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Biomedical subjects

J Park

Publications and source records attributed to J Park.

At least 73 records · Page 4Linked to original sources
PubMed2001

Chemosensitivity prediction by transcriptional profiling.

In an effort to develop a genomics-based approach to the prediction of drug response, we have developed an algorithm for classification of cell line chemosensitivity based on gene expression profiles alone. Using oligonucleotide microarrays, the expression levels of 6,817 genes were measured in a panel of 60 human cancer cell lines (the NCI-60) for which the chemosensitivity profiles of thousands of chemical compounds have been determined. We sought to determine whether the gene expression signatures of untreated cells were sufficient for the prediction of chemosensitivity. Gene expression-based classifiers of sensitivity or resistance for 232 compounds were generated and then evaluated on independent sets of data. The classifiers were designed to be independent of the cells' tissue of origin. The accuracy of chemosensitivity prediction was considerably better than would be expected by chance. Eighty-eight of 232 expression-based classifiers performed accurately (with P < 0.05) on an independent test set, whereas only 12 of the 232 would be expected to do so by chance. These results suggest that at least for a subset of compounds genomic approaches to chemosensitivity prediction are feasible.

Drug Resistance, Neoplasm↗
PubMed2001

2-(Hydroxycarbonyl)benzyl glycosides: a novel type of glycosyl donors for highly efficient beta-mannopyranosylation and oligosaccharide synthesis by latent-active glycosylation.

2-(Benzyloxycarbonyl)benzyl (BCB) glycosides were prepared by coupling of the corresponding tetraacetylglycosyl bromides and benzyl 2-(hydroxymethyl)benzoate. The BCB glycosides were converted almost quantitatively into the corresponding 2-(hydroxycarbonyl)benzyl (HCB) glycosides by selective hydrogenolysis of the benzyl ester functionality without affecting the benzylidene acetal and the benzyl ether. Treatment of the HCB 4,6-O-benzylidenemannopyranoside 4 with triflic anhydride in the presence of di-tert-butylmethylpyridine and subsequent addition of the glycosyl acceptor having a primary hydroxyl group afforded exclusively the disaccharide of the beta-mannopyranosyl linkage. Glycosylation of the compound 4 with secondary and tertiary alcohols also provided beta-mannopyranosides as the major products. Glycosylation of the HCB 4,6-O-cyclohexylidenemannoside 5 with primary alcohols was also highly beta-selective, and the HCB 2,3-O-cyclohexylidenemannoside 6 exhibited the moderate beta-selectivity. On the other hand, unlike the HCB mannosides, the HCB 4,6-O-benzylideneglucoside 7 gave exclusively the disaccharides of the alpha-glycopyranosyl linkage in the glycosylation with primary alcohols. The latent BCB-disaccharide 23, which was obtained from the HCB mannoside 4 as the donor and the BCB glucoside 12 as the acceptor by the present glycosylation method, was converted into the active HCB-disaccharide 39 by selective hydrogenolysis. Repetitive glycosylation of the donor 39 with the same acceptor 12 afforded the BCB-trisaccharide 40. Other BCB-trisaccharides 42 and 46 were also efficiently synthesized by employing the present methodology.

Carbohydrate Sequence↗
PubMed2001

Molecular cloning and functional expression of bovine brain GABA transaminase.

We isolated a cDNA that encodes the bovine brain gamma-aminobutyrate transaminase (GABA-T; EC 2.6.1.19) from the lambda gt 11 cDNA library, which showed a high degree of sequence similarity to the corresponding enzymes from various sources. Northern blot analysis revealed two differentially expressed GABA-T transcripts of approximately 2.0 and 6.0 kb in the bovine tissues. Southern blot analysis indicates that the two GABA-T transcripts are encoded in a greater-than 10-kb, single-copy gene. Bovine GABA-T cDNA was expressed in E. coli using the pGEX bacterial- expression vector system. The overexpressed GABA-T was enzymatically active after purification, and it had very similar kinetic parameters when compared with those of other mammalian GABA-Ts.

