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Myocardial blood flow distribution in miniature pigs during exercise.

Total and regional myocardial blood flow was measured in miniature pigs at rest and during two levels of treadmill exercise, including maximal exercise. Exercise increased the myocardial blood flow in a linear manner with heart rate (r = 0.87). At rest the endocardial/epicardial blood flow ratio was significantly greater than unity with flow favoring the endocardium. Exercise failed to appreciably alter the distribution of coronary blood flow. Thus the myocardium was capable of further dilatation and perfusion of blood without compromising endocardial flow even during the most severe level of exercise when maximal heart rates were attained.

Animals↗

Temporal feature estimation during walking using miniature accelerometers: an analysis of gait improvement after hip arthroplasty.

A new method for the detection of gait cycle phases using only two miniature accelerometers together with a light, portable digital recorder is proposed. Each subject is asked to walk on a walkway at his/her own preferred speed. Gait analysis was performed using an original method of computing the values of temporal parameters from accelerometer signals. First, to validate the accelerometric method, measurements are taken on a group of healthy subjects. No significant differences are observed between the results thus obtained and those from pressure sensors attached under the foot. Then, measurements using only accelerometers are performed on a group of 12 patients with unilateral hip osteo-arthritis. The gait analysis is carried out just before hip arthroplasty and again, three, six and nine months afterwards. A mean decrease of 88% of asymmetry of stance time and especially a mean decrease of 250% of asymmetry of double support time are observed, nine months after the operation. These results confirm the validity of the proposed method for healthy subjects and its efficiency for functional evaluation of gait improvement after arthroplasty.

Acceleration↗

Measurement of regional myocardial perfusion using collimated miniature radiation detectors and the method of 133-xenon washout.

A new approach to the measurement of local myocardial perfusion based on the application of highly-collimated miniature cadmium telluride radiation detectors to measure washout of 133-Xenon from well-defined tissue volumes is presented. Single-hole collimators with length/diameter ratios of 1 (L = 4 mm, D = 4 mm) and 4 (L = 12 mm, D = 3 mm) were employed as prototype designs. The probe field of view was characterized theoretically using a spherical model of the myocardium in conjunction with experimental point source response measurements for each collimator. Method evaluation using two medium resolution collimators (L/D = 1) was effected by performing left main and circumflex coronary artery perfusion studies at controlled but variable flows in the dog heart. An excellent correlation (r greater than 0.99) between actual and estimated perfusion determined from 65 washout curves over the flow range 0.38 to 3.18 ml/min/gm was demonstrated. The ability to resolve regional flow differences was verified by measuring tracer washout from circumflex and left anterior descending regions of the myocardium, using two high resolution collimators (L/D = 4), in a preparation where the left main coronary artery was cannulated and a snare was placed around the circumflex coronary artery to selectively reduce flow to that region.

Biomedical Engineering↗

Performance study of a miniature gamma ray scintillation vivo probe for tumor localization.

UNLABELLED: We have developed a miniature gamma-ray endoscopic probe consisting of dual BGO detector probes for tumor detection inside the body cavities. The dual detector system was coupled with random coincidence to decrease the distant background radiation and to improve its spatial resolution for tumor localization. METHOD: The performance of the probe was investigated with a point source and a water phantom. A solution of positron emitting 18F isotope was used as the source. Clinical trials of the probe were done to localize tumors on the skin surface of four subjects carrying tumors close to the body surfaces, into whom 67Ga-citrate and 18F-FDG radiopharmaceuticals were injected. RESULTS: Measurements indicated that the spatial resolution of the dual detector probes is around 1.5 times better than the single detector probe, and both single and dual detector endoscopic probe systems are capable of localizing a tumor on a large photon background. CONCLUSION: The endoscopic probe may be easier to insert inside body cavities due to the small crystal size and the flexible light guides. A single detector probe with higher sensitivity may be useful in searching for tumors over a wide intracavity area but a dual detector probe can be used for precise tumor localization. The detector probe may also be suitable for intraoperative observation.

Adult↗

Characterization of terminal-repeat retrotransposon in miniature (TRIM) in Brassica relatives.

