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Biomedical subjects

S Dong

Publications and source records attributed to S Dong.

At least 109 records · Page 6Linked to original sources
PubMed1996

Long-term follow-up of minimal residual disease in childhood acute lymphoblastic leukemia patients by polymerase chain reaction analysis of multiple clone-specific or malignancy-specific gene markers.

Two types of markers, namely the clone-specific markers including T-cell receptor (TCR) gamma, TCR delta, and Ig heavy-chain (IgH) gene rearrangements, and malignancy-specific fusion gene mRNA such as SIL-TAL-1, BCR-ABL, and HRX-partner genes, were investigated by molecular biology techniques in 65 Chinese patients with acute lymphoblastic leukemia (ALL). In combination, these markers were informative among 96% of patients. Minimal residual disease (MRD) was followed up in 23 of these patients with available materials over a period varying from 8 to 54 months with at least one leukemia-specific probe. In most children, MRD was decreased continuously to an ultimately undetectable level within 6 to 12 months after remission induction therapy. One patient exhibited low-level residual leukemic cells for 4 years before the MRD turned negative. Another patient remained in complete remission for 45 months, although a positive signal was detected at 34 months using TCR delta probe, but was negative with a TCR gamma marker which was positive at presentation. In three patients who relapsed, MRD either persisted through the clinical course or became positive and eventually increased 3-11 months before clinical relapse. These data suggested that the combined use of multiple gene markers is a valuable tool for the PCR-based MRD detection, since it can cover most ALL patients. Furthermore, long-term follow-up of MRD is helpful for determining the dosage as well as the period of maintenance chemotherapy and for predicting impending relapse.

Base Sequence↗
PubMed1996

Isolation and purification of functional bovine lung mast cells (BLMCs).

Purified pulmonary mast cells were obtained from bovine lung using a combination of enzymatic digestion of tissue, density gradient centrifugation using Percoll, and centrifugal elutriation. In the initial procedure, lung tissue was enzymatically digested with collagenase, hyaluronidase, protease and elastase in three 30 min incubations at 37 degrees C. Monodispersed cell suspensions contained between 2 and 6% mast cells. Further purification of these mast cells by Percoll gradients and elutriation consistently yielded mast cells of > 90% purity. These cells were morphologically intact, viable and functional, as determined by histamine release evoked by secretagogue challenge. Incubation of BLMCs with Pasteurella haemolytica A1 culture supernate containing leucotoxin (LCT) alone, resulted in increased histamine release compared to controls. LCT also potentiated calcium ionophore (CaI)-induced histamine release from BLMCs.

Animals↗
PubMed1996

[A preliminary study of the relation between topoisomerase I and translocation (15;17)].

The chromosomal translocation (15;17) which produced the PML-RAR alpha fusion gene had been found in acute promyelocytic leukemia (APL) cells. It was identified that the RAR alpha gene on chromosome 17 and PML gene on chromosome 15 were involved in this translocation. For study the molecular mechnisms of t(15:17) in APL, we cloned and sequenced the junctional region of the chromosomal reciprocal translocation in one APL patient. Furthermore, we compared 21 junctional sequences which had been reported with the consensus sequence of the DNA-topoisomerase I-binding sites. Thus, we proposed a hypothesis that topoisomerase I may play certain role in t(15;17) illegitimate recombination.

Adult↗
PubMed1996

[Immunoglobulin super-gene family and its related genes in detecting MRD in ALL].

