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C Lee

Publications and source records attributed to C Lee.

At least 217 records · Page 12Linked to original sources
PubMed2000

Prostatic luminal cell differentiation and prostatic steroid-binding protein (PBP) gene expression are differentially affected by neonatal castration.

BACKGROUND: Although normal prostatic development is androgen-dependent, the prostate continues to grow in the neonate despite castration. However, the manner in which neonatal growth of the prostate occurs, in the absence of the testis, remains largely unknown. The purpose of this study was to examine the differentiation of prostatic epithelial cells after neonatal castration. METHODS: Immunohistochemistry was utilized to detect the expression of differentiation products: basal-cell cytokeratin (CK 5), luminal-cell cytokeratin (CK 18), and prostatic steroid-binding protein (PBP), a ventral prostate-specific marker indicative of secretory function in luminal cells. The reverse transcription-polymerase chain reaction was used to detect transcription products of the three polypeptide subunits of PBP, designated C1, C2, and C3. Rats were castrated on day 5 after birth, and ventral prostates were collected on day 14. Dihydrotestosterone was injected (100 microg/animal every 2 days) in castrated animals to determine if PBP expression could be initiated by androgen. RESULTS: Although no major effects of castration were detected on the differentiation of stromal or basal cells (which differentiate prior to day 5), castration had a pronounced effect on luminal-cell differentiation. Castration inhibited PBP protein expression, but did not affect the expression of luminal-cell cytokeratin (CK 18) protein. Furthermore, castration reduced C1, C2, and C3 transcription. Androgen replacement to castrated animals allowed for the initiation of PBP expression, although its onset was delayed. CONCLUSIONS: These observations indicate that the testis is not necessary for prostatic luminal-cell differentiation, but is necessary for full expression of luminal-cell secretory phenotype. Furthermore, our study suggests that factors of testicular origin, in addition to androgen, are needed for proper timing of PBP expression. This investigation establishes that the cytological and the physiological differentiation of the rat prostate are differentially regulated.

Androgen-Binding Protein↗
PubMed2000

Inhibitory action of thimerosal, a sulfhydryl oxidant, on sodium channels in rat sensory neurons.

The effects of thimerosal, a sulfhydryl oxidizing agent, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion neurons were studied using the whole-cell patch clamp technique. Thimerosal blocked the two types of sodium channels in a dose-dependent manner. The inhibitory effect of thimerosal was much more pronounced in TTX-R sodium channels than TTX-S sodium channels. The effect of thimerosal was irreversible upon wash-out with thimerosal-free external solution. However, dithiothreitol, a reducing agent, partially reversed it. Thimerosal shifted the steady-state inactivation curves for both types of sodium channels in the hyperpolarizing direction. The voltage dependence of activation of both types of sodium channels was shifted in the depolarizing direction by thimerosal. The inactivation rate in both types of sodium channels increased after thimerosal treatment. All these effects of thimerosal would add up to cause a depression of sodium channel function leading to a diminished neuronal excitability.

Animals↗
PubMed2000

Absence of proximal duct apoptosis in the ventral prostate of transgenic mice carrying the C3(1)-TGF-beta type II dominant negative receptor.

BACKGROUND: Prostatic epithelial cells are sensitive to the inhibitory effects of TGF-beta. However, TGF-beta signaling in the prostate is dependent on androgenic status. Under the in vivo conditions, it is difficult to dissociate the effect of TGF-beta from that of androgen on the prostate. METHODS: The objective of the present study was to create and verify a transgenic mouse system in which epithelial cells of the ventral prostate are insensitive to the actions of TGF-beta. By using a modified prostate-specific promoter, C3(1), the TGF-beta dominant negative receptor is only expressed in the epithelial cells of the ventral prostate, and these cells are resistant to TGF-beta. Morphology of transgenic animal prostates was compared to wild-type animal prostates by immunohistochemistry and microscopy. RESULTS: The prostate of transgenic mice exhibited an abnormal morphology with multiple layers of epithelial cells lining the proximal ducts, in contrast to the simple cuboidal monolayer of cells seen in the normal prostate. This observation was accompanied by a loss of apoptosis in this region, as seen by TUNEL assay. There was no significant difference in serum levels of testosterone between the wild-type and transgenic animals. CONCLUSIONS: These results demonstrated that a loss of sensitivity to TGF-beta results in the accumulation of multiple layers of epithelial cells in the proximal region of the ventral prostate. This abnormal growth illustrates that TGF-beta plays an important role in regulating prostate growth. The current transgenic system can be used as an experimental model to study the functional role of TGF-beta in prostatic growth and function.

