Chih-Peng Chang

The effect of Chinese medicinal herb Zingiberis rhizoma extract on cytokine secretion by human peripheral blood mononuclear cells

The ethanolic extract of the Chinese medicinal herb Zingiberis rhizoma, the rhizome of Zingiber officinale Roscoe (Zingiberaceae), was found to show biphasic effects on secretion of cytokines by human peripheral blood mononuclear cells in vitro. In this study, the augmentative effect of Zingiberis rhizoma extract on cytokine secretion was shown to be time dependent. No significant secretion of cytokine was noted when the reaction time was 1 or 3 h. Secretion of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and granulocyte-macrophage colony-stimulating factor (GM-CSF) by the mononuclear cells was markedly increased in the presence of a low concentration of Zingiberis rhizoma extract, varying from 10-30 mg/ml, when the reaction time was 18 or 24 h. A higher concentration of the herbal extract did not show similar or stronger augmentative effect as did low concentration of the herbal extract.

Mutation analysis of the PTEN/MMAC1 gene in cancers of the digestive tract

The 10q23.3 gene PTEN (phosphatase and Tensin homologue deleted on chromosome 10) or MMAC1 (mutated in multiple advanced cancers 1) was recently reported to undergo frequent mutation, including mutations and deletions in multiple advanced cancers. This study showed that the aberrant transcripts of this gene are frequently found in cancers of the digestive tract, paired non-cancerous tissues and normal peripheral mononuclear cells. Sequence analysis of the aberrant transcripts revealed three types of deletions: (i) a deletion junction with a splicing-like donor or acceptor sequence; (ii) several-base homology near or between the donor acceptor site at the deletion junction; and (iii) deletion with insertion. From these results, it is suggested that aberrant transcripts of PTEN/MMAC1 found by nested reverse transcription-polymerase chain reaction are a common (or natural) phenomenon unrelated to oncogenesis. The mechanism producing these aberrant transcripts needs further investigation. Using single-strand conformation polymorphism and direct sequencing to analyse for small base changes of the genomic DNA of the PTEN/MMAC1 gene revealed no point mutations or small base changes.

Molecular analysis of SMN, NAIP and P44 genes of SMA patients and their families

Mutations of the telomeric survival motor neuron gene (SMN1) are related to spinal muscular atrophy (SMA). However, no phenotype-genotype correlation has been observed since the SMN1 gene is lacking in the majority of patients affected with either the severe form (type I) or the milder forms (types II and III). Here, we analyze the SMN, NAIP and P44 genes in 132 Chinese SMA patients and their families. At least three types of normal allele, and four types of mutant allele were found in this study. The combination of one normal allele with one mutant allele resulted in carriers of different types, and the combination of different mutant alleles accounted for the different genotypes among different types of SMA. Deletions of mutant alleles can be further subgrouped into four types, which includes involving SMN1, SMN1 and NAIP(T) (telomeric portion of NAIP gene), SMN1 and NAIP(T) and P44(T) (telomeric portion of P44 gene), and SMN1 and SMN2 (centromeric portion of SMN gene). Some of the severe (type I) SMA cases correlated with the extent of deletions in the SMN, NAIP and P44 genes or the dosage of SMN gene when both SMN1 and SMN2 are deleted. We also found two novel point mutations, an A insertion at codon 8 (AGT-->AAGT) and an A substitution at codon 228 (TTA-->TAA).

Molecular analysis of survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes of spinal muscular atrophy patients and their parents.