OBJECTIVE: To study the rearrangements of the immunoglobulin super-gene family and its related genes, as well as their application in the detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). METHODS: 15 ALL cases including 11 B-ALLs and 3 T-ALLs were studied separately by using immunophenotype detection and molecular clonging methods. RESULTS: Using DNA Southern blot analysis, we found that the rearrangement of IgH gene existed in 11 B-lineage ALL cases. The specific PCR band was visualized through amplification of the CDR3 region and J region of IgH gene. Furthermore, we sequenced the V-D-J junctional region of IgH gene in 3 B-lineage patients and synthesized two probes against the V-D-J junctional region in order to detect MRD. The hybridization results showing the sensitive of the two probes were 10(-4). In addition, we performed comparative study of MRD detection by using both T cell receptor (TCR) gamma gene as gene specific marker and SIL-TAL-1 fusion gene as tumor-specific marker in one T-ALL patient. CONCLUSION: The rearrangements of IgH gene, as well as TCR gene and tumor fusion gene can be used as specific markers in the detection of MRD in ALL.

Adolescent↗
PubMed1995

RT/PCR detection of SIL-TAL-1 fusion mRNA in Chinese T-cell acute lymphoblastic leukemia (T-ALL).

The TAL-1 gene is located on chromosome 1p32. In about 20% of T-cell acute lymphoblastic leukemias (T-ALL), this gene is disrupted in its 5' portion by a site-specific 100-kg deletion and is fused with the 5' part of the SIL gene, to form SIL-TAL-1 chimeric gene. In this study, we established a "nested" retrotranscriptase/polymerase chain reaction (RT/PCR) technique which allows detection of the SIL-TAL-1 transcriptional expression. A chimeric mRNA was observed in four of 17 T-ALL cases and has been shown to result from the fusion between the exon 1 of SIL and exon 3 of TAL. A sensitivity test showed that this RT/PCR procedure could detect one leukemic cell among 10(6) normal cells. A positive RT/PCR result was obtained in two cases during clinical remission, suggesting the presence of minimal residual disease (MRD). One patient developed clinical relapse 3 months after PCR positivity. Moreover, analysis of the Tald rearrangement by DNA-based PCR in four patients with SIL-TAL-1 fusion revealed the type A (Tald1) rearrangement in all cases. Sequence analysis demonstrated the presence of N region and non-random "P" nucleotide, as well as base deletions at the genomic SIL-TAL-1 joining site. These data indicate that detection of TAL-1 gene abnormality is important for diagnosis and monitoring of MRD in a subset of T-ALL.

Adolescent↗
PubMed1995

[Epidemiological studies on risk for adverse pregnancy outcomes in women neighboring a petrochemical works].

Data of 7695 deliveries during 1985-1992 in four general hospitals neighboring a petrochemical works in Guangzhou were collected. Relationship between air pollution surrounding the plant and adverse pregnancy outcomes (APO) in women living there, including congenital malformation, stillbirth, low birth weight and preterm birth, was analyzed with air monitoring data and exposure assessment indices. Results showed there was difference in incidence rates of APO between lying-in women living in the places with various distance from the plant, and the incidence lowered with the distance prolonging, with a P-value of less than 0.05. Unconditional logistic model was used to control confounding and to estimate relative risks in a study of 325 cases of APO and 390 randomly sampled normal controls. Results indicated the distance between the places where the women living and the plant related to incidence of APO. It suggested air pollution caused by the petrochemical works may be a risk factor for APO in neighbored women.

Air Pollutants↗
PubMed1995

V-J junctional sequences of T cell receptor gamma gene in acute lymphocytic leukemia.

T cell receptor TCR gamma gene rearrangement in a series of acute lymphocytic leukemia (ALL) patients was studied using PCR technique. 18 V-J junctional sequences (designated by N sequence) of TCR gamma gene were amplified with the V gamma and J gamma primers, using an unsymmetrical PCR and analysed by direct sequencing, demonstrating that in Chinese ALLs, the N sequences of TCR gamma gene are indeed clone-specific. Based on the known N sequences, several oligo-nucleotides were synthesized as probes, which were specific for the leukemic clone, and were used to detect the minimal residual disease (MRD) in 4 ALL cases. The sensitivity of this method was 0.1%-0.01%.

Adult↗
PubMed1995

[Junctional sequences of T cell receptor V delta 2-D delta 3 or D delta 2-D delta 3 rearrangements in acute lymphoblastic leukemia].

