occurring restriction site of MspI was used to detect the Hb Quong-Sze. For the deletion form of the Hb H disease, the differences among nonhomologous I, II and III of the rightward or leftward deletion were used to distinguish the mutations. For further characterization of the subtype of -alpha 3.7 deletion, the same primers for detecting termination codon mutations were used to amplify part of the alpha-globin gene, then the PCR product was digested by the restriction enzyme ApaI. In the 57 cases which were studied, 19 were deletion forms while 38 were nondeletion forms. In the deletion form cases, 13 were rightward deletion (-alpha 3.7) and the other 6 were leftward deletion (-alpha 4.2). However, all of the nondeletion form cases were alpha-thalassemia-1 with Hb CS. After the subtyping of -alpha 3.7 deletion, 11 out of 13 were type I deletions and the other 2 were type II deletions.

Adolescent↗
PubMed1993

"Hot spots" mutation analysis of p53 gene in gastrointestinal cancers by amplification of naturally occurring and artificially created restriction sites.

We developed a rapid, simple method to detect "hot spot" point mutations of the p53 gene. A DNA fragment from cancer tissue of a surgical specimen was selectively amplified with specific oligonucleotide primers and then digested with restriction enzymes that recognized artificial or naturally occurring restriction sites. To detect mutations in codons 273 and 245, we created artificial Bst U1 and BgI I sites by introducing a single nucleotide mismatch into the respective mutagenesis primers. We used the naturally occurring restriction sites of Msp I, Hae III, and Hha I to detect mutations in codons 248, 249, and 175, respectively. In 74 cases of gastrointestinal cancer, 5 of 35 colorectal cancers showed mutations; 1 of 15 cases of gastric cancers showed mutation; and 1 of 24 hepatocellular carcinomas showed mutation. This nonradioactive method is an accurate and clinically useful way to detect hot-spot point mutations of the p53 gene in gastrointestinal cancers.

Base Sequence↗
PubMed1993

Production and characterization of monoclonal antibodies against alpha and beta spectrin subunits.

Twenty monoclonal antibodies against human erythrocyte spectrin were generated using purified native spectrin as the immunogen. Thirteen out of 20 monoclonal antibodies reacted to the alpha subunit, and the remaining seven monoclonal antibodies reacted to the beta subunit in Western blot analysis. One anti-beta subunit and two anti-alpha subunit monoclonal antibodies cross-reacted with the cell lysate of the non-erythroid T. lymphoma, J. Jhan, cell line. Identification of 20 monoclonal antibodies to the respective tryptic domains was also achieved. The specificity of these antibodies to respective tryptic domains of spectrin confirms the alignments and uniqueness of each of the previously identified five domains in the alpha subunit and four domains in the beta subunit, and establishes the identity of proteolytic daughter peptide fragments.

Animals↗
PubMed1993

Prenatal and molecular diagnosis of beta-thalassemia major in Taiwan by naturally and amplified created restriction sites.

To characterize mutations rapidly in 43 patients with beta-thalassemia major in Taiwan, we utilized a method of natural and amplified created restriction site (ACRS) analysis for detection of beta-globin gene mutation. After analysis, eight different point mutations were found among 86 known chromosomes. IVS-2 nt 654 (C-->T), accounting for 40 of the 86 mutations with mutant beta-globin genes, is the most common mutation, followed by frameshift codons 41/42 (-TCTT) in 28 mutations, -28 mutation (A-->G) in 7 mutations, nonsense codon 17 (A-->T) in 5 mutations, frameshift codons 27/28 (insertion of C) in 2 mutations, IVS-1 nt 1 (G-->T) in 2 mutations, frameshift codons 71/72 (insertion of A) in 1 mutation, and IVS-1 3' end TAG-->GAG in 1 mutation. The first four mutations account for 80 of all 86 mutations of beta-thalassemia major in Taiwan. Furthermore, the beta-globin gene mutation was identified successfully in one chorionic villi biopsy for prenatal diagnosis and in specimen of blood from one patient who had received bone marrow transplantation (BMT). Complete diagnosis is possible in all of the Chinese families with beta-thalassemia in Taiwan, and the first trimester prenatal diagnosis can be achieved simply by using only 13 oligonucleotide primers and 10 restriction endonucleases. This non-radioactive assay was shown to be a rapid, sensitive, precise and safe method in detecting the mutations of beta-thalassemia in Taiwan.

