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Biomedical subjects

Y Pan

Publications and source records attributed to Y Pan.

At least 55 records · Page 3Linked to original sources
PubMed2001

PTEN induces chemosensitivity in PTEN-mutated prostate cancer cells by suppression of Bcl-2 expression.

The tumor suppressor gene PTEN (MMAC1/TEP1) is lost frequently in advanced prostate cancer (PCa). However, the function of PTEN in tumorigenesis is not understood fully. In this study, we demonstrate that expression of Bcl-2 in prostate tumors correlates with loss of the PTEN protein. This finding was verified by studies in the PCa cell lines DU145, PC-3, LNCaP, and an androgen-refractory subline of LNCaP. Transient transfection of PTEN into the PTEN-null cells resulted in decreased levels of Bcl-2 mRNA and protein. These effects appear to be mediated at the level of gene transcription, since a Bcl-2 promoter-reporter construct was down-regulated by ectopic expression of PTEN in LNCaP cells. The inhibition of Bcl-2 required the lipid-phosphatase activity of PTEN and was blocked by overexpression of a constitutively active form of Akt. Moreover, the transcription-regulatory protein cAMP-response element-binding protein (CREB) may be involved, since decreased phosphorylation of CREB at Ser(133) was detected following PTEN expression, and ectopic expression of CREB repressed completely the PTEN-induced inhibition of Bcl-2 promoter activity. Furthermore, cotransfection of Bcl-2 and PTEN expression vectors rescued PTEN-induced cell death but not G(1) cell cycle arrest. Finally, forced expression of PTEN sensitized LNCaP cells to cell death induced by staurosporine, doxorubicin, and vincristine, and this chemosensitivity was attenuated by exogenous expression of Bcl-2. Taken together, these data demonstrate that loss of PTEN leads to up-regulation of the bcl-2 gene, thus contributing to survival and chemoresistance of PCa cells. These findings suggest that the PTEN gene and its regulated pathway are potential therapeutic targets in prostate cancer.

Antineoplastic Agents↗
PubMed2001

Sterically stabilized polyplex: ligand-mediated activity.

Synthetic vectors have been considered as a safer and more versatile alternative to viral-based gene delivery systems. A variety of very simple synthetic vector systems, e.g., cationic lipid- and polymer-complexed plasmid DNA have activity in vivo but it appears to be mediated by non-specific electrostatic interactions limiting targeting. In order to avoid these problems, we designed a sterically stabilized layered colloidal system. The steric polymer coating reduces non-specific interactions. We have synthesized a PEG conjugate of PEI that complexes DNA to form small, stable colloids with a steric polymer coat on their surface. The polymer enhances colloidal stability and reduces non-specific binding and toxicity. It also renders the complex inactive presumably due to reduced binding. Ligands are then appended to the distal end of the steric polymer to restore cell binding and expression at target cells. We prepared conjugates with RGD peptide ligands appended to the distal end of the steric polymer. The resulting conjugates also form complexes but with ligands exposed on their surface restoring binding and activity. Labeled oligonucleotides and DNA were used to measure intracellular distribution. Oligonucleotides are found localized in the nucleus, whereas the labeled plasmid DNA remained in the cytoplasm. Import of plasmid DNA into the nucleus appears to be very inefficient yet sufficient for expression.

Chemical Phenomena↗
PubMed2001

Technetium(V) oxo complexes of substituted propylene diamine dioxime (PnAO) ligands: water-dependent interconversion between syn and anti isomers.

