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K Peck

Publications and source records attributed to K Peck.

At least 19 recordsLinked to original sources
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↗
PubMed1990

Is serum gamma-glutamyl transferase a good marker of alcohol intake in stroke patients?

Serial serum gamma-glutamyl transferase (GGT) levels were estimated in 23 consecutive patients admitted to hospital with a diagnosis of acute stroke. The proportion of patients with elevated GGT levels in the initial, 36-hour and 72-hour samples was 13%, 30% and 24% respectively, suggesting a transient rise following a stroke. Patients with a history of diabetes mellitus had an initial serum GGT level 21 IU/l (95% confidence interval 6 to 37) higher than non-diabetics. We conclude that GGT levels after a stroke may reflect a history of diabetes and cerebral damage as well as the usual more established causes. Physicians, therefore, should be wary of attributing all unexplained high GGT levels in stroke patients to alcohol.

Acute Disease↗
PubMed1989

Catalysis by the RNA subunit of RNase P--a minireview.

RNase P, an enzyme that contains both RNA and protein components, cleaves tRNA precursors to generate mature 5' termini. The catalytic activity of RNase P resides in the RNA component, with the protein cofactor affecting the rate of the cleavage reaction. The reaction is also influenced by the nature of the tRNA substrate.

Catalysis↗
PubMed1989

Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin.

A theory for single-molecule fluorescence detection is developed and then used to analyze data from subpicomolar solutions of B-phycoerythrin (PE). The distribution of detected counts is the convolution of a Poissonian continuous background with bursts arising from the passage of individual fluorophores through the focused laser beam. The autocorrelation function reveals single-molecule events and provides a criterion for optimizing experimental parameters. The transit time of fluorescent molecules through the 120-fl imaged volume was 800 microseconds. The optimal laser power (32 mW at 514.5 nm) gave an incident intensity of 1.8 x 10(23) photons.cm-2.s-1, corresponding to a mean time of 1.1 ns between absorptions. The mean incremental count rate was 1.5 per 100 microseconds for PE monomers and 3.0 for PE dimers above a background count rate of 1.0. The distribution of counts and the autocorrelation function for 200 fM monomer and 100 fM dimer demonstrate that single-molecule detection was achieved. At this concentration, the mean occupancy was 0.014 monomer molecules in the probed volume. A hard-wired version of this detection system was used to measure the concentration of PE down to 1 fM. This single-molecule counter is 3 orders of magnitude more sensitive than conventional fluorescence detection systems.

Lasers↗
PubMed1988

Optical errors in a liquid chromatography absorbance cell.

The shadowgraph method is used to visualize mobile phase flow through a liquid chromatography absorbance cell with "Z" flow geometry. It is shown that mixing is incomplete in the cell. Composition gradients persist throughout the elution of any sample. The gradients systematically change as the sample flows through the cell. Additionally, window wedge angles are measured in several cells. The implications for low noise absorbance detectors are discussed.

Azo Compounds↗
PubMed1988

Rapid scanning thermal lens/laser transmission densitometer.

An automated densitometer based on the thermal lens principle is described. The apparatus also operates as a conventional laser transmission densitometer. Comparison of the performance in both modes shows that thermal lens densitometry provides lower detection limits, but that transmission densitometry is more satisfactory at high optical densities. The instrument is characterized with proteins separated by SDS-PAGE and stained with Coomassie Brilliant Blue G250.

Densitometry↗
PubMed1984

Should chronic transfusions be matched for antigens other than ABO and Rho(D)?

It has been recommended that red blood cell transfusions to patients with hemoglobinopathy or aplastic anemia be matched for antigens other than ABO and Rho(D). We studied 1,010 patients with disorders that often lead to repetitive transfusion. The frequency of transfused patients with clinically important antibodies was not significantly different among the disease groups except for those with lymphocytic leukemia. The frequency of multiple red cell antibodies was about 3% overall. Most antibodies (71%) developed early in the transfusion course, before the 15th transfusion. From the standpoints of frequency of alloimmunization, multiplicity of antibodies, and time course of antibody development, patients with hemoglobinopathy and aplastic anemia were not significantly different from other transfused patients. Matching for antigens other than ABO or Rho(D) might increase costs in our hospital by 40,000-370,000 dollars per year for these patients. Because morbidity or mortality due to these antibodies is rare, antigen matching for other than ABO and Rho(D) is not cost-effective.

ABO Blood-Group System↗