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K Peck

Publications and source records attributed to K Peck.

At least 19 recordsLinked to original sources
PubMed2001

p53 amino acids 339-346 represent the minimal p53 repression domain.

The p53 tumor suppressor protein functions as an activator and also as a repressor of gene transcription. Currently, the mechanism of transcriptional repression by p53 remains poorly understood. To help clarify this mechanism, we carried out studies designed to identify the minimal repression domain that inhibits p53 transcriptional activities. We found only eight amino acids (339) of the COOH-terminal domain (termed P53MRD) that possess activities of repression. The exact location of this minimal domain is on the E6-binding region, and it lacks the ability of tetramerization. P53MRD is able to repress the transcription of p53 while not affecting VP16. The mutants (amino acids M340P and F341D) of native p53 also lost transcriptional repression of the thymidine kinase chloramphenicol acetyltransferase promoter. These results suggest that this eight-amino acid element is required for the repression of p53.

Binding Sites↗
PubMed2001

Acid-induced gene expression in Helicobacter pylori: study in genomic scale by microarray.

To understand the RNA expression in response to acid stress of Helicobacter pylori in genomic scale, a microarray membrane containing 1,534 open reading frames (ORFs) from strain 26695 was used. Total RNAs of H. pylori under growth conditions of pH 7.2 and 5.5 were extracted, reverse transcribed into cDNA, and labeled with biotin. Each microarray membrane was hybridized with cDNA probe from the same strain under two different pH conditions and developed by a catalyzed reporter deposition method. Gene expression of all ORFs was measured by densitometry. Among the 1,534 ORFs, 53 ORFs were highly expressed (> or = 30% of rRNA control in densitometry ratios). There were 445 ORFs which were stably expressed (<30% of rRNA in densitometry) under both pH conditions without significant variation. A total of 80 ORFs had significantly increased expression levels at low pH, while expressions of 4 ORFs were suppressed under acidic condition. The remaining 952 ORFs were not detectable under either pH condition. These data were highly reproducible and comparable to those obtained by the RNA slot blot method. Our results suggest that microarray can be used in monitoring prokaryotic gene expression in genomic scale.

Acids↗
PubMed2001

Correlation of gene expression with physiological functions: Examples of pulmonary blood vessel rheology, hypoxic hypertension, and tissue remodeling.

Microarray gene chip technology is a powerful invention looking for applications. A general principle is proposed here to direct the power of the technology toward physiology, medicine, and pharmacology. Our principle is to match quantitative measures of gene expression with the trend of mathematical parameters that describe biological functions. Mathematical parameterization is the heart. The procedure is illustrated by lung physiology, including the hypoxic hypertension, rheological properties of the tissues, and the remodeling of the pulmonary arterial wall under hypertensive stress. We show first how to reduce the experimental results on these physiological functions into mathematical formulas, and how the parameters of these formulas describe the functional trends precisely. Then under the assumption that the microarray reveals gene activities quantitatively, we match the trends of the gene activity with the trends of the functional parameters. Genes whose trends do match are interpreted as relevant to the functions. Those that do not match are considered irrelevant to the functions. The more functions we consider, the fewer will be the number of genes that are relevant to all functions. Thus we learn about the generality and specificity of the influence of genes on physiology.

Animals↗
PubMed2000

Profiling the downstream genes of tumor suppressor PTEN in lung cancer cells by complementary DNA microarray.

