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PubMed2001

Automating parallel peptide synthesis for the production of PNA library arrays.

A system was establishedfor the parallel synthesis of peptide library arrays in afully automated manner Synthesis takes place in blocks made of polyoxymethylene that hold during all synthesis steps a polypropylene membrane of 8 x 12 cm. Yields are in the nanomole range, obtained at a low consumption of reagents. The current setup is based on a commercially available pipetting robot and supports the generation of 1536 different oligomers/run. Much higher array densities are possible because the membranes are amicable to spot diameters of down to 200 microm, naturally at a cost of the absolute amount produced of each oligomer The method was put to use for the creation of arrayed libraries of peptide nucleic acids (PNAs). These can be employed both as a source of PNA molecules applied individually in experimentation subsequent to their release or as intact oligomer arrays in hybridization analyses.

Base Sequence↗

PubMed2002

Robotic high-throughput assay for isolating apoptosis-inducing genes.

The need to assign functions to genes will increase in upcoming years because of the accumulation of sequence information from various organisms. Here we present a genetic screen for the systematic functional analysis of genes in mammalian cells. The screen is based on high-throughput, transient transfections of individual cDNAs by a robotic system. This construction uses a moveable pipetting head with a novel 96-channel parallel tube-pump array and an integrated de-lidding device. Because of the robot design, the processing speed can reach 2,000 transfections/h. We demonstrate the suitability of the screen to isolate apoptosis-inducing genes as a first read-out. This is achieved in combination with co-transfected reporter plasmids to produce a novel and convenient assay for apoptosis induction.

Alkaline Phosphatase↗

PubMed2002

Systematic analysis of intrinsic factors affecting differential display.

Differential display (DD) is a widely used method for identifying differentially expressed genes. To improve further the efficiency and reproducibility of the method, this report systematically examines four critical parameters of standard DD-PCR. Specifically, the study determined the optimal annealing temperature, elongation time, dNTP concentration, and arbitrary primer concentration. By using a thermal cycler that was capable of displaying a temperature gradient across a PCR plate, it was possible to determine (in a single experiment) the effect of different annealing temperatures. The optimal annealing temperaturefor a 13-mer arbitrary primer fell within a broad range of 40 degrees C-50 degrees C. Elongation times over a range of 30-120 s worked best. The optimal concentration for dNTPs was within a very broad range of 2-50 microM, with higher amounts allowing for greater pipetting accuracy. The most favorable concentration for the arbitrary primer was also within a broad range of 0.1-2.0 microM. A primer concentration below this range greatly reduced the efficiency of the amplification process. In conclusion, the experimental findings delineated the best possible DD conditions for a more reliable assessment of differential gene expression.

Animals↗

PubMed2002

Fully automated radioligand binding filtration assay for membrane-bound receptors.

Here we describe a fully automated, hands-free radioligand filtration binding assay for dopamine D3 receptors. Three separate instruments were linked in tandem to perform the following operations: The Genmate and Genesis were linked to perform liquid handling, incubation, and the scheduling operations, while an automated harvester was used to perform rapid filtration. To minimize carryover of compounds, disposable tips were used for diluting and dispensing the compounds. A custom-designed tip holder was used to handle loading and pipetting by the Genmate 96-well pipettor. The assay for 84 compounds with six concentrations that spans six logs can be completed within 4 h. The reproducibility of the individual data point (cv < 10% between duplicates) and Ki (cv < 20%) is superior to that determined by manual procedures. Ki values of various dopamine ligands determined by the hands-free procedure are similar to published values. This technology reduces hands-on time (at least 70%), minimizes exposure to radioligands (up to 95%), and improves the reproducibility of results. The technology is applicablefor high-throughput screening and rapid determination of structure-activity relationship of compounds for many other membrane-bound receptors.

Animals↗

PubMed1997

Optimization of cellular ELISA for assay of surface antigens on human synoviocytes.

The cellular enzyme-linked immunosorbent assay (CELISA) permits assay of cell surface antigens on intact fixed cells. Using a monolayer of cells as the solid phase, the CELISA offers an inexpensive alternative to flow cytometry. In addition, this protocol has the decided advantages of miniaturization (small numbers of cells) and ease of replication (the 96-well cluster plate format). In efforts to optimize CELISA for detecting surface antigens on human fibroblastlike synoviocytes, the authors found that cell number, serum proteins, choice of culture plate, pipetting technique and fixatives may all impact the results of the CELISA.

