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Biomedical subjects

W T Cheng

Publications and source records attributed to W T Cheng.

At least 19 recordsLinked to original sources
PubMed2004

Dispersion of organic pigments using supercritical carbon dioxide.

This research describes dispersion of organic pigments using supercritical fluids. With low surface tension and high diffusivity of fluids in supercritical states, aggregated particles may be effectively wetted and swelled to form the primary constituent of the dispersing solution by volume. In this paper, the conditions of temperature and pressure are used to control the density of supercritical carbon dioxide subject to PGMEA as cosolvent for dispersing organic powder in a solution. As shown from measurement with a laser scattering particle analyzer, the average diameter of phthalocyanine green 36 with the haloid structure can be significantly reduced to 93.5 nm; for aminoanthraquinone red containing and amino group (-NH(2)) and phthalocyanine blue 15:6 with symmetry benzene and inner hydrogen bond, the mean particle sizes are 178.5 and 188.7 nm, respectively, using supercritical CO(2). Additionally, the transmittance of UV light is used to confirm the dispersing performance in this study.

Journal Article↗
PubMed2003

cDNA nucleotide sequence coding for stearoyl-CoA desaturase and its expression in the zebrafish (Danio rerio) embryo.

A cDNA sequence of stearoyl-CoA desaturase (SCD) was determined from zebrafish (Danio rerio) and compared to the corresponding genes in several teleosts. Zebrafish SCD cDNA has a size of 1,061 bp, encodes a polypeptide of 325 amino acids, and shares 88, 85, 84, and 83% similarities with tilapia (Oreochromis mossambicus), grass carp (Ctenopharyngodon idella), common carp (Cyprinus carpio), and milkfish (Chanos chanos), respectively. This 1,061 bp sequence specifies a protein that, in common with other fatty acid desaturases, contains three histidine boxes, believed to be involved in catalysis. These observations suggested that SCD genes are highly conserved. In addition, an oligonucleotide probe complementary to zebrafish SCD mRNA was hybridized to mRNA of approximately 396 bases with Northern blot analysis. The Northern blot and RT-PCR analyses showed that the SCD mRNA was expressed predominantly in the liver, intestine, gill, and muscle, while a lower level was found in the brain. Furthermore, we utilized whole-mount in situ hybridization and real-time quantitative RT-PCR to identify expression of the zebrafish SCD gene at five different stages of development. This revealed that very high levels of transcripts were found in zebrafish at all stages during embryogenesis and early development.

Amino Acid Sequence↗
PubMed2001

Secretory origin and temporal appearance of the porcine beta-microseminoprotein (sperm motility inhibitor) in the boar reproductive system.

A specific antiserum against the porcine sperm motility inhibitor (SMI) was used in Western blotting analysis of tissue homogenates to reveal the possible origin of SMI in the boar reproductive system at different ages. The ages of the boar used were day 0, day 15, day 30, day 60, day 100, day 120, day 135, day 150, and day 210. The tissue homogenates of the day 60 and older showed immunoreaction. The results were further checked by indirect immunohistochemical staining and observed under light microscope. The SMI antigen appeared in the epithelial cells and in the lumen of the secretory ducts of the prostate gland. These results indicate that porcine SMI is synthesized only by the postnatal prostate gland. The homogenate of the prostate gland of day 100 was also used for the purification of SMI. The prostatic SMI was co-eluted with the seminal SMI in the reversed phase HPLC. Mass spectrometric analysis of the prostatic SMI revealed a molecular weight of 10,066. These results indicate that the prostatic SMI is identical to that purified from seminal plasma (Jeng et al., 1993; Biochem Biophys Res Communi 191:435-440).

Animals↗
PubMed2001

In silico mining of EST databases for novel pre-implantation embryo-specific zinc finger protein genes.

