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PubMed1978

Common origin of rods, cores, miniature cores, and focal loss of cross-striations.

Twelve patients suffering from congenital neuromuscular disorders showed at least two of the following structural changes in their muscle biopsy specimens: rods, cores, miniature cores (M lesions), and focal loss of cross-strians (F lesions). There appeared to be no essential differences between M and F lesions except for the presence of vesicular nuclei in and around the latter. A striking finding was the presence of adjacent M lesions, F lesions, or both in two or several adjoining muscle fibers. Serial sections often showed a definite relationship of the lesions with blood vessels. The finding of rods cores, and M and F lesions, not only in one biopsy specimen but also in one muscle fiber, raised the intriguing possibility of common pathogenic mechanisms producing these lesions.

Adult↗

PubMed1998

Quantitative determination of Ecteinascidin 743 in human plasma by miniaturized high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A method was developed for the bio-analysis of Ecteinascidin 743 (ET-743) using miniaturized liquid chromatography (LC) coupled to an electrospray ionization sample inlet (TurbolonSpray) and two quadrupole mass analyzers (LC/ESI-MS/MS). Solid-phase extraction was used as a sample pretreatment procedure. Ecteinascidin 743 is a very potent anticancer compound and is administered in microgram m-2 dosages, which demands special requirements in terms of sensitivity for the analytical method supporting clinical pharmacokinetic studies. Using conventional LC/UV, a lower limit of quantitation (LLQ) of 1 ng ml-1 plasma was reached using a 500 microliters sample volume, but LC/ESI-MS/MS permitted an LLQ of 10 pg ml-1. The latter method was found to be accurate and precise, and provided a broad linear concentration range of 0.010-2.50 ng ml-1.

Antineoplastic Agents↗

PubMed1999

Miniaturized time-of-flight mass spectrometer for peptide and oligonucleotide analysis.

A matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer was developed which uses a novel reflectron composed of a grounded cylinder and an adjustable endcap electrode to provide high-order kinetic energy focusing for a miniaturized mass analyzer. The nearly quadratic potential form of the reflecting field focuses ions desorbed from a source of very small dimensions formed by placing the sample probe within the centered hole of the coaxial dual channel plate detector. At the same time, the depth of the reflectron can be adjusted to accommodate a short drift length between the source/detector and the reflectron. For larger drift lengths, in particular to allow the addition of an XY stage for the analysis of sample arrays, endcap reflectron focusing can be combined with time-delayed ion extraction to achieve good mass resolution. The instrument has been used for the analysis of peptides digested with trypsin or carboxypeptidase, and also small DNA oligomers.

Amino Acid Sequence↗

PubMed1998

Miniaturized hemoproteins.

The present paper highlights and reviews current research in the field of hemoprotein models. Hemoproteins have been extensively studied in order to understand structure-function relationships, and to design new molecules with desired functions. A wide number of synthetic analogues have been developed, using quite different approaches. They differ in molecular structures, ranging from simple meso-substituted tetraaryl-metalloporphyrins and peptide-porphyrin conjugates. In this paper we summarize the state of the art on peptide based hemoprotein models. We also report here the approach used by us to develop a new class of molecules, named mimochromes. They can be regarded as miniaturized hemoproteins, because mimochromes are low molecular weight compounds with some structural and functional properties common to those of the parent high molecular weight protein. The basic structure of mimochromes is a deuteroporphyrin ring covalently linked to two helical peptide chains. Two molecules of this series have been fully characterized. All the information derived from their structural analysis has been applied to the design of new analogues with additional functions.

Amino Acid Sequence↗

PubMed1999

A miniature specimen mechanical testing technique scaled to articulating surface of polyethylene components for total joint arthroplasty.

