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Biomedical subjects

D L Barker

Publications and source records attributed to D L Barker.

At least 19 recordsLinked to original sources
PubMed2000

Automated parallel DNA sequencing on multiple channel microchips.

We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated four-color confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect. We present experimental evidence consistent with the predictions of the theory.

Automation↗
PubMed2000

Mining the human genome using microarrays of open reading frames.

To test the hypothesis that the human genome project will uncover many genes not previously discovered by sequencing of expressed sequence tags (ESTs), we designed and produced a set of microarrays using probes based on open reading frames (ORFs) in 350 Mb of finished and draft human sequence. Our approach aims to identify all genes directly from genomic sequence by querying gene expression. We analysed genomic sequence with a suite of ORF prediction programs, selected approximately one ORF per gene, amplified the ORFs from genomic DNA and arrayed the amplicons onto treated glass slides. Of the first 10,000 arrayed ORFs, 31% are completely novel and 29% are similar, but not identical, to sequences in public databases. Approximately one-half of these are expressed in the tissues we queried by microarray. Subsequent verification by other techniques confirmed expression of several of the novel genes. Expressed sequence tags (ESTs) have yielded vast amounts of data, but our results indicate that many genes in the human genome will only be found by genomic sequencing.

Cell Line↗
PubMed1998

Analysis of multiplexed short tandem repeat (STR) systems using capillary array electrophoresis.

The profiling of polymorphic short tandem repeat (STR) markers is being applied to human identification, parentage testing and genetic mapping. Reliable genotyping of these markers is facilitated by polymerase chain reaction (PCR) amplification and high-resolution electrophoretic separation. Capillary array electrophoresis (CAE) offers very rapid, high-resolution separation of the amplified DNA and potential for automated sample processing not realized employing conventional slab-gel electrophoresis. The use of CAE to type DNA samples amplified at 11 genetic loci in multiplex profiles is presented. Two sets totaling 208 samples were amplified in a multiplex fashion using AmpFlSTR-Blue or AmpFlSTR-Green I and analyzed in a blind study using CAE. With the exception of one sample, the CAE genotyping results were in complete agreement with results obtained using a single-capillary system or two slab-gel electrophoresis systems. The sample, genotype TH01 7/10, migrated similar to TH01 6.3/9.3 allele sizes, which suggested a potential band migration shift. The recommended approach to such an observation is to analyze the sample again. The sample was rerun and correct genotype verified. Allelic ladder samples were analyzed multiple times by CAE to determine sizing accuracy and precision. The sizing of over 240 allelic ladder samples yielded an average within-run precision of +/- 0.13 bp and between-run precision of +/- 0.21 bp for fragments up to 350 bp. The CAE protocols permit processing of up to 96 multiplex STR samples in under 70 min.

Alleles↗
PubMed1998

Mini-haptics to improve capsular fixation of plate-haptic silicone intraocular lenses.

PURPOSE: To evaluate the effects of a new mini-haptic design on the strength and stability of capsular bag fixation of plate-haptic silicone intraocular lenses (IOLs) and determine whether this design encourages the growth of regenerating lens material or fibrous tissue around the haptic biomaterial and thus improves lens fixation in the capsular bag. SETTING: Center for Research on Ocular Therapeutics and Biodevices, Storm Eye Institute, Medical University of South Carolina, Department of Ophthalmology, Charleston, South Carolina, USA. METHODS: Six rabbits had bilateral continuous curvilinear capsulorhexis, phacoemulsification, and plate-haptic silicone IOL implantation. Each rabbit had a small-hole plate IOL (Chiron C10UB) implanted in the right eye and a mini-haptic plate IOL (Chiron C40UB) in the left eye. All rabbits were killed at 2 months. The force required to extract one haptic from the capsular bag was measured with a digital force gauge. Histopathologic analysis was performed on all specimens. RESULTS: The mini-haptic style IOLs required significantly more extraction force than the small-hole design (P = .011). Histopathologically, proliferating lens epithelial cells were observed growing circumferentially around the mini-haptics, causing a 360 degree synechia formation. This formation did not occur with the conventional small-hole plate IOLs used as the control. CONCLUSIONS: Lens epithelial cell proliferation around the mini-haptics significantly improved capsular bag fixation of the plate-haptic silicone IOL. This should decrease the incidence of clinical decentration and dislocation.

Animals↗
PubMed1998

The geographic variation in stroke incidence in two areas of the southeastern stroke belt: the Anderson and Pee Dee Stroke Study.

