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PubMed1994

Manifold sequencing: efficient processing of large sets of sequencing reactions.

Automated instruments for DNA sequencing greatly simplify data collection in the Sanger sequencing procedure. By contrast, the so-called front-end problems of preparing sequencing templates, performing sequencing reactions, and loading these on the instruments remain major obstacles to extensive sequencing projects. We describe here the use of a manifold support to prepare and perform sequencing reactions on large sets of templates in parallel, as well as to load the reaction products on a sequencing instrument. In this manner, all reaction steps are performed without pipetting the samples. The strategy is applied to sequencing PCR-amplified clones of the human mitochondrial D-loop and for detection of heterozygous positions in the human major histocompatibility complex class II gene HLA-DQB, amplified from genomic DNA samples. This technique will promote sequencing in a clinical context and could form the basis of more efficient genomic sequencing strategies.

Base Sequence↗
PubMed1991

Production of quail-chick chimaeras by blastoderm cell transfer.

1. Quail-chick chimaeras were produced by injecting dissociated quail blastoderm cells into chick embryos. 2. Quail blastoderms were removed from the yolk and the cells were dispersed by trypsin treatment or pipetting. The cell suspension (1 to 5 microliters) was injected into the subgerminal cavity of unincubated chick embryos. The chick embryos were then cultured in recipient eggshells. 3. Quail blastoderm cells injected into the chick embryos adhered to the chick embryonic cells. The rates of hatching were 8.6% (38 chicks from 441 eggs) and 40.3% (48 chicks from 119 eggs) when the volumes of the cell suspension injected were 3 to 5 microliters and 1 microliter, respectively. 4. Seven out of 86 hatched birds were clearly identified as being chimaeric because part of the feather colouring was of quail specificity. In addition to these chimaeric birds, there were 8 chimaeric embryos which died before hatching. The distribution patterns of the quail feathers were varied among the chimaeric birds and embryos. 5. This technique provides a basis for the investigation of chick embryo cryopreservation, genetic transformation and analysis of cell lineage of chickens.

Animals↗
PubMed1997

Effects of arm support on shoulder and arm muscle activity during sedentary work.

The aim of this study was to evaluate different arm supports by comparing the activity of shoulder and arm muscles during various work tasks, with and without the lower arm supported. Twelve female subjects, aged between 23 and 37 years, were asked to perform three types of tasks: typing, simulated assembly work (in two different positions), and pipetting. The supports used were: fixed arm support (FIX), horizontal movable arm support (HOR), and spring-loaded arm support (SLA). During the experiments, the electromyograms (EMG) of four muscles were simultaneously recorded: m. deltoideus anterior and lateralis, m. trapezius pars descendens and m. extensor carpi radialis brevis. Normalization was made against maximum isometric contraction. The mean values of the normalized EMG levels showed a reduced EMG level of the shoulder muscles when using arm supports in all the tasks, and for all muscles but the wrist extensor, compared to the EMG levels without arm supports. The horizontal movable support was more effective in reducing the EMG levels of the shoulder muscles than other arm supports, in tasks at table height. Thus, it is possible to reduce muscle activity of the shoulder region by using arm supports. Further research is needed to make biomechanical calculations to compare the EMG level of these muscles using suspension and the effects of inclination of work task.

Adult↗
PubMedDate not supplied

Aggregation of human sperm at higher temperature is due to hyperactivation.

