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L C Chan

Publications and source records attributed to L C Chan.

At least 91 records · Page 5Linked to original sources
PubMed1998

Optimising fed-batch production of recombinant proteins using the baculovirus expression vector system.

Fed-batch culture can offer significant improvement in recombinant protein production compared to batch culture in the baculovirus expression vector system (BEVS), as shown by Nguyen et al. (1993) and Bedard et al. (1994) among others. However, a thorough analysis of fed-batch culture to determine its limits in improving recombinant protein production over batch culture has yet to be performed. In this work, this issue is addressed by the optimisation of single-addition fed-batch culture. This type of fed-batch culture involves the manual addition of a multi-component nutrient feed to batch culture before infection with the baculovirus. The nutrient feed consists of yeastolate ultrafiltrate, lipids, amino acids, vitamins, trace elements, and glucose, which were added to batch cultures of Spodoptera frugiperda (Sf9) cells before infection with a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) expressing beta-galactosidase (beta-Gal). The fed-batch production of beta-Gal was optimised using response surface methods (RSM). The optimisation was performed in two stages, starting with a screening procedure to determine the most important variables and ending with a central-composite experiment to obtain a response surface model of volumetric beta-Gal production. The predicted optimum volumetric yield of beta-Gal in fed-batch culture was 2.4-fold that of the best yields in batch culture. This result was confirmed by a statistical analysis of the best fed-batch and batch data (with average beta-Gal yields of 1.2 and 0.5 g/L, respectively) obtained from this laboratory. The response surface model generated can be used to design a more economical fed-batch operation, in which nutrient feed volumes are minimised while maintaining acceptable improvements in beta-Gal yield.

Animals↗
PubMed1998

Exon scrambling of MLL transcripts occur commonly and mimic partial genomic duplication of the gene.

The MLL gene is frequently rearranged in acute human leukemia of both the myeloid and lymphoid lineages. Using a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we identified several abnormally spliced transcripts in which MLL exons were joined in an order different from the genomic orientation (scrambled exons). Mis-splicing of MLL was present in both normal and malignant tissues. Although the majority of these scrambled transcripts were joined accurately at consensus splice sites, there were several examples in which the junctions of exons spliced in aberrant order were at non-consensus sites. A number of features differentiate mis-splicing of MLL from the previously described cases of scrambled exons and circular RNAs. Some scrambled transcripts appear to be present in the polyadenylated fraction of RNA. No correlation of exon scrambling with exon skipping was found, and there was no particular tendency for the exons involved to be near large introns. Our data show that splicing of MLL is extremely complex. The presence of scrambled transcripts in both normal and leukemic cells, indistinguishable from transcripts resulting from genomic MLL rearrangements, precludes the use of nested RT-PCR as a screening method for detection of tandem duplication of tandem duplication of MLL.

Acute Disease↗
PubMed1998

Allogeneic bone marrow transplantation for adult acute lymphoblastic leukemia: a single-centre experience.

Between 1990 and 1997, we performed 29 allogeneic BMTs for acute lymphoblastic leukemia (ALL) patients with HLA-identical sibs. Their median age was 31 years (range 15 to 43); there were 15 males and 14 females. The conditioning protocol was Cy-TBI (n = 15), VP16-Cy-TBI(n = 12), CBV (n = 1) and Bu-Cy (n = 1). Cyclosporin and methotrexate were used for GVHD prophylaxis. The median disease-free survival (DFS) was 12 months (range 1 to 92) with an actuarial 4-years DFS of 42.3 per cent. Three patients died of transplant-related complications before 100 days. Relapse occurred in 11 cases at a median time of 5 months (range 3 to 14). All nine patients relapsing within one year died form resistant leukemia. Three patients died of late treatment-related complications. There were 13 survivors (median follow-up 38 months, range 12-98), with 12 in remission. Only four had limited cGVHD, and all had 100 per cent performance scores. One patient also cleared her chronic hepatitis B carrier status due to acquired immunity. The DFS rates amongst CR1 cases and R1/CR2 cases were comparable (p = 0.39). No long-term DFS is obtained from patients with resistant disease (n = 4). The survival results for BMT at CR1 were superior to those using intensive chemotherapy consolidation (p = 0.29), mainly due to poor late results in the chemotherapy arm. For young ALL patients with HLA-matched siblings, the option of BMT should be considered in light of local consolidation survival results.

