Biomedical subjects

R N Wong

Publications and source records attributed to R N Wong.

At least 19 recordsLinked to original sources

PubMed2001

Differentiation of Lycium barbarum from its related Lycium species using random amplified polymorphic DNA.

The RAPD (random amplified polymorphic DNA) technique was applied for the first time to distinguish Lycium barbarum from other closely related species of the same genus. In this study, eight samples were collected, including five species, two varieties and one cultivated variety. A total of fifty arbitrary primers were used in the RAPD analysis. Distinctive DNA fingerprints corresponding to different Lycium species were successfully obtained from ten primers. Similarity index (S.I.) analysis revealed that the values are higher between intraspecies than interspecies. These results confirmed that the RAPD technique can be employed for distinguishing closely related species of Lycium.

Base Sequence↗

PubMed2001

Expression of protein kinase C isoforms in euxanthone-induced differentiation of neuroblastoma cells.

Euxanthone, a potent neuritogenic compound isolated from the roots of the medicinal herb Polygala caudata, has recently been shown to induce the differentiation of murine neuroblastoma Neuro 2A (BU-1) cells. In this study, the role of protein kinase C (PKC) and the expression of various PKC isoforms in euxanthone-treated BU-1 cells were examined. mRNA phenotyping using the reverse-transcription polymerase chain reaction (RT-PCR) showed that BU-1 cells express six different PKC isoforms, namely PKC-alpha, -beta, -delta, -epsilon, -lambda, and -zeta. Differential regulation and expression of PKC isoforms was observed in BU-1 cells treated with 100 microM euxanthone. PKC-apha, -beta, -delta, -lambda and -zeta were all up-regulated, with 1.7- to 9.5-fold increase, at around 30 to 60 minutes after euxanthone treatment. The expression level of PKC-epsilon remained relatively constant during the treatment. PKC-gamma, -eta, and -theta were not detected in both untreated and euxanthone-treated BU-1 cells. Staurosporine, a broad spectrum PKC inhibitor, was found to inhibit both spontaneous and euxanthone-induced neuritogenesis in BU-1 cells. A significant reduction of the euxanthone-induced neuritogenic effect was also observed when the PKC isoform-specific inhibitor Go6976 was included in the culture. These results suggest that the euxanthone-induced differentiation of the neuroblastoma BU-1 cells may be mediated through the differential expression of PKC-alpha, -beta, -delta, -lambda and -zeta isoforms.

Animals↗

PubMed2000

Protective mechanism of metallothionein against copper-1, 10-phenanthroline induced DNA cleavage.

Metallothionein (MT) has been shown to protect DNA against cleavage induced by a variety of mutagenic agents. The mechanism has been attributed to its ability to either chelate transitional metals that participate in the Fenton reaction, or scavenge free radicals by means of the abundant cystenyl residues of the proteins. In the present study, the protective action of MT against DNA cleavage by the copper-1,10-phenanthroline [(OP)(2)Cu(+)] complex was studied in situ. At 0.1 microM, MT inhibited the (OP)(2)Cu(+) induced DNA cleavage by about 50% (IC(50) approximately 0.1 microM). At 2.5 microM, the cleavage activity was completely inhibited. Similar to MT, cysteine can protect against DNA cleavage by (OP)(2)Cu(+) (IC(50) of approximately 3 mM), however, its action was 1500-fold less efficient than MT. The combined action of MT and cysteine was additive. Reduced glutathione (1 and 10 mM) did not protect the (OP)(2)Cu(+) induced DNA cleavage. Sodium azide could inhibit the cleavage only at high concentrations (IC(40) approximately 25 mM). Spectrophotometric analysis showed that MT can inhibit the formation of the DNA[(OP)(2)Cu(+)] complex possibly by chelating Cu. It can also cause a dissociation of the complex after it was formed. In the later case, the mechanism through which MT protects against the DNA cleavage might occur when MT fitted in closely with the complex, competing with the hydroxyl groups of the nucleotides base for Cu, which, in turn, terminate the Fenton-like free radical reaction.

DNA↗

PubMed2000

Effects of euxanthone on neuronal differentiation.

