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Biomedical subjects

H S Kwan

Publications and source records attributed to H S Kwan.

At least 19 recordsLinked to original sources
PubMed2005

Differential gene expression identified in complete hydatidiform mole by combining suppression subtractive hybridization and cDNA microarray.

Complete hydatidiform mole (CHM) is a type of gestational trophoblastic disease with pure paternal chromosome contribution and unpredictable malignant potential. As an attempt to assess the molecular pathogenesis of CHM, suppression subtractive hybridization (SSH) combined with cDNA microarray was used to compare the gene expression pattern of CHM compared with normal first-trimester placenta of similar gestational ages. cDNA microarray analysis using tissue-specific chips constructed with subtracted cDNA libraries identified 13 differentially expressed gene transcripts. Quantitative real-time polymerase chain reaction (PCR) confirmed up-regulation of human chorionic gonadotropin beta subunit (CGB) (P=0.0008) and KIAA1200 (P=0.0005), a G-protein regulator, as well as down-regulation of osteopontin (SPP1) (P<0.0001) in 14 genotyped CHM when compared with 15 normal placentas. These candidate genes may contribute toward understanding the mechanism involved with the development and progression of CHM.

Adult↗
PubMed2002

Inhibition of ATPases by Cleistocalyx operculatus. A possible mechanism for the cardiotonic actions of the herb.

The water extract of the buds of Cleistocalyx operculatus, Roxb. (CO), a herb commonly used as an ingredient for tonic drinks in southern China, was shown to increase the contractility and decrease the frequency of contraction in an isolated rat heart perfusion system. CO was found to inhibit Na+/K(+)-ATPase activities in rat heart sarcolemma, as well as in a purified enzyme from porcine cerebral cortex. CO also inhibited Ca(2+)-dependent ATPase in mouse heart homogenate and in mouse heart sarcoplasmic reticulum at a similar dose. These enzyme inhibitory actions provide a possible explanation for the positive inotropic and negative chronotropic actions of CO on the perfused rat heart. This study suggests the presence of ATPase inhibitory compounds in CO with specificities different from that of ouabain.

Adenosine Triphosphatases↗
PubMed2001

Inotropic and chronotropic actions of Ilex latifolia inhibition of adenosine-5 '-triphosphatases as a possible mechanism.

Ilex latifolia is widely used as an ingredient to prepare traditional beverage drinks in southern China. In fact, various Ilex species have been used in Chinese folk medicine to treat coronary heart diseases. The mode of action is believed to be mediated by their coronary vasodilative effects. In this study, the water extract of the leaves of Ilex latifolia (IK-TP) was shown to increase the contractility and decrease the frequency of contraction in an isolated rat heart perfusion system. IK-TP was found to inhibit Na+/K+-ATPase activities in rat heart sarcolemma, rat brain microsomes and a purified enzyme from porcine cerebral cortex. IK-TP also inhibited Ca2+-dependent ATPase at a similar dose. Following exposure of the isolated rat heart to IK-TP at a dose that produces pronounced cardiac effects, inhibition of Na+/K+-ATPase activity can be readily detected in the heart. This study suggests the presence of ATPase inhibitory compounds in Ilex latifolia with specificities different from that of ouabain.

Animals↗
PubMed2000

Cloning and characterization of two hydrophobin genes differentially expressed during fruit body development in Lentinula edodes.

Hydrophobins play important roles in morphogenesis and pathogenesis in fungi and fruit development in mushrooms. Two genes encoding hydrophobins (Le.hyd1 and Le.hyd2) were isolated during sequencing of random clones from a primordial cDNA library of Lentinula edodes. The nucleotide sequences of these two genes were determined. These two genes are 760 and 738 bp in length and the deduced amino acid sequences are homologous to various fungal hydrophobins with characteristic cysteine spacing. These hydrophobin genes are Class I hydrophobins judging by their conserved domains and hydropathy patterns. The transcript level of Le.hyd1 is high in primordium and that of Le.hyd2 is high in dikaryotic mycelial tissues. Poor expression of these two genes in monokaryotic parents indicates that these two genes are under mating-type regulation. We thus suggest that differential expression of these two L. edodes hydrophobins during fruit development may contribute to their distinct roles in fruiting of this mushroom.

Amino Acid Sequence↗
PubMed2000

Identification by RNA fingerprinting of genes differentially expressed during the development of the basidiomycete Lentinula edodes.