The phosphatase and tensin homology deleted on chromosome 10 (PTEN) is a tumor suppressor gene with sequence homology to tyrosine phosphatases and the cytoskeletal proteins tensin and auxilin. PTEN has recently been shown to inhibit cell migration and the spreading and formation of focal adhesions. This study investigated the role of PTEN in carcinoma invasion in a lung-cancer cell line and examined the downstream genes regulated by PTEN. We have previously established a cell-line model in human lung adenocarcinoma with different invasive abilities and metastatic potentials. Examining PTEN gene expression in these cell lines, we found that a homozygous deletion in exon 5 is associated with high invasive ability. We then constructed stable constitutive and inducible wild-type PTEN-overexpressed transfectants in the highly invasive cell line CL(1-5). We found that an overexpression of PTEN can inhibit invasion in lung cancer cells. To further explore the downstream genes regulated by PTEN, a high-density complementary DNA (cDNA) microarray technique was used to profile gene changes after PTEN overexpression. Our results indicate a panel of genes that can be modulated by PTEN. PTEN overexpression downregulated genes, including integrin alpha(6), laminin beta(3), heparin-binding epidermal growth factor-like growth factor, urokinase-type plasminogen activator, myb protein B, Akt2, and some expressed sequence tag (EST) clones. In contrast, PTEN overexpression upregulated protein phosphatase 2A1B, ubiquitin protease (unph), secreted phosphoprotein 1, leukocyte elastase inhibitor, nuclear factor-kappaB, cyclic adenosine monophosphate response element binding protein, DNA ligase 1, heat shock protein 90, and some EST genes. Northern hybridization and flow cytometry analysis also confirmed that PTEN overexpression results in the reduced expression of the integrin alpha(6) subunit. The results of this study indicate that PTEN overexpression may inhibit lung cancer invasion by downregulation of a panel of genes including integrin alpha(6). The cDNA microarray technique may be an effective tool to study the downstream function of a tumor suppressor gene.

Adenocarcinoma↗
PubMed1999

Sensitivity issues in DNA array-based expression measurements and performance of nylon microarrays for small samples.

DNA or oligonucleotide arrays are widely used for large-scale expression measurements, using various implementations: macroarrays in which DNA is spotted onto nylon membranes of relatively large dimensions (with radioactive detection) on the one hand; microarrays on glass slides and oligonucleotide chips, both used with fluorescent probes, on the other hand. Nylon micro-arrays with colourimetric detection have also been described recently. The small physical dimensions of miniaturized systems allow small hybridization volumes (2-100 microl) and provide high probe concentrations, in contrast to macroarrays. We show, however, that actual sensitivity (defined as the amount of sample necessary for detection of a given mRNA species) is in fact similar for all these systems and that this is mostly due to the very different amounts of target material present on the respective arrays. We then demonstrate that the combination of nylon microarrays with(33)P-labelled radioactive probes provides 100-fold better sensitivity, making it possible to perform expression profiling experiments using submicrogram amounts of unamplified total RNA from small biological samples. This has important implications in basic and clinical research and makes this alternative approach particularly suitable for groups operating in an academic context.

Biosensing Techniques↗
PubMed1998

Profiling expression patterns and isolating differentially expressed genes by cDNA microarray system with colorimetry detection.

A high-density cDNA microarray with colorimetry detection system to simultaneously monitor the expression of many genes on nylon membrane is described and characterized. To quantify the expression of genes and to isolate differentially expressed genes, the southern hybridization process on filter membranes was employed. The levels of gene expression were represented by color intensities generated by colorimetric reactions in place of hazardous radioisotopes or costly laser-induced fluorescence detection. The gene expression patterns on nylon membranes were digitized by devices such as an economical flatbed scanner or a digital camera. The quantitative information of gene expression was retrieved by image analysis software. Quantitative comparison of the northern dot-blotting method with the microarray system is described. Applications employing single-color detection as well as dual-color detection to isolate differentially expressed genes among thousands of genes are demonstrated.

Blotting, Northern↗
PubMed1998

Detection and quantitation of circulating cancer cells in the peripheral blood of lung cancer patients.

Detection and quantitation of circulating cancer cells in peripheral blood may improve cancer staging and monitoring. This study explored the feasibility of using circulating cancer cell detection in peripheral blood for the rapid assessment of chemotherapeutic response. Cytokeratin 19 mRNA was amplified by nested reverse transcriptase-PCR in the peripheral blood of 29 healthy volunteers, 33 pneumonia patients, and 86 lung cancer patients. Circulating cancer cells in the peripheral blood were semiquantitatively determined by taking the ratio of cytokeratin 19 band intensity from the second round of nested PCR to the glyceraldehyde-3-phosphate dehydrogenase band intensity from the first round of PCR amplification. The detection limit of the method was 1 cancer cell in 107 peripheral blood mononuclear cells. The positive detection rate was 40% for lung adenocarcinoma patients of all stages, 41% for squamous carcinoma patients of all stages, and 27% for small cell lung cancer patients. Only one control sample from a pneumonia patient showed a positive result (1.6%). The quantitative method reliably and sensitively estimated cancer cell numbers in the peripheral blood of lung cancer patients. Serial measurement of the relative number of circulating cancer cells correlated with the tumor burden and treatment response of patients. This method may help rapidly assess the efficacy of anticancer treatment, redefine cancer staging, and facilitate the design of better therapeutic strategies for the treatment of cancer patients.