Progress in the understanding of early mammalian embryo development has been severely hampered by scarcity of study materials. To circumvent such a constraint, we have developed a strategy that involves a combination of in silico mining of new genes from expressed sequence tags (EST) databases and rapid determination of expression profiles of the dbEST-derived genes using a PCR-based assay and a panel of cDNA libraries derived from different developmental stages and somatic tissues. We demonstrate that in a random sample of 49 independent dbEST-derived zinc finger protein genes mined from a mouse embryonic 2-cell cDNA library, more than three-quarters of these genes are novel. Examination of characteristics of the human orthologues derived from these mouse genes reveals that many of them are associated with human malignancies. Expression studies have further led to the identification of three novel genes that are exclusively expressed in mouse embryos before or up to the 8-cell stage. Two of the genes, designated 2czf45 and 2czf48 (2czf for 2-cell zinc finger), are zinc finger protein genes coding for a RBCC protein with a RFP domain and a protein with three C2H2 fingers, respectively. The third gene, designated 2cpoz56, codes for a protein with a POZ domain that is often associated with zinc finger proteins. These three genes are candidate genes for regulatory or other functions in early embryogenesis. The strategy described in this report should generally be applicable to rapid and large-scale mining of other classes of rare genes involved in other biological and pathological processes. Mol. Reprod. Dev. 59:249-255, 2001.

Animals↗
PubMed2001

Rewards of automated testing. Identifying problems before rollout can avoid system disruption.

PROBLEM: Needed order management application that performed consistently and met high demands. SOLUTION: Load testing identified specific problem areas. RESULTS: Precise description of problems and their occurrence helped speed resolution and ensured successful implementation. KEYS TO SUCCESS: Testing of conditions that emulated expected demands at early stage of design and planning.

Cancer Care Facilities↗
PubMed2000

Growth hormone gene polymorphisms and growth performance traits in Duroc, Landrace and Tao-Yuan pigs.

This study investigated the restriction fragment length polymorphism (RFLP) of the porcine growth hormone (pGH) gene in Duroc, Landrace, and Tao-Yuan pigs and its effects on growth performance and levels of plasma growth hormone in peripheral circulation. Genomic DNA extracted from 81 Tao-Yuan, 60 Landrace and 48 Duroc pigs were subjected to Southern blot hybridization with a pGH cDNA probe. Polymorphism was detected with the restriction enzymes TaqI and DraI. A comparison of these three breeds showed significant differences in allelic frequencies. Blood samples for radioimmunoassay (RIA) of GH were collected biweekly during the experimental period from pigs 12 to 40 weeks of age. Tao-Yuan pigs showed a mean plasma GH level (2.51 +/- 1.23 ng/mL) that was much lower than that of the Landrace (3.80 +/- 1.52 ng/mL) and Duroc (4.20 +/- 1.03 ng/mL) pigs (P < 0.05). Moreover, the Tao-Yuan pigs also showed poorer growth performance than the Landrace and the Duroc pigs both in the daily weight gain (0.37 +/- 0.06 vs. 0.67 +/- 0.05 and 0.70 +/- 0.05 kg/day, P < 0.01) and feed efficiency (3.12 +/- 0.28 vs. 2.60 +/- 0.14 and 2.52 +/- 0.12, P < 0.05). These results suggest that the growth performance trait in these pigs is highly correlated with their growth hormone genotype.

Animals↗
PubMed2000

Growth enhancement of fowls by dietary administration of recombinant yeast cultures containing enriched growth hormone.

In present study the methylotrophic yeast, Pichia pastoris, was used to express a recombinant growth hormone (rGH) gene of swine. A synthetic secretion cassette was constructed using the promoter of the alcohol oxidase1 gene (AOX1), and a alpha-factor signal peptide. After electroporatic transformation and zeocin selection, several clones exhibited high levels of rGH protein expression constituting more than 20% of total yeast protein. Over 95% of rGH was shown to be export into the culture supernatant. Yeast transformant containing the highest recombinant growth hormone level (rGH yeast) and native GS115 Pichia pastoris (non-rGH yeast, as a control) were separately cultured, harvested and adsorbed by wheat bran. Yeast cultures of four dosages (0.05, 0.1, 0.2, and 0.4%) were mixed respectively with chick basal diet and fed to simulated country chickens for 9 weeks. The results showed that, when compared to control chicks, the percentage of body weight gain was improved significantly (P<0.05) in chicks fed with diets containing 0.1 or 0.2% rGH-rich yeast culture at brooding stage, and in chicks fed with 0.4% rGH-rich yeast culture at growing stage. The average weight gain in rGH yeast treated groups for the full-term (0 to 63d) and short term (43 to 63d) of growth were 10.6 and 9.4%, respectively, better than the non-rGH yeast control group. These experimental data suggest that the use of rGH-containing yeast as a supplement in fed provided an alternative approach for growth improvement in simulated country chickens.

