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Biomedical subjects

D W Li

Publications and source records attributed to D W Li.

At least 19 recordsLinked to original sources
PubMed2001

Human bcl-2 gene attenuates the ability of rabbit lens epithelial cells against H2O2-induced apoptosis through down-regulation of the alpha B-crystallin gene.

It is well established that the proto-oncogene, bcl-2, can prevent apoptosis induced by a variety of factors. Regarding the mechanism by which BCL-2 prevents cell death, one theory suggests that it acts by protecting cells from oxidative stress. In the lens system, oxidative stress-induced apoptosis is implicated in cataractogenesis. To explore the possibility of anti-apoptotic gene therapy development for cataract prevention and also to further test the anti-oxidative stress theory of BCL-2 action, we have introduced the human bcl-2 gene into an immortalized rabbit lens epithelial cell line, N/N1003A. The stable expression clones of both vector- and bcl-2-transfected cells have been established. Treatment of the two cell lines with H(2)O(2) revealed that bcl-2-transfected cells were less capable of detoxifying H(2)O(2) than the control cells. Moreover, bcl-2-transfected cells are more susceptible to H(2)O(2)-induced apoptosis. To explore why bcl-2-transfected cells have reduced resistance to H(2)O(2)-induced apoptosis, we examined the expression patterns of several relevant genes and found that expression of the alphaB-crystallin gene was distinctly down-regulated in bcl-2-transfected cells compared with that in vector-transfected cells. This down-regulation was specific because a substantial inhibition of BCL-2 expression through antisense bcl-2 RNA significantly restored the level of alphaB-crystallin and, moreover, enhanced the ability of the bcl-2-transfected cells against H(2)O(2)-induced apoptosis. Introduction of a mouse alphaB-crystallin gene into bcl-2-transfected cells also counteracted the BCL-2 effects. Down-regulation of alphaB-crystallin gene was largely derived from changed lens epithelial cell-derived growth factor activity. Besides, alphaB-crystallin prevents apoptosis through interaction with procaspase-3 and partially processed procaspase-3 to prevent caspase-3 activation. Together, our results reveal that BCL-2 can regulate gene expression in rabbit lens epithelial cells. Through down-regulation of the alphaB-crystallin gene, BCL-2 attenuates the ability of rabbit lens epithelial cells against H(2)O(2)-induced apoptosis.

Animals↗
PubMed2001

Caspase-3 is actively involved in okadaic acid-induced lens epithelial cell apoptosis.

Phosphorylation and dephosphorylation are important cellular events regulating major metabolic activities such as signal transduction, gene expression, cell cycle progression, and apoptosis. It is well documented that okadaic acid, a potent inhibitor of protein phosphatase-1 (PP-1) and -2A (PP-2A), can induce apoptosis in a variety of cell lines. Our recent studies have revealed that in the immortal rabbit lens epithelial cell line, N/N1003A, inhibition of PP-1, but not PP-2A, leads to rapid apoptosis of the lens epithelial cells. This induction of cell death is associated with up-regulated expression of a set of genes, including the tumor-suppressor gene, p53, and the proapoptotic gene, bax. In the present study, we demonstrate that inhibition of PP-1 by okadaic acid in the primary cultures of rat lens epithelial cells also leads to apoptotic death. Moreover, we show that the cysteine protease, caspase-3, is important in the execution of okadaic acid-induced apoptosis. Treatment of the primary cultures of rat lens epithelial cells with 100 nM okadaic acid up-regulates expression of caspase-3 at the mRNA, protein, and enzyme activity levels. Inhibition of the caspase-3 activity with a chemically synthesized inhibitor prevents okadaic acid-induced apoptosis in rat lens epithelial cells. Similar results are also observed in the immortal cell line N/N1003A. Furthermore, stable expression of the mouse gene encoding lens alphaB crystallin inhibits okadaic acid-induced apoptosis, and this inhibition is associated with repression of the okadaic acid-induced up-regulation of caspase-3 activity. Taken together, these results demonstrate that caspase-3 is actively involved in okadaic acid-induced lens epithelial cell apoptosis.

Animals↗
PubMed2001

Anti-inflammatory, analgesic and antipyretic activities of loxoprofen sodium given intramuscularly in animals.

The evaluation of the anti-inflammatory, analgesic and antipyretic activities of loxoprofen sodium given in intramuscular route was investigated as compared to oral application in rats and mice. The intramuscular ED50 values of loxoprofen sodium in carrageenan edema and vascular permeability tests are 1.15 and 7.8 mg/kg, respectively, which represent more potent than in case of oral application. Its therapeutic effects in adjuvant arthritis were shown at 6 mg/kg i.m. and 3mg/kg p.o. Analgesic effect was shown to be more potent as given intramuscularly. Similar potency of antipyretic effects was shown in both administration routes. Considerably weak gastric damages were observed in intramuscular application.