Adolescent↗
PubMed1992

Rapid diagnosis of beta-thalassemia mutations in Chinese by naturally and amplified created restriction sites.

We developed a rapid and simple method to diagnose the molecular defects of beta-thalassemia in Chinese patients. This method involves the selective amplification of a DNA fragment from human beta globin gene with specific oligonucleotide primers, followed by digestion with restriction enzymes that recognize artificially created or naturally occurring restriction sites. To detect the 4-nucleotide deletion of codon 41-42, we introduced a single mismatch nucleotide into the 3' end of the upstream primer to create an artificial Taq I restriction site. With a similar approach, an artificial Rsa I site was generated to detect the nucleotide 654 mutation (C-->T) of IVS-2, and Alu I restriction site was created to detect the codon 17 mutation (A-->T), and EcoRI restriction site was created for the -28 mutation (A-->G), a Rsa I restriction site was created for the nucleotide 5 mutation (G-->C) of IVS-1, and a Spe I restriction site was created to distinguish the codon 71 (+T) and codon 71/72 (+A) mutations from a normal sequence. The other eight rare mutations that occur in the genes of the Chinese people naturally create or abolish restriction sites. Using this kind of approach, we are able to provide a simple, rapid, accurate, and nonradioactive method to detect the genetic defects of beta-thalassemia in the Chinese population. It should be used not only for routine screening but also for prenatal diagnosis.

Base Sequence↗
PubMed1992

Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) deficiency by natural and amplification created restriction sites: five mutations account for most G6PD deficiency cases in Taiwan.

We have developed a rapid and simple method to diagnose the molecular defects of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Chinese in Taiwan. This method involves the selective amplification of a DNA fragment from human G6PD gene with specific oligonucleotide primers followed by digestion with restriction enzymes that recognize artificially created or naturally occurring restriction sites. Ninety-four Chinese males with G6PD deficiency were studied. The results show that 50% (47 of 94) were G to T mutation at nucleotide (nt) 1376, 21.3% (20 of 94) were G to A mutation at nt 1388, 7.4% (7 of 94) were A to G mutation at nt 493, 7.4% (7 of 94) were A to G mutation at nt 95, 4.2% (4 of 94) were C to T mutation at nt 1024, 1.1% (1 of 94) was G to T mutation at nt 392, and 1.1% (1 of 94) was G to A mutation at nt 487. These results show that the former five mutations account for more than 90% of G6PD deficiency cases in Taiwan. Aside from showing that G to T change at nt 1376 is the most common mutation, our research indicates that nt 493 mutation is a frequent mutation among Chinese in Taiwan. We compared G6PD activity among different mutations, without discovering significant differences between them.

Base Sequence↗
PubMed1992

Rapid molecular diagnosis of hemoglobin variants by RT-PCR of reticulocyte mRNA and direct sequencing.

We have developed a rapid and simple approach for the molecular characterization of hemoglobin variants by a one-step reverse transcription-polymerase chain reaction of reticulocyte mRNA and direct sequencing of the product. This method can selectively amplify the alpha 1- or alpha 2-globin gene or the beta-globin gene transcript. The amino acid substitution of Hb G-Taichung is due to a G----C mutation at codon 74 of the alpha 1-globin gene, that of Hb J-Meinung to a G----A substitution at codon 56 of the beta-globin gene, and that of Hb Kaohsiung (or New York) to a T----A substitution at codon 113 of the beta-globin gene. The amplified segment encompassed the sequence from upstream of the initial codon behind the Cap site to downstream of the terminal codon before the polyadenylation addition signal. Hence, all hemoglobin variants should be able to be characterized by this approach.

Base Sequence↗
PubMed1992

Suppression of tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6 and granulocyte-monocyte colony stimulating factor secretion from human monocytes by an extract of Poria cocos.