The small punch test was developed to investigate the mechanical behavior of polyethylene using miniature specimens (< 14 mg) measuring 0.5 mm in thickness and 6.4 mm in diameter. The objective of this study was to demonstrate the feasibility and reproducibility of the small punch test when applied to clinically relevant polyethylenes. Mechanical behavior was characterized during 66 tests performed on GUR4150HP and GUR4120 specimens following alternate sterilization methods and 4 weeks of accelerated aging at 80 degrees C. The small punch test was found to be highly reproducible with regard to characterizing the ductility, ultimate strength, and fracture resistance of sterilized and aged polyethylene. In the future, the small punch test can be used to directly measure mechanical properties near the articulating surface of retrieved components.

Air↗

PubMed2000

Prenatal protein malnutrition results in increased frequency of miniature inhibitory synaptic currents in rat CA1 pyramidal cells.

There is growing evidence for an effect of prenatal protein malnutrition on the GABAergic neurotransmitter system in the rat hippocampus and associated structures. In the present study, we examined the functional electrophysiological consequences of observed alterations in GABA(A) and benzodiazepine receptor systems. Whole-cell patch clamp recordings of spontaneous and of miniature inhibitory postsynaptic currents (mIPSCs) generated by CA1 pyramidal cells were performed in in vitro hippocampal slices prepared from control and prenatally protein malnourished adult male rats. The characteristics of spontaneous synaptic currents were unaltered by the prenatal insult, as were the amplitudes and kinetics of GABA(A) receptor-mediated mIPSCs. The frequency of mIPSCs, however, was significantly increased in CA1 pyramidal cells in slices prepared from prenatally malnourished vs. control rats. The effect of the benzodiazepine receptor agonist chlordiazepoxide on the characteristics of mIPSCs was also examined and found to be the same in cells from both nutritional groups. The increased frequency of mIPSCs together with the lack of a change in amplitude, kinetics, or modulation by benzodiazepines of mIPSCs in response to prenatal protein malnutrition indicate a presynaptic locus of effect of this insult.

2-Amino-5-phosphonovalerate↗

PubMed2000

Miniature time-of-flight mass spectrometer using a flexible circuitboard reflector

An innovative design for a miniature time-of-flight mass spectrometer has been developed employing several newly designed components. These include: (1) a gridless, focusing ion source allowing for the use of very high extraction energies in a maintenance-free design, (2) a new method of construction for an ion reflector using rolled flexible circuitboard material, and (3) an improved center-hole microchannel plate detector assembly that significantly reduces the noise (or 'ringing') inherent in the coaxial design. A prototype time-of-flight instrument was constructed and used to evaluate the performance of these components. Compared to previous designs, results indicate that background noise for data acquired on this instrument is substantially reduced, resolution is improved, and the potential for mass producing this instrument in an inexpensive and rugged package for field-portable and remote installations is significantly enhanced. Copyright 2000 John Wiley & Sons, Ltd.

Journal Article↗

PubMed2001

Miniaturized pyrosequencer for DNA analysis with capillaries to deliver deoxynucleotides.

As the human genome project proceeds, various types of DNA analysis tools are required for life sciences and medical sciences including DNA diagnostics. For example, a small DNA sequencer for sequencing a short DNA is required for bed-side DNA testing as well as DNA analysis in a small laboratory. Here, a new handy DNA sequencing system (pyrosequencer) based on the detection of inorganic pyrophosphate (PPi) released by polymerase incorporation is demonstrated. The system uses the bioluminescence detection system. The key point for the miniaturized DNA sequencer is to make a deoxynucleotide triphosphate (dNTP) delivery system small and inexpensive. It has been realized by using narrow capillaries to connect a reaction chamber and four dNTP reservoirs. Each dNTP is introduced into the reaction chamber by applying a pressure to the reservoir. Compared with other microdispensers, it is much cheaper and easier. By optimizing the conditions, an excellent sequencing ability is achieved while it is a simple and inexpensive system. In most cases, more than 40 bases can be successfully sequenced. A homopolymeric region, which can not be easily sequenced by a conventional gel-based DNA sequencer, is readily sequenced with this system. The new system is successfully applied to sequence a GC rich region or a region close to a priming region where misreading frequently occurs. A rapid analysis for a short DNA was easily achieved with this small instrument.