Analgesics, Non-Narcotic↗
PubMed2001

Anti-inflammatory activity of methanol extract of Kalopanax pictus bark and its fractions.

The methanol extract of Kalopanax pictus bark was evaluated on anti-inflammatory and anti-nociceptive activities in animal models. The extract produced a significant inhibition of vascular permeability at doses of 1 and 3 g/kg, p.o. in mice and of leucocyte emigration at doses of 0.15 and 0.3 g/rat, s.c., in CMC-pouch of rats. However, the extract (0.25 and 3 g/kg, p.o.) did not show anti-inflammatory activity in carrageenan induced edema of rats. The extract at a dose of 2.5 g/kg, p.o. inhibited writhing syndromes, whereas it did not exhibit anti-nociceptive in Randall-Selitto assay. The methanol extract was then partitioned with n-hexane, chloroform, ethyl acetate and butanol to give each soluble fraction and finally water soluble fraction. Among those fractions, the inhibitory effect on vascular permeability in mice was produced by ethyl acetate soluble fraction in this activity-guided fractionation. The methanol extract showed low acute toxicity in mice. These results suggest that the methanol extract of Kalopanax pictus bark has an anti-inflammatory activity which is distributed in the ethyl acetate fraction.

Analgesia↗
PubMed2001

Inhibition of lens epithelial cell growth by induction of apoptosis: potential for prevention of posterior capsule opacification.

As a model of the cell proliferation occurring in posterior capsule opacification (PCO), lens epithelial cells (LEC) from human and rabbit capsulotomies, and a rabbit LEC line (N/N1003A) were grown in Dulbecco's Minimal Essential Media (MEM) with 10% fetal calf serum. LEC were exposed to the calcium ionophore, calcimycin, and viability was assessed by trypan blue staining, growth by 3H-thymidine incorporation and apoptosis by annexin/propidium iodide staining, calcein AM/ethidium bromide staining and DNA laddering. Human capsulotomy samples were similarly exposed to calcimycin, and apoptosis assayed by calcein AM/ethidium bromide staining. Calcimycin exposure induced apoptosis in both rabbit LEC cultures and human LEC, and changes leading to apoptosis could be detected within 30 minutes of calcimycin treatment. The decrease in viability and growth in human and rabbit LEC was dose-dependent. These data support the further evaluation of apoptosis induction as a possible treatment mechanism to prevent development of PCO following primary cataract surgery in humans.

Animals↗
PubMed2001

Analysis of expression patterns of protein phosphatase-1 and phosphatase-2A in rat and bovine lenses.

PURPOSE: The reversible phosphorylation and dephosphorylation at the serine and threonine residues on proteins play distinct roles in regulating multiple cellular activities. Whereas the protein serine-threonine kinases have been well studied in the lens system, very little is known about the expression and function of the serine-threonine phosphatases. The present article reports the expression patterns of protein phosphatase (PP)-1 and -2A in adult rat and bovine lenses. METHODS: Total RNAs and proteins were extracted from the epithelial and fiber cells of rat and bovine lenses. RT-PCR and Northern blot analysis were used to detect the mRNA expression levels in the epithelial cells and different fractions of fiber cells of these two types of lenses. Western blot was used to examine the protein expression levels in these different samples. An enzymatic assay was used to detect the activity distribution of PP-1 and -2A in these samples. RESULTS: The mRNAs for the PP-1 catalytic subunit (PP-1cs) and PP-2A catalytic subunit (PP-2Acs) were expressed in both epithelial and fiber cells of rat and bovine lenses. A detailed examination of the expression patterns of the two mRNAs in different fractions of fiber cells revealed that the cortical fiber cells (F1) contain the highest level of PP-1cs and -2Acs mRNAs (similar to those in the epithelial cells) among different fractions of fiber cells. The levels of the two mRNAs were sequentially decreased in the next layers of fiber cells (F2 and F3) and became barely detectable in the inner layers of fiber cells (F4 and N). In contrast to the mRNA expression patterns, the PP-1cs protein was mainly found in the epithelial cells. Among different layers of fiber cells, only cortical (F1) fiber cells contained detectable level of PP-1cs protein (bovine lenses contained a relatively higher level of PP-1cs than rat lenses in this region). In the remaining fiber cells, the PP-1cs protein was hardly detectable in rat lenses and slightly detectable in bovine lenses. The PP-2Acs protein was detectable only in the lens epithelial cells. Enzymatic assays revealed that the distribution patterns of PP-1 and -2A activities were similar to those of PP-1cs and -2Acs proteins. Furthermore, PP-1 activity was approximately four to five times higher than PP-2A activity in the lens epithelial cells. CONCLUSIONS: This study demonstrates that active PP-1 and -2A are mainly distributed in the lens epithelial cells, with PP-1 as a major phosphatase. The mRNAs and proteins for PP-1cs and -2Acs are differentially expressed in the epithelial and fiber cells of rat and bovine lenses.