BACKGROUND AND PURPOSE: South Carolina and the southeastern United States have maintained the highest stroke mortality in the country. The Anderson and Pee Dee Stroke Study is an assessment of cerebrovascular disease incidence in 2 geographically defined communities in the stroke belt. METHODS: Strokes were identified in the Anderson and Pee Dee areas of South Carolina. All hospitalized and out-of-hospital deaths occurring during 1990 among the residents of these 2 areas were included. Strokes were classified by an independent panel of neurologists using a standard protocol that included specific criteria for stroke and subtypes. RESULTS: The overall age-adjusted stroke incidence rates (per 100 000 population) were significantly higher in the Pee Dee population (293.1) compared with Anderson (211.2). The geographic differences were more dramatic in the younger age groups of 35 to 64 years. Likewise, incidence rates for blacks were nearly twice the rates for whites. The rates in the Pee Dee were higher than the rates from other studies in the United States and other parts of the world. Although the stroke subtypes did not vary between the 2 regions, race-sex differences were identified. CONCLUSIONS: High stroke incidence and disease rates persist for all 4 race-sex groups in the Southeast and reflect similar risks as mortality rates. However, geographic variability in stroke rates suggests that the pattern of disease in the region is not so much a "belt" of increased stroke in contiguous areas but rather more a "necklace" of different levels of risk. These results should be useful in the identification of factors associated with this geographic enigma.

Adult↗
PubMed1997

Short tandem repeat typing by capillary array electrophoresis: comparison of sizing accuracy and precision using different buffer systems.

Polymorphic microsatellite markers are widely used in gene discovery and mapping, human identification, agricultural genetics, and diagnosis of triplet-repeat expansion disorders. Reliable genotyping of these markers requires polymerase chain reaction (PCR) amplification and very-high-resolution electrophoresis. Capillary array electrophoresis offers extremely fast, high-resolution separation of DNA and more automated sample processing because labor-intensive slab-gel pouring and sample loading are eliminated. We report a simple, reliable procedure for preparing PCR samples for electrokinetic injection into capillaries using a 96-well tray and float dialysis. We developed an improved sizing standard for genotyping and used it to evaluate systematically the sizing accuracy and precision of low-viscosity, replaceable matrix formulations. Our study sizing over 28,000 alleles yielded an average precision of +/- 0.12 bp for fragments up to 350 bp. Low-viscosity formulations permit low-pressure matrix injection (40 psi) and a turnaround time of 70 min for 48-96 samples.

Alleles↗
PubMed1997

Versatile low-viscosity sieving matrices for nondenaturing DNA separations using capillary array electrophoresis.

The high-resolution separation of double-stranded DNA (dsDNA) has important applications in physical mapping strategies and in the analysis of polymerase chain reaction (PCR) products. Although high-resolution separations of dsDNA by capillary electrophoresis (CE) have been reported, pulsed fields were required to achieve complete resolution of DNA fragments beyond 23 kilobase pairs (kbp). Here, we report a single formulation to separate a broad range (80 bp-40 kbp) of DNA fragments without the use of pulsed fields. We used a low-viscosity sieving medium (ca. 5 cP, at 25 degrees C) based on polyethyleneoxide (PEO) to separate DNA fragments up to 40 kbp. The matrix contained a mixture of 0.5% PEO (Mn 10(6)) to separate fragments up to 1.5 kbp, combined with 0.1% PEO (Mn 8 x 10(6)) to separate fragments between 1-40 kbp, within a single run. All PEO matrix formulations tested were compatible with a variety of intercalating dyes and with two different capillary wall coating methods. We obtained a detection limit of 25 fg of a 200 bp DNA quantitation standard using Vistra Green in the matrix. Resolution was best using short injection times (5 s or less) and low field strengths (approximately 100 V/cm). Sample runs were complete in 70 min, and use of the capillary array electrophoresis (CAE) system permitted high-throughput DNA analysis. The size range separated is approximately 10 times greater than with conventional slab gel separations.

Coloring Agents↗
PubMed1997

Security of capsular fixation: small-versus large-hole plate-hepatic lenses.