The growth inhibitory and differentiation inducing effects of euxanthone (1,7-dihydroxyxanthone) from the medicinal plant Polygala caudata on the neuroblastoma (Neural 2A, subclone BU-1) were investigated. At the concentration range of 0-100 microM, euxanthone inhibits the growth of BU-1 cells in a dose dependent manner. The 50% growth inhibitory concentration (IC50) was 41 microM. Significant induction of morphological differentiation and neurite growth was observed at the concentration of 100 microM. Frequency of proliferative neuroblastoma cells was determined after induction of differentiation. The frequency of proliferating BU-1 cells was markedly reduced from 1/1.1 to <1/99. Confocal microscopy also confirmed that the morphological differentiation of BU-1 was associated with the expression of neurite specific marker MAP-2 protein in neurites. These data suggest that euxanthone may be one of the neuropharmacological active compounds in the medicinal plant Polygala caudata.

Animals↗

PubMed2000

Depletion of glutamate GluR2 receptor-containing neurons in the rat neostriatum by specific immunotoxin.

In the present study, a novel GluR2 receptor-specific immunotoxin was produced. The immunotoxin was produced by conjugation of molecules of trichosanthin, a ribosome inactivating protein, with goat anti-mouse immunoglobulin molecules. The secondary antibody was then combined with a commercially available GluR2 specific primary antibody to form an immunotoxin. The immunotoxins were unilaterally injected either into the neostriatum or into the lateral ventricle of rats. After one week, ipsilateral turning movements were observed after apomorphine treatments in those animals injected by the striatal route. In perfuse-fixed sections of the neostriatum, immunoreactivity for GluR2 was found to decrease in the striatal-lesioned animals. Most of the GluR2-immunoreactive perikarya in the neostriatum, the presumed medium spiny neurons, were depleted. In addition, immunoreactivity for GluR2/3, GluR5/6/7 and NMDAR1 was found to decrease to a different extent in the lesioned neostriatum. The number of GluR1-immunoreactive perikarya in the neostriatum, a group of striatal interneurons, was not affected by the GluR2 lesion. Ventricular administration of the GluR2 immunotoxin however, was found to be less potent. These results demonstrate for the first time that an indirect immunotoxin is useful for immunolesioning. A difference in potency was also observed in different routes of administration. The depletion of GluR2-containing medium spiny neurons in the neostriatum may upset the balance of the output systems of the basal ganglia and has a profound effect in movement control of the animals.

Animals↗

PubMed2000

Immunolesioning of glutamate receptor GluR1-containing neurons in the rat neostriatum using a novel immunotoxin.

1. To investigate the potency of a novel immunotoxin that is specific for glutamate receptor GluR1, a subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type receptor channel, immunolesioning was performed. 2. A ribosome-inactivating protein, trichosanthin (TCS), was isolated and conjugated to the goat anti-rabbit IgG antibody molecule. The anti-rabbit antibody-TCS complex was preincubated with GluR1-specific rabbit antibody to produce a GluR1-specific immunotoxin. The immunotoxin was unilaterally administered into either the neostriatum or the lateral ventricle of rats. 3. Immunoreactivity for GluR1 or GluR4 was revealed in perfuse-fixed sections of the neostriatum obtained from the lesioned and control animals by immunocytochemistry. After ventricular or striatal injections of the immunotoxin, depletions of GluR1-immunoreactive neurons, the presumed GABAergic interneurons in the neostriatum, were found. Depletions of GluR4-immunoreactive perikarya, the presumed same subpopulation of striatal interneurons, were also found. In addition, no change in the pattern of distribution of immunoreactivity for GluR2 or glial fibrillary acidic protein was found in the lesioned neostriatum. These results indicate that the novel GluR1 immunotoxin is potent and specific. 4. In addition, striatal application of the immunotoxin caused a greater depletion in the number of GluR1-immunoreactive neurons. The present results also indicate that the route of immunotoxin application may be important in producing specific lesions.

Animals↗

PubMed2000

[Study on thermal unfolding process of trichosanthin by FTIR spectroscopy].

Fourier-transform infared spectroscopy, combined with resolution-enhancement techniques including second-derivative spectroscopy, Fourier self-deconvolution and curvefitting technique, was used to investigate the thermally induced unfolding process of anti-HIV-I toxin protein trichosanthin. During heating from 25 degrees C to 85 degrees C, the peak of Amide I shifted to 1618 cm-1 while the secondary structural contents change with the temperature. Upon cooling the protein from 85 degrees C to 25 degrees C, the contour of the Amide I do not change. All these show that the thermal unfolding of trichosanthin is an irreversible intermolecular aggregation process between 25 degrees C and 85 degrees C. The changes of secondary structures with temperature suggest the presence of folding intermediates.