Alcohol Oxidoreductases↗
PubMed2000

Estrogen receptor is expressed in pig embryos during preimplantation development.

Although estrogen is recognized as essential for embryonic development and maintenance of pregnancy, it remains unclear whether it has a direct role in the embryos themselves. The aim of this study was to investigate whether estrogen can have any effect in pig embryos during preimplantation development. Since the function of estrogen is mediated through its specific receptor, estrogen receptor (ER), the presence of ER mRNA and protein in pig embryos collected in vivo at different stages of preimplantation development was determined and compared. Using reverse transcription polymerase chain reaction, ER RNA was detected at the one-cell, two-cell, and four-cell stages. The level became undetectable at the five- to eight-cell stages and the morula stages and then reappeared again at the blastocyst stage. To determine whether the ER message observed in the embryos was translated into ER protein, immunocytochemical analysis was performed and the presence of ER protein was detected in oocytes at one-cell and four-cell stages. However, the amount of ER protein in porcine embryos at the blastocyst stage was still below the detection limit. The presence of ER mRNA at the blastocyst stages suggests that estrogen may start to act directly on pig embryos afterwards, and our results provide a basis for determining the direct role of estrogen in preimplantation pig embryos.

Animals↗
PubMed2000

Identification of genes expressed in the epithelium of porcine oviduct containing early embryos at various stages of development.

As a first step toward elucidation of the action of factors secreted by the epithelium of oviduct, differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used in this study to identify transcripts of such oviductal factors in gilts carrying various stages of early embryo development post hormone-induced ovulation. A total of 13 differentially expressed transcripts were identified between 50 and 120 hr post-hCG injection (between 1- and 8-cell embryonic stages). Twelve of these transcripts were found to be initially expressed at 96 hr post-hCG injection (at 4-cell embryonic stage) and beyond. Three of such genes were shown by sequence analysis to be the porcine transforming growth factor-alpha, the porcine transforming growth factor-beta-binding protein II and a porcine astral natriuretic factor receptor-like transcript. Only one differentially expressed gene was detected between 50-60 and 85 hr post-hCG injection, and this gene turned out to be the porcine follicle-stimulating hormone receptor. The remaining eight transcripts detected by DDRT-PCR were novel. Moreover, most of these newly expressed genes were found to be turned on at a time coincidental with that of the 4-cell block of porcine embryos cultured in vitro. Our results demonstrate that DDRT-PCR is a feasible approach for rapid identification of genes that are differentially expressed in oviductal epithelium. Some of the genes thus identified may be important for unhindered development of embryos in the oviduct.

Animals↗
PubMed2000

Progesterone receptor gene expression in preimplantation pig embryos.

OBJECTIVE: It is not known whether progesterone, which plays a key role in establishing and maintaining pregnancy, acts directly on embryos or indirectly through the mother's reproductive tract. Since the physiological effects of progesterone are mediated by progesterone receptors (PR), the expression of PR during the preimplantation stages of pig embryos was determined. DESIGN AND METHODS: Preimplantation pig embryos at different developmental stages were examined using reverse transcription-polymerase chain reaction techniques for the purpose of determining PR gene expression. Immunocytochemistry procedures were used to determine whether PR mRNA is translated into PR protein in preimplantation embryos. RESULTS: PR mRNA was found in pig embryos at the two-cell stage, but levels started to decline at the four-cell stage; none was detected at the five- to eight-cell stage, nor at any time during the morula and blastocyst stages. Results showed that PR protein was immunostained in pig oocytes and embryos at the 4-cell stage, but that no significant immunostaining occurred during the morula and blastocyst stages. CONCLUSION: These results indicate that the effects of PR on early embryogenesis appear to be indirect, perhaps via PR-regulated growth-promoting factors produced in the maternal reproductive tract.