Chemotaxis of sperm cells to chemicals and hormones, such as progesterone, helps us to understand the concept of sperm transport. Here, the hypothesis was that heat increased sperm hyperactive motility, which caused the sperm to aggregate at the higher temperature. The objectives were (1) to determine the concentration of sperm at both halves of an artificial female reproductive tract made from a hermetically sealed cryopreservation straw filled with culture medium and placed with each end at different temperatures, and (2) to analyze the motility or kinematic parameters and hyperactivation of sperm found at the different temperatures. Cryopreserved-thawed human donor sperm (N = 6) were pooled and processed through 2-layer colloid solution. Analyses of the motile sperm were carried out and the washed sperm were homogeneously mixed and pipetted into several 0.5-mL French cryopreservation straws and heat-sealed. The control substance, consisting of acid-treated sperm, was also placed in several straws. The plastic straws of sperm were placed half at 23 degrees C and half was at either 37 or 40 degrees C. After 4 h, sperm at different sections of the straws were analyzed using the Hamilton Thorn motility analyzer (HTM-C). After 4 h of incubation, the concentration of sperm was doubled at the 40 degrees C heated half of the straw when compared with the other half of the straw at 23 degrees C. There were no differences in sperm concentration in the straw kept half at 37 degrees C and half at 23 degrees C. There were significantly higher percent motility, mean average path velocity, straight line velocity, lateral head displacement, and percent hyperactivation in sperm at the 40 degrees C temperature. The aggregation of sperm at the higher temperature of 40 degrees C may be due to enhanced motility, increased sperm velocities, and a 10-fold increase in hyperactivation at that temperature. The 37 degrees C temperature was not sufficient to attract sperm. Sperm cells migrating into the higher temperature site of ovulation begin nonprogressive hyperactivation movement, which is the physiological "brake" to detain the sperm at the site of ovulation.

Cell Aggregation↗
PubMed1981

A simple solid-phase radioimmunoassay for morphine and related opiates using a mixture of labelled morphine and antiserum pre-precipitated by polyethylene glycol.

A novel, solid-phase radioimmunoassay for morphine and related opiates is described in which radiolabelled morphine, antiserum and polyethylene glycol are premixed before being added to the samples or standards. The mixture can be stored at 2 degrees C and used for several months until decay of the 125I label leads to excessively long counting times. The method was developed using the Roche Abuscreen radioimmunoassay for morphine and has the particular advantages of simplicity (only 2 pipetting steps required instead of 5) and economy (800 assay tubes can be prepared from a 100-tube Abuscreen plus a 50% saving in operator time). The procedure can be applied to other assays providing the polyethylene glycol-precipitated antiserum is stable and that the antibody affinity for the labelled compound is not so high as to prevent displacement of the labelled compound from the antibody binding sites by unlabelled drug.

Barbiturates↗
PubMed2004

Comparison of gravimetric and gas chromatographic methods for assessing performance of textile materials against liquid pesticide penetration.

Penetration of liquid pesticides through textile materials is a criterion for determining the performance of protective clothing used by pesticide handlers. The pipette method is frequently used to apply liquid pesticides onto textile materials to measure penetration. Typically, analytical techniques such as Gas Chromatography (GC) are used to measure percentage penetration. These techniques are labor intensive and costly. A simpler gravimetric method was developed, and tests were conducted to compare the gravimetric and GC methods of analysis. Three types of pesticide formulations and 4 fabrics were used for the study. Diluted pesticide formulations were pipetted onto the test specimens and percentage penetration was measured using the 2 methods. For homogeneous formulation, the results of the two methods were fairly comparable. However, due to the filtering action of the textile materials, there were differences in the percentage penetration between the 2 methods for formulations that were not homogeneous.

Analysis of Variance↗
PubMedDate not supplied

Standardization of clinical cholinesterase measurements.

