Biomedical subjects

K W Tsim

Publications and source records attributed to K W Tsim.

At least 19 recordsLinked to original sources

PubMed2003

Authentication of Panax notoginseng by 5S-rRNA spacer domain and random amplified polymorphic DNA (RAPD) analysis.

The great majority of Panax species are well-known herbal medicines in the Orient, and many of them share a close resemblance in appearance and chemical composition. Among these Panax species, the root of P. notoginseng (Sanqi) is a unique herb that has distinct clinical usage. Here, the 5S-rRNA spacer domains were isolated from P. notoginseng, P. japonicus var. major, P. stipuleanatus, P. quinquefolius, P. ginseng, P. zingiberensis, and P. wangianus, and four common adulterants of P. notoginseng including Curcuma wenyujin, Curcuma longa, Bletilla striata and Gynura segetum. The spacer domains were sequenced and compared, which showed over 75 % DNA identity among all Panax species, but not for the adulterants. In addition, random amplification of polymorphic DNA (RAPD) analysis was used to distinguish different members of Panax genus as well as the morphological variants of P. notoginseng. These molecular methods could be used in the authentic identification of P. notoginseng from other Panax species.

DNA Primers↗

PubMed2001

Expression of the P2Y1 nucleotide receptor in chick muscle: its functional role in the regulation of acetylcholinesterase and acetylcholine receptor.

In vertebrate neuromuscular junctions, ATP is stored at the motor nerve terminals and is co-released with acetylcholine during neural stimulation. Here, we provide several lines of evidence that the synaptic ATP can act as a synapse-organizing factor to induce the expression of acetylcholinesterase (AChE) and acetylcholine receptor (AChR) in muscles, mediated by a metabotropic ATP receptor subtype, the P2Y(1) receptor. The activation of the P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphates and intracellular Ca(2+) mobilization in cultured chick myotubes. P2Y(1) receptor mRNA in chicken muscle is very abundant before hatching and again increases in the adult. The P2Y(1) receptor protein is shown to be restricted to the neuromuscular junctions and colocalized with AChRs in adult muscle (chicken, Xenopus, and rat) but not in the chick embryo. In chicks after hatching, this P2Y(1) localization develops over approximately 3 weeks. Denervation or crush of the motor nerve (in chicken or rat) caused up to 90% decrease in the muscle P2Y(1) transcript, which was restored on regeneration, whereas the AChR mRNA greatly increased. Last, mRNAs encoding the AChE catalytic subunit and the AChR alpha-subunit were induced when the P2Y(1) receptors were activated by specific agonists or by overexpression of P2Y(1) receptors in cultured myotubes; those agonists likewise induced the activity in the myotubes of promoter-reporter gene constructs for those subunits, actions that were blocked by a P2Y(1)-specific antagonist. These results provide evidence for a novel function of ATP in regulating the gene expression of those two postsynaptic effectors.

Acetylcholinesterase↗

PubMed2001

Determination of nucleosides in natural Cordyceps sinensis and cultured Cordyceps mycelia by capillary electrophoresis.

Cordyceps sinensis is a well-known traditional Chinese medicine, and some of the active components are nucleosides. The analysis of nucleosides in Cordyceps material has been performed by reversed-phase high-performance liquid chromatography (HPLC) with gradient elution or by spectrometry. Here, we have explored the possibility of using capillary electrophoresis to determine the content of three major nucleosides (adenosine, guanosine and uridine) in Cordyceps. Capillary electrophoresis needs no gradients, and it provides a better separation due to its higher efficiency. In order to optimize the resolution, the separation of adenosine, guanosine and uridine was determined in Cordyceps with respect to the variation of buffer concentration, pH, temperature, and voltage. By using the calibrated electrophoresis system, the separation was achieved for the three nucleosides in less than 10 min with a background electrolyte consisting of 0.2 M boric acid-sodium hydroxide buffer, pH 8.5. The nucleoside contents of various types of natural Cordyceps and cultured Cordyceps mycelia were determined and compared. There was a great variation of nucleoside content in different sources of Cordyceps; the cultured Cordyceps mycelia, however, contains a much higher concentration than the natural Cordyceps.