T-cell receptor (TCR) delta chain gene rearrangements were studied by polymerase chain reaction (PCR) analysis in 46 patients with acute lymphoblastic leukemia (ALL). Sixteen patients were found to have incomplete rearrangements of the TCR delta genes. Among them, 13 patients displayed V delta 2-D delta 3 rearrangement, while 3 had both V delta 2-D delta 3 and D delta 2-D delta 3 rearrangements. To determine the junctional sequence of TCR delta gene, PCR products from the 16 patients were sequenced directly or after M13 cloning. The results showed the junctional sequences of TCR delta gene are highly specific for each allele. This sequence diversity resulted from several factors including deletion of the 3' end of V delta 2 or D delta 2 segment and 5' end of D delta 3 segment, the presence of D delta 1 or D delta 2 sequences, insertion of N nucleotides and the association of P nucleotides with intact V delta 2 and D delta 3 segments. In addition, analysis of N-nucleotide contents revealed that the amount of GC was much larger than that of AT (70%: 30%), indicating the insertion of N nucleotide was not fully random. Our sequence data confirmed that the imcomplete rearrangement of TCR delta gene is an early event in the lymphoid cell ontogenesis, and its N sequences in V-(D)-J junctional region may be used as a specific marker of clonality to detect the minimal residual disease (MRD) in ALL.

Adolescent↗
PubMed1995

Proteolytic processing of the MHV polymerase polyprotein. Identification of the P28 cleavage site and the adjacent protein, P65.

The polymerase gene of Mouse Hepatitis Virus strain JHM (MHV-JHM) encodes a polyprotein larger than 750 kilodaltons. This polyprotein is proposed to be processed by several viral proteinases into functional subunits. The amino-terminal subunit is p28, which is cleaved by the first viral papain-like proteinase domain. In this study, we identified the cleavage site of this papain-like cysteine proteinase by amino acid sequencing of radiolabeled polypeptide adjacent to p28. Proteolysis occurs between the glycine-247 and valine-248 dipeptide bond. To determine which amino acid residues are critical for proteolysis, we preformed site-directed mutagenesis on the coding sequences surrounding the cleavage site and assayed for the efficiency of cleavage of p28 in an in vitro transcription and translation system. We report that glycine-247 and arginine-246 are the most critical residues for efficient processing of p28.

Amino Acid Sequence↗
PubMed1994

Determinants of the p28 cleavage site recognized by the first papain-like cysteine proteinase of murine coronavirus.

The murine coronavirus polymerase gene is 22 kb in length with the potential to encode a polyprotein of approximately 750 kDa. The polyprotein has been proposed to encode three proteinase domains which are responsible for the processing of the polyprotein into mature proteins. The proteolytic activity of the first proteinase domain has been characterized and resembles the papain family of cysteine proteinases. This proteinase domain acts autoproteolytically to cleave the amino terminal portion of the polymerase polyprotein, releasing a 28-kDa protein designated p28. To identify the cleavage site of this papain-like cysteine proteinase, we isolated the peptide adjacent to p28 and determined the amino terminus sequence by Edman degradation reaction. We report that proteolysis occurs between the Gly-247 and Val-248 dipeptide bond. To determine the role of the amino acid residues surrounding the cleavage site, we introduced a total of 42 site-specific mutations at the residues spanning the P5 to P3' positions and assessed the effects of the mutations on the processing of p28 in an in vitro transcription and translation system. The substitutions of Gly-247 at the P1 position or Arg-246 at the P2 position resulted in a dramatic decrease of proteolytic activity, and the mutations of Arg-243 at P5 position also led to considerable reduction in p28 cleavage. In contrast, the substitutions of amino acids Gly-244 (P4), Tyr-245 (P3), Val-248 (P1'), Lys-249 (P2'), and Pro-250 (P3') had little or no effect on the amount of p28 that was released. This work had identified Gly-247-Val-248 as the cleavage site for the release of p28, the amino-terminal protein of the murine coronavirus polymerase polyprotein. Additionally, we conclude that the Gly-247 and Arg-246 are the major determinants for the cleavage site recognition by the first papain-like cysteine proteinase of murine coronavirus.