Previous studies showed that commonly used kits for measuring cholinesterases were not optimal for determining acetylcholinesterase (AChE) activity. Clinical use of different kits and methodologies resulted in AChE levels being reported in different units and activities that were not reproducible among laboratories. Findings such as these led to a revision in California regulations (covering AChE measurements for pesticide worker safety) calling for clinical laboratories to standardize their findings. The laboratories were contacted and invited to participate in a split-sample study of human blood AChE and nonspecific cholinesterase (BChE) assays. Participating laboratories measured erythrocyte (RBC) AChE and/or plasma BChE from undiluted and 50% diluted blood, according to their practices. Aliquots of blood samples were shipped to University of California Davis for measurement, using an optimized semiautomated plate reader version of the method of Ellman. Nine of 25 laboratories sent samples for comparison. Two others performed their own comparisons and submitted data to the state. Best correlations were obtained with BChE activity. Correlations (r(2)) were .88 or above for four of five laboratories for BChE, and above .9 for two of seven laboratories for AChE. Reasons for poor correlations may include difficulties in pipetting RBCs, storage, and processing. A bovine AChE RBC ghost "standard" was devised and tested. Activity of the preparation was maintained at -70 degrees C for approximately 11 months. A test with an East coast laboratory resulted in a high correlation, demonstrating the reliability of the RBC ghost standard and that one laboratory can replicate the AChE findings of another. The overall poor correlation of interlaboratory cholinesterase results points to the need to further standardize sample handling and assay methods.

Acetylcholinesterase↗
PubMed2003

Efficacy of a technique for exposing the mouse lung to particles aspirated from the pharynx.

Recent studies have demonstrated that the mouse lung can be exposed to soluble antigens by aspiration of these antigens from the pharynx. This simple technique avoids the trauma associated with intratracheal instillation. In this study, the pharyngeal aspiration technique was validated for exposing the mouse lung to respirable particles. Using respirable fluorescent amine-modified polystyrene latex beads and beryllium oxide particles, we investigated the localization of aspirated particles within the lung and the relationship between the amount of material placed in the pharynx and the amount deposited in the lung. For exposure, mice were anesthetized with isoflurane in a bell jar, placed on a slant board, and the tongue was gently held in full extension while a 50-microl suspension of particles was pipetted onto the base of the tongue. Tongue restraint was maintained until at least two breaths were completed. Less than a minute after exposure, all mice awoke from anesthesia without visible sequela. There were no significant differences in particle distribution between the left and right side of the lung (p=.16). Particles were widely disseminated in a peribronchiolar pattern within the alveolar region. There was a linear and significant correlation (r2=.99) between the amount administered and the amount deposited in the lung. In beryllium-exposed mice, measurable lung beryllium was 77.5 to 88.2% of the administered beryllium. These findings demonstrate that following aspiration of pharyngeal deposited particles, exposures to the deep lung are repeatable, technically simple, and highly correlated to the administered dose.

Animals↗
PubMed2001

Reliability of isoelectrofocusing for the detection of Hb S, Hb C, and HB D in a pioneering population-based program of newborn screening in Brazil.

Out of 128,326 newborns in the first 6-month period of a population-based screening program in Minas Gerais, Brazil, a second sample was obtained at the age of 6 months from 4,635 carriers of Hbs AS, AC, and AD which were detected by isoelectrofocusing. Discordance in results occurred in only 27 cases (0.6%): in seven there was a history of hemotransfusion; errors during pipetting or transcription of results occurred in seven cases; it was difficult to differenciate between Hbs S and D in eight patients; and the causes were not elucidated in five patients. The incidence of Hbs FS and FSC for the total population was 1:2,800 and 1:3,450, respectively. Isoelectrofocusing is a very reliable method for distinguishing AS, AC, or AD carriers from patients presenting with [corrected] variant hemoglobin and beta(+)-thalassemia combinations, and may be widely used in massive newborn screening programs.

Anemia, Sickle Cell↗
PubMedDate not supplied

A simple method for the purification of human peripheral blood antigen presenting cells (dendritic cells, monocytes/macrophages, and B lymphocytes).

An adherence method was developed that enriches for antigen presenting cells (dendritic cells, monocytes/macrophages, and B lymphocytes) from peripheral blood mononuclear cell (PBMC) preparations. This method utilizes the cells' natural adherence to polystyrene tissue culture dishes and their subsequent removal with K3EDTA after incubation at 4 degrees, with gentle pipeting. Flow cytometric analysis revealed that on average, the enrichment of CD83+ dendritic cells, CD14+ monocytes/ macrophages, and CD19+ B cells increased by 12.5 to 20, 2, and 4 fold, respectively, compared to their initial numbers present in PBMC preparations. Cell viability, determined by trypan blue exclusion, was between 90 and 98%. After the enrichment procedure, the cells could still be activated by tetanus toxoid and this was shown by flow cytometric analysis, as enhancement of class II major histocompatibility complex (MHC) (31% increase) (after antigen treatment). This is a fast and economical alternative to other established methods for the preparation of pure, functionally competent antigen presenting cells derived from peripheral blood.