Hot Temperature↗

PubMed2000

[Study on unfolding process of neo-trichosanthin in the presence of GdnHCl].

Fluorescence spectroscopy and circular dichroism are effective instruments to determine the chemical unfolding process of protein solution. In this paper, both of them were selected to investigate the unfolding processes of neo-trichosanthin in the presence of GdnHCl. One GdnHCl is added, both spectra of fluorescence and circular dichroism changed remarkedly. The denaturation curve of CD ellipticity meets that of fluorescence intensive excitation at 295 nm very well. The result shows that, in the presence of GdnHCl, the unfolding of neo-trichosantin matches the two-state process well. The model is different from the model of trichosanthin that others reported previously. The mechanism will be further probed later.

Circular Dichroism↗

PubMed1999

Immunolesioning of nerve growth factor p75 receptor-containing neurons in the rat brain by a novel immunotoxin: anti-p75-anti-mouse IgG-trichosanthin conjugates.

In the present study, a comparison of potency between a commercially available immunotoxin, 192-immunoglobulin-SAP (192-IgG), and a novel immunotoxin produced in our laboratory, anti-p75-anti-mouse IgG-trichosanthin conjugates (p75-TCS), was conducted. Both of the immunotoxins were specific for nerve growth factor p75 receptor. Cholinergic neurons in the rat basal forebrain and in the neostriatum were depleted after the injection of either 192-IgG or p75-TCS. These indicate that both types of immunotoxins are potent and useful in performing immunolesioning experiments. In addition, there were variations in potency among the two immunotoxins in different routes of administration. The 192-IgG was more potent than the p75-TCS in the case of ventricular injections. In case of striatal injections, 192-IgG caused serious tissue necrosis and considerable tissue damage in the brain region. In contrast, p75-TCS was potent and caused a selective and specific depletion of cholinergic neurons in the neostriatum. These results indicate that indirect immunotoxins may be more useful for performing immunolesioning experiments in case of brain parenchyma administration.

Animals↗

PubMed1999

Role of TYR70 in the N-glycosidase activity of neo-trichosanthin.

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) which possesses rRNA N-glycosidase activity. TCS has long been used as an abortifacient in China. In recent years, its immunomodulatory, anti-tumor and anti-HIV properties have attracted more and more attention. An isoform of trichosanthin, neo-trichosanthin (n-TCS), has been cloned and expressed as recombinant protein. The biochemical studies revealed that n-TCS has virtually the same rRNA N-glycosidase activity as TCS. The crystal structure of n-TCS is similar to TCS. The crystal of Y70A n-TCS, the mutant of recombinant n-TCS, was soaked in sodium citrate buffer (pH 5.5) containing 25% KCl and AMP (10 mg/ml) prior to data collection. After structure determination and refinement, no electron density corresponding to adenine can be detected around the active pocket. Furthermore, the reaction products of Y70A n-TCS and AMP incubated at various reaction times were analyzed using HPLC. No adenine can be detected. These results suggest that Tyr70 is crucial to n-TCS for its substrate recognition, binding and perhaps N-glycosidase activity.

Adenine↗

PubMed1999

Role of Arg163 in the N-glycosidase activity of neo-trichosanthin.

Three mutant crystals of neo-trichosanthin (n-TCS), R163K, R163H and R163Q, were obtained by the hanging drop vapor diffusion method. Structure determination indicated that there are no significant differences between the mutants and n-TCS except in the active pocket. All of them were also soaked in sodium citrate buffer (pH 4. 5) containing 20% KCl and 10 mg/ml AMP. Structure determination suggests that in the active pocket of the crystals of R163K and R163H, parallel to the aromatic ring of Tyr70, each mutant possesses an adenine. The relationship between structure and function is discussed. Biochemical analysis reveals that the mutants R163K and R163H have N-glycosidase activity, while R163Q does not. This suggests that R163 is a crucial residue for the enzyme activity of n-TCS, and its role is providing proton.