Abstract We have assayed deletions of two candidate genes for spinal muscular atrophy (SMA), the survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes, in 101 patients from 86 Chinese SMA families. Deletions of exons 7 and 8 of the telomeric SMN gene were detected in 100%, 78.6%, 96.6%, and 16.7%, in type I, II, III, and adult-onset SMA patients, respectively. Deletion of exon 7 only was found in eight type II and one type III patient. One type II patient did not have a deletion of either exon 7 or 8. The prevalence of deletions of exons 5 and 6 of the NAIP gene were 22.5% and 2.4% in type I and II SMA patients, respectively. We also examined four polymorphisms of SMN genes and found that there were only two, SMN-2 and CBCD541-2, in Chinese subjects. In our study, analysis of the ratio of the telomeric to centromeric portion (T/C ratio) of the SMN gene after enzyme digestion was performed to differentiate carriers, normals, and SMA patients. We found the T/C ratio of exon 7 of the SMN gene differed significantly among the three groups, and may be used for carrier analysis. An asymptomatic individual with homozygous deletion of exons 7 and 8 of the SMN gene showed no difference in microsatellite markers in the SMA-related 5q11.2–5q13.3. In conclusion, SMN deletion in clinically presumed child-onset SMA should be considered as confirmation of the diagnosis. However, adult-onset SMA, a heterogeneous disease with phenotypical similarities to child-onset SMA, may be caused by SMN or other gene(s).

Analysis of the mRNA transcripts of the survival motor neuron (SMN) gene in the tissue of an SMA fetus and the peripheral blood mononuclear cells of normals, carriers and SMA patients

Spinal muscular atrophy (SMA) is a disorder characterized by degeneration of the anterior horn cells of the spinal cord. The gene most highly associated with SMA is the survival motor neuron (SMN) gene. In this study, we present an analysis of messenger RNA (mRNA) expression of the SMN gene in peripheral blood mononuclear cells in normal subjects, SMA carriers and patients from 20 SMA families. We found at least 6-8 different transcripts of SMN gene formed by alternative splicing involving exons 3, 5 and 7. We compared transcripts from the different types of SMA and found no definite differences in transcript patterns and amounts. Normal subjects with the telomeric SMN (SMN(T)) gene only had variable splicing resulting in several transcripts, the most dominant being a transcript containing all coding regions. However, SMA patients with the centromeric SMN (SMN(C)) gene only had a higher degree of splice variation and tended to show little or no exon 7. These results demonstrate that SMN(T) and SMN(C) genes participate in alternative splicing phenomena. The different splicing patterns support the view that the SMN(T) gene is responsible for SMA disease. We also analyzed the transcripts from several tissues of an SMA fetus who had a homozygous SMN(T) gene deletion. Different splicing patterns were also found in these tissues, and were similar to the splicing pattern of leukocytes. We compared the major transcripts from exons 4 to 8 of both the SMN(T) and SMN(C) genes and found that the relative proportion varied among normal subjects, SMA carriers and patients. This approach could be used as a novel diagnostic method. We suggest that analyzing the mRNA expression of the SMN gene in peripheral blood mononuclear cells offers an apparently reliable technique for separating SMA patients, carriers, and normal individuals.

Mutation analysis of the putative tumor suppressor gene PTEN/MMAC1 in hepatocellular carcinoma.

Abstract Loss of heterozygosity of chromosome 10q has been reported in hepatoma. Areas with a high rate of loss of genetic material could harbor putative tumor suppressor genes. PTEN/MMAC1, a candidate tumor suppressor gene located at chromosome 10q23.3, has recently been identified and found to be homozygously deleted or mutated in several different types of human tumors. To determine whether the PTEN/MMAC1 gene is a target of 10q loss of heterozygosity in hepatoma, we examined 42 primary hepatomas for mutations in PTEN/MMAC1 by using nested reverse transcriptase polymerase chain reaction (RT-PCR) of the RNA and single-stranded conformation polymorphism (SSCP) analysis of all genomic exons. Although 2 of 42 hepatoma tissues had aberrant transcripts, 5 matched noncancerous liver tissues also had aberrant transcripts. Southern blot analysis of the entire genomic DNA revealed no genomic change. Therefore, like the TSG101 or FHIT gene, aberrant transcripts of PTEN/MMACl using the nested RT-PCR method were a common phenomenon for both cancerous and noncancerous liver tissues, which may not be related to oncogenesis. None of the 42 cases had small deletions, point mutations, or insertions. Our results suggest that the PTEN/MMAC1 gene may not play a role in the pathogenesis of hepatoma.