Adult↗
PubMed2005

A simple, efficient, and economical method for recovering DNA from agarose gel.

A simple method of recovering DNA from agarose gel that is fast, inexpensive, and friendly both to operators and environment is described. Two rows of wells are made in an agarose gel, and a DNA sample is loaded into the well nearest to the negative pole for separation by electrophoresis. Recovery is accomplished by pipetting the DNA-containing TAE buffer from the well near the positive pole after target DNA fragments have migrated into the well. A recovery rate of up to 94 +/- 2.3% was observed with this method.

DNA↗
PubMed2000

Selective uncoupling of p120(ctn) from E-cadherin disrupts strong adhesion.

p120(ctn) is a catenin whose direct binding to the juxtamembrane domain of classical cadherins suggests a role in regulating cell-cell adhesion. The juxtamembrane domain has been implicated in a variety of roles including cadherin clustering, cell motility, and neuronal outgrowth, raising the possibility that p120 mediates these activities. We have generated minimal mutations in this region that uncouple the E-cadherin-p120 interaction, but do not affect interactions with other catenins. By stable transfection into E-cadherin-deficient cell lines, we show that cadherins are both necessary and sufficient for recruitment of p120 to junctions. Detergent-free subcellular fractionation studies indicated that, in contrast to previous reports, the stoichiometry of the interaction is extremely high. Unlike alpha- and beta-catenins, p120 was metabolically stable in cadherin-deficient cells, and was present at high levels in the cytoplasm. Analysis of cells expressing E-cadherin mutant constructs indicated that p120 is required for the E-cadherin-mediated transition from weak to strong adhesion. In aggregation assays, cells expressing p120-uncoupled E-cadherin formed only weak cell aggregates, which immediately dispersed into single cells upon pipetting. As an apparent consequence, the actin cytoskeleton failed to insert properly into peripheral E-cadherin plaques, resulting in the inability to form a continuous circumferential ring around cell colonies. Our data suggest that p120 directly or indirectly regulates the E-cadherin-mediated transition to tight cell-cell adhesion, possibly blocking subsequent events necessary for reorganization of the actin cytoskeleton and compaction.

Animals↗
PubMed1963

STUDIES ON CILIA. THE FIXATION OF THE METACHRONAL WAVE.

Upon excision into spring water, the lateral cilia of the gill of the freshwater mussel Elliptio complanatus (Solander) stop beating, but 0.04 M potassium ion can activate the gill so that these cilia again beat with metachronal rhythm. One per cent osmium tetroxide quickly pipetted onto a fully activated gill fixes the lateral cilia in a pattern that preserves the form and arrangement of the metachronal wave, and permits the cilia to be studied with the electron microscope in all stages of their beat cycle. Changes are seen in the fixed active preparation that are not present in the inactive control, i.e., in the packing of the cilia, the position of the axis of the ciliary cross-section, and the diameter of the ring of peripheral filaments. Analysis of these parameters may lead to new correlations between ciliary fine structure and function.

Animals↗
PubMed1969

The effects of endotoxin on vascular endothelium.