Adenosine↗

PubMed2001

The cyclic AMP-mediated expression of acetylcholinesterase in myotubes shows contrasting activation and repression between avian and mammalian enzymes.

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent signalling pathway has been proposed to regulate acetylcholinesterase (AChE) expression in chick muscle; however, its role in mammalian enzyme is not known. We provide several lines of evidence to suggest that the cAMP-mediated AChE expression in myotube is oppositely regulated between avian and mammalian enzymes. Human AChE promoter was tagged with luciferase, namely Hp-Luc, which was transfected into cultured chick myotubes. Application of cAMP and forskolin induced the expression of chick AChE but reduced human AChE promoter-driven luciferase activity. Transfection of cDNAs encoding active mutants of G proteins altered the intracellular cAMP level in myotubes as well as the expression of chick and human AChE. When the constitutively active forms of Activating Transcription Factor-1 (EWS/ATF-1 oncogene) were over expressed in Hp-Luc transfected myotubes, the expression of chick AChE transcript and protein increased from approximately 1.8- to approximately 2.5-fold, but the luciferase activity was decreased by over 60%. Overexpression of cAMP-responsive element binding protein (CREB) in Hp-Luc transfected myotubes markedly enhanced the cAMP-mediated AChE expression in up- and downregulated chick and human enzymes, respectively. In addition, CREB bound the CRE sequence of human AChE promoter. Mutation on the CRE site markedly enhanced the expression of the promoter-driven luciferase; however, its response to cAMP inhibition in cultured myotubes was still retained. These findings suggest that a cAMP-dependent pathway is contrasting activation and repression of AChE expression in chick and human muscles.

Acetylcholinesterase↗

PubMed2001

Chips and Qi: microcomponent-based analysis in traditional Chinese medicine.

Over the last 50 years or so Traditional Chinese medicine (TCM) has been subject to intensive basic and clinical research. Although the effectiveness and remarkable safety of TCM have been documented after controlled clinical studies, there are several herbal and animal parts that are toxic or difficult to identify. DNA polymorphism-based assays have recently been developed for the identification of herbal medicines. In this approach, small amounts of DNA are amplified by the polymerase chain reaction and the reactions products are analyzed by gel electrophoresis, sequencing, or hybridization with species-specific probes. With the DNA based identification of TCM materials as an example, chip-based analytical micro devices were developed with the goal of fabricating an integrated device that will enable sample preparation, amplification, and analysis on a single microchip-based device ("lab-on-a-chip"). The application of a silicon-based polymerase chain reaction microreactor and a DNA microarray for the DNA sequence-based identification of toxic medicinal plants is reported here.

DNA, Plant↗

PubMed2001

Cdk5 is involved in neuregulin-induced AChR expression at the neuromuscular junction.

Here we describe an important involvement of Cdk5/p35 in regulating the gene expression of acetylcholine receptor (AChR) at the neuromuscular synapse. Cdk5 and p35 were prominently expressed in embryonic muscle, and concentrated at the neuromuscular junction in adulthood. Neuregulin increased the p35-associated Cdk5 kinase activity in the membrane fraction of cultured C2C12 myotubes. Co-immunoprecipitation studies revealed the association between Cdk5, p35 and ErbB receptors in muscle and cultured myotubes. Inhibition of Cdk5 activity not only blocked the NRG-induced AChR transcription, but also attenuated ErbB activation in cultured myotubes. In light of our finding that overexpression of p35 alone led to an increase in AChR promoter activity in muscle, Cdk5 activation is sufficient to mediate the up-regulation of AChR gene expression. Taken together, these results reveal the unexpected involvement of Cdk5/p35 in neuregulin signaling at the neuromuscular synapse.