Adolescent↗
PubMed1998

Genetic influence of the R/Q353 genotype on factor VII activity is overwhelmed by environmental factors in Chinese patients with Type II (non-insulin-dependent) diabetes mellitus.

We studied the effects of genetic and environmental influences on factor VII coagulant activity (VIIc) in Chinese diabetic patients (263 with Type II [non-insulin-dependent] diabetes mellitus, 78 with Type I [insulin-dependent] diabetes mellitus) and 143 normal control subjects. VIIc was measured by a one-stage biological assay. The R/Q353 or Msp1 polymorphism at codon 353 of the factor VII gene was detected after Msp1 digestion of polymerase chain reaction-amplified genomic DNA. In both diabetic and control subjects the allele frequencies of the R (M1) and Q (M2) alleles were 0.96 and 0.04; the corresponding reported frequencies in Caucasians being 0.90 and 0.10: VIIc were 21% lower in Chinese control subjects and Type I diabetic patients with R/Q, compared with R/R subjects (p < 0.001 and p < 0.05). The corresponding difference was 4% for Type II diabetc patients (p = NS). Type II diabetic patients had higher mean VIIc levels than control subjects and Type I diabetic patients (p < 0.01); they were also older, and had higher serum creatinine and triglyceride (all p < 0.01). They also had higher VIIc levels than an age-matched older control group (p < 0.01; n = 182) in whom the genotype effect was clearly seen. On stepwise linear regression analysis, the significant independent determinants of VIIc were serum triglyceride (contributing 20% and 25% to variance in control subjects and diabetic patients), the R/Q353 genotype (contributing to 12% of the variance in control subjects but only 1% in diabetic patients), age and total cholesterol in all subjects, and in the diabetic patients female sex, urinary albumin excretion rate and serum creatinine. VIIc was higher in diabetic patients with macroangiopathy and retinopathy (both p < 0.0001). We conclude that compared with Caucasians, the Q allele frequency is significantly lower in these Chinese subjects. Plasma VIIc is determined by both genetic and environmental influences such that in Chinese Type II diabetic patients, the effect of environmental factors predominates, almost negating the influence of the R/Q353 genotype. High VIIc may contribute to the increased cardiovascular risk in Type II diabetic patients.

Adult↗
PubMed1998

Trisomy 5 in two cases of acute monocytic leukemia with hyperdiploid clones.

Although numerical chromosomal aberrations are commonly seen in acute myeloid leukemia (AML), trisomy 5 (+ 5) is very rarely detected. We report two patients, both of whom suffered from acute monocytic leukemia, in which + 5 was found in hyperdiploid clones. A review of the English literature shows 17 additional cases of AML with + 5 in at least one of the abnormal clones, making a total of 19 such cases including ours. Trisomy 5 has been reported in all FAB subtypes of AML except acute promyelocytic leukemia. In the 19 cases identified in this report, + 5 was found in association with other numerical changes (four cases), structural changes (five cases) or both (eight cases). Trisomy 5 as a sole karyotypic abnormality was exceedingly rare (two cases). Its biologic and prognostic significance remains to be determined.

Adult↗
PubMed1998

Near-haploid common acute lymphoblastic leukaemia of childhood with a second hyperdiploid line: a DNA ploidy and fluorescence in-situ hybridization study.

Near-haploidy is a rare cytogenetic finding in childhood acute lymphoblastic leukaemia (ALL) and is associated with a poor prognosis. A second hyperdiploid line, occurring presumably by endoreduplication of the near-haploid stemline, is often observed. We present a case of common ALL in relapse characterized morphologically by a dual population of small and large lymphoblasts. Cytogenetic analysis supplemented with fluorescence in-situ hybridization (FISH) studies localized near-haploidy and hyperdiploidy to the small and large blast population respectively. DNA ploidy determination confirmed two abnormal clones with near-haploidy as the predominant one. A novel t(9;12)(q11;q13) was present in the near-haploid clone and was duplicated in the hyperdiploid clone. This finding identified cells bearing near-haploidy to be the clonogenic population following malignant transformation and confirmed endoreduplication as the mechanism for the presence of associated hyperdiploidy.

Child↗
PubMed1998

Clinicopathological features of megaloblastic anaemia in Hong Kong: a study of 84 Chinese patients.

