Biomedical subjects

Yan Xu

Publications and source records attributed to Yan Xu.

At least 217 records · Page 12Linked to original sources

PubMed2002

Application of cDNA microarrays to generate a molecular taxonomy capable of distinguishing between colon cancer and normal colon.

In order to discover global gene expression patterns characterizing subgroups of colon cancer, microarrays were hybridized to labeled RNAs obtained from seventeen colonic specimens (nine carcinomas and eight normal samples). Using a hierarchical agglomerative method, the samples grouped naturally into two major clusters, in perfect concordance with pathological reports (colon cancer versus normal colon). Using a variant of the unpaired t-test, selected genes were ordered according to an index of importance. In order to confirm microarray data, we performed quantitative, real-time reverse transcriptase-polymerase chain reaction (TaqMan RT-PCR) on RNAs from 13 colorectal tumors and 13 normal tissues (seven of which were matched normal-tumor pairs). RT-PCR was performed on the gro1, B-factor, adlican, and endothelin converting enzyme-1 genes and confirmed microarray findings. Two hundred and fifty genes were identified, some of which were previously reported as being involved in colon cancer. We conclude that cDNA microarraying, combined with bioinformatics tools, can accurately classify colon specimens according to current histopathological taxonomy. Moreover, this technology holds promise of providing invaluable insight into specific gene roles in the development and progression of colon cancer. Our data suggests that a large-scale approach may be undertaken with the purpose of identifying biomarkers relevant to cancer progression.

Aged↗

PubMed2002

Lysophosphatidylcholine as a ligand for immunoregulation.

Despite the recognized effects of lysophosphatidylcholine upon cells of the immune system and its association with inflammatory processes, its mechanism of action has remained poorly characterized. Our recent identification of the first lysophosphatidylcholine receptor as an immunoregulatory G protein-coupled receptor named G2A whose genetic ablation results in the development of inflammatory autoimmune disease has, therefore, provided a new perspective on the role of this lysophospholipid as a modulator of immune responses. This commentary discusses the biological properties of lysophosphatidylcholine as an immunoregulatory ligand for cells of the innate and adaptive arms of the immune system. Although we focus primarily on ligand interactions with G2A, we also discuss the issue of possible functional redundancy with other receptors with recently established ligand specificities towards phosphorylcholine-containing lysolipids including lysophosphatidylcholine.

Adjuvants, Immunologic↗

PubMed2002

Phosphorylation of Bcl-2 in G2/M phase-arrested cells following photodynamic therapy with hypericin involves a CDK1-mediated signal and delays the onset of apoptosis.

The role of Bcl-2 in photodynamic therapy (PDT) is controversial, and some photosensitizers have been shown to induce Bcl-2 degradation with loss of its protective function. Hypericin is a naturally occurring photosensitizer with promising properties for the PDT of cancer. Here we show that, in HeLa cells, photoactivated hypericin does not cause Bcl-2 degradation but induces Bcl-2 phosphorylation in a dose- and time-dependent manner. Bcl-2 phosphorylation is induced by sublethal PDT doses; increasing the photodynamic stress promptly leads to apoptosis, during which Bcl-2 is neither phosphorylated nor degraded. Bcl-2 phosphorylation involves mitochondrial Bcl-2 and correlates with the kinetics of a G(2)/M cell cycle arrest, preceding apoptosis. The co-localization of hypericin with alpha-tubulin and the aberrant mitotic spindles observed following sublethal PDT doses suggest that photodamage to the microtubule network provokes the G(2)/M phase arrest. PDT-induced Bcl-2 phosphorylation is not altered by either the overexpression or inhibition of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH(2)-terminal protein kinase 1 (JNK1) nor by inhibiting the extracellular signal-regulated kinases (ERKs) or protein kinase C. By contrast, Bcl-2 phosphorylation is selectively suppressed by the cyclin-dependent protein kinase (CDK)-inhibitor roscovitine, completely blocked by the protein synthesis inhibitor cycloheximide and enhanced by the overexpression of CDK1, suggesting a role for this pathway. However, in an in vitro kinase assay, active CDK1/cyclin B1 complex failed to phosphorylate immunoprecipitated Bcl-2, suggesting that this protein kinase may not directly modify Bcl-2. Mutation of serine-70 to alanine in Bcl-2 abolishes PDT-induced phosphorylation and restores the caspase-3 activation to the same levels of the vector-transfected cells, indicating that Bcl-2 phosphorylation may be a signal to delay apoptosis in G(2)/M phase-arrested cells.