Animals↗

PubMed2001

Authentic identification of stigma Croci (stigma of Crocus sativus) from its adulterants by molecular genetic analysis.

Stigma Croci, stigma of Crocus sativus L., is a precious traditional Chinese medicine, which is commonly used to activate blood circulation and to dissipate blood stasis. Three plant species, Carthamus tinctorius L., Hemerocallis fulva (L.) L. and Hemerocallis citrina Baroni, could carry the name Stigma Croci in the commercial markets of South East Asia. However, C. sativus is the only one that has proven its effectiveness, while the others could act as adulterants. The authentic identification of C. sativus on the market is difficult. By using molecular genetic method, the spacer domains of 5S-rRNA were cloned from the genomic DNAs that were isolated from C. sativus, C. tinctorius, H. fulva and H. citrina. The cDNAs encoding the spacer domains, about 300 to 500 bp, were sequenced. The nucleotide sequences of these four species showed great diversity, which could serve as markers for authentic identification of Stigma Croci to distinguish from its substitution and counterfeit.

Drugs, Chinese Herbal↗

PubMed2001

Anti-oxidation activity of different types of natural Cordyceps sinensis and cultured Cordyceps mycelia.

Cordyceps, one of the well-known traditional Chinese medicines, consists of the dried fungus Cordyceps sinensis growing on the larva of the caterpillar. It is commonly used for the replenishment of body health. One of the known pharmacological effects is its anti-oxidation activity. However, there is a great variation of the quality in different sources of Cordyceps. Here, the water extracts of various sources of natural C. sinensis and cultured Cordyceps mycelia were analyzed for their anti-oxidation activity by using three different assay methods such as the xanthine oxidase assay, the induction of hemolysis assay and the lipid peroxidation assay. The results showed that Cordyceps, in general, possesses a strong anti-oxidation activity in all assays tested. However, both natural and cultured Cordyceps showed the lowest inhibition in the lipid peroxidation when compared with the other two assay methods. The cultured Cordyceps mycelia had equally strong anti-oxidation activity as compared to the natural Cordyceps. Besides, the anti-oxidation activities were increased to 10-30 folds in the partially purified polysaccharide fractions from the cultured Cordyceps mycelia, which suggested that the activity could be derived partly from Cordyceps polysaccharides.

Animals↗

PubMed2001

[The contents and their change of nucleosides from natural Cordyceps sinensis and cultured cordyceps mycelia].

AIM: To compare the content of nucleosides from natural Cordyceps sinensis and cultured Cordyceps mycelia, and the effect of humidity and heat on the contents of nucleosides. METHODS: The contents of nucleosides were determined using high performance capillary electrophoresis (HPCE). Beckman P/ACE System 5010 apparatus equipped with a UV detector and a Beckman untreated fused-silica capillary (57 cm x 75 microns, 50 cm effective length) were used. Before sample injection, the capillary was rinsed with 1 mol.L-1 sodium hydroxide and running buffer for 5 minutes, respectively. 20 kV was applied during separation. Pressure injection was 586 kPa for 6 seconds, and the wavelength of detector was 254 nm. The running time was 20 minutes at 20 degrees C. The effect of humidity and heat on the content of nucleosides from natural Cordyceps sinensis and cultured Cordyceps mycelia was observed for 1, 3, 5 and 10 days at temperature 40 degrees C, and relative humidity 75%. RESULTS: The contents of nucleosides from cultured Cordyceps mycelia were higher than that of those from natural Cordyceps sinensis. The contents of nucleosides from freshly collected natural Cordyceps sinensis, were under the detection limit. The contents of nucleosides from natural Cordyceps sinensis were significantly increased by effect of humidity and heat, the phenomenon was not observed in cultured Cordyceps mycelia. CONCLUSION: There are difference in nucleosides content between natural Cordyceps sinensis and cultured Cordyceps mycelia. The nucleosides in natural Cordyceps sinensis may be derived from the degradation of macro-molecular nucleic acids. That means it is probably controversial that adenosine was used for quality control of natural Cordyceps sinensis.

