Biomedical subjects

C M Lai

Publications and source records attributed to C M Lai.

At least 19 recordsLinked to original sources

PubMed2006

Long-term global retinal microvascular changes in a transgenic vascular endothelial growth factor mouse model.

AIMS/HYPOTHESIS: Vascular endothelial growth factor (VEGF) plays a pivotal role in the pathogenesis of diabetic retinopathy. We investigated whether transgenic mice with moderate VEGF expression in photoreceptors (trVEGF029) developed changes similar to diabetic retinopathy and whether retinopathy progressed with time. MATERIALS AND METHODS: Human VEGF(165) (hVEGF(165)) expression was analysed using ELISA and quantitative RT-PCR; serum glucose levels were also measured. Fundus fluorescein angiography (FA) was used to screen the degree of retinopathy from 6 weeks. Dynamic changes in the density of retinal microvasculature, as well as other changes similar to diabetic retinopathy, including retinal leucostasis, capillary endothelial cell and pericyte loss, and numbers of acellular capillaries, were quantified. RESULTS: trVEGF029 mice were normoglycaemic and showed a moderate, short-term hVEGF(165) upregulation for up to 3 weeks. Changes in the retinal microvasculature not only mimicked those seen in diabetic retinopathy, but also showed similar pathological progression with time. FA at 6 weeks identified two phenotypes, mild and moderate, which were distinguished by the extent of vascular leakage. Quantitative analysis of diabetic retinopathy-like changes revealed that these parameters were tightly correlated with the initial degree of vascular leakage; low levels reflected slow and limited retinal microvascular changes in mild cases and high levels reflected more rapid and extensive changes in moderate cases. CONCLUSIONS/INTERPRETATION: The data suggest that even an early short-term elevation in hVEGF(165) expression might set a train of events that lead to progressive retinopathy. Induction of many features characteristic of diabetic retinopathy in trVEGF029 enables mechanisms leading to the disease state to be examined, and provides a relevant animal model for testing novel therapeutics.

Animals↗

PubMed2005

A rapid HPLC-ESI-MS/MS for qualitative and quantitative analysis of saponins in "XUESETONG" injection.

'XUESETONG' injection, one of the most widely used proprietary medicines in traditional Chinese medicine, consists of total saponins made from Panax notoginseng, which is a highly valued and important Chinese medicinal herb. It is used to treat cardiovascular diseases. In order to control the quality of XUESETONG injection, a rapid HPLC-ESI-MS/MS method was developed for qualitative and quantitative determination of the saponins. The analyses were performed on SB-C18 column using gradient elution in 25 min. Full scan and time programmed selected reaction monitoring (SRM) were used for qualitative and quantitative analysis of saponins, respectively. Twenty-seven saponins were identified and nine of them including notoginsenoside R1, ginsenoside Rb1, Rb2, Rb3, Rc, Rd, Re, Rf and Rg1 were quantified. Ten XUESETONG injections were analyzed and compared. The results showed that there is a great variation among different samples. In conclusion, the developed method is rapid, accurate and sensitive for qualitative and quantitative analysis of saponins in XUESETONG injection. Moreover, it also can be used for the quality control of P. notoginseng raw material and its preparations.

Chromatography, High Pressure Liquid↗

PubMed2005

Simultaneous determination of nine saponins from Panax notoginseng using HPLC and pressurized liquid extraction.

A HPLC and pressurized liquid extraction (PLE) method was developed for simultaneous determination of nine saponins, including notoginsenoside R1, ginsenoside Rg1, Re, Rf, Rb1, Rc, Rb2, Rb3 and Rd in Panax notoginseng. The analysis was performed on C18 column with water-acetonitrile gradient elution and the investigated saponins were authenticated by comparing retention time and mass spectra with their reference compounds. Several methods including PLE, ultrasonication, soxhlet extraction and immersion were used for sample preparation and their extraction efficiency was compared. The results showed that PLE has the highest extraction efficiency and repeatability, which would be valuable on standardization of sample preparation for quality control of Chinese medicines. The developed HPLC and PLE is an effective approach for simultaneously quantitative determination of sapoinins in P. notoginseng, which could be used for quality control of P. notoginseng and its preparations.