Adenosine Monophosphate↗

PubMed1998

Refolding of denatured trichosanthin in the presence of GroEL.

The stability of trichosanthin (TCS), a 27-kDa ribosome-inactivating protein, was investigated in the presence of guanidinium chloride (GdnHCl). The process of unfolding was monitored by CD and fluorescence spectroscopy. Both methods show the presence of partially folded intermediates. Unfolding of TCS is attained in 6M GdnHCl, but the inactive species recover a good deal of its DNase activity upon dilution with buffer containing GroEL and ATP. The mechanism of recognition of unfolded TCS by GroEL was studied by fluorescence spectroscopy.

Adenosine Triphosphate↗

PubMed1998

Synthesis, DNA cleavage and cytotoxicity of some novel cyclic peptide-2,6-dimethoxyhydroquinone-3-mercaptoacetic acid conjugates containing D-amino acids.

This paper reports an ongoing study of the use of small-ring-size cyclic peptides as carriers of a potential antitumor agent: 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid (DMQ-MA). Three new cyclic tripeptide-DMQ-MA conjugates--cyclo[D-Val-Lys(DMQ-MA)-gamma-aminobutyric acid (GABA)-], cyclo[Val-Lys(DMQ-MA)-GABA-] and cyclo[D-Val-D-Lys(DMQ-MA)-GABA-]--were synthesized. The isomeric cyclic tripeptide-DMQ-MA conjugates were designed and synthesized to study the effect of stereoisomerism of the conjugates on cytotoxicity. The cyclic peptides were synthesized by coupling protected amino acids in solution and the final cyclization performed by the pentafluorophenyl ester method as described previously. After removing the lysyl-Z protecting group of the cyclic peptides the conjugation was achieved by reacting with the pentafluorophenyl ester of DMQ-MA. Electron spin resonance (ESR) studies of these three cyclic tripeptide-DMQ-MA conjugates showed that hydroxyl radicals were generated as a non-linear function of L-ascorbic acid (AH2) concentration. The IC50 of the cyclic tripeptide-DMQ-MA conjugates against a human pulmonary carcinoma cell line (PC-9 cells) under the synergistic activation of AH2 ranges from 0.4 to 1.6 microM, which is significantly lower than the parent compound DMQ-MA (6.1 microM). Agarose gel electrophoresis showed that DMQ-MA and these cyclic peptide-DMQ-MA conjugates are capable of cleaving supercoiled plasmid DNA to open circular and linear forms, even in the absence of AH2. The effects of enantiomeric and diastereomeric variations of these cyclic tripeptide-DMQ-MA conjugates on the cytotoxicity against PC-9 cells were discussed.

Amino Acids↗

PubMed1997

Cellular localization of GluR1, GluR2/3 and GluR4 glutamate receptor subunits in neurons of the rat neostriatum.

Glutamate excitocytotoxicity is implied in the cause of neuronal degeneration in the neostriatum, in which the toxicity may be mediated by different families of glutamate receptors. The precise cellular localization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate receptor subunits (GluR1-4), one of the major family that involves in the mechanisms of glutamate excitocytotoxicity, in different populations of striatal neurons is therefore of special interest. Immunoreactivity for GluR2/3 subunits was detected in the medium-sized spiny neurons. By double labelling experiments, immunoreactivity for GluR1 and GluR4 was detected only in aspiny striatal neurons that display parvalbumin immunoreactivity, but not in the other neuron populations that display choline acetyltransferase or muscarinic m2 receptor immunoreactivity, nor neurons that display nitric oxide synthase immunoreactivity or nicotinamide adenine dinucleotide phosphate-diaphorase activity. These results indicate that GluR1 and GluR4 immunoreactivity is displayed only in the GABAergic interneurons in the neostriatum. In addition, almost all of the GluR1-immunoreactive neurons were found to display GluR4 immunoreactivity. This finding indicates for the first time that the striatal GABAergic interneurons co-express GluR1 and GluR4 subunits. The results of the present study indicate that there is a differential localization of AMPA-type glutamate receptor subunits in different populations of striatal neurons and they may have a different susceptibility to glutamate excitocytotoxicity.

Animals↗

PubMed1997

Myelin basic protein is a zinc-binding protein in brain: possible role in myelin compaction.