As part of an ongoing project to understand the molecular mechanisms of fruit body development in Lentinula edodes (Shiitake mushroom), RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) was used to identify differentially expressed genes in RNA populations from four stages of L. edodes development vegetative mycelium, primordium, young fruit body and mature fruit body. From 30 RNA fingerprints, we cloned and sequenced 33 RAP fragments after their differential expression patterns had been verified by reverse Northern dot-blot hybridization. Thirteen RAP fragments show high sequence similarity to known gene products which are involved in (1) transport across the plasma membrane (drug efflux pump and sugar transporter); (2) cell cycle control (cyclin B); (3) signal transduction and transcriptional regulation (mitogen-activated protein kinase, Cdc39/Not1, PriA, Jun-D); (4) intracellular molecule trafficking (ubiquitin, plasma membrane proton ATPase, and alpha-adaptin); (5) mitochondrial biogenesis (mitochondrial processing peptidase beta-subunit, mitochondrial glycerol-3-phosphate dehydrogenase); and (6) intermediary metabolism (fructose 1,6 bisphosphatase). The transcript levels for plasma membrane proton ATPase and alpha-adaptin remained constant, whereas the other eleven genes were differentially expressed during L. edodes. development. The expression profiles of the genes suggest that transport across the plasma membrane is important in the mycelial stage. Specific signal transduction and transcriptional controls may play important roles during the initiation of primordia and the formation of young fruiting bodies. When the mushroom matures, expression of genes involved in metabolic pathways becomes prominent. The isolation of these genes indicates their involvement in homobasidiomycete development and suggests new directions for molecular studies on mechanisms of mushroom development.

Amino Acid Sequence↗
PubMed2000

Molecular cloning, characterization, and differential expression of a glucoamylase gene from the basidiomycetous fungus Lentinula edodes.

The complete nucleotide sequence of putative glucoamylase gene gla1 from the basidiomycetous fungus Lentinula edodes strain L54 is reported. The coding region of the genomic glucoamylase sequence, which is preceded by eukaryotic promoter elements CAAT and TATA, spans 2,076 bp. The gla1 gene sequence codes for a putative polypeptide of 571 amino acids and is interrupted by seven introns. The open reading frame sequence of the gla1 gene shows strong homology with those of other fungal glucoamylase genes and encodes a protein with an N-terminal catalytic domain and a C-terminal starch-binding domain. The similarity between the Gla1 protein and other fungal glucoamylases is from 45 to 61%, with the region of highest conservation found in catalytic domains and starch-binding domains. We compared the kinetics of glucoamylase activity and levels of gene expression in L. edodes strain L54 grown on different carbon sources (glucose, starch, cellulose, and potato extract) and in various developmental stages (mycelium growth, primordium appearance, and fruiting body formation). Quantitative reverse transcription PCR utilizing pairs of primers specific for gla1 gene expression shows that expression of gla1 was induced by starch and increased during the process of fruiting body formation, which indicates that glucoamylases may play an important role in the morphogenesis of the basidiomycetous fungus.

Amino Acid Sequence↗
PubMed1999

Characterization, molecular cloning, and differential expression analysis of laccase genes from the edible mushroom Lentinula edodes.

The effect of different substrates and various developmental stages (mycelium growth, primordium appearance, and fruiting-body formation) on laccase production in the edible mushroom Lentinula edodes was studied. The cap of the mature mushroom showed the highest laccase activity, and laccase activity was not stimulated by some well-known laccase inducers or sawdust. For our molecular studies, two genomic DNA sequences, representing allelic variants of the L. edodes lac1 gene, were isolated, and DNA sequence analysis demonstrated that lac1 encodes a putative polypeptide of 526 amino acids which is interrupted by 13 introns. The two allelic genes differ at 95 nucleotides, which results in seven amino acid differences in the encoded protein. The copper-binding domains found in other laccase enzymes are conserved in the L. edodes Lac1 proteins. A fragment of a second laccase gene (lac2) was also isolated, and competitive PCR showed that expression of lac1 and lac2 genes was different under various conditions. Our results suggest that laccases may play a role in the morphogenesis of the mushroom. To our knowledge, this is the first report on the cloning of genes involved in lignocellulose degradation in this economically important edible fungus.

Amino Acid Sequence↗
PubMed1998

Cloning and characterization of the gene encoding beta subunit of mitochondrial processing peptidase from the basidiomycete Lentinula edodes.