Antigens, Surface↗

PubMed1998

TurboPrep II: an inexpensive, high-throughput plasmid template preparation protocol.

In an effort to reduce plasmid template preparation costs for large-scale genomic sequencing projects, a boiling minipreparation protocol has been developed that enables a single individual to easily prepare 768 sequence-quality templates in 8 h, without automation. The maximum throughput for one individual using one centrifuge in the manual configuration is 1920 templates in about 8 h. The most time-consuming manual steps of this method involve pipetting, which can be automated, resulting in a significant increase in throughput and about a 60% increase in yield. This method in the fully manual configuration yields sufficient double-stranded template for two sets of cycle sequencing reactions, membrane spotting for hybridization analysis and host cell transformation for the recovery of the original recombinant. The current materials cost per template using this method is less than twenty cents. The quality of the sequence generated has been evaluated by manual 35S radioactive cycle sequencing. Initial results have shown templates prepared by this method to yield greater than 300 bp of readable sequence when the radioactively labeled products were resolved on 6% modified denaturing polyacrylamide gels.

DNA, Recombinant↗

PubMed1998

Quantitative detection of t(14;18)-positive cells by real-time quantitative PCR using fluorogenic probes.

To detect t(14;18)-positive cells present in human lymphoma tissue, bone marrow aspirates and peripheral blood mononuclear cells (PBMNC), we have established an automated, real-time quantitative PCR using double-labeled fluorogenic probes. In relation to t(14;18)-positive genomic DNA or a cloned t(14;18)-DNA fragment, highly reproducible results can be obtained with initial copy numbers between 10 to 10(5). The detection of single copies has been verified by the stochastic multiple-tube approach. PBMNC cells obtained during clinical follow-up of patients with follicular lymphoma were analyzed by the one-step, real-time quantitative PCR and a two-step, semi-nested PCR combined with a limiting dilution assay. The quantitative results obtained by both assays correlate very well. Real-time quantitative PCR has several advantages: (i) it involves less critical pipetting steps, (ii) is less time-consuming and (iii) UTP, in combination with uracil-N-glycosylase, can be used to control carryover contamination. The higher specificity is due to optimized primer annealing conditions and MgCl2 concentration and the use of AmpliTaq Gold. The sensitivity is at least as high as by the two-step PCR. Real-time quantitative PCR will be very helpful in large epidemiological studies and in research for molecular staging and the detection of minimal residual tumor cells, including the analysis of blood stem-cell preparations to be used for transplantation after myelo-ablative therapy.

Cloning, Molecular↗

PubMed1999

High-throughput DNA extraction from old paraffin-embedded biopsies.

We have developed a rapid and reliable microporous spin-column-based method for high-throughput extraction of DNA from old paraffin-embedded biopsies. The method was developed specifically for PCRs and molecular genetic studies. De-waxing and DNA extraction from tissues is performed in only one vessel, minimizing pipetting steps and eliminating supernatant/pellet procedures. All steps are performed at 60 degrees, 55 degrees and 95 degrees C minimize deleterious DNase activity. The amount of the extracted DNA was measured spectrophotometrically, and the quality of the extracted DNA and the PCR products was controlled on ethidium bromide-stained 3% agarose gel. The presented method for DNA extraction is less laborious and more convenient than previously published methods. It provides means by which extensive, paraffin-embedded material can be subjected to molecular genetic studies.

Actins↗

PubMed1996

Expression of insulin receptor mRNA and insulin receptor substrate 1 in pancreatic islet beta-cells.

The expression of insulin receptor mRNA was examined in rat pancreatic islet cells by single-cell reverse transcriptase (RT)-polymerase chain reaction (PCR). Single cells from disaggregated islets were individually isolated in a microcapillary pipet, and the beta-cells were identified by amplification of the mRNA for insulin. We found that in single beta-cells, the mRNA for the insulin receptor was also expressed. The fraction of single islet cells expressing both insulin receptor and insulin mRNAs corresponds closely to the fraction of beta-cells in the disaggregated islet cell preparation. These results indicate that normal beta-cells have the potential to express authentic insulin receptors. Immunohistochemical analysis was insufficiently sensitive for assaying insulin receptor protein; however, insulin receptor substrate 1 (IRS-1) was readily immunolocalized in islet beta-cells. Since IRS-1 links several cell surface receptors, including those for insulin and IGF-I, to distal signal transduction pathways, our observations indicate that hormonal regulation of islet beta-cells potentially involves the same signal transduction pathway that mediates insulin and growth factor signaling in peripheral insulin target tissue cell types.