Calibration↗

PubMed2004

Simultaneous determination of ergosterol, nucleosides and their bases from natural and cultured Cordyceps by pressurised liquid extraction and high-performance liquid chromatography.

A simple method is described for the simultaneous determination of ergosterol, nucleosides and their bases in Cordyceps. The samples were extracted by using pressurised liquid extraction (PLE). The effects of experimental variables, such as solvent, temperature, static extraction time and cycles, on PLE efficiency have been studied. The results showed a strong influence of the solvent and temperature on extraction efficiency of PLE. The determination was achieved by high-performance liquid chromatography (HPLC) using a Zorbax NH2 analytical column (250 x 4.6 mm i.d., 5 microm) with diode-array detector (DAD). The automated preparation of the sample permits a very fast analysis which is an important goal for routine purpose.

Chromatography, High Pressure Liquid↗

PubMed2002

Potential long-term inhibition of ocular neovascularisation by recombinant adeno-associated virus-mediated secretion gene therapy.

Neovascularisation (NV) within the eye often results in visual loss. Vascular endothelial growth factor (VEGF) has been implicated in the development of ocular NV. Previous studies have shown that VEGF antagonists successfully suppressed retinal and choroidal NV in animal models. However, the systemic approach and transient nature of the delivery systems used in these studies hinder therapeutic application. To achieve stable and localised ocular anti-angiogenic therapy, we explored the use of recombinant adeno-associated virus (rAAV)-mediated secretion gene therapy (SGT). In this study, we generated a rAAV vector encoding soluble VEGF receptor 1, sFlt-1 (AAV-CMV.sflt) and determined its ability to inhibit cautery-induced corneal NV and laser-induced choroidal NV. Delivery of AAV-CMV.sflt into the anterior chamber resulted in transgene expression in the iris pigment epithelium and corneal endothelium, which reduced the development of corneal NV in the stroma of cauterised rats by 36% compared with cauterised control groups (P = 0.009). Subretinal delivery of AAV-CMV.sflt near the equator of the eye also suppressed choroidal NV at the laser lesions around the optic nerve by 19% (P = 0.002), indicating that there was diffusion of the secreted anti-angiogenic protein across the retina. Both results suggest that the long-term suppression of ocular NV is possible through the use of stable rAAV-mediated SGT.

Adenoviridae↗

PubMed2001

Inhibition of angiogenesis by adenovirus-mediated sFlt-1 expression in a rat model of corneal neovascularization.

Pathological angiogenesis, or the production of new capillary vessels from preexisting vasculature, within the eye is a serious event that often leads to blindness. Upregulation of vascular endothelial growth factor (VEGF) has been linked to neovascularization in the eye, suggesting that it could be a suitable target to inhibit angiogenic changes. This work investigated whether the presence of a proven antiangiogenic factor, the soluble variant of the VEGF receptor, sFlt-1, in the anterior chamber is sufficient to inhibit new vessel formation in the cornea in an animal model of corneal neovascularization. A recombinant adenovirus vector that can mediate efficient in vivo gene transfer and expression in ocular cells was selected as a delivery agent. We have shown that after the injection of Ad.betagal into the anterior chamber of normal and cauterized rat eyes, corneal endothelial cells and cells of the trabecular meshwork were efficiently transduced and that beta-galactosidase (beta-Gal) expression was maintained up to 10 days postinjection. Cauterization significantly increased the amount of immunoreactive VEGF in vehicle- or Ad.null-injected animals (t test, p < 0.001 and p < 0.001, respectively). However, when cauterization was combined with Ad.sflt injection there was no statistically significant increase in the amount of immunoreactive VEGF (p = 0.12). The injection of Ad.sflt into the anterior chamber slowed or inhibited VEGF-induced angiogenic changes. After cauterization, 100% of uninjected and vehicle-injected and 82% of Ad.null-injected animals developed moderate to severe corneal angiogenesis in contrast to 18% of Ad.sflt-injected animals. These in vivo results suggest that the transient presence of antiangiogenic agents in the anterior chamber can be successfully used to inhibit the development of corneal angiogenesis.

Adenoviridae↗

PubMed2001

Identification and characterization of S2V, a novel putative siglec that contains two V set Ig-like domains and recruits protein-tyrosine phosphatases SHPs.