PURPOSE: To assess the effect of relatively large positioning holes on the security of capsular bag fixation of plate-haptic silicone intraocular lenses (IOLs). SETTING: Center for Research on Ocular Therapeutics and Biodevices, Department of Ophthalmology, Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina, USA. METHODS: This study tested the hypothesis that larger holes allow ingrowth of lens material, fibrous tissue, or both through them, which helps fixate the lens more firmly in the capsular bag. Five rabbits had bilateral continuous curvilinear capsulorhexis, phacoemulsification, and implantation of a plate-haptic silicone IOL. An IOL with a small, round positioning hole (Staar AA-4203V) was implanted in the right eye in each rabbit, and a large-hole IOL (Staar AA-4203VF) was implanted in the left eye. After 2 months, all rabbits were killed. The force required to extract one haptic from the capsular bag was measured with a digital force meter. All eyes had histopathological analysis. RESULTS: It was slightly more difficult to extract a large-hole IOL from the capsular bag, although this trend was not statistically significant. However, histopathological analysis consistently showed 360 degree synechia formation through the holes, showing that the IOL could be securely fixed in position. CONCLUSIONS: Proliferation of lens epithelial cells through a large positioning hole in a plate-haptic silicone IOL may improve the long-term security of capsular bag fixation. This will help reduce the incidence of IOL decentration and dislocation.

Animals↗
PubMed1996

Sensitivity, reproducibility, and accuracy in short tandem repeat genotyping using capillary array electrophoresis.

The Human Genome Initiative has increased significantly the rate at which disease-causing genes are being mapped and sequenced. New cost-effective methods to locate the genes and to characterize disease-causing mutations require robust, reproducible, and accurate protocols for measuring DNA fragment lengths. Capillary array electrophoresis (CAE) offers rapid, high-resolution separations, high throughput, and sensitive detection. To assess the utility of CAE for the accumulation of genetic information, we tested both sizing accuracy and reproducibility using 48-capillary prototype systems. Two multiplex PCR allelic ladder standards and several CA-repeat markers were analyzed in > 100 runs. Reproducibility in typing > 8000 genotypes reveals a standard deviation of less than 0.2 bp on these systems under optimized conditions. However, sequence-dependent migration anomalies were observed at most simple sequence loci even when analyzed under denaturing conditions, resulting in a systematic bias in estimated fragment sizes. We show here that, by normalizing results to known typing controls, one can obtain locus-averaged accuracies of < 0.06 bp and normalized results within 1 bp of actual. We detect as little as a 1:30,000 dilution of a DNA quantitation standard stained with highly sensitive intercalating dyes, indicating an 80-zeptomole sensitivity limit. However, to obtain reproducible electrokinetic injection, approximately 200 attomoles of fluorescein-labeled DNA is required. These sensitivity limits, sizing precision, and accuracy, together with the 1-hr run times for 48-96 samples, indicate that CAE is a viable method for high-throughput genetic analysis of simple sequence repeat polymorphisms.

Capillary Action↗
PubMed1994

Dietary carnitine did not influence performance and carcass composition of broiler chickens and young turkeys fed low- or high-fat diets.

Two experiments were conducted to determine the influence of supplemental dietary carnitine on performance and carcass composition of young turkeys and broiler chickens. Experiments 1 and 2 were done with poults to 21 d of age and broilers to 45 d of age, respectively. Three dietary concentrations of L-carnitine (0, 50, or 100 mg/kg) were fed in a factorial arrangement with two concentrations of animal-vegetable fat (A-V fat), 2.25 or 8% in Experiment 1 and 1 or 5% in Experiment 2. L-Carnitine did not affect (P > .05) BW gain or feed efficiency in either experiment, irrespective of concentration of dietary fat. Similarly, proximate composition of 21-d-old poults and 45-d-old broilers was not changed by L-carnitine. Increasing levels of fat supplementation improved feed efficiency of poults and improved 45-d BW and feed efficiency of broilers. Carcass fat of poults and broilers was increased (P < or = .05) by supplemental fat at the expense of carcass water and protein.

Animal Feed↗
PubMed1993

Research note: vitamin E status of turkey poults as influenced by different dietary vitamin E sources, a bile salt, and an antioxidant.