Animals↗
PubMed2000

hTERT can function with rabbit telomerase RNA: regulation of gene expression and attenuation of apoptosis.

Telomerase is a specialized DNA polymerase that adds telomeric sequences onto chromosome ends. The functional telomerase complex contains a telomerase reverse transcriptase (TERT) and also a telomerase RNA (TR). Although it is well established that the human telomerase reverse transcriptase (hTERT) can function well in different human cell lines, it has not been shown whether it is compatible with telomerase template RNA from other species. Here we report that the expressed hTERT is functionally compatible with rabbit telomerase template RNA (rTR) as demonstrated by TRAP assay. The direct interaction between hTERT and rTR is further confirmed by immunoprecipitation-linked RT-PCR in which rTR is detected from the complex immunoprecipitated by an anti-hTERT antibody. The hTERT expressed in rabbit lens epithelial cells demonstrates two major functions: modulation of expression of other genes and attenuation of apoptosis. Thus, telomerase has a variety of functions besides telomere synthesis, and the template RNA is functionally conserved between human and rabbit.

Animals↗
PubMed1998

Okadaic acid-induced lens epithelial cell apoptosis requires inhibition of phosphatase-1 and is associated with induction of gene expression including p53 and bax.

It is well established that phosphorylation and dephosphorylation are key cellular events which regulate important metabolic activities such as gene expression, cell cycle progression, and apoptosis. The polyether fatty acid, okadaic acid has been shown previously to activate apoptosis in a variety of cell lines. Although this marine sponge toxin is known to inhibit protein phosphatase (PP)-2A and PP-1, it is not certain in most cases whether inhibition of PP-1 or PP-2A is necessary to activate apoptosis. Furthermore, it is not clear how inhibition of these phosphatases leads to apoptosis. Here we present evidence that inhibition of PP-2A by okadaic acid does not activate apoptosis in the lens system. However, when PP-1 is inhibited by okadaic acid, rabbit lens epithelial cells undergo rapid apoptosis. Associated with this process is the several-fold up-regulation of the tumor suppressor gene p53 and the pro-apoptotic gene bax at both mRNA and protein levels. Analyses of the temporal pattern of expression of the two genes reveal that the up-regulation is maximized in a few hours after treatment with okadaic acid, when the majority of the treated cells become committed to apoptosis. A brief treatment of the cells with a protein synthesis inhibitor can abolish okadaic acid-induced up-regulation of both P53 and Bax proteins. Concomitant with this inhibition, okadaic acid-induced apoptosis is also temporarily blocked. These results suggest that okadaic acid-induced expression of p53, bax, and other genes are necessary for the activation of the apoptotic programs in lens systems.

Animals↗
PubMed1998

The immune status of the myelin deficient rat and its immune responses to transplanted allogeneic glial cells.

This study examined the immunological responsiveness of the myelin deficient (md) rat, and its immune response to transplanted allogeneic glial cells, with and without immunosuppression therapy. Skin grafts from an ACI strain of rat were found to be acutely rejected by Wistar md rats. Anti-donor cytotoxic antibody was produced and alloreactive T helper cells were expanded in these mutants after skin sensitization. Equivalent high frequencies of precursor cytotoxic T lymphocytes (pCTLs) specific to the ACi MHC antigens were observed in both normal controls and mutants. An immune response was noted when allogeneic glial cells were transplanted into the spinal cords of md rats, which was effectively suppressed for 2 weeks post transplantation by treatment with either cyclosporin A (CsA) or a monoclonal antibody to the interleukin-2 receptor (IL-2R). These results demonstrate that md rats are immunocompetent, but that CNS allograft rejection can be prevented by immunosuppressive agents.

Animals↗
PubMed1997

Hydrogen peroxide-induced expression of the proto-oncogenes, c-jun, c-fos and c-myc in rabbit lens epithelial cells.

The involvement of H2O2 in cataract development has been established in both human patients and animal models. At the molecular level H2O2 has been observed to cause damage to DNA, protein and lipid. To explore the oxidative stress response of the lens system at the gene expression level, we have examined the effects of H2O2 on the mRNA change of the proto-oncogenes, c-jun, c-fos and c-myc in a rabbit lens cell line, N/N1003A. H2O2 treatment of the rabbit lens epithelial cells for 60 min induces quick up-regulation of both c-jun and c-fos mRNAs. The maximal induction is 38 fold for c-jun at 150 microM and 72 fold for c-fos at 250 microM H2O2. Treatment of N/N1003A cells with 50-250 microM H2O2 for 60 min leads to a 2-5 fold increase of the c-myc mRNA level. H2O2 also induces an up-regulation in transactivity of the activating protein-1 (AP-1) as shown with a reporter gene driven by a prolactin gene promoter with 4 copies of AP-1 binding sites inserted in the upstream of the promoter. Maximal induction occurs with 150 microM H2O2. In the same system, the antioxidants, N-acetyl-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) at concentrations shown to up-regulate the mRNAs of both c-jun and c-fos, also enhance the transactivity of AP-1. NAC and PDTC have different effects in modulating the induction of AP-1 activity by H2O2 and TPA. These results reveal that oxidative stress regulates expression of various regulatory genes in lens systems, which likely affects cell proliferation, differentiation and viability and thus affect normal lens functions.