4-Aminobutyrate Transaminase↗
PubMed2001

Self-assembled monolayer of a redox-active calix[4]arene: voltammetric recognition of the Ba2+ ion in aqueous media.

The Redox-active monolayer of a novel calix[4]arene recognizing redox-inactive ionic species by voltammetry is reported. Calix[4]arene-disulfide-diquinone, which is not only redox-active but is also a highly selective ionophore for the Ba2+ ion, spontaneously forms a stable and dense monolayer film on gold. The redox-active calixarene monolayer selectively recognizes Ba2+ ion in aqueous media, and the voltammetric signals are proportional to the ionic concentration. A new voltammetric peak can be detected by square-wave voltammetry upon adding a dilute solution containing Ba2+ ion having a concentration as low as 1.0 x 10(-6) M. The Langmuir plot (1/ip vs 1/[Ba2+]) shows a linear slope in the range from 1.0 x 10(-6) M to 1.0 x 10(-4) M. This modified electrode does not show any significant interference from alkali and alkaline earth metal ions except for Sr2+ and Ca2+. Only 100- and 500-fold concentrations of Sr2+ and Ca2+ ions, respectively, can lead to voltammetric responses comparable to that of Ba2+.

Journal Article↗
PubMed2001

Identification of tyrosine phosphorylation sites on 3-phosphoinositide-dependent protein kinase-1 and their role in regulating kinase activity.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287-292). Here, we report that PDK1 becomes tyrosine-phosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5- to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased approximately 6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinase in vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2001

Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells.

The effects of Escherichia coli heat-stable enterotoxin (ST) and uroguanylin were examined on the proliferation of T84 and Caco2 human colon carcinoma cells that express guanylyl cyclase C (GC-C) and SW480 human colon carcinoma cells that do not express this receptor. ST or uroguanylin inhibited proliferation of T84 and Caco2 cells, but not SW480 cells, in a concentration-dependent fashion, assessed by quantifying cell number, cell protein, and [(3)H]thymidine incorporation into DNA. These agonists did not inhibit proliferation by induction of apoptosis, assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dNTP-biotin nick end labeling of DNA fragments) assay and DNA laddering, or necrosis, assessed by trypan blue exclusion and lactate dehydrogenase release. Rather, ST prolonged the cell cycle, assessed by flow cytometry and [(3)H]thymidine incorporation into DNA. The cytostatic effects of GC-C agonists were associated with accumulation of intracellular cGMP, mimicked by the cell-permeant analog 8-Br-cGMP, and reproduced and potentiated by the cGMP-specific phosphodiesterase inhibitor zaprinast but not the inactive ST analog TJU 1-103. Thus, GC-C agonists regulate the proliferation of intestinal cells through cGMP-dependent mechanisms by delaying progression of the cell cycle. These data suggest that endogenous agonists of GC-C, such as uroguanylin, may play a role in regulating the balance between epithelial proliferation and differentiation in normal intestinal physiology. Therefore, GC-C ligands may be novel therapeutic agents for the treatment of patients with colorectal cancer.

Bacterial Toxins↗
PubMed2001

Silyl derivatization of alkylphenols, chlorophenols, and bisphenol A for simultaneous GC/MS determination.

A fast silyl derivatization technique for simultaneous GC/ MS analysis of alkylphenols, chlorophenols, and bisphenol A was developed. The analytes were silylized with an excess amount of bis(trimethylsilyl)trifluoroacetamide (BSTFA) followed by hydrolysis of excess silyl reagent with water. Reaction rates of derivatization were studied in various solvents and found to be fastest in acetone. Derivatization reaction in acetone was completed quantitatively within 15 s at room temperature while it took more than 1 h in other solvents studied. Similar results were obtained in mixed solvents with acetone if the content of acetone was higher than 60% (v/v). Since water-immiscible solvents such as dichloromethane or hexane are frequently used in the extraction of phenolic analytes in various sample matrixes, acetone can be added to the extracts in order to accelerate the reaction rate of derivatization. Stability of the derivatives in sample for long-term storage was ensured by hydrolyzing excess derivatizing reagent, BSTFA, with a spike of water followed by dehydration using anhydrous sodium sulfate. On the basis of the above results, a derivatizing treatment kit was designed to improve the convenience of analysis. It was possible to treat sample within several minutes successfully by using the kit. So fast simultaneous determination of those anlaytes by GC/MS was possible with improved convenience as well as sensitivity and reproducibility.