We describe the molecular cloning and characterization of S2V, a novel sialic acid binding immunoglobulin-like lectin. The cDNA of S2V encodes a type 1 transmembrane protein with four extracellular immunoglobulin-like (Ig-like) domains and a cytoplasmic tail bearing a typical immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like motif. A unique feature of S2V is the presence of two V-set Ig-like domains responsible for the binding to sialic acid, whereas all other known siglecs possess only one. S2V is predominantly expressed in macrophage. In vivo S2V was tyrosine-phosphorylated when co-expressed with exogenous c-Src kinase. Upon tyrosine phosphorylation, S2V recruits both Src homology 2 (SH2) domain-containing protein-tyrosine phosphatases SHP-1 and SHP-2, two important inhibitory regulators of immunoreceptor signal transduction. These findings suggest that S2V is involved in the negative regulation of the signaling in macrophage by functioning as an inhibitory receptor. When expressed in COS-7 cells, S2V was able to mediate sialic acid-dependent binding to human red blood cells, suggesting that S2V may function through cell-cell interaction.

Amino Acid Sequence↗

PubMed2001

Carbon dioxide flux density in cultivated rice paddy field.

The carbon dioxide flux density in a subtropical cultivated rice paddy field were determined over four crop seasons (two consecutive years) at five stages of rice plant development, transplanting, active tillering, booting, flowering and ripening, and an idle period between crops. During 24 h sampling periods at those stages, the CO2 flux densities were measured and found to be -2,000 to 55,000 mg CO2/m2/day in the first year and 600 to 29,000 mg CO2/m2/day in the second year in acrylic chambers covering rice plants. The CO2 flux densities in glass domes without plants but with soil were -700 to 6,000 mg CO2/m2/day in the first year and 147 to 4,500 mg CO2/m2/day in the second year. The data for the CO2 flux density over an entire year are extrapolated. Although our non-controlled environment experiments have inherent drawbacks, the collected data indicate that continuous rice cultivation has a tendency to result in increased CO2 emissions.

Carbon Dioxide↗

PubMed2000

Controlled production of active cathepsin D in retinal pigment epithelial cells following adenovirus-mediated gene delivery.

The transduction of a low cathepsin D-producing retinal pigment epithelial cell line with a recombinant adenovirus, Ad.proCatD, carrying a viral promoter and the precursor form of the lysosomal enzyme cathepsin D, procathepsin D, led to the upregulation of proCatD expression. However, the resultant aspartic protease activity did not exceed that observed in normal primary human retinal pigment epithelial cells. Following the injection of Ad. proCatD into rat eyes, immunohistochemistry and Western blot analysis localized the expression of procathepsin D to the retinal pigment epithelial cell layer and to the sclera/choroid/retinal epithelial cell layers, respectively. This upregulation of procathepsin D expression was accompanied by a limited increase in aspartic protease activity. The injected eyes did not demonstrate any of the retinal changes that have been associated with the overproduction and secretion of active cathepsin D. Immunoelectronmicroscopy of Ad.proCatD-transduced retinal pigment epithelial cells demonstrated the presence of cathepsin D not only in cytoplasmic vesicles and lysosomes but also in the nucleoli and, less strongly, elsewhere in euchromatic regions of some 10% of cells. In spite of the upregulated expression of procathepsin D, the production of active cathepsin D in Ad.proCatD-transduced retinal pigment epithelial cells was strictly controlled. It is proposed that active cathepsin D production is controlled at the point of posttranslational modification by an intranuclear feedback mechanism initiated by the relative excess of procathepsin D in Ad. proCatD-transduced retinal pigment epithelial cells.

Adenoviridae↗

PubMed2000

The use of adenovirus-mediated gene transfer to develop a rat model for photoreceptor degeneration.