Animals↗
PubMed1999

Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene.

A sensitive technique for the sexing of bovine embryos was developed using polymerase chain reaction (PCR) amplification of the bovine amelogenin (bAML) gene on the X- and Y-chromosomes of Holstein dairy cattle. Cloning and DNA sequencing showed a 45.1% homology between the fifth intron of the bAML-X and bAML-Y gene with multiple deletions. A pair of sex-specific primers was designed to allow amplification of a single fragment of 467-bp from the X-chromosome of female cattle and two fragments of 467-bp and 341-bp from the X- and Y-chromosomes of male cattle. The primers were successfully applied to bovine sexing from single blastomeres isolated from day-6 to day-7 cow embryos by direct cell lysis and PCR. Our protocol of embryo sexing should be applicable to the diagnosis of defective genes in vitro in human embryos and in other domestic or recreational animals.

Amelogenin↗
PubMed1998

Use of nonautologous microencapsulated fibroblasts in growth hormone gene therapy to improve growth of midget swine.

The aim of the present study was to investigate the expression activity, both in vitro and in vivo, of the porcine growth hormone complementary DNA (pGH cDNA) in porcine fetal fibroblast (PFF) cells. The pGH gene had been constructed inside the bicistronic retroviral vector PSN and subsequently transfected into PFF cells further encapsulated with immunoprotective microcapsules. This would provide a way to evaluate the improvement in growth performance of Tao-Yuan swine by the use of nonautologous microencapsulated fibroblasts carrying the pGH cDNA via the technique of somatic gene therapy. Results from Southern blot analysis confirmed that the full length of the pGH cDNA was completely integrated into the genome of the PFF cells after they had been infected one to four times using a PSN retroviral vector. Moreover, Northern blot analysis showed that high transcription activity was present in clones infected twice, and exogenous pGH secretion was found when the pGH-infected PFF had been further cultured for 48 hr in vitro and subjected to immunoblot assay. Encapsulation of the pGH-PFF with an alginate-poly-L-lysine-alginate membrane did not show any deterioration in their proliferation and survival both in vitro and in vivo. The pGH gene in encapsulated recombinant fibroblasts was fully expressed after it had been transplanted into the peritoneal cavity of the Tao-Yuan swine, and reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed on the microcapsules retrieved 1 month later. The feasibility of pGH gene therapy to improve midget Tao-Yuan swine growth enhancement is further supported by the fact that transplantation of the encapsulated recombinant fibroblast cells resulted in a much more significant increase in weight gain than in those swine in either the age-matched untreated control group or in those that had been transplanted with uncapsulated recombinant PFF cells (10.56 +/- 1.01 kg versus 6.95 +/- 0.94 and 5.27 +/- 1.30 kg; p < 0.05). These experimental data suggest that growth hormone gene therapy did provide an alternative approach for growth improvement in midget Tao-Yuan swine.

Animals↗
PubMed1996

Molecular analysis of cellular loci disrupted by papillomavirus 16 integration in cervical cancer: frequent viral integration in topologically destabilized and transcriptionally active chromosomal regions.

To discern the structural features of cellular loci that are disrupted by type 16 human papillomavirus (HPV-16) integration in cervical cancer, a polymerase chain reaction (PCR)-based strategy was employed for direct amplification and sequence analysis of four such cellular loci in cancer biopsy samples. One of the HPV-16-disrupted loci was found to be the microtubule-associated protein (MAP-2) gene and the other three loci were uncharacterized and were designated PID-1 to -3 (for papillomavirus integration-disrupted). The junctional sequences of the viral integration sites in the four loci analyzed are bracketed by long tracts of homogeneous purine or pyrimidine or alternating purine-pyrimidine which are known to destabilize the B-form conformation of the DNA structure. Using a panel of human/hamster hybrid cell DNAs and PCR analysis, the four loci were assigned to chromosomes 2 (MAP-2), 9 (PID-1), 1 (PID-2) and 8 (PID-3), respectively. These chromosomes carry numerous other previously determined viral integration and chromosomal fragile sites and the myc oncogenes. The PID-1 locus was further found in Southern analysis to be rearranged and amplified in another cervical cancer biopsy and a cervical carcinoma cell line (CaSki). On Northern analysis, the PID-1 and -3 probes detected a 3.0- and a 3.6-kb transcript, respectively, in normal cervical cells and in cervical cancer cell lines. The findings suggest that HPV-16 genome integrates frequently into topologically destabilized and transcriptionally active chromosomal sites. It remains to be elucidated whether the MAP-2 and the PID loci contribute to the pathogenesis of cervical cancer.