Anthracenes↗

PubMed2002

Instabilotyping reveals unique mutational spectra in microsatellite-unstable gastric cancers.

Microsatellite instability (MSI) within coding regions causes frameshift mutations (FSMs). This type of mutation may inactivate tumor suppressor genes in cancers with frequent MSI (MSI-H cancers). To identify novel FSMs in gastric carcinogenesis in an unbiased and comprehensive manner, we screened for this type of mutation at 154 coding region repeat loci in 18 MSI-H gastric cancers. We also compared FSM rates and spectra in MSI-H gastric versus colorectal cancers. Thirteen novel loci showed FSMs in >20% of gastric tumors. Novel loci with the highest mutation frequencies included the activin type 2 receptor gene (44.4%), DKFZp564K112 (a homologue of the Drosophila tumor suppressor gene multi-sex-combs; 41.2%), and an endoplasmic reticulum chaperone protein gene SEC63 (37.5%). The mutational spectra for genes with high mutation frequencies were also significantly different between MSI-H gastric and colorectal cancers.

Colorectal Neoplasms↗

PubMed2002

Artificial neural networks and gene filtering distinguish between global gene expression profiles of Barrett's esophagus and esophageal cancer.

cDNAmicroarrays, combined with bioinformatics analyses, are becomingincreasingly used in current medical research. Existing analytic methods,particularly those that are unsupervised, often have difficulty recognizing subtle differences among predefined subgroups. In contrast, supervised methods, such as Artificial Neural Networks (ANNs), are able to recognize subtly different biological entities. We applied ANNs in a proof-of-principle study of cDNA microarray data in esophageal cancer (CA) and premalignancy. cDNA microarrays, each containing 8064 clones, were hybridized to RNAs from 22 esophageal lesions, including 14 Barrett's esophagus (BA) metaplasias and 8 esophageal carcinomas (3 squamous cell carcinomas and 5 adenocarcinomas). Scanned cDNA microarray data were analyzed using the bioinformatics software Cluster/TreeView, Significance Analysis of Microarrays (SAM), and ANNs. Cluster analysis based on all 8064 clones on the microarrays was unable to correctly distinguish BA specimens from CA specimens. SAM then selected 160 differentially expressed genes between Barrett's and cancer. Cluster analysis based on this reduced set still misclassified 2 Barrett's as cancers. The ANN was trained on 12 samples and tested against the remaining 10 samples. Using the 160 selected genes, the ANN correctly diagnosed all 10 samples in the test set. Finally, the 160 genes selected by SAM may merit further study as biomarkers of neoplastic progression in the esophagus, as well as in elucidating pathological mechanisms underlying BA and CA.

Adenocarcinoma↗

PubMed2002

Sphingosylphosphorylcholine and lysophosphatidylcholine: G protein-coupled receptors and receptor-mediated signal transduction.

In recent years, certain lysophospholipids (lyso-PLs) have been recognized as important cell signaling molecules. Among them, two phosphorylcholine-containing lyso-PLs, sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC), have been shown to be involved in many cellular processes and are produced under physiological and pathological conditions. Although signaling properties of SPC and LPC have been studied in a variety of cellular systems, specific cell membrane receptors for SPC and LPC have not been identified previously. Recently, ovarian cancer G protein-coupled receptor 1 (OGR1, also known as GPR68), G protein-coupled receptor 4 (GPR4), and G2A have been identified as receptors for SPC and LPC. The signaling and ligand-binding properties of these receptors are reviewed here. These discoveries provide an intriguing opportunity and a novel approach in studying the pathophysiological roles of SPC and LPC and their receptors.

Animals↗

PubMed2002

Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine.

