Biomedical subjects

H Pang

Publications and source records attributed to H Pang.

At least 19 recordsLinked to original sources

PubMed1999

DNA sequencing using 96-capillary array electrophoresis.

Practical DNA sequencing in a rugged capillary array electrophoresis system coupled directly to 96-well microtiter plates is demonstrated. A CCD detector was used to monitor all capillaries simultaneously with laser-induced fluorescence at 1.75 frames per second. The reconstructed electropherograms show good signal-to-noise ratios and resolution for the entire capillary array. The system used standard dye labeling and image splitting to obtain fluorescence intensities in two wavelength regions to allow calling up to 410 bases for the DNA sequence. The use of a replaceable poly(ethylene oxide) matrix and a protective poly(vinylpyrrolidone) coating allows high separation speed and short turnaround time for high throughput DNA sequencing. Critical evaluation of the system performance over repeated runs with base calling is presented.

Automation↗

PubMed1999

Determination of ginsenoside Rg3 in plasma by solid-phase extraction and high-performance liquid chromatography for pharmacokinetic study.

A method using high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE) is described for the determination of ginsenoside Rg3 in human plasma. A 2.5-ml volume of plasma was mixed with 2.5 ml 60% methanol aqueous solution, and centrifuged at 1100 g for 10 min, the supernatant fluid was further purified by SPE with 200 mg/5 ml 40 microns octadecyl silica and separation was obtained using a reversed-phase column under isocratic conditions with ultraviolet absorbance detection. The intra- and inter-day precision, determined as relative standard deviations, were less than 5.0%, and method recovery was more than 97%. The lower limit of quantitation, based on standards with acceptable RSDs, was 2.5 ng/ml. No endogenous compounds were found to interfere with analyte. A good linear relationship with a regression coefficient of 0.9999 in the range of 2.5 to 200 ng/ml was observed. This method has been demonstrated to be suitable for pharmacokinetic studies in humans. Method development for determination of drug with low UV absorption by SPE and HPLC is also discussed.

Calibration↗

PubMed1999

The fusion gene at the ABO-secretor locus (FUT2): absence in Chinese populations.

The fusion gene (se(fus)) is a null allele of the secretor type alpha (1, 2) fucosyltransferase gene (FUT2) and was first found in a Japanese population. It has not yet been reported in any other ethnic population. In the present study, we investigated the distribution of the fusion gene of the FUT2 locus in five populations from three ethnic groups in East Asia. The fusion gene was found in two additional Japanese populations with a high frequency (0.0551 in Okinawa and 0.0792 in Akita) and, for the first time outside Japan, in a Korean population, at a very low frequency (0.0063 in Seoul). In contrast, we found no fusion gene in two Chinese populations. These findings showed that the FUT2 fusion gene was ubiquitous in Japanese, but was rare in neighboring populations, suggesting that the FUT2 fusion gene had emerged from within the Japanese. Additionally, a new null allele with a C-to-T substitution at nucleotide 658 was found in one individual native of southern China.

ABO Blood-Group System↗

PubMed1999

Allelic diversity of the human plasma alpha(1,3)fucosyltransferase gene (FUT6).

The 1080-bp coding region of the human plasma alpha(1,3)fucosyltransferase gene (FUT6) was sequenced in a total of 161 individuals (322 chromosomes) drawn from three populations, involving 56 Africans (Xhosa), 52 European-Africans of South Africa, and 53 Japanese. In addition to six reported base substitutions, eleven new base substitutions and a single base insertion were found in the coding region of the FUT6. Eleven functional and four null alleles were encountered, of which 10 alleles were novel alleles identified in this study. Two null alleles have been identified previously, whereas two novel null alleles, which contained a single base (cytosine) insertion at nucleotide 499, were found in a Xhosa population. The allelic distributions of FUT6 were different among these three populations. The heterozygosity of FUT6 was 0.860, 0.699, and 0.632, in Xhosa, European-African (South Africa), and in Japanese populations, respectively. The extensive DNA sequence diversity of the FUT6 may be suitable for application as a tool in genetic studies for modern human evolution.

Alleles↗

PubMed1999

Lewis (FUT3) genotypes in two different Chinese populations.