Megaloblastic anaemia is uncommon in Hong Kong. Eighty-four consecutive Chinese patients with megaloblastic anaemia were studied. There were 48 males and 36 females, with a median age at presentation of 67 years. Vitamin B12 deficiency was found in all cases, with none of the patients showing folate deficiency. The frequency of pernicious anaemia in our patients was higher than in other south-east Asian series but comparable with western ones. When compared with patients in the West, our cases showed the following main differences: virtual absence of folate deficiency, even in alcoholics; absence of associated gastric malignancies; and a high frequency of tuberculosis.

Adult↗
PubMed1998

Myelodysplastic syndrome with myelofibrosis and basophilia: detection of trisomy 8 in basophils by fluorescence in-situ hybridization.

We report a case of myelodysplastic syndrome (MDS) with myelofibrosis and pulmonary tuberculosis who had marked basophilia in the peripheral blood. A clonal karyotypic abnormality characterized by trisomy 8 was demonstrated by cytogenetic analysis. By correlation of cell morphology with results of fluorescence in situ hybridization using a chromosome 8 probe, we demonstrated that the basophils were not reactive but belonged to the neoplastic MDS clone.

Adult↗
PubMed1998

Lack of structural rearrangement in c-kit and stem cell factor genes in Hong Kong Chinese patients with myelodysplastic syndromes or acute myeloid leukaemia.

Stem cell factor is a haemopoietic growth factor that interacts with the c-kit-encoded transmembrane tyrosine kinase receptor during signal transduction in haemopoietic progenitor stem cells. We have screened 127 Chinese patients with myelodysplastic syndromes or acute myeloid leukaemia for structural rearrangements in the stem cell factor and c-kit genes using Southern blot analysis. No structural rearrangements were detected in any of the bone marrow samples that were tested. It seems that structural rearrangements in the stem cell factor and c-kit genes are rare in Hong Kong patients who have a haematological malignancy.

Journal Article↗
PubMed1997

Low frequency of c-kit expression and detection of an aberrant Kit message among Hong Kong Chinese myelogenous leukaemia patients.

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor. It is expressed by the primitive CD34 positive haemopoietic stem cells and interacts with the Kit ligand for signal transduction. It was reported to be expressed in over 80% of acute myelogenous leukaemia (AML) patients in North America and Japan. We analyzed 20 AML patients for c-kit expression using either Northern blot analysis or flow cytometry with the YB5.B8 anti-c-kit antibodies. Only 6 out of 20 AML patients expressed the c-kit mRNA or protein product. However, a previously unreported abnormal sized 1.7-1.9 kb transcript was detected in the blast cells of 1 AML patient, 1 acute mixed lineage leukaemia patient and 1 chronic myelogenous leukaemia (CML) patient in myeloblastic transformation. Our data suggested that in most Hong Kong Chinese AML patients, leukaemia transformation may have occurred at a c-kit negative stage. Alternatively, the abnormal sized c-kit transcript that was detected in some Chinese myeloid leukaemia patients may represent an aberrant c-kit receptor that plays an important role in leukaemogenesis.

Blotting, Southern↗
PubMed1997

Frame shift mutation, exon skipping, and a two-codon deletion caused by splice site mutations account for pyruvate kinase deficiency.

Three novel splice site mutations and two novel missense mutations were identified by molecular analysis of pyruvate kinase (PK) deficiency associated with hereditary nonspherocytic hemolytic anemia. A Nepalese PK variant, PK Kowloon, was found to have a homozygous transversion at the 5'-splice site of the seventh intervening sequence (IVS) of the L-type PK gene (Ivs7[+1]gt --> tt). Using a reverse transcription polymerase chain reaction (RT-PCR) assay, we showed that the R-type PK mRNA in the proband's reticulocytes included the seventh IVS between the seventh and eighth exon, introducing a stop codon 3 nucleotides downstream of the mutated site. Consequently, the translational product may lack 44% of the R-PK polypeptide. A transition at the last nucleotide of exon 9 (1269GCG --> GCA) was found in a Japanese PK variant, PK 'Kamata.' The mutation did not alter the amino acid sequence, but caused skipping of the ninth exonic sequence in the R-PK transcripts. As a result, the affected R-type PK lost 51 amino acid residues (373Met-423Ala del). A transversion at the splice acceptor site of the third IVS (Ivs 3[-2]ag --> tg) was identified in PK 'Aomori.' The mutation resulted in aberrant splicing at a cryptic splice site within exon 4, causing deletion of two codons in the aberrant R-PK transcript (95 Gly-96 Pro --> del). Both PK 'Kamata' and PK 'Aomori' had a missense mutation on the other allele, 1044AAG --> AAT (348Lys --> Asn) and 1075CGC --> TGC (359Arg --> Cys), respectively. Although both 348Lys and 359Arg were located in the sixth loop of A domain (beta/alpha)8 barrel, which has been shown to contain the substrate and cation binding sites, the degree of anemia was much more severe in PK 'Kamata' than PK 'Aomori,' possibly because the 51 amino acid deletion of PK 'Kamata' but the 2 amino-acid deletion of PK 'Aomori' may abolish PK catalytic activity.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1997

Prevalence and genotypes of alpha- and beta-thalassemia carriers in Hong Kong -- implications for population screening.