Amino Acid Sequence↗
PubMed1994

Gene structure prediction by linguistic methods.

The higher-order structure of genes and other features of biological sequences can be described by means of formal grammars. These grammars can then be used by general-purpose parsers to detect and to assemble such structures by means of syntactic pattern recognition. We describe a grammar and parser for eukaryotic protein-encoding genes, which by some measures is as effective as current connectionist and combinatorial algorithms in predicting gene structures for sequence database entries. Parameters of the grammar rules are optimized for several different species, and mixing experiments are performed to determine the degree of species specificity and the relative importance of compositional, signal-based, and syntactic components in gene prediction.

Animals↗
PubMed1994

Gamma and delta chain gene rearrangement of T cell receptor in acute lymphoblastic leukemia.

The immunophenotype, rearrangements of T cell receptor (TCR) gamma and delta chain genes as well as the immunoglobulin heavy chain (IgH) gene were studied in 37 cases of morphologically defined acute lymphoblastic leukemia (ALL). According to the expression of differentiation antigens, 8 cases were classified as T-ALL, 26 B lineage ALL, 2 acute undifferentiated leukemia (AUL) and myeloid phenotype. An order of TCR gene rearrangements was observed in T-ALL, with the rearrangement of delta gene preceding that of gamma gene. Both genes were also found frequently rearranged and/or deleted in high proportions of the ALL of B cell lineage. However, the patterns of gene rearrangements were somewhat different between the T and B lineage ALLs. In contrast, the IgH gene rearrangements were observed only in the B lineage ALL. The immunogenotype analysis of ALL proved to be a useful marker of the clonality and provided us with important information on early human lymphoid differentiation. We conclude that the determination of TCR gamma gene V-J junctional sequence can be used as clonal marker for detecting the minimal residual disease during clinical remission.

Adolescent↗
PubMed1993

Breakpoint clusters of the PML gene in acute promyelocytic leukemia: primary structure of the reciprocal products of the PML-RARA gene in a patient with t(15;17).

DNA studies of the translocation t(15;17) in acute promyelocytic leukemia (APL) have shown that the retinoic acid receptor alpha (RARA) gene on chromosome 17 is juxtaposed to the promyelocytic leukemia (PML) gene on chromosome 15. The PML breakpoints have been mapped to 3 clusters: bcr1, bcr2, and bcr3. We have examined the PML breakpoint distribution in a series of 33 Chinese patients with APL. Twenty-two patients fell within bcr1, 2 within bcr2, and 9 within bcr3. The primary structure of the reciprocal chromosome translocation joints of one patient and that of their normal counterparts have been determined and compared to those of 2 previously reported cases. These studies revealed possible topoisomerase II cleavage sites close to the breakpoints and suggested implications of DNA attachment sites to nuclear matrix. We propose that these features are relevant to the process of illegitimate recombination generating the translocation.

Base Sequence↗
PubMed1993

Identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus.