Adenosine↗

PubMed2000

The promoter of human acetylcholinesterase is activated by a cyclic adenosine 3',5'-monophosphate-dependent pathway in cultured NG108-15 neuroblastoma cells.

Different transcription elements have been proposed to play a role in the regulation of acetylcholinesterase (AChE) in muscle and neuron, and cyclic adenosine 3',5'-monophosphate (cAMP)-dependent pathway is one of them. In order to test the possible role of cAMP in regulating the expression of human AChE, an approximately 2.2 kb DNA fragment of human AChE promoter was linked up stream to a luciferase reporter. The chimeric DNA was transfected into cultured NG108-15 neuroblastoma cells. Application of Bt(2)-cAMP and forskolin increased the promoter driven luciferase activity over 2-fold in the transfected NG108-15 cells; the increase was parallel to the activation of endogenous AChE protein and enzymatic activity. The intracellular cAMP level was increased in the Galpha(sQL) (constitutively active mutant of Galpha(s)) cDNA transfected NG108-15 cells. The Galpha(sQL) cDNA transfected cells showed an increase of over 10-fold in the luciferase activity. In addition, a constitutively active mutant of activating transcription factor-1 (ATF-1) was able to turn on human AChE promoter by approximately 4-fold when they were co-expressed in the neuroblastoma cells. These results support the involvement of a cAMP-dependent pathway in regulating the expression of human AChE.

Acetylcholinesterase↗

PubMed2000

The cAMP-dependent protein kinase mediates the expression of AChE in chick myotubes.

Calcitonin gene-related peptide (CGRP), a neuropeptide synthesized by motor neurons, stimulates the expression of AChR and AChE at the vertebrate neuromuscular junctions. The signaling mechanism of CGRP-induced AChE expression in muscle was determined both in vitro and in vivo. In cultured chick myotubes, the intracellular cAMP-dependent protein kinase (PKA) activity increased to approximately 2-fold after the application of CGRP or PKA activators; the induction was blocked by PKA inhibitors. in vivo transfection analysis on chick gastrocnemius muscles showed that the transfection of cDNA encoding constitutively active mutant Galphas increased the expression of AChE mRNA and protein to approximately 2-fold, while the constitutively active mutant Galphai cDNA transfection showed an opposite effect. The induced catalytic subunit of AChE at approximately 105 kDa was determined by specific antibody. These findings indicate that the CGRP-induced AChE expression in chick muscle is mediated by a PKA-dependent pathway.

Acetylcholinesterase↗

PubMed2000

Species identification of Radix Astragali (Huangqi) by DNA sequence of its 5S-rRNA spacer domain.

About 300 species and varieties of Astragalus are identified in China, making the identification of the origin of a particular Astragalus species on the consumer market difficult. A molecular genetic approach was developed to identify various species of Astragalus. Although the 5S-rRNA coding sequence is conserved in higher eukaryotes, the spacer domain of the 5S-rRNA gene has great diversity among different species. The 5S-rRNA spacer domain was amplified by polymerase chain reaction (PCR) from the isolated genomic DNA, and the PCR products (approximately 300 bp) covering the 5S-rRNA spacer domain were sequenced. The nucleotide sequences of Astragalus membranaceus, A. membranaceus var. mongholicus, A. lehmannianus, A. hoantchy, and of one closely related species Hedysarum polybotrys (Hongqi), were determined. Diversity in DNA sequence and restriction enzyme mapping among various species was found in their 5S-rRNA spacer domains. This is the first report on the detection of 5S-rRNA spacer region sequence of Astragalus, and the results could be used for genetic identification of Huangqi.

Astragalus propinquus↗

PubMed2000

Authentication of an animal crude drug, Zaocys, by diagnostic PCR.