Activin Receptors, Type I↗
PubMed2000

Prostatic ductal system in rats: changes in regional distribution of extracellular matrix proteins during castration-induced regression.

BACKGROUND: The extracellular matrix (ECM) is an intricate network composed of an array of molecules that play an integral role in the regulation of cell function, differentiation, and tissue-specific gene expression in various epithelia. In the present study, we examined the distribution of collagen type IV and laminin along the rat ventral prostatic duct before and after castration. METHODS: Mature Sprague-Dawley rats were castrated and their prostates processed for immunocytochemistry of ECM proteins, laminin, and collagen type IV. Tissue sections were also processed for apoptosis staining, using the 3' end-labeling technique. To examine the effect of ECM proteins on epithelial growth, rat ventral epithelial cells were cultured on ECM-coated surfaces. RESULTS: In the intact rat, laminin was localized in the basement membrane along all regions of the ventral prostate ductal system. Collagen type IV was found to be distributed evenly in the basement membrane of the distal and intermediate regions but was absent or poorly organized in the proximal region, where apoptosis in the epithelium occurs at a high rate. In the regressing prostate after castration, there was a shift in apoptosis from the proximal region to the distal intermediate regions of the prostatic duct. Associated with the shift was a remodeling of basement membrane proteins due to the specific loss of collagen type IV in the distal and intermediate regions. Collagen type IV reappeared underneath the epithelium 7 days after castration, when apoptosis in the epithelium stopped. In vitro, collagen type IV enhanced the growth of ventral prostatic epithelial cells, as assessed by cell number. CONCLUSIONS: Collagen basement membrane type IV mediates growth of rat ventral prostate epithelium, and its loss during tissue remodeling after castration is associated with cell death.

Animals↗
PubMed2000

Identification and characterization of a novel polycystin family member, polycystin-L2, in mouse and human: sequence, expression, alternative splicing, and chromosomal localization.

Polycystins-1, -2, -L, and -REJ are the four known members of the polycystin family of proteins. In this study, we describe a fifth member of the family, polycystin-L2, encoded by PKD2L2 in human and Pkd2l2 in mouse. Full-length cDNA sequences for both mouse and human polycystin-L2 were obtained from testis cDNA. Sequence analysis predicts that the mouse and human polycystin-L2 proteins consist of 621 and 624 amino acid residues, respectively. Polycystin-L2 has significant homology with polycystins-L and -2, with similarities of 58 and 59%, respectively. Both human and murine polycystin-L2 proteins are predicted to have seven putative transmembrane (TM) domains, and, by comparison with transient receptor potential channels, the six carboxyl-terminal TM domains are likely to constitute an ion channel subunit. Northern blot analysis indicated that mouse Pkd2l2 has an abundant approximately 2.5-kb transcript in testis and an approximately 2.2-kb transcript in heart. RT-PCR analysis showed that the full-length transcript is expressed in human brain, kidney, testis, and HepG2 cells, and there are three alternatively spliced variants that were differentially expressed. PKD2L2 consists of 17 exons spanning approximately 50 kb of genomic DNA. PKD2L2 was mapped to human chromosome 5q31 and Pkd2l2 to mouse chromosome 18 in band C.

Alternative Splicing↗
PubMed2000

EWS-FLI1, EWS-ERG, and EWS-ETV1 oncoproteins of Ewing tumor family all suppress transcription of transforming growth factor beta type II receptor gene.