Adult↗
PubMed1997

Restriction mapping of genes by capillary electrophoresis with laser-induced fluorescence detection.

Restriction mapping is one of the essential steps in gene analysis and molecular biology studies. Slab gel electrophoresis is the traditional way to separate DNA fragments for restriction mapping. However, slab gel electrophoresis does not provide sufficient resolution as required in many mapping applications, and the use of radioisotopes in traditional mapping methods creates health hazards. In the present study, capillary electrophoresis coupled with laser-induced fluorescence detection and a modified partial digestion mapping procedure was developed to map DNA fragments. By using capillary electrophoresis, a restriction map of genomic lambda phage clone of human interleukin 5 receptor alpha chain (IL5R alpha) gene was constructed. The IL5R alpha gene was analyzed to have five XbaI enzyme cutting sites at locations 1370, 2290, 2950, 5430, and 9330. The system was further characterized by using pBluescript SK(+) phagemid DNA as a model. Using a sequence-derived map as a reference, the pBluescript SK(+) restriction map constructed by capillary electrophoresis had an accuracy greater than 90%.

DNA↗
PubMed1997

Comparative pharmacokinetics of caffeine and three metabolites in clinically normal horses and donkeys.

OBJECTIVE: To determine whether clearance of capacity-limited drugs in horses differs from that in donkeys by comparing the serum disposition of caffeine and its metabolites, theophylline, theobromine, and paraxanthine after i.v. administration of caffeine to horses and donkeys. ANIMALS: 4 healthy horses and 5 healthy donkeys. PROCEDURE: Blood samples were collected from each animal at time 0 (before) and 5, 10, 15, 20, 30, and 45 minutes, and 1, 2, 3, 4, 6, 8, 12, 24, 30, 36, 48, 54, 60, 72, and 96 hours after IV administration of a bolus of caffeine. Serum was analyzed in triplicate by high-performance liquid chromatography to determine caffeine, theophylline, theobromine, and paraxanthine concentrations. The serum concentration-time curves for each animal were analyzed separately to estimate model-independent pharmacokinetic variables. RESULTS: Mean pharmacokinetic values for caffeine, theophylline, and paraxanthine did not differ significantly in horses, compared with donkeys. Mean peak serum concentration of theobromine was significantly higher in donkeys, compared with horses. CONCLUSION: Clearance of the capacity-limited drug caffeine does not appear to differ in horses, compared with donkeys. CLINICAL RELEVANCE: For some drugs that undergo hepatic metabolism, the dose and dose interval used for horses may be appropriate for use in donkeys.

Animals↗
PubMed1994

Agonist-induced down-regulation of platelet-activating factor receptor gene expression in U937 cells.

Prolonged exposure (8-24 h) of human promonocytic U937 cells to 100 nM 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (carbarmyl-PAF), a non-metabolizable analogue of platelet-activating factor (PAF), reduced the numbers of PAF receptors by 50-75%, as determined by the radioligand-binding assay. To clarify whether the down-regulation of receptor numbers is due to decreased expression level of the PAF-receptor gene, the effect of carbamyl-PAF on the steady-state level of PAF-receptor mRNA was examined by a highly sensitive reverse-transcriptase PCR method. A 50% decline in the level of PAF-receptor mRNA was observed in U937 cells pretreated with 100 nM carbamyl-PAF for 24 h. The effect of carbamyl-PAF was dose-dependent, with an EC50 value around 10 nM. PAF-receptor antagonist, SRI-63675, was able to attenuate the effect of carbamyl-PAF. Furthermore lysoPAF, at 1 uM, was unable to induce a significant decrease in PAF-receptor mRNA after incubation for 24 h, indicating that the effect of carbamyl-PAF was specific. The half-life of the PAF-receptor mRNA measured in the presence of actinomycin D was unaffected by carbamyl-PAF treatment. In contrast, nuclear run-off experiments demonstrated that the transcription rate of the PAF-receptor gene in carbamyl-PAF-treated cells was about 65% of that in control cells. These results suggest that the PAF receptor in U937 cells is subject to down-regulation by agonist, at least partly, at the transcriptional level.