The zinc-binding proteins (ZnBPs) in porcine brain were characterized by the radioactive zinc-blot technique. Three ZnBPs of molecular weights about 53 kDa, 42 kDa, and 21 kDa were identified. The 53 kDa and 42 kDa ZnBPs were found in all subcellular fractions while the 21 kDa ZnBP was mainly associated with particulate fractions. This 21 kDa ZnBP was identified by internal protein sequence data as the myelin basic protein. Further characterization of its electrophoretic properties and cyanogen bromide cleavage pattern with the authentic protein confirmed its identity. The zinc binding properties of myelin basic protein are metal specific, concentration dependent and pH dependent. The zinc binding property is conferred by the histidine residues since modification of these residues by diethyl-pyrocarbonate would abolish this activity. Furthermore, zinc ion was found to potentiate myelin basic protein-induced phospholipid vesicle aggregation. It is likely that zinc plays an important role in myelin compaction by interacting with myelin basic protein.

Animals↗

PubMed1996

Modeling of the three-dimensional structure of luffin-alpha and its simulated reaction with the substrate oligoribonucleotide GAGA.

A fundamental problem in biochemistry and molecular biology is understanding the spatial structure of macromolecules and then analyzing their functions. In this study, the three-dimensional structure of a ribosome-inactivating protein luffin-alpha was predicted using a neural network method and molecular dynamics simulation. A feedforward neural network with the backpropagation learning algorithm were trained on model class of homologous proteins including trichosanthin and alpha-momorcharin. The distance constraints for the C alpha atoms in the protein backbone were utilized to generate a folded crude conformation of luffin-alpha by model building and the steepest descent minimization approach. The crude conformation was refined by molecular dynamics techniques and a simulated annealing procedure. The interaction between luffin-alpha and its analogous substrate GAGA was also simulated to understand its action mechanism.

Algorithms↗

PubMed1996

A highly efficient procedure for purifying the ribosome-inactivating proteins alpha- and beta-momorcharins from Momordica charantia seeds, N-terminal sequence comparison and establishment of their N-glycosidase activity.

A new purification scheme, involving two successive ion exchange chromatographic steps on DEAE-cellulose and Mono-S FPLC, was developed for the isolation of the ribosome-inactivating proteins, alpha- and beta-momorcharins, from the Chinese herb Kuquazi (seeds of Momordica charantia). This simple and rapid procedure yielded 3.1 and 1.7 mg of alpha- and beta-momorcharins, respectively, from 2.5 g of decorticated seeds in only two days. The N-terminal amino acid sequence of beta-momorcharin was found to be DVNFDLSTATAKTYTKFIED. It differed from that of alpha-momorcharin (DVSFRLSGADPRSYGMFIKD) in 10 out of the 20 positions investigated. Like other ribosome-inactivating proteins, the purified momorcharins showed specific N-glycosidase activity at nanomolar concentrations, when rRNA from rabbit reticulocyte lysate was used as substrate. The N-glycosidase activity of both momorcharins was optimal at pH7, not inhibited by K+ and not appreciably affected by NH4+. The activity of alpha-momorcharin was not drastically altered by Mn2+ but (1-10mM) Mn2+ inhibited the activity of beta-momorcharin by about 40%.

Amino Acid Sequence↗

PubMed1996

Demonstration of ribonuclease activity in the plant ribosome-inactivating proteins alpha- and beta-momorcharins.

Alpha- and beta-momorcharins, ribosome-inactivating proteins from Momordica charantia seeds, were utilized in this investigation. Ribonucleolytic cleavage was observed after naked rRNA was incubated with either momorcharin. Beta-momorcharin, and to a lesser extent alpha-momorcharin, also acted on tRNA to release acid-soluble UV-absorbing products. Such activity was optimal at pH around 5.5. Using polyhomoribonucleotides as substrate, it was found that the momorcharins preferentially acted on polyU, but exerted negligible effects on polyA, polyC and polyG. Chromatographic analysis of the reaction product indicated that mono and/or oligo-ribonucleotides, but not free base, were generated from polyU, suggesting that the enzymatic action involved ribonucleolytic cleavage. Similar to the results obtained with tRNA as substrate, beta-momorcharin was about 15-fold more active than alpha-momorcharin on polyU.

Plant Proteins↗