This study was undertaken to develop a technique for the preparation of sheets of endothelial cells and to investigate the effects of bacterial endotoxin on large numbers of cells from continuous sheets of vascular endothelium. Rabbits were divided into one control and two experimental groups. The experimental animals received intracardially an LD(50) dose of Escherichia coli endotoxin. 1 and 24 hr postinjection, the vessels of the animals were perfused with glutaraldehyde in Millonig's buffer with methylene blue as a marker. Pieces of mesentery containing arteries were postfixed in buffered glutaraldehyde, dehydrated, and placed in acetone (to remove fat deposits). The surrounding connective tissue was stripped from the mesenteric arteries, and segments of the vessels were slit longitudinally, flattened out, and firmly affixed to a sheet of cork with fine mounting pins. A 3% solution of Formvar in ethylene dichloride was pipetted onto the luminal surfaces of the vessels. The endothelial cells were impregnated with and adhered to the Formvar and, after soaking overnight in 10 N NaOH, could be stripped from the vessel walls as monolayers. Sheets of Formvar-impregnated cells were temporarily mounted on glass slides in aqueous methylene blue and examined by phase and bright-field microscopy. Methylene blue stained the nuclei a deep blue and the cytoplasm faintly, but cell outlines were indistinct. Endothelial sheets from control rabbits had smooth, elliptical nuclei oriented parallel to the longitudinal axis of the cells and irregularly distributed over a smooth background with faint longitudinal striations. Essentially every cell in endothelial sheets from endotoxin-injected animals appeared to be severely damaged. Cell sheets from 24 hr posttreatment animals exhibited the same type of, but more extensive, damage than that observed in 1 hr posttreatment animals. The most prominent features of the damaged endothelium were distorted nuclei, apparent nuclear vacuolization, and missing nuclei. Unstained platelets and plaques were present on the surfaces of the specimens from the experimental animals only. Stained and unstained red blood cells were also dispersed over the luminal surfaces of the endotoxin-treated vessels.

Animals↗
PubMed1999

Gating of single N-type calcium channels recorded from bullfrog sympathetic neurons.

For many neurons, N-type calcium channels provide the primary pathway for calcium influx during an action potential. We investigated the gating properties of single N-type calcium channels using the cell-attached patch technique. With 100 mM Ba2+ in the pipet, mean N-channel open probability (Po, measured over 100 ms) increased with depolarization, but the range at a single voltage was large (e.g., Po at +40 mV ranged from 0.1 to 0.8). The open dwell time histograms were generally well fit by a single exponential with mean open time (tauo) increasing from 0.7 ms at +10 mV to 3.1 ms at +40 mV. Shut time histograms were well fit by two exponentials. The brief shut time component (taush1 = 0.3 ms) did not vary with the test potential, while the longer shut time component (taush2) decreased with voltage from 18.9 ms at +10 mV to 2.3 ms at +40 mV. Although N-channel Po during individual sweeps at +40 mV was often high ( approximately 0.8), mean Po was reduced by null sweeps, low Po gating, inactivation, and slow activation. The variability in mean Po across patches resulted from differences in the frequency these different gating processes were expressed by the channels. Runs analysis showed that null sweeps tended to be clustered in most patches, but that inactivating and slowly activating sweeps were generally distributed randomly. Low Po gating (Po = 0.2, tauo = 1 ms at +40 mV) could be sustained for approximately 1 min in some patches. The clustering of null sweeps and sweeps with low Po gating is consistent with the idea that they result from different modes of N-channel gating. While Po of the main N-channel gating state is high, the net Po is reduced to a maximum value of close to 0.5 by other gating processes.

Animals↗
PubMed2000

The role of steady phosphodiesterase activity in the kinetics and sensitivity of the light-adapted salamander rod photoresponse.