Base Sequence↗
PubMed1994

Relative overexpression of collagen type I and collagen type III messenger ribonucleic acids by uterine leiomyomas during the proliferative phase of the menstrual cycle.

Uterine leiomyomas contain abundant quantities of extracellular matrix (ECM). We characterized the localization of three ECM proteins, collagen type I, collagen type III, and fibronectin, in leiomyomas and adjacent normal myometrium. We further examined the expression of messenger ribonucleic acid (mRNA) levels for these proteins from 1) women who were in various stages of the menstrual cycle and 2) multiple leiomyomas from the same patient. Immunohistochemical staining showed that fibronectin was localized primarily around individual smooth muscle cells (SMC). Collagen type I was distributed across the ECM and also in the cytoplasm of SMC. Collagen type III was found primarily in the ECM. Leiomyomas showed more intense staining for collagen types I and III than corresponding normal myometrium. Northern blot and densitometric analysis showed that both collagen type I and III mRNAs were consistently elevated in leiomyomas relative to the adjacent myometrium in patients who were in the proliferative phase of the menstrual cycle (P < 0.02 for both). These differences were not evident in the secretory phase. Fibronectin mRNA levels were quite variable, and there were no significant differences between the two tissues at any stage of the menstrual cycle. We conclude that leiomyomas show increased levels of mRNA for collagen types I and III relative to the normal myometrium. However, this difference is only manifested during the proliferative phase of the menstrual cycle. These findings suggest that leiomyomas may be more sensitive to the hormonal changes that occur during the menstrual cycle than is normal myometrium.

Blotting, Northern↗
PubMed1992

Immobilization chemistries suitable for use in the BIAcore surface plasmon resonance detector.

Surface plasmon resonance detectors, such as the BIAcore instrument produced by Pharmacia, show promise for the detection and quantitation of macromolecular interactions in a label-free mode. Such detectors rely on the covalent immobilization of one of the interacting species onto the sensing surface. To date, the only published chemistry for this purpose is reaction of primary amino-containing ligands with an N-hydroxysuccinimide (NHS) ester-activated surface. In an effort to increase the versatility of the BIAcore with respect to immobilizing ligands, we undertook an investigation of activation chemistries compatible with this system. Using readily available reagents, we demonstrated that the carboxylated dextran-coated sensing surface could be easily converted to functions other than NHS-esters, including amine-activated, hydrazine-activated, and sulfhydryl-activated surfaces. In addition, use was made of the streptavidin/biotin interaction to probe chemical modifications of the sensing surface, by employing specifically modified biotin derivatives.

Avidin↗
PubMed1992

Localization of HPV-16 DNA sequence in CaSki cells by electron microscopic hybridocytochemistry.

We investigated the HPV-16 DNA sequence in the CaSki cervical carcinoma cell line by electron microscopic hybridocytochemistry using biotinylated HPV-16/18 probes. At the light microscopic level, reaction product of hybridized HPV-16 DNA sequence was not seen in the cytoplasm but appeared as spots or rods randomly distributed in the nuclei. By electron microscopy, reaction product was seen aggregated in several regions in the nuclei. Most of the stained areas did not reveal particular architecture but showed part of the chromatin structure. In other nuclei, reaction product was observed to be associated with strings of loop-like structure, and some stained loops were seen to be connected directly to the nuclear filamentous chromatin structure. The skeletonized images of hybridized HPV-16 DNA in the nuclei were illustrated by computerized image analysis. In conclusion, we have demonstrated the HPV-16 DNA sequence in the nuclei of CaSki cells by electron microscopy. The identification of stained areas localized only in the chromatin suggests an integrated form of HPV-16 DNA sequence in the cells. This method could be used to identify an integrated or episomal form of viral DNA in the virus-containing cells.

Biotin↗
PubMed1991

High-sensitivity two-color detection of double-stranded DNA with a confocal fluorescence gel scanner using ethidium homodimer and thiazole orange.