The allelic frequencies of the alpha (1,3/4)fucosyltransferase gene (FUT3) in two different Chinese populations (138 individuals in Shenyang and 154 in Guangzhou) were investigated using PCR-RFLP and nucleotide sequencing methods. The common alleles in the Oriental population, Le (wild type allele), le59,508 (with the mutations at nucleotide (nt) 59T-->G and nt 508G-->A) and le59,1067 (with the mutations at nt 59T-->G and nt 1067T-->A) were encountered, and also the rare alleles, le1067 (with the mutation at nt 1067T-->A) and Le59 (with the mutation at 59T-->G), were observed in these Chinese populations. In addition, the common allele in Caucasians, le202,314 (with the mutations at nt 202T-->C and nt 314C-->T), was found in the Oriental population for the first time. The allelic frequencies of the Le, Le59, le59,508, le59,1067, le202,314, and le1067, were 0.750, 0.011, 0.145, 0.054, 0.036, and 0.004 in the Shenyang population and 0.675, 0.026, 0.14, 0.123, 0.026, and 0.010 in the Guangzhou population, respectively. The presence of the alleles containing either the 59 mutation (Le59) or the 1067 mutation (le1067) suggested that the allele le59,1067 may have originated by recombination between them.

Asian People↗

PubMed1998

Sp3 regulates fas expression in lung epithelial cells.

By transducing an apoptotic signal in immune effector cells, Fas has been directly implicated in the control of immunological activity. Expression and functional results, however, have also suggested a role for Fas in regulating cell turnover in specific epithelial populations. To characterize factors responsible for Fas expression in epithelial cells, approximately 3 kb of the 5' flanking region of the mouse Fas gene was isolated. By rapid amplification of cDNA ends and primer extension, transcriptional start sites were identified within 50 bp upstream of the translation start site. Transient transfection of promoter-luciferase constructs in a mouse lung epithelial cell line, MLE-15, localized promoter activity to the first 77 bp of upstream sequence. By using a 60 bp DNA probe (-18 to -77) in electrophoretic mobility-shift assays, three shifted complexes were found. Incubation with excess cold Sp1 oligonucleotide or an anti-Sp3 antibody inhibited complex formation. Site-directed mutagenesis of the Sp1 site resulted in 60-70% loss of promoter activity. In Drosophila SL-2 cells, promoter activity was markedly increased by co-transfection of an Sp3 expression construct. These results show that the Sp3 protein is involved in regulating Fas gene expression in lung epithelial cells.

3T3 Cells↗

PubMed1998

Nucleotide sequence homology to bovine viral diarrhea virus 2 (BVDV 2) in the 5' untranslated region of BVDVs from cattle with mucosal disease or persistent infection in Japan.

Cytopathogenic and non-cytopathogenic bovine viral diarrhea viruses (BVDVs) were isolated from cattle with mucosal disease or persistent infection in Japan. These isolates were compared for antigenic properties by cross-neutralization tests with Japanese reference strains of BVDV belonging to classical type 1. Significantly low cross-reactivity to reference strains was noted, indicating the viruses to possibly represent a new serotype in Japan. Thus, to determine the genotype of the isolates, nucleotide sequences of the 5' untranslated region were determined and compared with those of previously reported BVDV 1 and 2. The isolates were clearly shown to belong to BVDV 2, not to BVDV 1.

Animals↗

PubMed1998

Five novel missense mutations of the Lewis gene (FUT3) in African (Xhosa) and Caucasian populations in South Africa.

Five novel missense mutations, viz., C304 A, T370 G, G484 A, G667 A, and G808 A, in the Lewis gene (FUT3) were detected in African (Xhosa) and Caucasian individuals in South Africa. These single base substitutions may result in changes in amino acid residues from Gln102 to Lys in the 304 mutation, Ser124 to Ala in the 370 mutation, Asp162 to Asn in the 484 mutation, Gly223 to Arg in the 667 mutation, and Val270 to Met in the 808 mutation. Out of the five novel mutations identified in this investigation, four new alleles (le484,667, le484,667,808, Le304, and Le370) were determined in the Xhosa population and two new alleles (le202,314,484 and Le304) in the Caucasian population. The determination of alpha(1,3/1,4)fucosyltransferase activity, after transfection of plasmids containing the new alleles into COS7 cells, suggested that alleles le484,667 and le484,667,808 encoded an inactive enzyme, and that alleles Le304 and Le370 encoded a functional enzyme. In addition, we also examined the incidence of five common alleles, Le59, le59,508 le59,1067, le202,314, and le1067 in two populations by the polymerase chain reaction/restriction fragment length polymorphism method and compared differences in the allele frequencies of FUT3 among three ethnic groups (Orientals, Africans, and Caucasians).