Gas Chromatography-Mass Spectrometry↗
PubMed2001

The ATM-related domain of TRRAP is required for histone acetyltransferase recruitment and Myc-dependent oncogenesis.

The ATM-related TRRAP protein is a component of several different histone acetyltransferase (HAT) complexes but lacks the kinase activity characteristic of other ATM family members. We identified a novel function for this evolutionarily conserved domain in its requirement for the assembly of a functional HAT complex. Ectopic expression of TRRAP protein with a mutation in the ATM-related domain inhibits Myc-mediated oncogenic transformation. The Myc-binding region of TRRAP maps to a separable domain, and ectopic expression of this domain inhibits cell growth. These findings demonstrate that the ATM-related domain of TRRAP forms a structural core for the assembly and recruitment of HAT complexes by transcriptional activators.

Acetylation↗
PubMed2001

Adenomatous polyposis coli gene promoter hypermethylation in non-small cell lung cancer is associated with survival.

Methylation of 5' CpG islands in promoter and upstream coding regions has been identified as a mechanism for transcriptional inactivation of tumor suppressor genes. The purpose of this study was to determine whether hypermethylation of the adenomatous polyposis coli (APC) gene promoter occurs in primary non-small cell lung cancer (NSCLC), and whether hypermethylated APC has any relationship with survival. APC promoter 1A methylation was determined in normal and corresponding tumor tissue from 91 NSCLC patients and in a control group of 10 patients without cancer, using a quantitative fluorogenic real-time PCR (Taqman) system. APC promoter methylation was detectable in 86 (95%) of 91 tumor samples, but also in 80 (88%) of 91 normal samples of NSCLC patients, and in only two (20%) of 10 normal lung tissues of the control group. The median level of APC promoter methylation was 4.75 in tumor compared to 1.57 in normal lung tissue (P<0.001). Patients with low methylation status showed significantly longer survival than did patients with high methylation status (P=0.041). In a multivariate analysis of prognostic factors, APC methylation was a significant independent prognostic factor (P=0.044), as were pT (P=0.050) and pN (P<0.001) classifications. This investigation shows that APC gene promoter methylation occurs in the majority of primary NSCLCs. High APC promoter methylation is significantly associated with inferior survival, showing promise as a biomarker of biologically aggressive disease in NSCLC.

Adenocarcinoma↗
PubMed2001

Constitutively active phosphatidylinositol 3-kinase and AKT are sufficient to stimulate the epithelial Na+/H+ exchanger 3.

Phosphatidylinositol 3-kinase (PI 3-kinase) is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors and has been shown to be involved in regulation of stimulated exocytosis and endocytosis. One of the downstream signaling molecules activated by PI 3-kinase is the protein kinase Akt. Previous studies have indicated that PI 3-kinase is necessary for basal Na(+)/H(+) exchanger 3 (NHE3) transport and for fibroblast growth factor-stimulated NHE3 activity in PS120 fibroblasts. However, it is not known whether activation of PI 3-kinase is sufficient to stimulate NHE3 activity or whether Akt is involved in this PI 3-kinase effect. We used an adenoviral infection system to test the possibility that activation of PI 3-kinase or Akt alone is sufficient to stimulate NHE3 activity. This hypothesis was investigated in PS120 fibroblasts stably expressing NHE3 after somatic gene transfer using a replication-deficient recombinant adenovirus containing constitutively active catalytic subunit of PI 3-kinase or constitutively active Akt. The adenovirus construct used was engineered with an upstream ecdysone promoter to allow time-regulated expression. Adenoviral infection was nearly 100% at 48 h after infection. Forty-eight hours after infection (24 h after activation of the ecdysone promoter), PI 3-kinase and Akt amount and activity were increased. Increases in both PI 3-kinase activity and Akt activity stimulated NHE3 transport. In addition, a membrane-permeant synthetic 10-mer peptide that binds polyphosphoinositides and increases PI 3-kinase activity similarly enhanced NHE3 transport activity and also increased the percentage of NHE3 on the plasma membrane. The magnitudes of stimulation of NHE3 by constitutively active PI 3-kinase, PI 3-kinase peptide, and constitutively active Akt were similar to each other. These results demonstrate that activation of PI 3-kinase or Akt is sufficient to stimulate NHE3 transport activity in PS120/NHE3 cells.