Base Sequence↗
PubMed1995

Frequent deletions and sequence aberrations at the transgene junctions of transgenic mice carrying the papillomavirus regulatory and the SV40 TAg gene sequences.

Exogenous DNA microinjected into one-cell mouse zygotes either integrates into the host genome within a short time span, or is rapidly degraded. On integration, a transgene sequence is frequently reiterated. In this report, we describe the enzymatic amplification analysis of transgene junctions of 12 transgenic mice carrying different copy numbers of the same transgene with dissimilar ends. The transgene was composed of the regulatory sequence of the type 18 human papillomavirus linked to the TAg gene of the SV40 virus. Nucleotide sequences of 36 of these junctions were also determined. Deletions were found in 33 (91.7%) of the junctions analysed. At the crossover regions, 55.6% contained short overlapping sequences of one to six nucleotides. Insertions of 2-6 extraneous nucleotides were also found in 8.3% of the transgene junctions. Within a 10-nucleotide sequence on both sides of the transgene junctions, topoisomerase I (topo I) cleavage sites, runs of homogeneous purines or pyrimidiens, alternating purine-pyrimidine tracks and (A-T)-rich sequences were found frequently. Stringent control experiments were also performed to ascertain that the observations made were not artefacts resulting from the polymerase chain reaction. Our data therefore indicate that damage had occurred quite frequently and extensively in our transgene construct. Such transgene damage may also occur to various extents in mice carrying other transgenes. Primary structure of the nucleotide sequences of the injected DNA seems to influence the process of transgene reiteration and aberration.

Animals↗
PubMed1994

Analysis of deletion of the integrated human papillomavirus 16 sequence in cervical cancer: a rapid multiplex polymerase chain reaction approach.

A protocol for a rapid physical mapping of the integrated type 16 human papillomavirus (HPV16) sequences in biopsied and paraffin-embedded archival cervical cancer samples is described. The procedure involves the use of an anchor primer and a mixture of indicator primers in a multiplex polymerase chain reaction (PCR). A minimal conserved region of viral integration of 2,745 bp in length has been mapped between nucleotide (nt) 6102-941, containing the entire regulatory region and the E6 and E7 open reading frames (ORFs). A general deletion domain of 1,465 bp in the integrated viral genome has been defined between nt 1417-2881, covering most of the E1 ORF at the 3'-half and 60 bp at the 5' terminus of the E2 ORF. This common deleted sequence contains an ATPase active domain speculated to be associated with a DNA helicase function essential for the viral replication, and it also falls within the actively spliced E1-E2 segment of the primary RNA transcripts. Detection of the loss of the 3'-half of the E1 ORF would be an ideal marker for PCR-based rapid determination of HPV integration in cervical cancer cells.

Chromosome Mapping↗
PubMed1993

Hypermethylation of the human papillomavirus transgenome in transgenic mice.

We have previously reported an observation that the regulatory sequence of the human papillomavirus (HPV) type 18 genome elicits only basal and unregulated transcriptional activities in transgenic mice [Choo et al., Virology (1992) 188:378-383]. We have now extended the study to other transgenic mice carrying an entire or a subgenomic fragment of HPV-16 and -18. Extremely low levels of HPV transcripts were detected in various organs of the mice only by reverse transcriptase-based polymerase chain reaction. On analysis of the methylation status of the HPV transgenome sequences, hypermethylation was clearly observed, therefore suggesting a possible explanation for low level HPV transgenome transcriptional activity in HPV transgenic mice.

Animals↗