Fu-Ling, the sclederma of Poria cocos (Schw.) Wolf, has long been used as a sedative and diuretic. However, data in this report suggest that Fu-Ling is a potential suppressor of cytokine secretion from human peripheral blood monocytes under in vitro condition. Monocyte culture medium containing 10% of Fu-Ling extract significantly inhibited secretion of TNF-alpha, IL-beta, IL-6 and GM-CSF from the monocyte monolayer. However, as Fu-Ling extract content was gradually reduced, cytokine secretion was augmented in comparison with the cytokine secretion in drug-free controls. This augmentative effect resulted from the trace amount (1.24 ng/ml in 0.62% of Fu-Ling extract) of lipopolysaccharide (LPS) which contaminated the Fu-Ling extract during the preparation process, since TNF-alpha, IL-1 beta and IL-6 secretion induced by 0.62% Fu-Ling extract could be significantly inhibited by polymyxin B, an LPS inhibitor. Furthermore, the amounts of TNF-alpha IL-1 beta and IL-6 induced by 1 ng/ml of LPS without the presence of drug were more than that induced by 0.62% of Fu-Ling extract. Thus, cytokine secretion induced by LPS contamination (1.24 ng/ml) in the Fu-Ling extract was partially suppressed by 0.62% of the Fu-Ling extract itself. GM-CSF secretion in the medium containing 0.62% of Fu-Ling extract was not induced by LPS since: a) GM-CSF induced by 0.62% Fu-Ling extract could not be inhibited by polymyxin B; b) LPS at 1 ng/ml showed no activity indicating induction of GM-CSF secretion.

Cytokines↗
PubMed1992

Detection of bcr/c-abl mRNA in chronic myelogenous leukemia by polymerase chain reaction to identify chromosome translocation.

The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia chromosome (Ph1) which is caused by a translocation of the c-abl gene from chromosome 9 to the breakpoint cluster region (bcr) on chromosome 22. Polymerase chain reaction (PCR) can be used to detect the chimeric bcr/c-abl mRNA as evidence of the translocation. We applied a very simple and quick method of isolating cytoplasmic RNA, as well as reverse transcription and PCR in detecting bcr/c-abl mRNA of seven CML patients in various stages. Our results showed that only a small amount of either peripheral blood or bone marrow material was required for the expression of the bcr/c-abl mRNA. This is a very fast and time-saving method since a one-step method of cytoplasmic RNA isolation is used instead of the several steps in total RNA isolation as published in other literature.

Base Sequence↗
PubMed1992

PCR amplified HLA DQ alpha and DQ beta DNA typed by dot-blot hybridization and direct sequence for suspect.

For more effective screening of suspects, we combined two methods to individualize the biologic evidence from a crime scene: polymerase chain reaction (PCR) dot-blot hybridization for DNA typing of HLA Dq alpha; and PCR direct sequencing for DNA typing of HLA DQ beta. PCR-DQ alpha was typed by an AmpliType HLA DQ alpha kit from Cetus. PCR-DQ beta was typed by direct sequence using the dideoxy chain termination method. In 20 DNA samples, complete separation was demonstrated by these two polymorphic DNAs. This will be of considerable use in screening suspects in crime investigation work. The sensitivity of PCR is useful for trace evidence, and the rapid results of dot-blot hybridization and the precise results of direct sequencing have significant advantages.

Base Sequence↗
PubMed1992

Detection of hemoglobin E heterozygotes by using polymerase chain reaction and direct DNA sequencing: report of a case.

From an overseas Chinese born in Thailand, an extraordinarily high level of A2 + E band (20.2%) was found during routine cellulose acetate Hb electrophoresis. With a suspicion of Hb E, the beta-globin gene was studied by using the polymerase chain reaction (PCR) and direct DNA sequencing method. A 1.4 kb fragment covering the whole beta-globin gene was amplified by PCR. Sequence analysis of the amplified product revealed a GAG > AAG transversion at codon 26, which resulted in an amino acid substitution of lysine for glutamic acid. A normal sequence at the corresponding codon was noted in the other allele; hence, this patient is a heterozygote of Hb E. PCR and direct DNA sequencing provide a rapid method for detection of Hb E from a small amount of DNA.

Adult↗