99mTc and (99)Tc complexes of PnAO (propylene diamine dioxime) ligands monosubstituted in the 6-position [PnAO-6-R] were prepared and studied. Ligands substituted with an alkyl group or with no substituent (R = H, CH(3), or CH(2)CH(CH(3))(2)), gave only one Tc complex. However, for several other nonalkyl substituents (R = COOCH(3), OH, OCH(3), OCH(2)CH(3), F, CN, NHCOCH(3), and NHCOCH(2)CH(3)), two Tc complexes A and B were formed. Products A and B were assigned to the anti and syn TcO(PnAO-6-R) species, respectively, based on (1)H NMR results. X-ray structure analyses supported these assignments. The A (anti) isomer of TcO(PnAO-6-OH) had the chemical formula TcC(13)H(25)N(4)O(4) and crystallized in an orthorhombic system with space group P2(1)2(1)2(1) and Z = 4; a = 12.744(2) A, b = 13.591(2) A, c = 9.976(2) A. The B (syn) isomer of TcO(PnAO-6-CN) had the chemical formula TcC(14)H(24)N(5)O(3) and was a 1:4 mixture of two monoclinic polymorphs: individual rectangular prisms (space group P2(1)/c, Z = 4) and clusters of intergrown twinned rectangular rods (space group Cc, Z = 8). For the prisms, a = 12.457(1) A, b = 13.932(1) A, c = 10.336(1) A, and for the rods, a = 31.344(5) A, b = 6.993(1) A, c = 21.657(2) A. The syn and anti isomers interconverted in the presence of water; nonequilibrium mixtures of epimers remained unchanged under dry conditions. The HPLC behavior under reversed phase conditions was consistent with on-column interconversion (poor resolution), whereas the two isomers were cleanly resolved under drier normal phase conditions. An oxo inversion mechanism involving trans water attack is proposed for the interconversion process. Water also influenced the position of equilibrium of the two isomers. The syn isomer was stabilized in water relative to the anti isomer.

Chromatography, High Pressure Liquid↗
PubMed2001

Characterization of chromosomal abnormalities in uroepithelial carcinomas by G-banding, spectral karyotyping and FISH analysis.

Chromosome analysis by G-banding, spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) was performed on 24 short-term cultured transitional cell bladder carcinomas and 5 cell lines established from bladder carcinomas. Except for one tumor with an apparently normal chromosomal constitution, clonal chromosome abnormalities were detected in all examined cases by the combined approach. The application of SKY and FISH techniques improved the karyotypic descriptions, originally based on G-banding only, by identifying 32 additional numerical changes, by establishing the chromosomal origin of 27 markers and 2 ring chromosomes, by redefining 53 aberrations and by detecting 15 hidden chromosomal rearrangements. No recurrent translocation, however, was detected. The most prominent karyotypic feature was thus the occurrence of deletions and losses of whole chromosome copies indicating the importance of tumor suppressor genes in transitional cell carcinoma pathogenesis. Invasive carcinomas were karyotypically more complex than were low grade superficial tumors. Specific losses of material from chromosome 9 and from chromosome arms 11p and 8p, and gains of 8q and 1q seem to be early changes appearing in superficial tumors, whereas losses from 4p and 17p and the formation of an isochromosome for 5p were associated with more aggressive tumor phenotypes.

Aged↗
PubMed2001

Direct comparison of experimental and calculated folding free energies for hydrophobic deletion mutants of chymotrypsin inhibitor 2: free energy perturbation calculations using transition and denatured states from molecular dynamics simulations of unfolding.

Previous molecular dynamics (MD) simulations of thermal denaturation of chymotrypsin inhibitor 2 (CI2) have provided transition-state models in good agreement with experiment. Unfortunately, however, the comparisons have been necessarily indirect. The simulations have provided detailed structural information but not energetics, while from experiment, structure is inferred from a ratio of free energy changes upon mutation (Phi values). Here, direct comparison with experimental free energies is obtained by performing free energy perturbation calculations of hydrophobic deletion mutants of CI2 using transition- and denatured-state structures from various denaturation MD simulations. The agreement between the calculated and experimental DeltaDeltaG and Phi values is quite good (R = 0.8-0.9). In addition, given the availability of realistic atomic models for the denatured protein, the common approach of using small peptides to represent the denatured state in stability calculations can now be evaluated. To this end, two different extended tripeptide models were used: one using the sequence from the protein with the residue to be mutated in the center and the other with this residue surrounded by Ala residues. The results for the two peptides agree neither with one another nor with the different full-length denatured-state models, which do provide results in good agreement with experiment. This finding is noteworthy because the denatured state of CI2 is very disrupted with little residual structure, such that the peptides might have been expected to serve as reasonable models. Overall the calculations presented here validate our previous MD-generated transition- and denatured-state models and therefore the simulated unfolding pathways and their relevance to refolding.