PURPOSE: To investigate the effects of recombinant adenovirus-mediated downregulation of cathepsin S (CatS) on the retinal pigment epithelium and/or neural retina in vivo. METHODS: The expression of green fluorescent protein (gfp) after subretinal injection of a recombinant adenovirus, Ad.gfp, into rat eyes was first established by in vivo fundus fluorescence photography and fluorescence microscopy. The autofluorescent debris accumulation in Ad.CatSAS (recombinant adenovirus carrying the antisense CatS gene)injected rat eyes was monitored by fluorescence microscopy, and the antisense CatS RNA expression was demonstrated by in situ hybridization. Changes in the retinal morphology were assessed by light microscopy. ResuLTS. The gfp expression was present in 30% to 90% of the injection area at 3 days and was absent 9 days after Ad.gfp injection. In Ad.CatSAS-injected eyes, the expression of antisense CatS RNA was demonstrated by in situ hybridization. Autofluorescent debris accumulation was significantly higher in Ad.CatSAS-injected eyes than in control eyes. The shortening of photoreceptor outer segments in Ad.CatSAS-injected eyes coincided with intense autofluorescent debris accumulation. The number of layers of photoreceptor cells decreased with time and were 11, 9, and 8 at 7, 14, and 28 days after Ad.CatSAS injection, respectively. In control eyes, the number of layers of photoreceptor cells (14) remained unchanged. CONCLUSIONS: These results demonstrate that recombinant adenovirus-mediated transient modulation of gene expression in retinal pigment epithelial (RPE) cells could induce changes in the retina, and, in spite of the low expression of endogenous CatS in RPE cells, this enzyme plays an important role in maintenance of normal retinal function.

Adenoviridae↗

PubMed1999

Preferential adenovirus-mediated transduction of cells at the sites of laser photocoagulation in the rat eye.

PURPOSE: This study aimed to investigate the feasibility of recombinant adenovirus-mediated gene transfer into cells implicated in the development of choroidal neovascularization (CNV). METHODS: A rat model of CNV which used laser photocoagulation was developed. Gene delivery into the laser spots was investigated following subretinal injection of recombinant adenoviruses, AdRSVlacZ, AdCMVlacZ or AdCMVgfp. Immunohistochemical analysis was performed using a proliferating cell nuclear antigen antibody and a cytokeratin-specific antibody to identify the cell types transduced by the recombinant adenoviruses. RESULTS: At 7 days post-injection, lacZ expression was detected in 51.6 +/- 13.2% and 71.2 +/- 19.3% of laser spots in AdRSVlacZ- and AdCMVlacZ-injected eyes, respectively. By 28 days post-injection, lacZ expression was only present in AdCMVlacZ-injected eyes. In vivo fundus fluorescent photography of AdCMVgfp-injected eyes detected gfp expression in 79.9 +/- 12.7% and 35.6% +/- 19.7% of laser spots at 4 and 7 days post-injection, respectively. Although fundus fluorescent photography did not detect the gfp signal at 10 days post-injection, fluorescent microscopy revealed a gfp signal in 81.3 +/- 6.0% of laser spots. Immunohistochemical analysis detected retinal pigment epithelial (RPE) cells as the most predominant proliferating cell type in the laser spots, although several other proliferating cell types were also identified. X-gal staining showed that the majority of transduced cells were those present in the laser spots. CONCLUSIONS: It is proposed that following laser photocoagulation, proliferating RPE cells are susceptible to adenovirus-mediated gene delivery and that they may be suitable targets for the delivery of antiangiogenic factors by recombinant adenoviruses in order to inhibit developing CNV membranes.

Adenoviridae↗

PubMed1999

Expanded-spectrum nonnucleoside reverse transcriptase inhibitors inhibit clinically relevant mutant variants of human immunodeficiency virus type 1.

A research program targeted toward the identification of expanded-spectrum nonnucleoside reverse transcriptase inhibitors which possess increased potency toward K103N-containing mutant human immunodeficiency virus (HIV) and which maintain pharmacokinetics consistent with once-a-day dosing has resulted in the identification of the 4-cyclopropylalkynyl-4-trifluoromethyl-3, 4-dihydro-2(1H)quinazolinones DPC 961 and DPC 963 and the 4-cyclopropylalkenyl-4-trifluoromethyl-3, 4-dihydro-2(1H)quinazolinones DPC 082 and DPC 083 for clinical development. DPC 961, DPC 963, DPC 082, and DPC 083 all exhibit low-nanomolar potency toward wild-type virus, K103N and L100I single-mutation variants, and many multiply amino acid-substituted HIV type 1 mutants. This high degree of potency is combined with a high degree of oral bioavailability, as demonstrated in rhesus monkeys and chimpanzees, and with plasma serum protein binding that can result in significant free levels of drug.