Heterologous prime/boost regimens have the potential for raising high levels of immune responses. Here, we report that DNA priming followed by a recombinant modified vaccinia Ankara (rMVA) booster has controlled a highly pathogenic immunodeficiency virus challenge in a Rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency virus proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster. These highly promising findings provide hope that a relatively simple multiprotein DNA/MVA vaccine can help to control the AIDS epidemic.

AIDS Vaccines↗

PubMed2002

Carboxyester hydrolysis catalyzed by a novel dicopper(II) complex with an alcohol-pendant macrocycle.

A novel hexaaza macrocycle bearing two hydroxyethyl pendants (L), 3,6,9,16,19,22-hexaaza-6,19-bis(2-hydroxyethyl)tricyclo[22,2,2,2(11,14)]triaconta-1,11,13,24,27,29-hexaene, was synthesized as a potential binucleating ligand. The corresponding Cu(II) complex [Cu(2)LCl(2)]Cl(2) small middle dot5.5H(2)O was isolated as a blue crystal, triclinic, space group P with a= 9.4920(19) A, b = 4.783(3) A, c = 16.553(3) A, alpha = 63.87(3) degrees, beta = 86.10(3) degrees, gamma = 83.8(3) degrees, V = 2072.8(7) A(-3), Z = 2, R1 = 0.0658, and wR2 = 0.1839. Both Cu ions adopt the geometry of a distorted trigonal bipyramid in a pentacoordinated environment. A complexation study on the novel title complex has revealed that the alcoholic OH groups of the complex Cu(2)L exhibit an obvious acidity with rather low pK(a) values at 25 degrees C. The Cu(II)-bound alkoxides, which act as reactive nucleophiles toward the hydrolysis of 4-nitrophenyl acetate in 10% (v/v) CH(3)CN at 25 degrees C, with I = 0.10 (NaNO(3)) and pH 9.3, have shown a second-order rate constant, 0.41 +/- 0.02 M(-1) s(-1), a value that is approximately 10 times greater than the corresponding value for the mononuclear Cu(II) complex formed by a relatively simple tripodal ligand (L1). The pH-rate profile gave a sigmoidal curve. The possible catalytic mechanism has been proposed, and the reason for the high catalytic activity of the title complex has been discussed.

Journal Article↗

PubMed2002

Photodynamic therapy with hypericin induces vascular damage and apoptosis in the RIF-1 mouse tumor model.

Hypericin, a polycyclic quinone obtained from plants of the genus Hypericum, has been proven to be a potent photosensitizer. The mechanism of tumor eradication and mode of cell death induced by in vivo photodynamic therapy (PDT) with hypericin were investigated in the present study using 2 therapeutic protocols. RIF-1 tumors were exposed to laser light at either 0.5 hr or 6 hr after hypericin administration (5 mg/kg, i.v.). A significant reduction in tumor perfusion, as determined by the retention of fluorescein in the tumor tissue, was detected immediately after both PDT treatments. Further decrease in tumor perfusion was observed in the hours after treatment. The re-establishment of tumor perfusion, however, occurred 24 hr after 6 hr-interval PDT, but not after 0.5 hr-interval PDT. The kinetics of tumor cell survival estimated by the in vivo/in vitro clonogenic assay revealed no or limited cell death when tumors were explanted immediately after irradiation, whereas a delayed but progressive cell death was detected when tumors remained in situ after both PDT treatments. The detection of nucleosomal DNA fragmentation by agarose gel electrophoresis or TUNEL assay and the assessment of cell morphology by light microscopy indicated that apoptosis was the most prominent tumor response to hypericin-mediated PDT. Furthermore, immunohistochemical analysis of the tumor tissue showed an increased expression of both Fas and Fas ligand after irradiation, suggesting that this cell death pathway might contribute to the overall PDT-induced apoptotic response. In conclusion, our results demonstrate that apoptosis, likely occurring as a result of vascular damage, is responsible for the tumor eradication by PDT with hypericin in this tumor model.

Animals↗

PubMed2002

Hypermethylation of the p14(ARF) gene in ulcerative colitis-associated colorectal carcinogenesis.