BACKGROUND: The thalassemias are common in southern China. We determined the prevalence of heterozygous carriers of these genetic disorders in Hong Kong and assessed the feasibility of a community-based screening program. METHODS: An educational and screening program for the thalassemias was carried out in three high schools with a total of 2420 students. Seventy-five percent of the students agreed to undergo screening, which consisted of blood counts, hemoglobin electrophoresis, serum ferritin measurements, and DNA analyses. RESULTS: Of the 1800 blood samples tested, 150 (8.3 percent) had microcytosis (mean corpuscular volume, <80 microm3). Ninety students (5.0 percent) were carriers of alpha-thalassemia, of whom 81 (4.5 percent) were carriers of the Southeast Asian type of deletion, in which both alpha-globin genes on the same chromosome 16 are deleted. Sixty-one students (3.4 percent) were carriers of either beta-thalassemia or the mutation coding for hemoglobin E. Six students were carriers of both alpha- and beta-thalassemias. On the basis of these figures, the estimated numbers of pregnancies in Hong Kong in which the fetus is at risk for homozygous alpha-thalassemia and beta-thalassemia major or intermedia are 145 and 80 per year, respectively. In Hong Kong the actual numbers of women referred for prenatal diagnoses of these disorders are approximately 95 and 40 per year, respectively. CONCLUSIONS: Despite the availability of hospital-based screening and prenatal diagnosis for many years in Hong Kong, many women carrying fetuses at risk for thalassemia are not referred for genetic counseling. A community-based program of education, screening, and counseling is needed in Hong Kong and southern China.

Adolescent↗
PubMed1997

EEN encodes for a member of a new family of proteins containing an Src homology 3 domain and is the third gene located on chromosome 19p13 that fuses to MLL in human leukemia.

The MLL gene, the closest human homologue to the Drosophila trithorax gene, undergoes chromosomal translocation with a large number of different partner genes in both acute lymphoid and acute myeloid leukemias. We have identified a new partner gene, EEN, fused to MLL in a case of acute myeloid leukemia. The gene is located on chromosome 19p13, where two other MLL partner genes, ENL and ELL/MEN have also been identified. The deduced protein of 368 aa contains a central alpha-helical region and a C-terminal Src homology 3 (SH3) domain most similar to the C-terminal SH3 domain found in the Grb2/Sem-5/Drk family of genes. Sequence analysis of the fusion MLL/EEN transcript in our patient reveals that exon 6 of MLL is fused to the N-terminal end of EEN, a fusion that would create a chimeric protein that includes the major functional domain of EEN. EEN is expressed in a variety of tissue types and encodes a protein of approximately 46 kDa. The EEN protein is the human homologue of a member of a recently described murine SH3 domain-containing protein family. It is also highly related to a putative gene identified in Caenorhabditis elegans, and a number of similar sequences are present in the EST databases of several species.

Amino Acid Sequence↗
PubMed1997

MLL self fusion mediated by Alu repeat homologous recombination and prognosis of AML-M4/M5 subtypes.

Fifty-six patients with de novo acute myeloid leukemia M4/M5 subtypes were studied for rearrangements of the mixed lineage leukemia gene, MLL (also called HRX, Htrx-1, or ALL-1). Ten patients (18%) showed rearrangements of the MLL gene, 9 in a major breakpoint cluster region within a centromeric 8.3-kb BamHI fragment, whereas rearrangement in one patient was the result of a direct tandem duplication of exons 2-6 of MLL. Analysis of sequences at the duplication junction revealed that the points of MLL fusion within introns 6 and 1 both lie within Alu elements. This suggests the involvement of Alu repeat mediated homologous recombination in MLL self fusion. For the 10 rearranged samples, cytogenetics analysis revealed a normal karyotype in 3, and 3 had abnormalities other than 11q23. Survival analysis of patients revealed no difference between those with rearrangement of MLL and those showing the germ-line configuration.

Adult↗