A pair of diagnostic primers for distinguishing the Chinese crude drug Zaocys (Zaocys dhumandes) from its substitutes was designed based on the sequence data of the original animal of the drug and substitutes. Total DNAs were extracted from genuine crude drug and 5 of its substitutes, as well as from 12 species of original animal of the snake crude drug. Diagnostic PCRs were performed using the primers with these total DNAs as a template, annealing at 60-65 degrees C. Positive amplifications were obtained from all DNA templates of Zaocys, whereas negative amplifications were obtained from that of others. The results indicate that Zaocys samples could be definitely distinguished from its substitutes by diagnostic PCR, and no incorrect discrimination was found under the same reaction conditions. The advantages of the method in the authentication of crude drugs are also discussed in the present paper.

Animals↗

PubMed2000

[Application of RAPD in the taxonomy of the genus Fritillaria].

OBJECTIVE: To provide the molecular data for the right application of the Chinese medicine "Beimu". METHOD: Using the technology of RAPD, we studied the relationship among 12 samples of Beimu. RESULT: The total genomic DNA of all the samples are about 21 Kb in size. Among 20 primers used, the five primers can reatedly generate a certain specified amplified band type, 27 bands were recored from all amplified products and 25 polymorphic fragments were found in it. The size of amplified fragments is between 450 bp and 1904 bp. CONCLUSION: The similarity within species is higher than those between species. The relationship of Fritillaria anhuiensis and F. puqiensis is the farest, while F. thunbergii and F. puqiensis is the closest.

DNA Primers↗

PubMed1999

Lack of allelic association of dopamine D4 receptor gene polymorphisms with Parkinson's disease in a Chinese population.

Parkinson's disease (PD) is a neurodegenerative disease caused by a multitude of environmental, neurochemical, and genetic factors. The gene for human dopamine D4 receptor (DRD4) has been considered as a plausible candidate for the pathogenesis of PD. Different dopamine D4 receptor allelic forms have variable affinity toward certain neuroleptics such as clozapine, suggesting a role for dopamine D4 receptors in neurologic disorders. To test the hypothesis that the DRD4 polymorphism is associated with the susceptibility to Parkinson's disease, we have examined differences in allele frequencies of different DRD4 polymorphisms in 101 Chinese patients with PD and in 105 age-matched control subjects in Hong Kong. The DRD4 gene was analyzed by a non-radioactive polymerase chain reaction-based Southern hybridization with chemiluminescence detection. The number of polymorphic 48 base pair tandem repeats in exon 3 was identified in each study subject. The DRD4 alleles with high frequencies in the control subjects are 4-repeat allele (72.4%), 2-repeat allele (21.4%), and 7-repeat allele (3.8%) which accounted for over 97% of the total alleles in the elderly Chinese population. The most prevalent genotype in the control subjects is the 4/4 (47.6%), followed by 4/2 (38.6), 4/7 (7.6%), and 2/2 (3.0%). None of the variable number tandem repeat polymorphism showed evidence for genetic association with Parkinson's disease.

Aged↗

PubMed1999

A cysteine-rich form of Xenopus neuregulin induces the expression of acetylcholine receptors in cultured myotubes.

Neuregulin-1 (NRG-1) has diverse functions in neural development, and one of them is to up regulate the expression of acetylcholine receptors (AChRs) at muscle fibers during the formation of neuromuscular junctions. NRG-1 has two prominent alternative splicing sites at the N-terminus; it could be an immunoglobulin (Ig)-like domain named Ig-NRG-1 or an apolar cysteine-rich domain (CRD) named CRD-NRG-1. cDNAs encoding Xenopus CRD-NRG-1 were isolated by cross-hybridization with Xenopus Ig-NRG-1 cDNA fragment. The amino acid sequence of Xenopus CRD-NRG-1 is 45 to 70% identical to the human, rat, and chick homologs. Similar to Ig-NRG-1, two variation sites within CRD-NRG-1 were identified at the spacer domain with 0 or 43 amino acids inserted and at the C-terminus of the EGF-like domain to derive either alpha or beta isoform. Two transcripts encoding CRD-NRG-1, approximately 7.5 and approximately 9.0 kb, were revealed in adult brain and spinal cord, but the expression in muscle was below the detectable level. The recombinant Xenopus CRD-NRG-1 when applied onto cultured myotubes was able to induce the tyrosine phosphorylation of ErbB receptors and the expression of AChR. The AChR-inducing activity of CRD-NRG-1 was precipitated by anti-NRG-1 antibody but not by heparin. In situ hybridization showed a strong expression of CRD-NRG-1 mRNA in developing brain, spinal cord, and myotomal muscles of Xenopus embryo. Similar to the results in other species, both CRD-NRG-1 and Ig-NRG-1 may play a role in the developing Xenopus neuromuscular junctions.