Rapid diagnosis of β‐thalassaemia by mutagenically separated polymerase chain reaction (MS‐PCR) and its application to prenatal diagnosis

Summary. We have developed a rapid and simple PCR‐based method which is modified from the mutagenically separated polymerase chain reaction (MS‐PCR) to detect the molecular defects of β‐thalassaemia. We can use this technique to amplify normal and mutant alleles of the β‐globin gene in the same reaction tube, using different‐sized allele‐specific primers. This mutagenesis separates the amplification reactions of alleles performed in the same tube, Subsequent gel electrophoresis shows at least one of the two allelic products at the same locus or at least two of the several allelic products at different loci. Therefore, in addition to simple handling, MS‐PCR provides a within‐assay quality control for the exclusion of false negative results. The five most common mutations of β‐thalassaemia and haemoglobin E which occur in the Taiwanese population were tested, and 14 prenatal samples were checked with accurate results. This method is simple, rapid and accurate, and can be used routinely in prenatal diagnosis. The principle used here can also be applied to other genetic diseases.

New anastomotic gun for biofragmentable anastomotic ring in low anterior resection.

PURPOSE: The biofragmentable anastomotic ring remains difficult to use for low rectal anastomosis. The authors report their experience of clinical application of the biofragmentable ring in low anterior resection with a newly designed instrument. METHODS: In this series, 31 patients underwent sphincter-preserving low anterior resections for rectal tumors from May 1993 to November 1994. With the assistance of a self-developed anastomotic instrument (biofragmentable anastomotic ring gun), biofragmentable ring anastomoses were performed following low anterior resection. RESULTS: There was no operative mortality. One patient had clinical evidence of anastomotic leakage. In postoperative follow-up, there was no anastomotic stenosis or incontinence. CONCLUSION: Therefore, we believe biofragmentable ring rectal anastomosis is a safe and reliable alternative to other anastomotic methods in rectal surgery.

The Effect of Evodia Rutaecarpa Extract on Cytokine Secretion by Human Mononuclear Cells in vitro

The effect of Evodia rutaecarpa extract on cytokine secretion by human mononuclear cells in vitro was investigated. Evodia rutaecarpa extract of various concentrations in mononuclear cell culture medium showed biphasic effects on the secretion of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF) by the mononuclear cells. Generally speaking, a low to medium level of Evodia rutaecarpa extract, in concentrations ranging from 10% to 30%, showed significant stimulating effects on the secretion of IL-1 beta, IL-6, TNF-alpha and GM-CSF. On the other hand, high level of Evodia rutaecarpa extract, with concentration more than 40%, lost its stimulating effects. Moreover, reaction time affected the stimulating effects of Evodia rutaecarpa extract on cytokine secretion by mononuclear cells. Mononuclear cell culture medium containing Evodia rutaecarpa extract that was allowed to react for 18 or 24 hours showed significantly better stimulating effects than that reacted for 1 or 3 hours.

Valtrac®-secured intracolonic bypass device

PURPOSE: The intracolonic bypass tube has been used both experimentally and clinically to protect the anastomotic site. A newly designed intracolonic bypass, the Valtrac®-secured intracolonic bypass, which consisted of a biofragmentable anastomosis ring (BAR) and was connected with a soft, thin vinyl tube, was used in the colon approximately 5 to 10 cm proximal to the anastomotic site. The distal end of the vinyl tube is passed through the colonic anastomosis to the anus to bypass the fecal stream. METHODS: Eighteen piglets were divided into three groups of six each. Group A piglets underwent colon resection and rough anastomosis with large gaps between sutures, followed by Valtrac®-secured intracolonic bypass. Group B piglets underwent the same procedures, but a colonic outlet obstruction also was done with pursestring sutures tied over the anus. Group C piglets underwent colon resection and rough anastomosis, but no intracolonic bypass tube was inserted (as in the control group). RESULTS: All Group A and Group B piglets survived. Passage of the BARs occurred approximately two weeks later. As the barium enema passed through the bypass tube, it showed a patent BAR-secured tube and intact anastomosis with no leakage. In Group C, anastomotic leakage occurred in four of six piglets, three of which died. Barium enema showed leakage at the anastomotic site. CONCLUSIONS: In the animal model we used, our new intracolonic bypass device proved to be a simple, safe, reliable means of protecting the anastomotic site and, thereby, eliminated the need for a diverting colostomy. Still we need further steps to test its potential in clinical use.