Base Sequence↗

PubMed2001

Microchip isoelectric focusing using a miniature scanning detection system.

A miniature scanning fluorescent detector has been developed for plastic microchannel isoelectric focusing (mIEF) analysis. The detector, comprised of a lamp and photomultiplier tube (PMT) on a moving stage, measured the real-time distribution of fluorescently labeled peptides subjected to gel-free mIEF. During the run, the effective length of the 6-cm channel was scanned every 9 s. Analysis was completed within 5 min while still obtaining high resolution and sensitivity. In addition, the scanning detector was used to characterize peptide migration properties within the channel by providing simultaneous temporal and spatial measurements.

Amino Acid Sequence↗

PubMed1990

Segregation distortion in inheritance of progressive rod cone degeneration (prcd) in miniature poodle dogs.

Segregation distortion was observed in inheritance of progressive rod-cone degeneration (prcd) in a colony of Miniature Poodle dogs. Breeding results, from both retrospective records and prospectively planned matings, were classified into five mating types: (1) affected to affected, (2) homozygous normal sire to any dam, (3) heterozygous to heterozygous, (4) heterozygous sire to affected dam, and (5) affected sire to heterozygous dam. For all but the last category, results were in accord with mendelian expectations for autosomal-recessive inheritance. However, litters of mating type 5 had fewer affected pups (20/77) than expected. The observed segregation ratio for this mating type (0.26) was significantly (P less than 0.001) less than the expected (0.50). The segregation distortion could not be accounted for by either pre- or postnatal loss of affected pups, as litter size and litter survivability were uniform among litters of different mating types. Either the prcd locus, or a linked locus, would appear to influence either gametic or zygotic fitness in the heterozygous mother. Comparison is drawn to the inheritance of retinitis pigmentosa in humans, in which decreased segregation ratios are also recognized.

Animals↗

PubMed1987

Developmental studies of Hanford miniature swine exposed to 60-Hz electric fields.

Evaluations of reproductive and developmental toxicology, including teratology, were included as part of a broad screening study in Hanford Miniature swine (HMS) to detect effects of exposure to electric fields. One group (E) was exposed to a uniform, vertical, 60-Hz, 30-kV/m electric field for 20 h/day, 7 days/week; sham-exposed (SE) swine were housed in a separate, environmentally equivalent building. The first generation (F0) gilts were bred after 4 months of study; some were killed for teratologic assays at 100 days of gestation (dg), and the others produced an F1 generation of offspring. The pooled incidence of terata in these litters (teratologic assays and live births) was similar in the E and SE groups. The F0 females, which produced the F1 generation, were bred again after 18 months of exposure and were killed at 100 dg. Malformation incidence in E litters (75%) was significantly greater than in SE litters (29%). No consistent differences in litter size, fetal mass, or mass of fetal organs were detected. The F1 gilts were bred at 18 months of age; defective offspring were found in significantly more of the E litters (71%) than in SE litters (33%). These F1 females were bred again 10 months later and teratologic assays were performed on their second litters at 100 dg. The percentage of litters with malformed fetuses was essentially identical in the E and SE groups (70% and 73%, respectively). There appears to be an association between chronic exposure to a strong electric field and developmental effects in swine, although the change in incidence of malformations between generations and between the first and second breedings makes it impossible to conclude unequivocally that there is a cause-and-effect relation.

Animals↗

PubMed1991

Miniature-probe measurements of electric fields and currents induced by a 60-Hz magnetic field in rat and human models.