An experiment was conducted to determine the effects of level and chemical form of dietary vitamin E on alpha-tocopherol status of poults. The effects of a dietary bile salt and an antioxidant on concentrations of alpha-tocopherol in serum and liver were also tested. Six dietary treatments were obtained by supplementing a corn-soybean meal diet with 12 IU of DL-alpha-tocopheryl acetate (TA)/kg (LE), 12 IU of TA plus 800 mg of sodium taurocholate/kg (LB), 12 IU of TA plus 500 mg of ethoxyquin/kg (LS), 12 IU of D-alpha-tocopheryl polyethylene glycol 1,000 succinate (TPGS)/kg (LT), 100 IU of TA/kg (HE), and 100 IU of TPGS/kg (HT). Growth rate and feed efficiency of poults were unaffected (P > .05) by dietary treatments. The HE diet increased alpha-tocopherol in liver (P < .01) at 14 and 21 days of age. Liver and serum alpha-tocopherol concentrations were unaffected by dietary TPGS (LT and HT diets) at any age. Serum alpha-tocopherol concentration was unaffected by dietary treatments at 5 days of age. The HE diet, however, increased (P < .01) serum alpha-tocopherol at 9, 14, and 21 days of age. Age-related changes in alpha-tocopherol concentration were observed. Both liver and serum alpha-tocopherol decreased markedly from 1 to 14 days of age. The HE diet only partly alleviated the reduction of alpha-tocopherol in liver and serum. The water-soluble form of vitamin E, TPGS, dietary sodium taurocholate, or dietary ethoxyquin, did not prevent the marked decline in alpha-tocopherol concentration of liver and serum during the 21-day experiment.

Animals↗
PubMed1990

Autoradiography using storage phosphor technology.

We describe the application of photostimulable storage phosphor imaging plates to autoradiography of samples labeled with 32P, 14C and 35S. Imaging plates can detect a 32P-labeled sample at an exposure level of 1 disintegration/mm2, and 14C and 35S at 25 disintegrations/mm2. Compared to X-ray film, imaging plates offer increased sensitivity of 15- to 250-fold for 32P and 20- to 100-fold for 14C and 35S. Resolution is in the 0.3 mm range and is therefore suitable for any gel or blot application.

Autoradiography↗
PubMed1988

Muscarinic antagonist enhances one-trial food-aversion learning in the mollusc Pleurobranchaea.

One hour before training, in two replicate studies on the sea slug Pleurobranchaea californica, all animals (N = 114) received body-cavity injections of scopolamine, oxotremorine, or the equivalent volume of the saline/seawater vehicle that was used to inject the drugs. The low drug doses (2 mumol/kg) were near the threshold for generating observable neurophysiological responses, but did not affect feeding thresholds arising to a stimulus derived from beer (Sbr) and to one derived from squid (Ssq). Before training, the animals did not discriminate between Sbr and Ssq, as indicated by similar thresholds to both stimuli. During training, experimental animals in each injection group received Sbr alone for 10 sec and then paired with electric shocks for 50 sec; control animals received shocks 1 hr after Sbr. Postconditioning tests began 12 hr after training and were repeated once daily thereafter. After training, all experimental groups exhibited the expected aversive behavior to Sbr, as indicated by 100- to 1000-fold increases in feeding thresholds, and retained low thresholds to Ssq, but the scopolamine animals were better able to discriminate between Sbr and Ssq than either of the other experimental groups. The aversive responses to Sbr increased over a 3-day period, but there appeared to be no difference between injection groups on such a long-duration "consolidation" phase. Of the control groups, only the scopolamine animals exhibited low feeding thresholds to both Sbr and Ssq. The other control groups exhibited similar behavior as the experimental animals, indicating that associative factors relating to the 1-hr separation between Sbr and shocks may have produced the behavior in these control animals. Thus, by comparison to the other injections, scopolamine 1) increased the ability of the experimental animals to make the discrimination between Sbr and Ssq, and 2) prevented learning to avoid Sbr in the control animals. An accompanying paper provides a detailed characterization of muscarinic receptor pharmacology in Pleurobranchaea.

Animals↗
PubMed1988

Migration and differentiation of neural crest and ventral neural tube cells in vitro: implications for in vitro and in vivo studies of the neural crest.

During vertebrate development, neural crest cells migrate from the dorsal neural tube and give rise to pigment cells and most peripheral ganglia. To study these complex processes it is helpful to make use of in vitro techniques, but the transient and morphologically ill-defined nature of neural crest cells makes it difficult to isolate a pure population of undifferentiated cells. We have used several established techniques to obtain neural crest-containing cultures from quail embryos and have compared their subsequent differentiation. We confirm earlier reports of neural crest cell differentiation in vitro into pigment cells and catecholamine-containing neurons. However, our results strongly suggest that the 5-HT-containing cells that develop in outgrowths from thoracic neural tube explants are not neural crest cells. Instead, these cells arise from ventral neural tube precursors that normally give rise to a population of serotonergic neurons in the spinal cord and, in vitro, migrate from the neural tube. Therefore, results based on previously accepted operational definitions of neural crest cells may not be valid and should be reexamined. Furthermore, the demonstration that cells from the ventral (non-neural crest) part of the neural tube migrate in vitro suggests that the same phenomenon may occur in vivo. We propose that the embryonic "neural trough," as well as the neural crest, may contribute to the PNS of vertebrates.