Amino Acid Sequence↗

PubMed1999

Xenopus muscle-specific kinase: molecular cloning and prominent expression in neural tissues during early embryonic development.

A muscle-specific receptor tyrosine kinase, designated MuSK, mediates agrin-induced aggregation of acetylcholine receptors at the vertebrate neuromuscular junction. cDNAs encoding Xenopus MuSK were isolated from embryonic cDNA libraries. The full-length MuSK cDNA encodes for a polypeptide of 948 amino acids and possesses the features unique to mammalian MuSK, including four Ig-like domains, C6 box, transmembrane region and an intracellular tyrosine kinase domain. Interestingly, Xenopus MuSK also contains a kringle domain similar to that previously reported for Torpedo MuSK. The overall amino acid sequence identity of Xenopus MuSK with mammalian MuSK is approximately 65%. Northern blot analysis demonstrated the presence of three MuSK transcripts (approximately 1 kb, approximately 3 kb and approximately 7 kb) which were differentially expressed during development. The expression of the approximately 7 kb MuSK transcript remained as the predominant species in adult tissues, e.g. skeletal muscle, spleen and lung. Immunocytochemical analysis with a MuSK-specific antibody revealed that Xenopus MuSK was colocalized with AChRs at neuromuscular junctions as well as in spontaneous acetylcholine receptor hot spots of cultured muscle cells. In situ hybridization revealed prominent expression of MuSK transcripts in neural tissues and myotomal muscle during the period of neurulation and synaptogenesis. The MuSK transcript detected at abundant levels in the central nervous system (CNS) was localized to the brain, spinal cord and eye vesicles during early embryonic development. In addition, the MuSK protein in the developing eye was found to be prominently expressed during embryonic stages of 32 and 35. These findings raise an intriguing possibility that, in addition to the known function in the formation of the neuromuscular junctions, MuSK may be involved in neural development.

Agrin↗

PubMed1999

Molecular diversity of 5S-rRNA spacer domain in Fritillaria species revealed by PCR analysis.

Beimu (bulbs of Fritillaria) is an important traditional Chinese herbal medicine commonly used as an antitussive and expectorant. There are about 25 species and varieties of Fritillaria that carry the name Beimu on commercial markets. The price for each Beimu may differ by more than 100-fold. However, the identification of the origin of a particular species on the market is difficult. Here, a molecular method was used to identify various species of Fritillaria regardless of their geographical origin. The 5S-rRNA coding sequence is highly conserved in higher eukaryotes, but the spacer sequence of the 5S-rRNA gene is variable among different species. Total genomic DNA from fresh leaves and bulbs of Fritillaria cirrhosa, F. puqiensis, F. anhuiensis, and F. thunbergii was extracted. The 5S-rRNA spacer region of the extracted DNAs was amplified by PCR with a pair of primers located within the conserved coding region. The isolated cDNA clones (approximately 600 bp) covering the 5S-rRNA spacer domain were sequenced. By aligning the isolated nucleotide sequences of the four Fritillaria species, sequence diversity was found in the spacer region. Furthermore, a unique EcoR I site was used for the rapid identification of different species of Fritillaria. To our knowledge, this is the first report on the detection of 5S-rRNA spacer domain sequences of Fritillaria and their use to identify species.

Base Sequence↗