The murine coronavirus mouse hepatitis virus gene 1 is expressed as a polyprotein, which is cleaved into multiple proteins posttranslationally. One of the proteins is p28, which represents the amino-terminal portion of the polyprotein and is presumably generated by the activity of an autoproteinase domain of the polyprotein (S. C. Baker, C. K. Shieh, L. H. Soe, M.-F. Chang, D. M. Vannier, and M. M. C. Lai, J. Virol. 63:3693-3699, 1989). In this study, the boundaries and the critical amino acid residues of this putative proteinase domain were characterized by deletion analysis and site-directed mutagenesis. Proteinase activity was monitored by examining the generation of p28 during in vitro translation in rabbit reticulocyte lysates. Deletion analysis defined the proteinase domain to be within the sequences encoded from the 3.6- to 4.4-kb region from the 5' end of the genome. A 0.7-kb region between the substrate (p28) and proteinase domain could be deleted without affecting the proteolytic cleavage. However, a larger deletion (1.6 kb) resulted in the loss of proteinase activity, suggesting the importance of spacing sequences between proteinase and substrate. Computer-assisted analysis of the amino acid sequence of the proteinase domain identified potential catalytic cysteine and histidine residues in a stretch of sequence distantly related to papain-like cysteine proteinases. The role of these putative catalytic residues in the proteinase activity was studied by site-specific mutagenesis. Mutations of Cys-1137 or His-1288 led to a complete loss of proteinase activity, implicating these residues as essential for the catalytic activity. In contrast, most mutations of His-1317 or Cys-1172 had no or only minor effects on proteinase activity. This study establishes that mouse hepatitis virus gene 1 encodes a proteinase domain, in the region from 3.6 to 4.4 kb from the 5' end of the genome, which resembles members of the papain family of cysteine proteinases and that this proteinase domain is responsible for the cleavage of the N-terminal peptide.

Amino Acid Sequence↗
PubMed1993

[Molecular study of the mechanism of chromosomal translocation (15;17) in acute promyelocytic leukemia (APL)].

Molecular studies of chromosomal translocation (15;17) in acute promyelocytic leukemia (APL) have shown that retinoic acid receptor A (RARA) gene on chromosome 17 is juxtaposed to the PML gene on chromosome 15. This results in a PML-RARA chimeric gene. Our work has demonstrated that the PML breakpoints in APL patients are clustered in two limited regions, PML-bcr1 and PML-bcr2, separated from each other by about 10 kb. DNA sequence of PML-bcr1 and primary structure of the junctional region of reciprocal chromosomal translocation in a patient have been determined in this paper. Compared to those of two previously reported cases abroad, we found that the breakpoint may be situated in the topoisomerase II cleavage site. A working model has been proposed for the mechanism of DNA illegitimate recombination in t (15;17).

Base Sequence↗
PubMed1993

A domestic cell bioreactor and its application in virus culture.

A cell culture bioreactor (CellCul-20) and its application in cell and virus culture are described in this paper. It has been evaluated with strict aseptic tests and one-year's operation shown that CellCul-20 bioreactor can keep its aseptic condition after being autoclaved. It can meet the requirement for the control of the main parameters for cell and virus culture and the finely adjustment of the main parameters to meet the changing conditions of the cultivation. A high cell density and a high level of virus titre were reached respectively for Vero cells and Japanese encephalitis virus (JEV) while they were cultured in this bioreactor. It is the first report on large-scale culture of JEV-infected Vero cells to prepare primary JEV vaccine. Some suggestions are made for the improvement of CellCul-20.

Animals↗
PubMed1993

Molecular study on the chromosome 15 breakpoints in the translocation t(15; 17) in acute promyelocytic leukemia (APL).

Chromosomal translocation t(15; 17) is a specific marker of acute promyelocytic leukemia (APL). In this study, molecular cloning of the t(15; 17) breakpoint was carried out in a Chinese APL patient. It has been shown that the retinoic acid receptor alpha (RARA) gene, normally located on chromosome 17, was fused with a new transcription unit PML, normally localized on chromosome 15. We have subsequently cloned a portion of the PML gene and generated a panel of probes. A PML gene rearrangement was detected in 33 out of 36 APL cases studied. 24 rearrangements were clustered in a 4.4 kb region, designated here as PMLbcr1 whereas 9 rearrangements were concentrated in a 6.5 kb region, defining another breakpoint cluster region (PMLbcr2). These two types of rearrangement constitute the basis for the heterogeneity of the PML-RARA fusion gene and its possible biological significance remains to be explored.

Adolescent↗