The p14(ARF) protein directly inhibits the MDM-2 oncoprotein, which mediates degradation of the p53 protein. It has been shown that p14(ARF) expression is frequently down-regulated by p14(ARF) gene hypermethylation in colorectal cancer. To determine whether p14(ARF) inactivation was involved in ulcerative colitis (UC)-associated carcinogenesis, the frequency and timing of p14(ARF) methylation was investigated in four different histological stages of UC-associated carcinogenesis. Methylation-specific PCR and bisulfite sequencing were used to determine the prevalence of p14(ARF) gene methylation. p14(ARF) methylation was observed in 19 of 38 (50%) adenocarcinomas, 4 of 12 (33%) dysplasias, and 3 of the 5 (60%) nonneoplastic UC mucosae. In contrast, 3 of 40 (3.7%) normal tissues showed p14(ARF) methylation (chi(2) test: P = 0.0003). Bisulfite sequencing was used to analyze 28 CpGs of p14(ARF) gene in 20 samples. The number of methylated CpGs ranged from 0 to 4, 0 to 20, and 0 to 28 in the normal, dysplastic, and carcinomatous samples, respectively (Kruskall-Wallis test: P = 0.0005). Densely methylated alleles were detected only in carcinomas by bisulfite sequencing. In conclusion, our data suggest that methylation of p14(ARF) is a relatively common early event in UC-associated carcinogenesis. p14(ARF) offers potential as a biomarker for the early detection of cancer or dysplasia in UC. Finally, analyses of p14(ARF) methylation in other organs should explore not only frank cancers but other premalignant lesions.

Base Sequence↗

PubMed2002

ADC characterization of region-specific response to cerebral perfusion deficit in rats by MRI at 9.4 T.

Region-specific cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) of water in the rat brain were quantified in vivo by high-field MRI (9.4 T) for 6-7 h after middle cerebral artery occlusion (MCAO). Upon occlusion, average CBF fell from about 1.5-2 ml/g/min to below 0.5 ml/g/min in cortical areas and the amygdala, and below 0.2 ml/g/min in the caudate putamen. CBF in some of the homologous contralateral areas also decreased by 20-30%. Average ADC decreased from about 8 center dot 10(-4) to 5 center dot 10(-4) mm(2)/s in the caudate putamen and parietal cortex. Corresponding changes in ADC were lower in the frontal cortex and negligible in the piriform cortex, suggesting that the perfusion threshold for ADC decrease may be different for different brain regions in the same animal. The area of decreased ADC correlated well with the infarction area revealed by 2,3,5-triphenyltetrazolium chloride (TTC) staining of brain slices in vitro. A better understanding of the mechanisms linking ADC and CBF changes to ischemic cell disorders may prove useful in characterizing the degree of tissue damage, and in developing and evaluating treatment strategies.

Analysis of Variance↗

PubMed2002

Activation of the esophagin promoter during esophageal epithelial cell differentiation.

Esophagin is a member of the small proline-rich protein family of cell envelope precursor proteins, which are expressed during squamous cell differentiation. Esophagin is expressed at high levels in normal esophageal epithelium, but its expression is absent from esophageal squamous cell carcinomas and adenocarcinomas. Moreover, loss of esophagin expression is present in areas of dysplasia or normal mucosa adjacent to carcinomas, suggesting that absence of esophagin may constitute a harbinger of early esophageal malignant transformation. A greater understanding of transcriptional control of esophagin may provide valuable insights into esophageal malignancy. Therefore, this study was undertaken in order to isolate and carry out initial characterization of a functional promoter for esophagin. A genomic clone containing esophagin was isolated and sequenced, including 2.7 kb of the esophagin promoter region. Esophagin expression was studied in response to various treatments of primary cultured human esophageal epithelial cells and squamous cell carcinoma cell lines. Calcium was the strongest inducer of the endogenous esophagin promoter, with induction occurring at 12-72 hours. In primary cultured esophageal epithelial cells, a region spanning 116 bp upstream of the transcriptional start site to 45 bp downstream was sufficient to direct low, basal, in vitro esophagin expression. However, responsiveness of primary esophageal cells to calcium required inclusion of promoter elements 1688 bp upstream of the transcriptional start site. Site-directed mutagenesis studies suggested a putative role for C/EBP-beta, OCT-1, and OCT-3 transcription factor binding sites in the minimal promoter region. In conjunction with published human in vivo studies, these data support the hypothesis that esophagin is a biomarker of esophageal squamous cell differentiation and provide an in vitro model to evaluate regulatory factors involved in this differentiation process.