A miniaturized probe was designed and built to provide detailed data on fields induced by a uniform 60-Hz magnetic field in homogeneous models of rat and human. The probe employed three silver wires twisted and potted in an 8-cm hypodermic needle. The exposed tips of the wires formed three sensing electrodes with a centered ground; highly sensitive voltage measurements were enabled by a lock-in amplifier. Tests were conducted in a 1-mT rms field that was uniform within +/- 5%. The models were made by casting 1.5% agar at 1-S/m conductivity into plastic-foam molds. The rat model was scaled 1:1 as an adult (22 cm length; mass about 640 g). The human model was scaled 1:4 as an adult (height = 46.5 cm; mass 1.4 kg). The probe was inserted into each model in several regions, and readings of induced fields were made under different exposure geometries. Maximal strengths of fields induced near the surface of the torso were as high as 120 microV/cm in the laterally exposed rat model. Data extrapolated from the quarter-scale human model revealed that an induced field as high as 700 microV/cm could occur at the torso of a frontally exposed human adult. An overall size-scale factor of about 5 appears to be appropriate for experimental exposures of rats that are intended to simulate currents induced in human beings by magnetic fields. The average strength of electric fields induced in the torso by a 1-mT magnetic field is comparable to that by a vertical electric-field at 60 kV/m and 28 kV/m, respectively, for the rat and human.

Animals↗

PubMed2004

Metabolic network analysis of lysine producing Corynebacterium glutamicum at a miniaturized scale.

We present a straightforward approach comprising (13)C tracer experiments at 200-microL volume in 96-well microtiter plates with on-line measurement of dissolved oxygen for quantitative high-throughput metabolic network analysis at a miniaturized scale. This method was successfully applied for cultivation and (13)C metabolic flux analysis of two mutants of lysine producing Corynebacterium glutamicum (ATCC 13287 and ATCC 21543). Microtiter-plate cultivations showed excellent accordance in kinetics and stoichiometry of growth and product formation as well as in intracellular flux distributions as compared with parallel shake-flask experiments. These cultivations further allowed clear identification of strain-specific flux differences such as increased flux toward lysine, increased flux through the pentose phosphate pathway (PPP), decreased flux through the tricarboxylic (TCA) cycle, and increased dihydroxyacetone formation in C. glutamicum ATCC 21543 compared with ATCC 13287. The present approach has strong potential for broad quantitative screening of metabolic network activities, especially those involving high-cost tracer substrates.

Carbon Isotopes↗

PubMed2003

Miniaturizing the comet assay with 3D vertical comets.

BACKGROUND: The comet or single-cell gel electrophoresis assay is a sensitive method for the detection of DNA damage. The main drawback of comet sampling is the low cell density necessary to prevent nucleus overlap after electrophoresis, which limits large-scale high throughput screening. Another problem may be inconsistent comet focusing. We investigated whether an approach based on three-dimensional (3D) confocal microscopy might be beneficial for these concerns. METHODS: A vertical comet assay enabling three-dimensional confocal comet imaging of nuclei seeded at very high density was developed together with dedicated software algorithms to retrieve quantitative data at the single cell level. RESULTS: Three-dimensional confocal comet imaging greatly relieved the user interactions of our nonautomated two-dimensional comet sampling procedure. Batches of comets were blindly sampled, and confocal sectioning improved the clarity of the images and the accuracy of comet sampling. A 1-Gy dose response was readily established. The sampling speed was competitive with that of commercial packages. CONCLUSIONS: Vertical comet imaging is a new concept for fast and user-friendly comet sampling that allows miniaturization of the assay. It may become an essential step toward high throughput screening and exploit the benefits of confocal imaging.

Algorithms↗

PubMed1989

Human alpha-1-antitrypsin subtyping by hybrid isoelectric focusing in miniaturized polyacrylamide gel.

The polymorphism of alpha-1-antitrypsin (PI) has been studied by hybrid isoelectric focusing in miniaturized immobilized pH gradient gels, with an interelectrode distance of 55 mm, in two narrow ranges of pH 4.35-4.75 and 4.35-4.55), following rehydration with pH 4.2-4.9 carrier ampholytes. The use of the separator N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) in combination with carrier ampholytes for gel rehydration has been shown to enhance PI band sharpness. The influence of different additives (sucrose, sorbitol and glycerol) on the PI band pattern has also been evaluated. Glycerol has been shown to be responsible for the change in the relative mobility of the M3 band. The analysis of the minor M-7 isoprotein zone by hybrid isoelectric focusing followed by silver staining has permitted a more reliable classification of PIM subtypes. A population study carried out with 164 unrelated individuals living in Spain is also presented.