Animals↗
PubMed2001

PartsList: a web-based system for dynamically ranking protein folds based on disparate attributes, including whole-genome expression and interaction information.

As the number of protein folds is quite limited, a mode of analysis that will be increasingly common in the future, especially with the advent of structural genomics, is to survey and re-survey the finite parts list of folds from an expanding number of perspectives. We have developed a new resource, called PartsList, that lets one dynamically perform these comparative fold surveys. It is available on the web at http://bioinfo.mbb.yale.edu/partslist and http://www.partslist.org. The system is based on the existing fold classifications and functions as a form of companion annotation for them, providing 'global views' of many already completed fold surveys. The central idea in the system is that of comparison through ranking; PartsList will rank the approximately 420 folds based on more than 180 attributes. These include: (i) occurrence in a number of completely sequenced genomes (e.g. it will show the most common folds in the worm versus yeast); (ii) occurrence in the structure databank (e.g. most common folds in the PDB); (iii) both absolute and relative gene expression information (e.g. most changing folds in expression over the cell cycle); (iv) protein-protein interactions, based on experimental data in yeast and comprehensive PDB surveys (e.g. most interacting fold); (v) sensitivity to inserted transposons; (vi) the number of functions associated with the fold (e.g. most multi-functional folds); (vii) amino acid composition (e.g. most Cys-rich folds); (viii) protein motions (e.g. most mobile folds); and (ix) the level of similarity based on a comprehensive set of structural alignments (e.g. most structurally variable folds). The integration of whole-genome expression and protein-protein interaction data with structural information is a particularly novel feature of our system. We provide three ways of visualizing the rankings: a profiler emphasizing the progression of high and low ranks across many pre-selected attributes, a dynamic comparer for custom comparisons and a numerical rankings correlator. These allow one to directly compare very different attributes of a fold (e.g. expression level, genome occurrence and maximum motion) in the uniform numerical format of ranks. This uniform framework, in turn, highlights the way that the frequency of many of the attributes falls off with approximate power-law behavior (i.e. according to V(-b), for attribute value V and constant exponent b), with a few folds having large values and most having small values.

Cysteine↗
PubMed2001

Mapping protein family interactions: intramolecular and intermolecular protein family interaction repertoires in the PDB and yeast.

In the postgenomic era, one of the most interesting and important challenges is to understand protein interactions on a large scale. The physical interactions between protein domains are fundamental to the workings of a cell: in multi-domain polypeptide chains, in multi-subunit proteins and in transient complexes between proteins that also exist independently. To study the large-scale patterns and evolution of interactions between protein domains, we view interactions between protein domains in terms of the interactions between structural families of evolutionarily related domains. This allows us to classify 8151 interactions between individual domains in the Protein Data Bank and the yeast Saccharomyces cerevisiae in terms of 664 types of interactions, between protein families. At least 51 interactions do not occur in the Protein Data Bank and can only be derived from the yeast data. The map of interactions between protein families has the form of a scale-free network, meaning that most protein families only interact with one or two other families, while a few families are extremely versatile in their interactions and are connected to many families. We observe that almost half of all known families engage in interactions with domains from their own family. We also see that the repertoires of interactions of domains within and between polypeptide chains overlap mostly for two specific types of protein families: enzymes and same-family interactions. This suggests that different types of protein interaction repertoires exist for structural, functional and regulatory reasons.

Binding Sites↗