Epithelium↗

PubMed2002

Protective effect of GSH on PD model induced by 6-OHDA in vitro.

To study the effects of 6-hydroxydopamine (6-OHDA) and reduced glutathione (GSH) on the nigral dopaminergic neurons in brain slices in vitro, immolunohistochemical technique was used to observe the changes of TH-stained neurons, including cell bodies and the dendrites, in the substantia nigra (SN) of midbrain slices of rats after incubation for 1 h in the presence of GSH 15 min before and during the period of incubation with 6-OHDA. The results showed that cell bodies remained intact but dendrites were fragmented and truncated after treatment with 6-OHDA. The antioxidant GSH alone did not significantly affect the dendrites of SN neurons but prevented 6-O-HDA-induced damage of dendrites. It was concluded that glutathione may prevent 6-OHDA-induced dopaminergic neurodegeneration and play a protective role in dopaminergic neurons.

Animals↗

PubMed2002

NMR structures of the second transmembrane domain of the human glycine receptor alpha(1) subunit: model of pore architecture and channel gating.

Glycine receptors (GlyR) are the primary inhibitory receptors in the spinal cord and belong to a superfamily of ligand-gated ion channels (LGICs) that are extremely sensitive to low-affinity neurological agents such as general anesthetics and alcohols. The high-resolution pore architecture and the gating mechanism of this superfamily, however, remain unclear. The pore-lining second transmembrane (TM2) segments of the GlyR alpha(1) subunit are unique in that they form functional homopentameric channels with conductance characteristics nearly identical to those of an authentic receptor (Opella, S. J., J. Gesell, A. R. Valente, F. M. Marassi, M. Oblatt-Montal, W. Sun, A. F. Montiel, and M. Montal. 1997. Chemtracts Biochem. Mol. Biol. 10:153-174). Using NMR and circular dichroism (CD), we determined the high-resolution structures of the TM2 segment of human alpha(1) GlyR and an anesthetic-insensitive mutant (S267Y) in dodecyl phosphocholine (DPC) and sodium dodecyl sulfate (SDS) micelles. The NMR structures showed right-handed alpha-helices without kinks. A well-defined hydrophilic path, composed of side chains of G2', T6', T10', Q14', and S18', runs along the helical surfaces at an angle approximately 10-20 degrees relative to the long axis of the helices. The side-chain arrangement of the NMR-derived structures and the energy minimization of a homopentameric TM2 channel in a fully hydrated DMPC membrane using large-scale computation suggest a model of pore architecture in which simultaneous tilting movements of entire TM2 helices by a mere 10 degrees may be sufficient to account for the channel gating. The model also suggests that additional residues accessible from within the pore include L3', T7', T13', and G17'. A similar pore architecture and gating mechanism may apply to other channels in the same superfamily, including GABA(A), nACh, and 5-HT(3) receptors.

Anesthetics↗

PubMed2002

Integrated approaches to the action of general anesthetics and alcohol.

The use of inhalational anesthetics was first publicly demonstrated in 1846. Alcohol has been consumed for centuries and is now our most costly drug abuse problem. Despite widespread use, the molecular mechanism of action of these drugs has remained an enigma. The prevailing theory suggests that alcohol and anesthetics interact directly with neuronal membrane proteins to cause their effects. Our laboratories are using a variety of cutting edge approaches to gain insight into the mechanism of action of these drugs. Biophysical approaches such as high-resolution nuclear magnetic resonance (NMR) spectroscopy and genetic engineering approaches such as creation of designer mice are currently being used by our laboratories. These approaches are providing exciting insight into how these drugs exert their effects. This research could ultimately result in safer and better anesthetics, may lead to treatments for alcoholism, and may provide insight into basic biologic processes such as consciousness.

Anesthetics, General↗

PubMed2002

NMR studies of drug interaction with membranes and membrane-associated proteins.