Ewing sarcoma-specific chromosomal translocations fuse the EWS gene to a subset of ets transcription factor family members, most commonly the FLI1 gene and less frequently ERG, ETV1, E1A-F, or FEV. These fusion proteins are thought to act as aberrant transcription factors that bind DNA through their ets DNA binding domain. Recently, we have shown (K-B. Hahm et al., Nat. Genet., 23: 222-227, 1999) that the transforming growth factor beta (TGF-beta) type II receptor (TGF-beta RII), a putative tumor suppressor gene, is a target of the EWS-FLI1 fusion protein. Here, we also examined effects of EWS-ETV1 and EWS-ERG on expression of the TGF-beta RII gene. We show that relative to the control, NIH-3T3 cell lines stably transfected with the EWS-FLI1, EWS-ERG, or EWS-ETV1 gene fusion express reduced levels of TGF-beta RII mRNA and protein, and that these cell lines have reduced TGF-beta sensitivity. Cotransfection of these fusion genes and the TGF-beta RII promoter suppresses TGF-beta RII promoter activity and also FLI1-, ERG-, or ETV1-induced promoter activity. These results indicate that transcriptional repression of TGF-beta RII is an important target of the EWS-FLI1, EWS-ERG, or EWS-ETV1 oncogene, and that EWS-ets fusion proteins may function as dominant negative forms of ets transcription factors.

3T3 Cells↗
PubMed2000

dCLOCK is present in limiting amounts and likely mediates daily interactions between the dCLOCK-CYC transcription factor and the PER-TIM complex.

In Drosophila melanogaster four circadian clock proteins termed PERIOD (PER), TIMELESS (TIM), dCLOCK (dCLK), and CYCLE (CYC/dBMAL1) function in a transcriptional feedback loop that is a core element of the oscillator mechanism. dCLK and CYC are members of the basic helix-loop-helix (bHLH)/PAS (PER-ARNT-SIM) superfamily of transcription factors and are required for high-level expression of per and tim and repression of dClk, whereas PER and TIM inhibit dCLK-CYC-mediated transcription and lead to the activation of dClk. To understand further the dynamic regulation within the circadian oscillator mechanism, we biochemically characterized in vivo-produced CYC, determined the interactions of the four clock proteins, and calculated their absolute levels as a function of time. Our results indicate that throughout a daily cycle the majority of the dCLK present in adult heads stably interacts with CYC, indicating that CYC is the primary in vivo partner of dCLK. dCLK-CYC dimers are bound by PER and TIM during the late evening and early morning, suggesting the formation of a tetrameric complex with impaired transcriptional activity. Although dCLK is present in limiting amounts and CYC is by far the most abundant of the four clock proteins that have been examined, PER and TIM appear to interact preferentially with dCLK. Our results suggest that dCLK is the main component regulating the daily abundance of transcriptionally active dCLK-CYC complexes.

ARNTL Transcription Factors↗
PubMed2000

N-Ethylmaleimide modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons.

The effects of N-ethylmaleimide (NEM), an alkylating reagent to protein sulfhydryl groups, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. When currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV NEM decreased the amplitude of TTX-S sodium current, but exerted little or no effect on that of TTX-R sodium current. The inhibitory effect of NEM on TTX-S sodium channel was mainly due to the shift of the steady-state inactivation curve in the hyperpolarizing direction. NEM did not affect the voltage-dependence of the activation of TTX-S sodium channel. The steady-state inactivation curve for TTX-R sodium channel was shifted by NEM in the hyperpolarizing direction as that for TTX-S sodium channel. NEM caused a change in the voltage-dependence of the activation of TTX-R sodium channel unlike TTX-S sodium channel. After NEM treatment, the amplitudes of TTX-R sodium currents at test voltages below -10 mV were increased, but those at more positive voltages were not affected. This was explained by the shift in the conductance-voltage curve for TTX-R sodium channels in the hyperpolarizing direction after NEM treatment.

Animals↗
PubMed2000

Recombinant Escherichia coli cell for d-p-hydroxyphenylglycine production from d-N-carbamoyl-p-hydroxyphenylglycine.

Recombinant Escherichia coli cell containing D-amidohydrolase was employed to convert D-N-carbamoyl-p-hydroxyphenylglycine (D-CpHPG) to D-p-hydroxyphenylglycine (D-pHPG). Biotransformations under pH 7 and 40 degrees C allowed to complete conversion of D-CpHPG into D-pHPG. Under the same reaction pH, the D-amidohydrolase activity of the cell in the phosphate buffer was higher than that in the Tris buffer. The activity decreased with the increase of phosphate buffer concentration. Instead of using buffer, the reaction pH maintained constant at 7 by titrating with 1 N HCl resulted in a higher D-pHPG production rate. Flocculating the cell suspension with chitosan and cross-linked by glutaraldehyde made the cell recovery for repeated use much easier. Both the cross-linking and (PMSF; a protease inhibitor) treatments could increase the cell reusability and storage stability. However, the cross-linking decreased the D-amidohydrolase activity of the cell to about 50%. The D-amidohydrolase activities of free and cross-linked cell were inhibited at substrate concentration higher than 150 mM and 100 mM, respectively. The conversion of 150 mM D-CpHPG to D-pHPG could be completed within 7 h for the free cell at the concentration of 10% (wet weight/volume).