Alleles↗

PubMed1998

Extensive polymorphism of the FUT2 gene in an African (Xhosa) population of South Africa.

The human secretor type alpha(1,2)fucosyltransferase gene (FUT2) polymorphism was investigated in Xhosa and Caucasian populations of South Africa by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing. Six new base substitutions were found in the coding region of FUT2. A single base (C) deletion at nucleotide 778, which led to a frame shift and produced a stop codon at codon 275, was responsible for the enzyme inactivation. Three nonsynonymous base substitutions, A40G (lle14Val), C379T (Arg127Cys), and G481A (Asp161Asn), and two synonymous base substitutions, A375G (Glu125) and C480T (His160), were also identified in functional alleles. As a result, seven new alleles, Se40, Se481, Se40,481, Se357,480, Se357,379,480, Se375, and se357,480,778 were identified. Population studies revealed that an allele containing a nonsense mutation G428A (Trp143stop) (se428) was the common null allele in both Xhosa and Caucasian populations, whereas an allele containing a missense A385T (Ile129Phe) mutation (se357,385), which is the common null allele in Orientals, was found to be absent from both populations. The heterozygosity rates of FUT2 genotypes were as high as 0.75 in the Xhosa population and 0.65 in the Caucasian population. Therefore, the extensive polymorphism and race specificity of the FUT2 gene make it suitable for application as a new tool in genetic studies of modern human evolutionary history.

Alleles↗

PubMed1998

Genetic heterogeneity of env gene of feline immunodeficiency virus obtained from multiple districts in Japan.

Feline immunodeficiency virus (FIV) infection is widespread in many countries. FIV isolates have been classified into five distinct subtypes, A, B, C, D and E based on their env gene sequences. Several reports indicate that most of the FIVs isolated in Japan belong to subtype B which includes the first Japanese isolate, TM2 strain. To examine the distribution of FIV subtypes in Japan, proviral DNA sequences of the env gene were directly amplified by nested PCR from FIV-infected cats that had been kept in multiple districts throughout Japan. Phylogenetic analysis of the 11 strains showed that four FIV subtypes, A, B, C and D, were present in Japan. Among these subtypes, subtypes B and D were the two most common subtypes in Japan, and they were mainly distributed in the eastern and western parts of Japan, respectively. The present study provides information that is fundamental for development of a vaccine to protect against FIV infection in cats.

Amino Acid Sequence↗

PubMed1998

Significance of each of three missense mutations, G484A, G667A, and G808A, present in an inactive allele of the human Lewis gene (FUT3) for alpha(1,3/1,4)fucosyltransferase inactivation.

Recently, we found three novel missense mutations, G484A (Asp162Asn), G667A (Gly223Arg), and G808A (Val270Met), present in a Lewis-negative allele (le484,667,808) from an African (Xhosa) population. To define the relative contribution of each of the three mutations in the le484,667,808 allele for inactivation of the FUT3-encoded enzyme, we made chimeric FUT3 containing each of the three mutations. A transient expression study indicated that COS7 cells transfected with the FUT3 construct containing the G484A mutation expressed the Lewis antigen and had about 20% enzyme activity as compared with COS7 cells transfected with the wild type FUT3 allele, whereas COS7 cells transfected with the FUT3 construct containing either the G667A mutation or the G808A mutation did not express the Lewis antigen and showed no detectable alpha(1,3/1,4)fucosyltransferase activity. These results suggest that the G667A and/or the G808A missense mutations of FUT3 alleles are responsible for the inactivation of the FUT3-encoded enzyme.