Alanine↗
PubMed2001

Plasticity in serotonin uptake in primary neuronal cultures of serotonin transporter knockout mice.

The cross talk between dopaminergic and serotonergic systems in the brain has multiple neurophysiological and behavioral implications. Primary neuronal cultures of embryonic wild type (+/+) and serotonin transporter knockout (-/-) mice were used as a model to elucidate the possibility of plasticity at the level of serotonin uptake. Serotonergic neurons were identified in midbrain-hindbrain cultures of both wild type and knockout mice, using polyclonal anti-serotonin antibodies. Adding serotonin (10 microM) to wild type midbrain-hindbrain cultures increased the intensity of serotonin immunostaining, but did not change the number of serotonergic neurons. This increased intensity of serotonin staining was blocked by the serotonin transporter inhibitors fluoxetine and imipramine, but not with the dopamine transporter inhibitor nomifensine. In serotonin transporter knockout cultures, however, serotonin increased both the intensity of serotonin immunostaining and the number of serotonin positive neurons, and nomifensine decreased the number of serotonin-labeled neurons. Uptake of [3H]serotonin to wild type midbrain-hindbrain cultures was completely blocked by 1 microM fluoxetine, whereas nomifensine had a very small effect. In contrast, [3H]serotonin uptake to serotonin transporter knockout cultures, although very weak, was better inhibited by nomifensine than fluoxetine. The results imply that midbrain-hindbrain neuronal cultures of knockout mice, that do not express serotonin transporters, acquire the capacity to take up serotonin, apparently via dopamine transporters.

Adrenergic Uptake Inhibitors↗
PubMed2001

Two-step high resolution sequence-based HLA-DRB typing of exon 2 DNA with taxonomy-based sequence analysis allele assignment.

A two-step high resolution sequence-based DRB typing method was developed. The system needs only one polymerase chain reaction (PCR) to type all functional DRB alleles of a given individual. It uses a pair of generic PCR primers to amplify exon 2 DNA of all functional DRB genes and a first-step taxonomy-based sequence analysis (FSTBSA) method to assign allele groups after sequencing the PCR products with a generic primer. In the second step, group-specific primers are used to sequence the same PCR products and a taxonomy-based sequence analysis (TBSA) is used to assign alleles. Thus, both low and high resolution DRB typing can be done with PCR amplified exon 2 DNA from a single PCR reaction. Correct allele group assignment by FSTBSA was confirmed by sequencing the PCR products with group-specific primers and correctly assigned all 158 DNA samples including 34 samples pre-typed by PCR-sequence-specific primer or PCR-sequence-specific oligonucleotide probe. FSTBSA correctly assigned 116 heterozygous combinations of 81 DRB1-DRB3/4/5 haplotypes. Sixty-seven DRB1, 6 DRB3, 1 DRB4, and 3 DRB5 alleles were identified in this study. TBSA successfully resolved all heterozygous allele combinations including 31 heterozygous combinations of 33 alleles of DRB1*03, 08, 11, 12, 13, and 14 allele groups, and six heterozygous combinations of six DRB3 alleles.

Alleles↗
PubMed2001

Constituents of Salvia moorcroftiana.

A new aromatic ester, 3-oxodecan-1-o1 p-hydroxybenzoate (1) was isolated from Salvia moorcroftiana aerial parts along with four other constituents.

Alkanes↗
PubMed2001

New diterpene from Hedychium villosum.

The stems of Hedychium villosum yielded the new diterpenoid 1, named villosin, along with coronarin E and beta-sitosterol. Their structures were elucidated by spectroscopic methods including mono- and bi-dimensional NMR.