We have isolated the gene encoding the beta subunit of mitochondrial processing peptidase (beta-MPP) from the shiitake mushroom Lentinula edodes. It is a nuclear gene with two small introns. Comparison with known beta-MPP genes revealed that the L. edodes gene is most closely related with that from Neurospora crassa, with 60.8% identities and 87% similarity in the amino-acid sequences. The deduced L. edodes beta-MPP peptide sequence contains the inverse zinc-binding motif (H-X-E-H) that has been found in a large family of zinc-binding metalloproteinases including bacterial proteinases, insulin degrading enzymes and beta-MPPs. The two histidines are thought to contribute two of the three residues for zinc binding. The expression of L. edodes beta-MPP is higher during the development of the fruiting bodies, suggesting that higher mitochondrial activities may be required to meet the energy demand in the rapid growth of the fruiting bodies.

Amino Acid Sequence↗
PubMed1998

Identification and molecular characterization of Charybdis feriatus tropomyosin, the major crab allergen.

BACKGROUND: Crab sensitivity is one of the most common seafood allergies. However, to date, there has been no report on the molecular characterization of crab allergens and no comparative analysis with other seafood allergens. OBJECTIVE: This study was undertaken to clone, identify, and determine the primary structure of a major IgE-reactive molecule in crab. METHODS: We constructed an expression cDNA library from a common crab, Charybdis feriatus. This library was then screened with the use of sera from subjects with a well-documented history of type I hypersensitivity reactions upon ingestion of crab. An IgE-reactive clone was chosen and subcloned into plasmids for nucleotide sequence determination and expression in Escherichia coli. RESULTS: We identified a 1-kb cDNA designated as Cha f 1. Expression of Cha f 1 produces a 34-kd recombinant protein reactive to the IgE antibodies from patients with crab allergies but not from control subjects. Cha f 1 has an opening reading frame of 264 amino acids and demonstrates marked homology to the shrimp tropomyosin Met e 1. Absorption of allergic sera with Cha f I removes IgE reactivity to crab extract. Moreover, absorption of allergic sera with recombinant shrimp Met e 1 tropomyosin removes IgE reactivity to Cha f 1. CONCLUSIONS: This 34-kd protein, designated as Cha f 1, is the first identified major allergen of crab. Nucleotide and amino acid comparison shows that this protein is the crab tropomyosin. The molecular basis of shrimp and crab allergy is readily demonstrated at the nucleotide and amino acid level.

Allergens↗
PubMed1998

Identification of genes differentially expressed in nasopharyngeal carcinoma by messenger RNA differential display.

We have applied the mRNA differential display method to compare and analyze mRNAs prepared from five normal nasopharyngeal epithelial cell cultures and five nasopharyngeal carcinoma cell lines. A total of 24 differential display experiments was performed using different combinations of PCR primers. Sixty-nine cDNA fragments differentially expressed in either normal or malignant nasopharyngeal epithelial cells were identified. Subsequent cloning and sequencing of these differentially expressed cDNA fragments resulted in the identification of seventeen distinct sequences. Seven of these sequences were shown to be novel cDNA sequences not previously reported. Ten of the remaining cDNA fragments showed sequence homology to previously reported genes. Differential expression of four of these seventeen cDNA fragments in normal nasopharyngeal epithelial cells was confirmed by reverse Northern hybridization. One of these cloned cDNA fragments is a novel cDNA sequence while the other three matched to previously reported cDNA sequences involved in cell growth and migration. Homologous sequences identified to be differentially expressed in normal nasopharyngeal epithelial cells in this study are: human 26 kDa cell surface protein (TAPA-1) mRNA, NF-E2 like basic leucine zipper transcriptional activator and the human bullous pemphigoid antigen. The mRNA differential display is a useful tool to identify candidate genes involved in the pathogenesis of nasopharyngeal carcinoma.

Base Sequence↗
PubMed1996

IgE reactivity against a cross-reactive allergen in crustacea and mollusca: evidence for tropomyosin as the common allergen.