This review focuses on the recent developments in the study of drug interactions with biological membranes and membrane-associated proteins using nuclear magnetic resonance (NMR) spectroscopy and other spectroscopic techniques. Emphasis is placed on a class of low-affinity neurological agents as exemplified by volatile general anesthetics and structurally related compounds. The technical aspects are reviewed of how to prepare membrane-mimetic systems and of NMR approaches that are either in current use or opening new prospects. A brief literature survey covers studies ranging from drug distribution in simplified lipid matrix to specific drug interaction with neuronal receptors reconstituted in complicated synthetic membrane systems.

Anesthetics, General↗

PubMed2002

Artificial neural networks distinguish among subtypes of neoplastic colorectal lesions.

BACKGROUND & AIMS: There is a subtle distinction between sporadic colorectal adenomas and cancers (SAC) and inflammatory bowel disease (IBD)-associated dysplasias and cancers. However, this distinction is clinically important because sporadic adenomas are usually managed by polypectomy alone, whereas IBD-related high-grade dysplasias mandate subtotal colectomy. The current study evaluated the ability of artificial neural networks (ANNs) based on complementary DNA (cDNA) microarray data to discriminate between these 2 types of colorectal lesions. METHODS: We hybridized cDNA microarrays, each containing 8064 cDNA clones, to RNAs derived from 39 colorectal neoplastic specimens. Hierarchical clustering was performed, and an ANN was constructed and trained on a set of 5 IBD-related dysplasia or cancer (IBDNs) and 22 SACs. RESULTS: Hierarchical clustering based on all 8064 clones failed to correctly categorize the SACs and IBDNs. However, the ANN correctly diagnosed 12 of 12 blinded samples in a test set (3 IBDNs and 9 SACs). Furthermore, using an iterative process based on the computer programs GeneFinder, Cluster, and MATLAB, we reduced the number of clones used for diagnosis from 8064 to 97. Even with this reduced clone set, the ANN retained its capacity for correct diagnosis. Moreover, cluster analysis performed with these 97 clones now separated the 2 types of lesions. CONCLUSIONS: Our results suggest that ANNs have the potential to discriminate among subtly different clinical entities, such as IBDNs and SACs, as well as to identify gene subsets having the power to make these diagnostic distinctions.

Adenoma↗

PubMed2002

The conformational study of two carbocyclic nucleosides: why carbocyclic nucleic acids (CarNAs) form more stable duplexes with RNA than DNA does.

Replacement of the furanose moiety of DNA with a cyclopentane ring produces a modified sugar analogue: Carbocyclic nucleic acid (CarNA). UV melting-temperature experiments demonstrate that the incorporation of 2'-deoxycarbaguanosine ((c)G) and 2'-deoxyaristeromycin ((c)A) of carbocyclic nucleosides into a DNA strand increases the stability of the CarNA/RNA hybrid. Circular Dichroism (CD) study indicates that the CarNA/RNA hybrid adopts an A-like conformation. To elucidate the molecular basis of the increased stability of the CarNA/RNA, the conformation of (c)G and (c)A were examined by (1)H NMR conformational analysis of (3)J(HH) coupling constants and ab initio molecular orbital (MO) calculations. These results show that the populations of N-type of (c)G and (c)A are higher than those of dG and dA, respectively, at different temperatures [For example, 37% (N%) of (c)G vs. 28%of dG, 36% (N%) of (c)A vs. 25% of dA at 278 K], which suggest that the cyclopentane rings of (c)G and (c)A prefer the N-type conformation in two-state N-S pseudorotional equilibrium in comparison with the furanose rings of dG and dA. The DeltaH degrees of (c)G (DeltaH degrees = - 0.43 kcal mol(-1)) and (c)A (DeltaH degrees = - 0.41kcal mol(-1)) are lower than that of dG (dG = - 1.8 kcal mol(-1)) and dA (dA = - 1.0 kcal mol(-1)), respectively, which suggest that the gauche effect in the (c)A and (c)G driving N-S pseudorotional equilibrium to S-type is reduced by replacement of the 4'-oxygen by a CH(2) group. These results suggest that the preferred N-type of the (c)G and (c)A leads to the A-like conformation, which contributes to the stability of CarNA/RNA hybrid.

Acids, Carbocyclic↗