Alleles↗

PubMed1998

Identification of a mutation (A1879G) of transferrin from cDNA prepared from peripheral blood cells.

We have prepared a transferrin (TF) cDNA from total RNA of peripheral blood cells of an individual with a TF B variant (TF Bv) phenotype to analyze the molecular basis for the TF Bv. The TF B variant allele (TF Bv) was found to contain an A to G transition at nucleotide 1879 in the coding region that may result in substitution of glutamic acid for lysine at codon 627 located in exon 16. In addition, a transient expression study by transfection of each plasmid containing TF C1, TF C2 or TF Bv cDNA into monkey kidney (COS7) cells indicated that the products from specific TF alleles were able to be identified respective phenotypes by isoelectrofocusing electrophoresis and immunoblotting analyses. Based on this nucleotide substitution, we also established a PCR-RFLP method to detect the TF Bv allele from genomic DNA. Thus, peripheral blood cells were useful as an alternative source for preparation of cDNA from human liver for the molecular analysis of TF polymorphism.

Animals↗

PubMed1998

Evaluation of equine infectious anemia virus core proteins produced in a baculovirus expression system in agar gel immunodiffusion test and enzyme-linked immunosorbent assay.

Equine infectious anemia virus (EIAV) core proteins (Gag and p26) obtained from a baculovirus expression system were used in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) antigens to test seventy-six horse sera. Those sera showed false-positive reaction in AGID test using Nisseiken antigen. However, none of them showed false-positive reaction with both of the expressed antigens. The 76 horse sera were also tested by ELISA. The sera gave a high background in ELISA using Nisseiken antigen. Gag and p26 reacted strongly against positive sera from horses immunized with Nisseiken antigen.

Animals↗

PubMed1997

Lipo-oligosaccharides of Campylobacter jejuni serotype O:10. Structures of core oligosaccharide regions from a bacterial isolate from a patient with the Miller-Fisher syndrome and from the serotype reference strain.

Lipo-oligosaccharide (LOSa) was obtained by phenol-water extraction of bacterial cells of an isolate PG 836, identified as Campylobacter jejuni serotype O:10, from a patient who subsequently developed the Miller-Fisher syndrome (MFS). The product was separated into a water-insoluble gel of low Mr and a water-soluble component of high Mr. The structure of the core oligosaccharide region in LOSa is reported herein for comparison with LOSb from the C. jejuni O:10 reference strain, and is based on investigations carried out on: (1) O-deacylated LOSa; (2) the core oligosaccharide (OS 1a) liberated on acetic acid hydrolysis of the ketosidic linkages to lipid A, with accompanying loss of N-acetylneuraminic acid residues; (3) the product of the removal of phosphate residues from OS 1a to give OS 2a; and (4) the Smith degradation of OS 2a to yield a mixture of Os 3a and OS 4a. The results revealed that the core oligosaccharide region in LOSa from the MFS bacterial isolate had chains (1a), of which some were terminated by an N-acetylneuraminobiose [Neu5Ac(alpha 2-8)Neu5Ac] unit in a GD3 [Neu5Ac-Neu5Ac-Gal] epitope, and the inner regions of which were different from those of other C. jejuni serotypes. Similar experiments on LOSb from bacterial cells of the C. jejuni O:10 reference strain showed that the core oligosaccharide unit [1a, R = P (phosphoric monoester)] of LOSa from the MFS isolate was more uniformly complete than that of the O:10 reference strain [1b, R = AEP (2-aminoethylphosphate)] differing in the nature of the phosphate substituent at the inner heptose residue. The close structural relationship of LOSa from the MFS associated bacterium to LOSb from the O:10 reference strain runs parallel to that of the previously studied Guillain-Barré syndrome (GBS) associated bacterium typed as C. jejuni O:19 in comparison with the lipo-oligosaccharide from the reference strain. Preliminary studies on the high Mr components showed that those from the O:10 strains were indistinguishable from each other, but were structurally unrelated to those from the GBS associated C. jejuni serotype O:19 isolates and the O:19 reference strain [G.O. Aspinall, A.G. McDonald, and H. Pang, Biochemistry, 33 (1994) 250-255].