Animals↗

PubMed1993

Effect of pressure on in vitro percutaneous absorption of caffeine.

The effect of increased pressure, which is a mechanical property of massage, was investigated on the percutaneous absorption of an amphiphilic compound (caffeine) in vitro on Franz diffusion cells, using excised human skin. 50 microliters of either a 320 micrograms/ml or a 15 mg/ml acetone solution of caffeine were pipetted onto the surface of each skin sample, which represented caffeine skin deposits of 5 micrograms/cm2 and 240 micrograms/cm2 respectively. During each experiment, a pressure device delivering 0.25 bar over the atmospheric pressure was applied for the first 30 min on half of the cells. At 2, 4, 6, 8, 12 and 24 h the aqueous dermal bathing solution, containing 14 g/l albumin, was removed and chromatographed. With the applied dose of 5 micrograms/cm2 no statistical difference was found between the cumulated absorbed amount under atmospheric pressure and increased pressure. On the other hand, with the applied dose of 240 micrograms/cm2, the permeation of caffeine was 1.8 times higher under increased pressure than the permeation under atmospheric pressure (p < 0.05). This enhancing effect of increased pressure was probably connected to either an improved transappendageal route during the percutaneous absorption process or a higher stratum corneum filling-up.

Adult↗

PubMed1991

A new concept for reconstruction of atresias of larynx and trachea: lining of wound surfaces with autologous isolated respiratory epithelial cells.

A new method, first applied two years ago in our clinic, has proved to be reliable for achieving a rapid reepithelialisation of epithelial defects after scar removal inside the trachea. The defect is seeded with isolated respiratory epithelial cells harvested the day before from the ethmoid. Isolation of epithelial cells was achieved by keeping the mucosa in 0.25% trypsin buffer solution under room temperature for about 16 h. Afterwards, the epithelial layer was separated from the submucosa using small forceps and knife. Cells were then isolated by pipetting. For seeding the wound the surface was covered with silastic sheeting and the cell suspension then injected into the cleft between both of them. Cell distribution occurred by capillary attraction. The tracheal lumen was maintained by inserting a silastic stent for about three weeks. So far, 10 patients between 6 and 45 years have been treated in this way. In 4 patients the tracheal wall additionally had to be stabilized using rip cartilage. Only in one case above the tracheostoma, considerable scar formation occurred again requiring a second operation some months later. In 8 patients decannulation was meanwhile possible.

Adolescent↗

PubMed1990

The use of robots and computers in the organisation of studies on the circadian variation of beta 2-adrenoceptor sites in peripheral mononuclear leucocytes.

We have developed a partially automated method for the performance of equilibrium radioligand binding studies which is applied by our group in investigations on circadian variations and stimulation studies on beta 2-adrenoceptor sites in human peripheral mononuclear leucocytes (pMNL). Using a Tecan Robotic Sample Processor, binding assays with 12 concentrations of 125iodocyanopindolol (1-150 pmol/l, total binding in triplicates, unspecific binding in the presence of 10(-5) mol/l timolol in duplicates) are prepared automatically with all titer tubes per experiment arranged in a microtiterplate-sized rack. After incubation in a waterbath for 2hr at 37 degrees C, the whole rack is centrifuged at 5000g and transferred back to the lab robot. Bound radioactivity is separated from the unbound ligand by removing the supernatant by the machine. The radioactive counts are evaluated using personal computers. The lab robot enhances reproducibility of experimental results and frees lab workers from time-consuming pipetting jobs. Radioactive exposure is minimized to the time preparing the radioligand working solution and transferring the sample tubes from the robot to the waterbath, to the centrifuge and back to the robot. The variability of our software allows easy adaptation to other binding studies with intact cells.

Circadian Rhythm↗

PubMed1987

Problems with measurements caused by high concentrations of serum solids.