4-Butyrolactone↗
PubMed2001

Isolation and structure analysis of a glucomannan from the seeds of Libyan dates.

Polysaccharides extracted from seeds of Libyan dates with hot ethanol 80% (FI) and 0.1 M phosphate solution (FII) were fractionated and purified by ion-exchange and gel-filtration chromatography. According to methylation and hydrolysis analysis, the main chains of FI and FII consisted of (1-->4)-linked glucomannan; only traces of branched sugar residues were detected. This is the first report on the isolation of glucomannan from date seeds.

Cathartics↗
PubMed2001

Separation and determination of protocatechuic aldehyde and protocatechuic acid in Salivia miltorrhrza by capillary electrophoresis with amperometric detection.

Capillary electrophoresis with amperometric detection was applied to separate and determine protocatechuic aldehyde and protocatechuic acid in Salivia miltorrhrza preparations. The electrode used was a 0.3 mm diameter carbon disk electrode fixed in a wall-jet with amperometric detection. Under the optimum conditions, the two analytes were separated completely within 8 min. Excellent linearity was obtained in the concentration ranges of 0.25-100.0 microg ml(-1) and 0.50-100.0 microg ml(-1) for protocatechuic aldehyde and protocatechuic acid, respectively. The detection limits were 0.10 microg ml(-1) of protocatechuic aldehyde and 0.25 microg ml(-1) of protocatechuic acid, which were found to be lower than those of other methods that determine protocatechuic aldehyde (3,4-dihydroxybenzaldehyde) and protocatechuic acid (3,4-dihydrobenzoic acid) simultaneously. The mean recoveries of protocatechuic aldehyde and protocatechuic acid were 97.4% and 103.3%. This method has been successfully applied to monitor these two components in real samples such as Salivia miltorrhrza and its two traditional Chinese medicinal preparations.

Anticarcinogenic Agents↗
PubMed2001

Expression of a cloned ovine gastrointestinal peptide transporter (oPepT1) in Xenopus oocytes induces uptake of oligopeptides in vitro.

We determined the primary structure, tissue distribution and in vitro functional characterization of a peptide transporter, oPepT1, from ovine intestine. Ovine PepT1 (oPepT1) cDNA was 2829-bp long, encoding a protein of 707 amino acid residues with an estimated molecular size of 78 kDa and an isoelectric point (pI) of 6.57. Transport function of oPepT1 was assessed by expressing oPepT1 in Xenopus oocytes using a two-electrode voltage-clamp technique. The transport process was electrogenic and pH dependent, but independent of Na+, Cl- and Ca2+. The oPepT1 displayed a broad substrate specificity for transport of neutral and charged dipeptides and tripeptides. All dipeptides and tripeptides examined evoked inward currents in a saturable manner, with an affinity constant (Kt) ranging from 27 micromol/L to 3.0 mmol/L. No responses were detected from tetrapeptides or free amino acids. Northern blot analysis demonstrated that oPepT1 was expressed in the small intestine, omasum and rumen, but was not expressed in liver and kidney. The presence of the peptide transporter in the forestomach at such levels could provide nutritionally important amino acid nitrogen to ruminants.

Amino Acid Sequence↗
PubMed2001

Detection of tumorigenesis in rat bladders with optical coherence tomography.