Animals↗
PubMed1986

Characterization of muscarinic cholinergic receptors in the crab nervous system.

The selective muscarinic antagonist L-[3H]-quinuclidinyl benzilate (L-[3H]QNB) binds reversibly and with high affinity (KD = 0.3 nM) to a single population (Bmax = 105 fmol/mg protein) of specific sites in nervous tissue of the crab Cancer magister. The binding site is stereoselective; (-)QNB is over 200 times more potent than (+)QNB as an inhibitor of specific L-[3H]QNB binding. The muscarinic antagonists scopolamine and atropine are over 10,000 times more potent inhibitors of L-[3H]QNB binding than the nicotinic antagonists decamethonium and d-tubocurarine. The muscarinic agonists oxotremorine, pilocarpine, arecoline, and carbachol also compete effectively for the L-[3H]QNB binding site. This pharmacological profile strongly suggests the presence of classical muscarinic receptors in the crab nervous system. These receptors are localized to nervous tissue containing cell bodies and neuropil, whereas specific L-[3H]QNB binding is low or absent in peripheral nerve, skeletal muscle, and artery.

Animals↗
PubMed1985

Sprouting and functional regeneration of an identified serotonergic neuron following axotomy.

An identified serotonergic neuron (C1) in the cerebral ganglion of Helisoma trivolvis sprouts following axotomy and rapidly (seven to eight days) regenerates to recover its regulation of feeding motor output from neurons of the buccal ganglia. The morphologies of normal and regenerated neurons C1 were compared. Intracellular injection of the fluorescent dye, Lucifer Yellow, into neuron C1 was compared with serotonin immunofluorescent staining of the cerebral and buccal ganglia. The two techniques revealed different and complimentary representations of the morphology of neuron C1. Lucifer Yellow provided optimal staining of the soma, major axon branches, and dendritic arborization. Immunocytochemical staining revealed terminal axon branches on distant targets and showed an extensive plexus of fine fibers in the sheaths of ganglia and nerve trunks. In addition to C1, serotonin-like immunoreactivity was localized in approximately 30 other neurons in each of the paired cerebral ganglia. Only cerebral neurons C1 had axons projecting to the buccal ganglia. No neuronal somata in the buccal ganglia displayed serotonin-like immunoreactivity. Observations of regenerating neurons C1 demonstrated: Actively growing neurites, both in situ and in cell culture, displayed serotonin-like immunoreactivity; severed distal axons of C1 retained serotonin-like immunoreactivity for up to 28 days; axotomized neurons C1 regenerated to restore functional control over the feeding motor program.

Animals↗
PubMed1985

Stereoselective L-[3H]quinuclidinyl benzilate-binding sites in nervous tissue of Aplysia californica: evidence for muscarinic receptors.

The muscarinic antagonist L-[3H]quinuclidinyl benzilate (L-[3H]QNB) binds with a high affinity (Kd = 0.77 nM) to a single population of specific sites (Bmax = 47 fmol/mg of protein) in nervous tissue of the gastropod mollusc, Aplysia. The specific L-[3H]QNB binding is displaced stereoselectively by the enantiomers of benzetimide, dexetimide, and levetimide. The pharmacologically active enantiomer, dexetimide, is more potent than levetimide as an inhibitor of L-[3H]QNB binding. Moreover, the muscarinic cholinergic ligands, scopolamine, atropine, oxotremorine, and pilocarpine are effective inhibitors of the specific L-[3H]QNB binding, whereas nicotinic receptor antagonists, decamethonium and d-tubocurarine, are considerably less effective. These pharmacological characteristics of the L-[3H]QNB-binding site provide evidence for classical muscarinic receptors in Aplysia nervous tissue. The physiological relevance of the dexetimide-displaceable L-[3H]QNB-binding site was supported by the demonstration of the sensitivity of the specific binding to thermal denaturation. Specific binding of L-[3H]QNB was also detected in nervous tissue of another marine gastropod, Pleurobranchaea californica. The characteristics of the Aplysia L-[3H]QNB-binding site are in accordance with studies of numerous vertebrate and invertebrate tissues indicating that the muscarinic cholinergic receptor site has been highly conserved through evolution.

Animals↗