There have been numerous reports of spectrophotometric and volume problems caused by elevated levels of lipids in blood. The offending lipids, primarily triglycerides, not only cause turbidity leading to optical aberrations when added to analytical reagents, but also result in short-sampling errors leading to the measurement of inaccurate volumes of sample. Numerous methods have been developed to clear the lipemia, including ultracentrifugation organic solvent extraction, chemical precipitation and, most recently, enzymic hydrolysis. Although the latter procedures eliminate the optical problems, they do not deal with the volume dilution error created by the triglycerides. In turn, corrective mathematics have been developed to compensate for the inaccurate pipetting caused by the elevated lipids in a sample; however, these empirical calculations are not truly accurate at high concentrations of total lipids. This monograph will describe the problems caused by the presence of elevated lipids and the means available for treating them.

Blood Chemical Analysis↗

PubMed1990

Semiautomated preparation of DNA templates for large-scale sequencing projects.

The rate limiting step in a large-scale sequencing project is the generation of single-stranded DNA templates. We describe a fast, semiautomated procedure, using 96-well microtitre plates, in which 192 templates can be readily prepared in 1 day. The technique can be carried out manually or can be semiautomated using a robot pipetting device. We also provide evidence for the reliability and applicability of this method to a large-scale sequencing project.

Bacteriophages↗

PubMed2003

Optimization and validation of a rapid method to determine citrate and inorganic phosphate in milk by capillary electrophoresis.

Quantification of phosphate and citrate compounds is very important because their distribution between soluble and colloidal phases of milk and their interactions with milk proteins influence the stability and some functional properties of dairy products. The aim of this work was to optimize and validate a capillary electrophoresis method for the rapid determination of these compounds in milk. Various parameters affecting analysis have been optimized, including type, composition, and pH of the electrolyte, and sample extraction. Ethanol, acetonitrile, sulfuric acid, water at 50 degrees C or at room temperature were tested as sample buffers (SB). Water at room temperature yielded the best overall results and was chosen for further validation. The extraction time was checked and could be shortened to less than 1 min. Also, sample preparation was simplified to pipet 12 microl of milk into 1 ml of water containing 20 ppm of tartaric acid as an internal standard. The linearity of the method was excellent (R2 > 0.999) with CV values of response factors <3%. The detection limits for phosphate and citrate were 5.1 and 2.4 nM, respectively. The accuracy of the method was calculated for each compound (103.2 and 100.3%). In addition, citrate and phosphate content of several commercial milk samples were analyzed by this method, and the results deviated less than 5% from values obtained when analyzing the samples by official methods. To study the versatility of the technique, other dairy productssuch as cream cheese, yogurt, or Cheddar cheese were analyzed and accuracy was similar to milk in all products tested. The procedure is rapid and offers a very fast and simple sample preparation. Once the sample has arrived at the laboratory, less than 5 min (including handling, preparation, running, integration, and quantification) are necessary to determine the concentration of citric acid and inorganic phosphate. Because of the speed and accuracy of this method, it is promising as an analytical quantitative testing technique.

Animals↗

PubMed1977

Assay of prolactin after freezing cow's milk.

Concentrations of prolactin were similar in aliquots of the same milk sample stored for 2 days at 4 C or --23 C but averaged lower if prepared for assay at 30 C than if prepared at 40 or 50 C. Average deviations in prolactin between duplicate measurements relative to respective treatment averages were lowest generally when technique of mixing included vortexing for 5 s immediately prior to pipetting volumes of milk for assay. The average deviation of differences in prolactin among duplicate measurements relative to average concentrations of prolactin were about the same for colostrum, milk, and blood plasma (11, 17, and 14%) stored frozen. Prolactin can be measured reliably in frozen cow's milk provided samples are warmed to 40 C to 50 C and mixed thoroughly when prepared for assay.

Animals↗

PubMed1967

[Studies On Phosphatase Activity In Some Parasitic Helminths]