Optical coherence tomography (OCT) is a novel technique that enables noninvasive cross-sectional imaging of biological tissues. Because of its high resolution (approximately 10 microm), superior dynamic range (140 dB in our case) and up to 2-3 mm penetration depth, OCT is potentially useful for noninvasive screening of superficial lesions. Bladder cancer arises within the transitional epithelium. Despite the ability to visualize the epithelium via cystoscopy, it is often difficult to detect early epithelial cancers and to determine their penetration to the underlying layers. To investigate the potential of OCT to enhance imaging of bladder cancers and other epithelial lesions, we applied OCT to normal and diseased bladder epithelium, and correlated the results with histological findings. OCT images of porcine bladder (a close homolog of human bladder) confirm the ability of this method to image human tissues. To determine whether OCT can track the course of bladder cancer, a standard rat model of bladder cancer in which Fisher rats are exposed to methyl-nitroso-urea (MNU), was followed both with OCT and histological studies. Our results show that the micro morphology of porcine bladder such as the urothelium, submucosa and muscles is identified by OCT and well correlated with the histological evaluations. OCT detected edema, inflammatory infiltrates, and submucosal blood congestion as well as the abnormal growth of urothelium (e.g., papillary hyperplasia and carcinomas). By contrast, surface imaging, which resembles cystoscopy, provided far less sensitivity and resolution than OCT. This is the first OCT study of any tumor documented in a systematic fashion, and the results suggest the potential of OCT for the noninvasive diagnosis of both bladder inflammatory lesions and early urothelial abnormalities, which conventional cystoscopy often misses, by imaging characterization of the increases in urothelial thickening and backscattering. However, because of the depth limitation, OCT may have limited applications in staging the invasion of higher-state urothelial cancers, especially for papillary carcinomas.

Animals↗
PubMed2001

Human glioma cells transformed by IGF-I triple helix technology show immune and apoptotic characteristics determining cell selection for gene therapy of glioblastoma.

AIMS: Insulin-like growth factor type I (IGF-I) antisense cellular gene therapy of tumours is based on the following data: rat glioma or hepatoma cells transfected with the vector encoding IGF-I antisense cDNA lose their tumorigenicity and induce a tumour specific immune response involving CD8(+) T cells. Recently, using the IGF-I triple helix approach in studies of tumorigenicity, major histocompatibility complex class I (MHC-I) antigens were demonstrated in rat glioma transfected cells. This study used comparative IGF-I antisense and triple helix technologies in human primary glioma cells to determine the triple helix strategy that would be most appropriate for the treatment of glioblastoma. METHODS: The cells were transfected using the IGF-I triple helix expression vector, pMT-AG, derived from the pMT-EP vector. pMT-AG contains a cassette comprising a 23 bp DNA fragment transcribing a third RNA strand, which forms a triple helix structure within a target region of the human IGF-I gene. Using pMT-EP, vectors encoding MHC-I or B7 antisense cDNA were also constructed. RESULTS: IGF-I triple helix transfected glioma cells are characterised by immune and apoptotic phenomena that appear to be related. The expression of MHC-I and B7 in transfected cells (analysed by flow cytometry) was accompanied by programmed cell death (detected by dUTP fluorescein terminal transferase labelling of nicked DNA and electron microscopic techniques). Cotransfection of these cells with MHC-I and B7 antisense vectors suppressed the expression of MHC-I and B7, and was associated with a pronounced decrease in apoptosis. CONCLUSION: When designing an IGF-I triple helix strategy for the treatment of human glioblastoma, the transfected tumour cells should have the following characteristics: the absence of IGF-I, the presence of both MHC-I and B7 molecules, and signs of apoptosis.

Apoptosis↗
PubMed2001

Bayesian segregation analysis of somatic cell scores of Ontario Holstein cattle.

Bayesian segregation analysis using a Gibbs sampling approach was applied to four sets of simulated data and one set of field data to detect evidence of major genes affecting the evaluated trait. The substitution effect of a major gene and its allelic frequency were estimated for each set of data. For two datasets simulated with a model with no major gene effect, the resulting estimates of polygenic variance and heritability agreed with the simulated values and tests for the presence of a major gene were not significant. Analyses of two sets of data simulated with a major gene produced posterior distributions that gave significant evidence of major gene effects but underestimated the substitution values of the major gene. The segregation analysis of field data suggested that a major gene significantly affected somatic cell score (SCS) in the population of Ontario Holstein cattle. The estimated heritability of SCS was approximately 0.16. The major gene variance accounted for about 17% of the total genetic variance and the point estimate of the frequency of the allele having a positive effect on SCS was 0.30. However, the precision of these estimates is questionable based on the simulation results. The effect of the major gene may be underestimated.

Animals↗