Acetylcysteine↗
PubMed1995

Indoor aeromycota in relation to residential characteristics and allergic symptoms.

The indoor aeromycota in several rooms of each of 15 residences in Kitchener-Waterloo, Ontario, Canada was studied from December 1991 to September 1993. There were significant differences in airborne spore concentrations among the types of rooms investigated. Numbers of airborne propagules were highest in the living rooms, followed by family rooms, kitchens, bathrooms and bedrooms. The highest fungal diversity was found in kitchens. Generally, presence of dampness and of carpets led to increased numbers of airborne spores. Forced air heating systems, humidifiers, air filters and air conditioners reduced concentrations of airborne fungi. Patients with respiratory allergies and known sensitivity to moulds reported allergic symptoms significantly less severe than average in residences with air conditioners, air filters, humidifers and forced air heating systems. In damp residences, their symptoms were significantly more severe than the average.

Air Conditioning↗
PubMed1995

A year-round study on functional relationships of airborne fungi with meteorological factors.

Air sampling was conducted in Waterloo, Canada throughout 1992. Functional relationships between aeromycota and meteorological factors were analysed. The meteorological factors were, in descending order of importance: mean temperature, minimum temperature, maximum temperature, mean wind speed, relative humidity (RH), rain, maximum wind speed and snow. The most important airborne fungal propagules in descending order were total fungal spores, unidentified Ascomycetes, Cladosporium, Coprinus, unidentified Basidiomycetes, Alternaria and unidentified fungi. Most airborne fungal taxa and highly significant relationship with temperature, but Aspergillus/Penicillium, hyphal fragments were positively associated with wind speed. In comparison with other airborne fungal taxa, Leptosphaeria and unidentified Ascomycetes were more closely correlated with rain and RH during the growing season.

Air Microbiology↗
PubMed1989

A mouse thymidylate synthase pseudogene derived from an aberrantly processed RNA molecule.

A DNA fragment containing a mouse-thymidylate-synthase(TS) processed pseudogene was cloned and analyzed. Comparison with the sequences of the mouse TS-encoding gene (ts) and cDNA revealed that the pseudogene started at one of the normal 5' termini of TS mRNA, ended with a poly(A) tail, and was flanked by 16-nucleotide (nt) direct repeats. The region corresponding to the open reading frame was 97.3% identical to that of the cDNA. Two unusual features were observed. First, the poly(A) tail of the pseudogene was located 2 kb downstream from the normal location. Second, the final 10 nt of intron 5 were retained in the 'coding region' of the pseudogene. Therefore, it appears that the pseudogene was derived from a nonfunctional TS 'mRNA' that was aberrantly spliced and polyadenylated. Analysis of the sequence of intron 5 of the ts gene revealed the presence of an alternative 3' splice site 10 nt upstream from the normal splice site. S1-nuclease protection assays showed that about 10% of TS mRNA isolated from mouse cells was spliced at the alternative site.

Amino Acid Sequence↗
PubMed1986

Structure of the gene for mouse thymidylate synthase. Locations of introns and multiple transcriptional start sites.

We have isolated and analyzed the structure of the gene for thymidylate synthase from a 5-fluoro-2'-deoxyuridine-resistant 3T6 cell line that overproduces thymidylate synthase 50-fold by virtue of gene amplification. Three overlapping DNA segments containing the entire thymidylate synthase gene were identified in Charon 35 genomic libraries. Sequence analysis revealed that all of the coding regions were contained in a 12-kilobase segment of DNA. The gene has 6 introns ranging from 0.6 to 4.1 kilobases in length. The sequences at the 5' and 3' ends of each intron conformed to the consensus sequences for mammalian introns. S1 nuclease and primer extension assays showed that transcription of the thymidylate synthase gene initiates at several sites within a 66-nucleotide region. There are no TATAA or CCAAT sequences in the vicinity of the initiation sites. However, the region does contain DNA sequences that are known to stimulate binding of the transcription factors Sp1 and USF. These binding sites are adjacent to each other, suggesting that the two proteins may bind to the upstream region of the thymidylate synthase gene in a cooperative or competitive manner.

Animals↗