Journal Article↗
PubMed2000

Over-expression of ERT(ESX/ESE-1/ELF3), an ets-related transcription factor, induces endogenous TGF-beta type II receptor expression and restores the TGF-beta signaling pathway in Hs578t human breast cancer cells.

The epithelium-specific transcription factor, ERT/ESX/ESE-1/ELF3, binds to the TGF-beta RII promoter in a sequence specific manner and regulates its expression. In this study, we investigated whether ERT could regulate endogenous TGF-beta RII expression in Hs578t breast cancer cells. Analyses of the Hs578t parental cell line revealed low RII mRNA expression and resistance to the growth inhibitory effects of TGF-beta. Infection of this cell line with a retroviral construct expressing ERT induced higher levels of endogenous RII mRNA expression and protein expression relative to cells infected with chloramphenicol acetyltransferase (CATneo) as a control. Relative to control cells, the ERTneo-expressing Hs578t cells show approximately a 50% reduction in cell growth in the presence of exogenous TGF-beta1, as well as a fourfold higher induction of activation in transient transfection assays using the 3TP-luciferase reporter construct. When transplanted into athymic mice, ERT-expressing Hs578t cells showed decreased and delayed tumorigenicity compared with control cells. This data strongly suggests that ERT plays an important role as a transcriptional activator of TGF-beta RII expression, and that deregulated ERT expression may play a critical role in rendering Hs578t human breast cancer cells insensitive to TGF-beta's growth inhibitory effects.

Animals↗
PubMed2000

Clonality analysis of defined cell populations in paraffin-embedded tissue sections by RT-PCR amplification of X-linked G6PD gene.

This paper establishes a method of clonality analysis using the reverse transcription-polymerase chain reaction (RT-PCR) to amplify X-linked G6PD transcripts on defined cell populations microdissected from archival, paraffin-embedded tissue sections. Four known monoclonal low-grade B-cell lymphomas from females who were heterozygous (informative) at the 1131 exonic polymorphic locus of the G6PD gene were used to validate the method. Lymphoma and reactive lesions in each case were separated by microdissection. In order to preserve the intact RNA species in the lesion, sections were digested on the slides before microdissection. A one-step RT-PCR was performed with a single pair of primers, one of which contained a mismatched base adjacent to the polymorphic site, to generate a PvuI cutting site. Successful amplification and allele identification by PvuI digestion were achieved from all RNA samples studied. Three of four samples from non-neoplastic reactive lesions showed two bands with equal intensity, representing transcription of the two alleles of the G6PD gene, while the corresponding tumour samples demonstrated a biased intensity in one allele, indicating monoclonality. To assess the method further, the clonal nature of in situ and invasive breast cancers was examined, along with adjacent normal breast tissue and hyperplastic lesions from three informative females from our archives. Apart from the clusters of normal terminal duct-lobular units, all lesions were monoclonal. This result is in agreement with data derived from other X-linked gene studies and loss of heterozygosity (LOH) analyses of pre-invasive breast disease. The results suggest that the clonality analysis method presented here is simple and reliable, and is therefore potentially applicable in a wide range of pathological conditions.

Breast↗
PubMed2000

Induction of oxidative DNA damage in u937 cells by TNF or anti-Fas stimulation.

TNF and Fas signaling pathways are reported to induce mitochondrial damage associated with production of oxygen radicals. We examined whether such radical production elicited detectable nuclear DNA damage in U937 cells following treatment with TNF or with anti-Fas antibodies. Using GC-mass spectroscopy for analysing base oxidation, several oxidized species increased significantly following TNF treatment, whereas anti-Fas resulted in less detectable oxidative damage using this assay. Cytogenetic analysis showed that, in the presence of aphidicolin, which blocks several types of DNA repair, TNF induced extensive chromosomal damage. Aphidicolin also synergized with TNF and anti-Fas in inducing cell death which was prevented by reducing atmospheric oxygen or addition of n -acetyl cysteine, a scavenger of oxygen radicals. Thus, several lines of evidence point to the TNF and Fas pathways inducing extensive oxidative DNA damage and repair, and suggest potential roles for these pathways in mutagenesis and aging.