In order to obtain some informations on the nature and relative activity of the phosphatases present in various helminths, biochemical studies have been made in thirteen kinds of worm parasites including the adults and larvae (Fasciola hepatica, Eurytrema pancreaticum, Paramphistomum sp., Taenia solium, Taenia pisiformis, Dipylidium caninum, Diphyllobothrium mansoni, Cysticercus cellulosae, Cysticercus fasciolaris and Sparganum). A comparison based on the analysis of pH-activity curves was made among these helminths. The worm materials were mostly obtained alive from an abattoir and removed from the organs or tissues of the animal hosts naturally infected. Sparganum and Cysticercus cellulosae, however, are collected from the subcutaneous tissue of the patients by surgical removal. The worms thoroughly washed were weighed and transferred with 0.1 M Tris buffer to a chilled glass grinder (Capacity; 15 ml) and homogenized in the cold. The homogenate was centrifuged at 5000 RPM for 30 minutes. The supernatant was pipetted off for determination of the phosphatase activity. Incubation mixtures consisted of 1 ml substrate, 1 ml buffer and 0.5ml extract. The buffers used were Tris (Hydroxymethyl) aminomethane and citric acid monohydrate and the substrate was paranitrophenyl phosphate (1 gm/25 ml). These mixtures were incubated at the temperature of 37 degrees C for 30 minutes in water bath. The absorbance or transferance of mixture was determined colorimetrically by "Spectronic 20 "spectrophotometer at 410 nm against a distilled water blank. The amount of phenol liberated was then calculated from a standard curve using phenol solutions. Controls consisted of unincubated mixtures. The results were deducted from this experiment. The phosphatase activity occurred over all parasitic helminths used in this experiment. In trematodes, pH-activity curves have demonstrated two peaks of phosphatase activity in Fasciola hepatica and Paramphistomum species. However the acid phosphatase activity was predominantly found and the alkaline phosphatase activity was found distinctly to be low in all three species. In Eurytrema pancreaticum, the pH-activity curves displayed two peaks in acid phosphatase activity, one at pH 5.0 and the other pH 9.0. In cestodes, both alkaline and acid phosphatase activity displayed the pH optima 5.0 and 9.0 to 10.0 in the adult tapeworms. However, major activity in the adults is due to the alkaline phosphtases. In contrast to the adults, Cysticercus and sparganum showed the higher activity in acid phosphatases which predominates in the larvae. In all cases of nematodes, the pH optimum for acid phosphatase was 4.0 to 6.0. A preponderance of acid phosphatase activity was shown in the extract of intestine of Ascaris lumbricoides. The aspect that phosphatases are correlated with phosphorylated passage of substances through the cuticle of helminths and may also be involved in carbohydrate metabolism is discussed.

Journal Article↗

PubMed1984

[Study on sensory papillae of Metagonimus yokogawai cercaria]

A number of studies on the papillae of cercariae of trematodes reported that the papillar patterns (or chaetotaxy) of cercariae might be an excellent method to attain better understanding of the digenetic trematodes (Richard, 1971; Short and Cartrett, 1973; Bayssade-Dufour, 1979). The present study was aimed to determine the number, distribution pattern and structure of the sensory papillae of Metagonimus yokogawai cercariae, and to elucidate the chaetotaxy of this digenetic trematode. M. yokogawai cercariae were pipetted from a vial in which infected snails (Semisulcospira libertica) had been kept for 3 hours. The snails were collected from an endemic area of M. yokogawai, Boseong river in west-southern part of Korea. Observations of papillae were based on light microscopy of those stained with silver nitrate, and on scanning electron microscopy. The results are summarized as follows: All papillae observed were uniciliated. Cilia in anterior tip were shorter than the others in other portions. The body papillae were arranged in essentially symmetrical patterns. Total number of the papillae was 126(63 pairs) in average; anterior tip 40(20 pairs), ventral 20(10 pairs), lateral 42(21 pairs), and caudal 8(4 pairs). The chaetotaxy of M. yokogawai cercaria was; C(I) cycle (3+3C(I)V, 2+2C(I)L, 2+3C(I)D), C(II) cycle(2C(II)V, 1C(II)L, 2C(II)D), C(III) cycle (1+lC(III)V, 1C(III)L), C(IV) cycle (1C(IV)V, 1C(IV)L) in cephalic region: A(I)(lA(I)V, l+2A(I)L, 1A(I)D), A(II)(1A(II)V, 1+3A(II)L, lA(II)D), A(III) {lA(III)V, l+lA(III)L, lA(III)D) and A(IV)(lA(IV)V, 2A(IV)L) in antacetabular region: 1M(I)V and 2M(I)L in median: 1+lP(I)L, 1P(II)L, lP(II)D lP(III)L, lP(IV)L and 1P(IV)D in postacetabular region: 2-2-2-2 in caudal region.

Journal Article↗