Amino Acid Substitution↗

PubMed1998

Determination of bisnafide, a novel bis-naphthalimide anticancer agent, in human plasma by high-performance liquid chromatography with UV detection.

A simple, specific, and sensitive high-performance liquid chromatographic (HPLC) assay utilizing ultraviolet (UV) detection for the determination of bisnafide in human plasma was developed, validated, and applied to plasma samples from patients undergoing cancer therapy. Plasma samples, containing an internal standard, XE842, were first deproteinized with 2.0 ml acetonitrile, and subsequently, 1.0 ml and pH 9 boric acid-potassium chloride-sodium hydroxide buffer (0.1 M) was added. To this mixture, 9.0 ml of ethyl ether was added then vortex mixed. Following centrifugation, the ether layer was back-extracted into 250 microliters of 0.1 M phosphoric acid, then removed by vacuum aspiration. A portion of the remaining acid layer was directly injected onto the HPLC. Bisnafide was quantified using a Shiseido Capcell Pak C8 HPLC column and ultraviolet detection (274 nm). The lower limit of quantification was 10 ng ml-1 using 1.0 ml plasma. The intraday precision (RSD) ranged from 2.7 to 8.6% over a concentration range of 10-1000 ng ml-1. The interday precision (RSD) ranged from 5.6 to 11.5%. Overall mean accuracy was +/- 5.2%. The drug was stable in frozen heparinized human plasma stored at -20 degrees C for at least 1 year and stable throughout at least two freeze-thaw cycles. This method was successfully utilized for quantifying plasma concentrations needed to study the clinical pharmacokinetics of bisnafide in patients undergoing cancer therapy.

Antineoplastic Agents↗

PubMed1998

Recombinant adenovirus-mediated gene delivery into the rat retinal pigment epithelium in vivo.

PURPOSE: The present paper describes changes following the subretinal injection of a recombinant replication-deficient adenovirus carrying the beta-galactosidase reporter gene construct into the rat retina. METHODS: Ad.RSV.betagal-mediated transduction of rat retinal pigment epithelium (RPE) cells in vitro and in vivo was examined following X-gal staining. RESULTS: There was a low level of beta-galactosidase expression in the RPE cells at 4 days postinjection. At 7 days postinjection, a strong transgene expression was present in RPE cells and the expression was maintained at 14 days postinjection. Except for the accumulation of cells at the site of the injection, the morphology of the rest of the retina remained normal. CONCLUSION: These results demonstrate that the RPE layer can be successfully targeted for gene delivery in the rat.

Adenoviridae↗

PubMed1997

Modulation of cathepsin D activity in retinal pigment epithelial cells.

This project used retinal pigment epithelial (RPE) cells to investigate the effects of up- and down-regulation of cathepsin D expression on the processing of cathepsin D and on the normal phagocytic and digestive function of these cells. RPE cells were transfected with a pHbetaApr-1-neo vector construct carrying the full-length sequence of the translated region of human cathepsin D in sense and antisense directions. Transfected cells were characterized for the presence and expression of the transgene by PCR amplification using transgene-specific primers. Total aspartic proteinase activity present in transformed RPE cells was measured by an enzyme assay using haemoglobin as substrate. Flow cytometry was used to quantify phagocytosis of fluorescein isothiocyanate-labelled rod outer segments (ROS), and lysosomal digestion of ROS was monitored by immunofluorescence. A 435 bp fragment was present in RPE cells carrying the cathepsin D transgene in sense and antisense orientations after PCR amplification. Expression of both 52 kDa procathepsin D and 34 kDa active cathepsin D was significantly up-regulated in sense cathepsin D-transfected RPE cells and down-regulated in RPE cells transfected with antisense cathepsin D. No other forms of cathepsin D were detected in the transfected cells, suggesting that, if pseudo-cathepsin D exists in RPE cells in vivo, it requires the presence of unknown specific regulatory elements. The up- and down-regulation of cathepsin D expression was further confirmed by enzyme assay. Transfected cells retained their phagocytosing ability after ROS challenge and maintained their ability to process ROS. The processing of ROS was significantly slower in RPE cells transfected with antisense than control vector or in sense-cathepsin D-transfected cells. These results demonstrate that cathepsin D is a major proteolytic enzyme participating in the lysosomal digestion of photoreceptor outer segments.