We investigated the kinetics and sensitivity of photocurrent responses of salamander rods, both in darkness and during adaptation to steady backgrounds producing 20-3,000 photoisomerizations per second, using suction pipet recordings. The most intense backgrounds suppressed 80% of the circulating dark current and decreased the flash sensitivity approximately 30-fold. To investigate the underlying transduction mechanism, we expressed the responses as a fraction of the steady level of cGMP-activated current recorded in the background. The fractional responses to flashes of any fixed intensity began rising along a common trajectory, regardless of background intensity. We interpret these invariant initial trajectories to indicate that, at these background intensities, light adaptation does not alter the gain of any of the amplifying steps of phototransduction. For subsaturating flashes of fixed intensity, the fractional responses obtained on backgrounds of different intensity were found to "peel off" from their common initial trajectory in a background-dependent manner: the more intense the background, the earlier the time of peeling off. This behavior is consistent with a background-induced reduction in the effective lifetime of at least one of the three major integrating steps in phototransduction; i.e., an acceleration of one or more of the following: (1) the inactivation of activated rhodopsin (R*); (2) the inactivation of activated phosphodiesterase (E*, representing the complex G(alpha)-PDE of phosphodiesterase with the transducin alpha-subunit); or (3) the hydrolysis of cGMP, with rate constant beta. Our measurements show that, over the range of background intensities we used, beta increased on average to approximately 20 times its dark-adapted value; and our theoretical analysis indicates that this increase in beta is the primary mechanism underlying the measured shortening of time-to-peak of the dim-flash response and the decrease in sensitivity of the fractional response.

Adaptation, Ocular↗
PubMed2001

Single-channel properties of recombinant acid-sensitive ion channels formed by the subunits ASIC2 and ASIC3 from dorsal root ganglion neurons expressed in Xenopus oocytes.

The acid-sensitive ion channels known as ASIC are gated by external protons. A set of these channels is expressed in dorsal root ganglion neurons where they may participate in the transduction of mechanical and nociceptive stimuli. Here, we have examined the single-channel properties of channels formed by the subunits ASIC2 and ASIC3 expressed in Xenopus oocytes using outside-out patches. The mean single-channel current-voltage relationship is linear with a slope conductance of 18 pS between -80 and -40 mV in 150 mM Na(+) outside and 150 mM K(+) inside the patch pipet. The selectivity for monovalent cations has the sequence Na(+) > Li(+) > K(+). Divalent cations such as Ca(2+) do not permeate, but instead block the channel when applied to the extracellular side. External protons increase the probability of channels being open to a maximum of 0.8 with an EC(50) of 16 +/- 4 microM and a Hill coefficient of 2.7 +/- 0.3, whereas the mean single-channel current amplitude is independent of external pH. Analysis of the kinetics of single channels indicates the presence of at least four modes of activity (Mod1 to Mod4) in addition to an inactivated state. Three of the modes exhibit distinct kinetics, and can be unambiguously identified on the basis of open probability (P(oMod1) = 0.5 +/- 0.05; P(oMod2) > 0.9 +/- 0.05; P(oMod3) < 0.1). Mode 4, which has a P(o) in the range of 0.5-0.8, may constitute a distinct mode or alternatively, it represents transitions between the other three modes of activity. Increasing [H(+)](o) increases the frequency of entering the modes with high P(o) (modes 1, 2, and 4) and the time the channel spends in the modes with high activity.

Acid Sensing Ion Channels↗
PubMed2003

Normalization of DNA-microarray data by nonlinear correlation maximization.

Signal data from DNA-microarray ("chip") technology can be noisy; i.e., the signal variation of one gene on a series of repetitive chips can be substantial. It is becoming more and more recognized that a sufficient number of chip replicates has to be made in order to separate correct from incorrect signals. To reduce the systematic fraction of the noise deriving from pipetting errors, from different treatment of chips during hybridization, and from chip-to-chip manufacturing variability, normalization schemes are employed. We present here an iterative nonparametric nonlinear normalization scheme called simultaneous alternating conditional expectation (sACE), which is designed to maximize correlation between chip repeats in all-chip-against-all space. We tested sACE on 28 experiments with 158 Affymetrix one-color chips. The procedure should be equally applicable to other DNA-microarray technologies, e.g., two-color chips. We show that the reduction of noise compared to a simple normalization scheme like the widely used linear global normalization leads to fewer false-positive calls, i.e., to fewer genes which have to be laboriously confirmed by independent methods such as TaqMan or quantitative PCR.

Algorithms↗