Ethidium homodimer (EthD; lambda Fmax 620 nm) at EthD:DNA ratios up to 1 dye:4-5 bp forms stable fluorescent complexes with double-stranded DNA (dsDNA) which can be detected with high sensitivity using a confocal fluorescence gel scanner (Glazer, A.N., Peck, K. & Mathies, R.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3851-3855). However, on incubation with unlabeled DNA partial migration of EthD takes place from its complex with dsDNA to the unlabeled DNA. It is shown here that this migration is dependent on the fractional occupancy of intercalating sites in the original dsDNA-EthD complex and that there is no detectable transfer from dsDNA-EthD complexes formed at 50 bp: 1 dye. The monointercalator thiazole orange (TO; lambda Fmax 530 nm) forms readily dissociable complexes with dsDNA with a large fluorescence enhancement on binding (Lee, L.G., Chen, C. & Liu, L.A. (1986) Cytometry 7, 508-517). However, a large molar excess of TO does not displace EthD from its complex with dsDNA. When TO and EthD are bound to the same dsDNA molecule, excitation of TO leads to efficient energy transfer from TO to EthD. This observation shows the practicability of 'sensitizing' EthD fluorescence with a second intercalating dye having a very high absorption coefficient and efficient energy transfer characteristics. Electrophoresis on agarose gels, with TO in the buffer, of preformed linearized M13mp18 DNA-EthD complex together with unlabeled linearized pBR322 permits sensitive fluorescence detection in the same lane of pBR322 DNA-TO complex at 530 nm and of M13mp18 DNA-EthD complex at 620 nm. These observations lay the groundwork for the use of stable DNA-dye intercalation complexes carrying hundreds of chromophores in two-color applications such as the physical mapping of chromosomes.

Benzothiazoles↗
PubMed1990

Optimization of high-sensitivity fluorescence detection.

We present general expressions for the number of photons emitted by a fluorescent chromophore as a function of the intensity and the duration of illumination. The aim is to find optimal conditions for detecting fluorescent molecules in the presence of both ground-state depletion and photodestruction. The key molecular parameters are the absorption coefficient epsilon, the excited singlet-state lifetime tau f, the excited triplet-state decay rate kT, the intersystem crossing rate kI, and the intrinsic photodestruction time tau d. When only singlet saturation and photochemistry are important, the signal-to-noise ratio depends on two fundamental variables: k, the ratio of the absorption rate ka to the observed fluorescence decay rate kf, and tau, the ratio of the duration of illumination taut to the intrinsic photodestruction time tau d. Equations are also developed for the more complicated cases when triplet formation and photochemistry are important. This theory was tested by measuring the fluorescence from a solution of beta-phycoerythrin flowed through a focused argon ion laser beam. The dependence of the fluorescence on the incident light intensity and the illumination time agrees well with the theoretical prediction for singlet saturation and photochemistry. The signal-to-noise ratio is optimal when the light intensity and the flow rate are adjusted so that both K and tau are close to unity (5 X 10(22) photons cm-2 s-1 and a transit time tau t of 700 mus). This analysis should be useful for optimizing fluorescence detection in DNA sequencing, chromatography, fluorescence microscopy, and single-molecular fluorescence detection.

Fluorescence↗
PubMed1990

A stable double-stranded DNA-ethidium homodimer complex: application to picogram fluorescence detection of DNA in agarose gels.

The complex between double-stranded DNA and ethidium homodimer (5,5'-diazadecamethylene)bis(3,8-diamino-6-phenylphenanthridini um) cation, formed at a ratio of 1 homodimer per 4 or 5 base pairs, is stable in agarose gels under the usual conditions for electrophoresis. This unusual stability allows formation of the complex before electrophoresis and then separation and detection in the absence of background stain. Competition experiments between the preformed DNA-ethidium homodimer complex and a 50-fold molar excess of unlabeled DNA show that approximately one-third of the dye is retained within the original complex independent of the duration of the competition. However, dye-extraction experiments show that these are not covalent complexes. After electrophoretic separation, detection of bands containing 25 pg of DNA was readily achieved in 1-mm thick agarose gels with laser excitation at 488 nm and a scanning confocal fluorescence imaging system. The band intensity was linear with the amount of DNA applied from 0.2 to 1.0 ng per lane and with the number of kilobase pairs (kbp) per band within a lane. Analysis of an aliquot of a polymerase-chain-reaction mixture permitted ready detection of 80 pg of a 1.6-kbp amplified fragment. The use of the ethidium homodimer complex together with laser excitation for DNA detection on gels is at least two orders of magnitude more sensitive than conventional fluorescence-based procedures. The homodimer-DNA complex exemplifies a class of fluorescent probes where the intercalation of dye chromophores in DNA forms a stable, highly fluorescent ensemble.

DNA↗