Animals↗

PubMed1997

The in vivo expression patterns of individual type I interferon genes in murine cytomegalovirus infections.

Type I interferon (IFN) family has more than 10 structurally related subtypes of alpha IFN (IFNA) genes and a single beta IFN (IFNB) gene. This study examined the expression of MuIFNA1, A4, A5, A6, A9 and MuIFNB mRNAs in the livers and spleens of MCMV-infected mice at 2, 4 and 6 h post infection. The three strains of inbred mice studied were C57BL/6, B6.C-H-28c and BALB/c. B6.C-H-28c strain is a congenic strain of C57BL/6 genomic background. Detection of the specific mRNAs was by an established semiquantitative procedure based on reverse transcription and PCR amplification followed by differential hybridization to specific oligonucleotides. Similar expression patterns of the type I IFN mRNAs were found in C57BL/6 and B6.C-H-28c strains of mice. However, when the genotype was significantly different (BALB/c), a different expression pattern of IFN mRNAs was seen. Differences in the expression patterns of the type I IFN mRNAs was also seen between the livers and spleens of a given mouse strain. Thus, the present study indicates that mouse genotype appears to be a major determinant of the subtype response pattern seen in vivo and that tissue-type can influence the subtype response pattern seen within a given mouse genotype.

Animals↗

PubMed1997

The in vitro expression patterns of individual type I interferon genes in Newcastle disease virus infected murine splenocytes and fibroblasts.

Murine type I interferon levels present in mice sera following Newcastle disease virus infections are influenced by the If-1 locus. Sera interferon levels in C57BL/6 mice (If-1h allele) are 10- to 15-fold higher than in BALB/c mice (If-1(1) allele). The B6.C-H-28c strain, which carries BALB/c If-1(1) allele on C57BL/6 genomic background, has low interferon levels in sera. This study examined the expression of interferon alpha 1, alpha 4, alpha 5, alpha 6, alpha 9 and beta mRNAs at 7 hr after Newcastle disease virus infection of primary cells (splenocytes and mouse embryo fibroblasts) from C57BL/6, B6.C-H-28c and BALB/c mouse genotypes. Total RNA from these cells was reverse transcribed and all known type I interferon subtypes were amplified. The products were identified by differential hybridization to a panel of subtype specific oligonucleotides. The results show that the pattern of interferon subtypes examined in splenocytes did not differ between If-1h and If-1(1) allele carrying C57BL mice. However, when the genotype was different (BALB/c splenocytes) the pattern of type I interferon mRNAs seen was altered. This genotype-dependent expression was also seen in newcastle disease virus infected fibroblasts. Within a given mouse strain, there were also differences in the subtype response patterns detected in fibroblasts compared with those seen in splenocytes. In conclusion, the present study indicates that mouse genotype appears to be a major determinant of the subtype response pattern seen and tissue specific pattern differences are present within a given mouse genotype.

Animals↗

PubMed1996

Alpha/beta interferons increase host resistance to murine AIDS.

Murine AIDS (MAIDS) is caused by a defective retrovirus present in the LP-BM5 murine leukemia virus mixture. Strains of inbred mice differ in resistance to MAIDS development; some are susceptible (e.g., C57BL/6), while others are resistant (e.g., CBA and B10.BR). As an early block to viral replication in resistant mice has been demonstrated previously by PCR studies, we postulated that alpha/beta interferons (IFN-alpha/beta) may be involved in resistance to MAIDS. Susceptible C57BL/6 mice infected with LP-BM5 were treated with IFN-alpha/beta or Newcastle disease virus. Newcastle disease virus induces high endogenous IFN-alpha/beta production in mice. Both treatments delayed the development of MAIDS, as assessed by splenomegaly and T- and B-cell proliferation. In addition, an IFN-alpha/beta response was detected by reverse transcription-PCR and dot blotting 3, 6, and 9 h after LP-BM5 infection in resistant mice but not in susceptible mice. These results suggest that the ability to produce IFN-alpha/beta in response to LP-BM5 infection may contribute to host resistance to MAIDS.

Animals↗