BACKGROUND: Although cross-reactivity between mollusks and other crustaceans in shrimp-sensitive subjects has been reported, the mechanism of this allergenic cross-reactivity has not been studied in detail. OBJECTIVE: To investigate this cross-reactivity in vitro, we have taken advantage of a complementary DNA that expresses tropomyosin, the immunodominant shrimp allergen. METHODS: Serum IgE from nine patients with known anaphylaxis to shrimp and five normal volunteers were analyzed by immunoblotting against 13 distinct crustaceans and mollusks. As additional antigens, muscle preparations were isolated from grasshopper, cockroach, fruit fly, chicken, and mouse. RESULTS: Sera from all patients, but not control subjects, reacted specifically with a 38 kd protein in all crustaceans and mollusks studied. In addition, 8 of 9 sera from patients, but from none of the normal control subjects, recognized proteins of various other molecular weights among the mollusk extracts studied. The 38 kd protein was identified as tropomyosin and was shown to share immunodominant epitopes among all species of crustaceans and mollusks tested by specific absorption studies. Moreover, sera from all nine subjects with shrimp allergy demonstrated IgE reactivities against grasshopper, cockroach, and fruit fly but not chicken or murine muscle. CONCLUSION: The allergic epitopes on tropomyosin are conserved among invertebrates including not only shellfish but also insects. This latter observation suggests that persons sensitive to shrimp should undergo further study for potential cross-reactive inhalant or ingested insect sensitivity.

Allergens↗
PubMed1994

Cloning, expression, and primary structure of Metapenaeus ensis tropomyosin, the major heat-stable shrimp allergen.

Shrimp is a common cause of seafood hypersensitivity. To study the mechanism of seafood hypersensitivity at the molecular level, we have determined the primary structure of the major heat-stable allergen of shrimp by cloning, expression, nucleotide sequencing, and amino acid sequence determination of an IgE-reactive cDNA clone, Met e I, isolated from a Metapenaeus ensis expression library in lambda gt 11. We first constructed a cDNA library from the shrimp M. ensis in lambda gt 11. We then screened the library with sera from patients with hypersensitivity reactions to shrimp and identified a positive IgE-reactive clone, designated as Met e I. This cDNA was purified to homogeneity and subsequently expressed in the plasmid pGEX. Serum antibodies from patients with shrimp allergy demonstrated positive IgE reactivity by immunoblotting to a protein encoded by the clone Met e I; sera from nonallergic control subjects were not reactive. The nucleotide sequence of this cDNA clone revealed an open reading frame of 281 amino acid residues, coding for a protein of 34 kd. Comparison of the Met e I amino acid sequence with the Genbank database showed that Met e I is highly homologous to multiple isoforms of tropomyosin.

Allergens↗
PubMed1993

The deduced amino-acid sequence of the cloned cpxR gene suggests the protein is the cognate regulator for the membrane sensor, CpxA, in a two-component signal transduction system of Escherichia coli.

The cpxA gene of Escherichia coli K-12 encodes a membrane-associated sensor element of a two-component signal transduction system in bacteria. The cognate regulator element, however, has not yet been definitively identified. A 2.1-kb segment upstream from cpxA was amplified by polymerase chain reaction, cloned and sequenced. An open reading frame encoding 232 amino acids was found. It showed high homology to the regulator elements of two-component transduction systems. The newly identified gene, designated as cpxR, may encode the cognate protein receiving signals from CpxA.

Amino Acid Sequence↗
PubMed1992

Transcription of glpT of Escherichia coli K12 is regulated by anaerobiosis and fnr.

The transcriptional expression of the sn-glycerol 3-phosphate transport gene, glpT, was studied with a glpT-lac fusion contained on a low-copy-number plasmid pFZY1. The fusion was isolated during a 'shot gun' cloning of promoters expressed under anaerobic growth conditions. It was shown that glpT was induced six-fold by anaerobiosis. Furthermore, aerobic expression of glpT was induced by the substrate glycerol 3-phosphate, while the anaerobic expression of glpT was decreased by nitrate, glucose and a fnr mutation. However, nitrate repression on glpT expression was relieved if the upstream region from -290 of the transcription site was removed.

Anaerobiosis↗
PubMed1989

Gene replacement and retrieval with recombinant M13mp bacteriophages.

We have developed an allele exchange system for shuttling sequences of DNA to and from their original chromosomal loci. Cloned segments of the histidine operon of Salmonella typhimurium and the lactose operon of Escherichia coli served as target sequences and were used to develop the system. Replacement and retrieval of target sequences used the phage M13mp vectors and proceeded through an M13 lysogen intermediate. The intermediates and products of allele exchange were characterized by genetic and hybridization analyses. Several unique properties of M13 lysogens were exploited to devise positive selections to detect integration and excision. These positive selections were used to manipulate phenotypically silent alleles.

Chromosome Deletion↗