Ampholyte Mixtures↗

PubMed1990

Haptoglobin subtyping by isoelectric focusing in miniaturized polyacrylamide gels rehydrated in presence of 2-mercaptoethanol.

A fast isoelectric focusing method for routine haptoglobin (Hp) subtyping is presented. This method is based on isoelectric focusing, under reducting conditions, of neuraminidase-treated plasma samples by using dry miniaturized (interelectrode distance: 55 mm) polyacrylamide gel, rehydrated in presence of 2-mercaptoethanol and a mixture of pharmalyte carrier ampholytes (pH 4-6.5 and pH 6-8) followed by immunoblotting. The presence of 2-mercaptoethanol in the gel prevented refolding of the Hp alpha and Hp beta chains during focusing, making it possible to obtain a sharp Hp band pattern with a clear separation of the different Hp alpha allelic products (1S, 1F, 2FS, 2SS and 2FF). A population study carried out with 250 unrelated individuals living in Central Spain is also presented.

Acrylic Resins↗

PubMed1991

Efficient, small scale electroelution of high molecular weight DNA from agarose gels by a miniature vertical electrophoresis cell.

A miniature, vertical electrophoretic system is described that allows quick elution and efficient harvesting of double-stranded, high molecular weight DNA fragments from agarose gels. A gel slice containing the band of interest is excised from the agarose gel after submarine electrophoresis and placed into an electrophoresis cell, assembled by inserting a truncated micropipette tip into a 2.0 mL polypropylene tube. The tip supports the agarose slice and is connected to dialysis tubing. Platinum wire electrodes are placed over the tip and at the bottom of the test tube. DNA molecules are quickly eluted into the dialysis bag (60 s to 10 min for bands ranging from 2 to 23 kb) and then easily recovered. The method is highly efficient and allows considerable time saving. Of special interest is its applicability to small starting amounts of DNA (less than 0.5 micrograms per band). Purity of the DNA is comparable, if higher, to that obtainable by conventional electroelution on dialysis bags and DEAE-cellulose membrane electrophoresis. The system is simple enough to be used as a routine method for eluting multiple bands in short times.

Bacteriophage lambda↗

PubMed2004

DNA sequencing and genotyping in miniaturized electrophoresis systems.

Advances in microchannel electrophoretic separation systems for DNA analyses have had important impacts on biological and biomedical sciences, as exemplified by the successes of the Human Genome Project (HGP). As we enter a new era in genomic science, further technological innovations promise to provide other far-reaching benefits, many of which will require continual increases in sequencing and genotyping efficiency and throughput, as well as major decreases in the cost per analysis. Since the high-resolution size- and/or conformation-based electrophoretic separation of DNA is the most critical step in many genetic analyses, continual advances in the development of materials and methods for microchannel electrophoretic separations will be needed to meet the massive demand for high-quality, low-cost genomic data. In particular, the development (and commercialization) of miniaturized genotyping platforms is needed to support and enable the future breakthroughs of biomedical science. In this review, we briefly discuss the major sequencing and genotyping techniques in which high-throughput and high-resolution electrophoretic separations of DNA play a significant role. We review recent advances in the development of technology for capillary electrophoresis (CE), including capillary array electrophoresis (CAE) systems. Most of these CE/CAE innovations are equally applicable to implementation on microfabricated electrophoresis chips. Major effort is devoted to discussing various key elements needed for the development of integrated and practical microfluidic sequencing and genotyping platforms, including chip substrate selection, microchannel design and fabrication, microchannel surface modification, sample preparation, analyte detection, DNA sieving matrices, and device integration. Finally, we identify some of the remaining challenges, and some of the possible routes to further advances in high-throughput DNA sequencing and genotyping technologies.

Animals↗