Campylobacter jejuni↗

PubMed1997

Antigenic and genetic comparisons of Japanese and Australian Simbu serogroup viruses: evidence for the recovery of natural virus reassortants.

The antigenicity and RNA genome structures of five Simbu serogroup bunyaviruses isolated in Japan and Australia were analyzed using monoclonal antibodies (Mabs) raised to Akabane (AKA) virus and oligonucleotide fingerprinting. The virion surface glycoprotein (G1) and the nucleocapsid (N) protein of heterologous viruses showed no reactivity to the Mabs, while the AKA-derived anti-G1 Mab (2F1) reacted with Peaton virus and all three AKA anti-N Mabs reacted with Tinaroo (TIN) virus at almost the same antibody titers as the homologous virus. Oligonucleotide fingerprinting analyses indicated that the three RNA species of all the viruses were unique and distinguishable. However, AKA and TIN viruses exhibited very similar S RNA oligonucleotide fingerprints, while the L and M RNA fingerprints were quite different. The S RNA sequence of TIN virus has been determined and compared with that of AKA and Aino viruses. The results revealed 95.1% S sequence homology between the AKA and TIN viruses. The antigenic and genetic comparisons of AKA and TIN viruses suggest that the two viruses may represent naturally occurring reassortant viruses.

Amino Acid Sequence↗

PubMed1997

Sequence determination and phylogenetic analysis of the Akabane bunyavirus S RNA genome segment.

The nucleotide sequence of the small (S) RNA segment of Akabane (AKA) bunyavirus was determined. The segment is 858 nucleotides long and contains two overlapping open reading frames (ORFs), which encode the nucleocapsid (N) and nonstructural (NSs) proteins, consistent with other bunyaviruses. Comparisons with the Aino virus S RNA sequence indicated that there is 73.5% identity in nucleotide sequence. However, the sequence identity of the 5' non-coding region of the genomic RNA between these two viruses is only 55%. The N ORFs from 20 Japanese and 2 Australian isolates of AKA virus were sequenced and subjected to phylogenetic analysis. This suggested that AKA virus has evolved in multiple lineages. Twenty-three isolates were grouped into three major clusters, and the cluster which includes recent isolates was subdivided into two branches. Thus, phylogenetic analysis of the AKA virus N protein gene gives a greater insight into bunyavirus evolution.

Genome, Viral↗

PubMed1997

Application of equine infectious anemia virus core proteins produced in a baculovirus expression system to serological diagnosis.

Equine infectious anemia virus (EIAV) core proteins were obtained from a baculovirus expression system. Recombinant baculoviruses (rBVs) highly expressed the Gag precursor and p26 antigens in an rBV-infected Sf21 cell culture supernatant. Enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) were conducted using the expressed proteins to detect antibodies from experimentally infected horses. The expressed antigens showed low background levels, high specificity and sensitivity in ELISA and AGID. The results of the serological tests using the expressed antigens were identical to those using a manufactured trial antigen. rBVs containing gag and p26 genes were found to express high quality and large quantities of Gag and p26 antigens, respectively. The antigens were quite useful for detecting anti-EIAV antibodies from virus-infected horses.

Animals↗

PubMed1997

Spontaneous growth in Chinese patients with Turner's syndrome and influence of karyotype.

The pretreatment mixed cross-sectional and longitudinal height measurements of 203 patients with Turner's syndrome (TS) were analysed. Only one observation was included per year per child and a total of 858 observations formed the basis of the growth study. The mean and SD values were fitted separately by a second-degree polynomial function, giving smoothed growth curves. The spontaneous growth pattern of Chinese girls with TS runs parallel to published Caucasian TS growth curves. The mean final height of Chinese patients with TS was 142 cm compared to 147 cm and 139 cm observed in Northern European and Japanese patients, respectively. Patients with the 46, X, i(Xq) karyotype were found to be significantly shorter, whereas children with the 45, X/46, XY and 46, X, del(Xq) karyotypes were significantly taller among this group of patients. Patients with TS who were disomic for Xp are significantly taller than patients who were monosomic for Xp. Our results suggest that karyotype can have a significant effect on the growth of children with TS.

Adolescent↗