Adenosine↗
PubMed2000

Directed evolution of toluene dioxygenase from Pseudomonas putida for improved selectivity toward cis-indandiol during indene bioconversion.

Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S,2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S,2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.

Amino Acid Substitution↗
PubMed2000

Experience with Four French Catheters for Outpatient Coronary Angiography.

Previous studies have demonstrated the efficacy and safety of outpatient cardiac catheterization on stable patients at low risk. We present our experience with four French catheters in 104 patients (72 male, 32 female, mean age 51 years old) with coronary angiography done as an outpatient procedure. No heparin was given during the procedure. After initial hemostasis had been attained, the patients returned to day center with vital signs monitored regularly. Patients were allowed ambulation after 4 hours' bed rest and were discharged the same afternoon. Normal coronary angiogram study was found in 76% of the study population. Single-vessel disease, double-vessel disease, triple-vessel disease, and left-main disease were found in 12%, 9.6%, 2%, and 1%, respectively. Nearly all of the patients demonstrated normal ventricular contraction (99%). Average procedural time was 20.2 +/- 4.4 minutes. Average hemostatic time was 8.4 +/- 3.1 minutes. No mortality directly attributed to the catheterization occurred in our study population. Moreover, there were no myocardial infarction, acute pulmonary edema, severe allergic reaction, and cerebrovascular accident. Femoral puncture site complication was only limited to superficial skin bruise. Quality of the cineangiogram was good in majority of the patients. Therefore, this study demonstrates that outpatient cardiac catheterization using four French Judkins catheter is a safe and cost-effective procedure.

Journal Article↗
PubMed2000

Properties of saccade-related neurons in the cat superior colliculus: patterns of movement fields and discharge timing.

The discharge characteristics of saccade-related neurons in the superior colliculi (SC) of trained cats were investigated in order to understand the involvement of the SC in dynamic control of saccades. In particular, two aspects of saccade-related collicular activity in relation to visually guided saccades were quantitatively analyzed: the bounded pattern of amplitude tuning and the timing of neural activity in relation to saccade offset. In order to determine whether the amplitude tuning of SC cells is bounded or unbounded, we characterized it with a quantitative index, the unbounded-field (UF) index. Analysis of the characteristics of the distribution of this index, including the absence of unbounded fields for cells within the central 10 degrees, suggested that all cells form a single class and that apparent unbounded amplitude tuning is an artifact occurring when the range of gaze shift is truncated by restraining the head. This interpretation is supported by the results of a computer simulation based on truncation of gaze-shift range that closely replicated the experimental data. Analyses of relative location of peak motor activity in the spatial and temporal dimensions revealed that the peak discharge was more closely linked to the midpoint of the saccade than to the offset of saccade, regardless of the pattern of amplitude tuning. These results do not support the proposal advanced by the moving-hill hypothesis: the bounded pattern of amplitude tuning is inconsistent with rostral migration of the activity locus during execution of a saccade, and the relative location of the peak motor activity is inconsistent with the idea that the SC activity encodes dynamic motor error (the difference between desired and current gaze directions).

Action Potentials↗
PubMed2000

Distribution of gamma satellite DNA on the human X and Y chromosomes suggests that it is not required for mitotic centromere function.

The bulk of the DNA found at human centromeres is composed of tandemly arranged repeats, the most abundant of which is alpha satellite. Other human centromeric repetitive families have been identified, one of the more recent being gamma satellite. To date, gamma satellite DNAs have been reported at the centromeres of human chromosomes 8 and X. Here, we show that gamma-X satellite DNA is not interspersed with the major DZX1 alpha-X block, but rather is organised as a single array of approximately 40-50 kb on the short-arm side of the alpha satellite domain. This repeat array is absent on two mitotically stable Xq isochromosomes. Furthermore, a related repeat DNA has been identified on the human Y chromosome. Fluorescence in situ hybridisation has localised this satellite DNA to the long arm side of the major DYZ3 alpha-Y domain, outside the region previously defined as that required for mitotic centromere function. Together, these data suggest that while blocks of highly related gamma satellite DNAs are present in the pericentromeric regions of both human sex chromosomes, this repeated DNA is not required for mitotic centromere function.

Animals↗