We have newly identified five Terminal-repeat retrotransposon in miniature (TRIM) families, four from Brassica and one from Arabidopsis. A total of 146 elements, including three Arabidopsis families reported before, are extracted from genomics data of Brassica and Arabidopsis, and these are grouped into eight distinct lineages, Br1 to Br4 derived from Brassica and At1 to At4 derived from Arabidopsis. Based on the occurrence of TRIM elements in 434 Mb of B. oleracea shotgun sequences and 96 Mb of B. rapa BAC end sequences, total number of TRIM members of Br1, Br2, Br3, and Br4 families are roughly estimated to be present in 660 and 530 copies in B. oleracea and B. rapa genomes, respectively. Studies on insertion site polymorphisms of four elements across taxa in the tribe Brassiceae infer the taxonomic lineage and dating of the insertion time. Active roles of the TRIM elements for evolution of the duplicated genes are inferred in the highly replicated Brassica genome.

Base Sequence↗

Toxicity of inorganic compounds in the Spirotox test: a miniaturized version of the Spirostomum ambiguum test.

The Spirostomum ambiguum toxicity test has been intensively studied in the Department of Environmental Health Sciences, Warsaw University of Medicine for the last 5 years. The purpose of the present work was to develop and evaluate a miniaturized microplate version of the test, called the Spirotox test, and to estimate the toxicity of selected inorganic compounds to the Spirostomum ambiguum. The test was carried out in conventional 24-well (6 x 4) polystyrene multiwell plate. Preliminary test was one control and 11 toxicant concentrations with two duplicates. Definitive test was one control and five toxicant concentrations with three duplicates per concentration. Dilution of the sample was made directly in the plate. Toxicity series of heavy metals based on 24-h LC50 may be established as follows: Cu > Ag > Hg > Cr > Cd > Zn > Ni > Pb > Co > Mn. The series may be divided into four classes: extremely toxic: below 0.1 ppm (Cu, Ag, Hg); very toxic: 0.1-1.0 ppm (Cr, Cd, Zn, Ni); toxic: 1.0-10 ppm (Pb, Co); and low toxic: above 10 ppm (Mn). Anions were much less toxic to S. ambiguum than cations. Using the same classification, only cyanide (CN) was toxic, other anions were low toxic. Toxicity series based on 24-h LC50 may be established as follows: CN > SeO3 > Cr2O7 > NO2 > S2O3 >WO4 > BO3.

Animal Testing Alternatives↗

Gene expression analysis of Escherichia coli grown in miniaturized bioreactor platforms for high-throughput analysis of growth and genomic data.

Combining high-throughput growth physiology and global gene expression data analysis is of significant value for integrating metabolism and genomics. We compared global gene expression using 500 ng of total RNA from Escherichia coli cultures grown in rich or defined minimal media in a miniaturized 50-microl bioreactor. The microbioreactor was fabricated out of poly(dimethylsiloxane) (PDMS) and glass and equipped to provide on-line, optical measurements. cDNA labeling for microarray hybridizations was performed with the GeniconRLS system. From these experiments, we found that the expression of 232 genes increased significantly in cells grown in minimum medium, including genes involved in amino acid biosynthesis and central metabolism. The expression of 275 genes was significantly elevated in cells grown in rich medium, including genes involved in the translational and motility apparatuses. In general, these changes in gene expression levels were similar to those observed in 1,000-fold larger cultures. The increasing rate at which complete genomic sequences of microorganisms are becoming available offers an unprecedented opportunity for investigating these organisms. Our results from microscale cultures using just 500 ng of total RNA indicate that high-throughput integration of growth physiology and genomics will be possible with novel biochemical platforms and improved detection technologies.

Bioreactors↗

A "miniaturized" method for the karyotypic analysis of bone marrow or blood samples in hematological malignancies.

Standard techniques of karyotypic analysis of bone marrow or peripheral blood cells generally use large numbers of cells. Thus, the quantity of cells harvested from one bone marrow or blood puncture frequently represents a limiting factor for other assays such as molecular biology or flow cytometry, which are often essential for the diagnosis of hematological diseases. To resolve this problem, we developed a "miniaturized technique" of cytogenetic analysis that we tested on bone marrow (BM) cells from 20 patients with multiple myeloma (MM), 5 patients with acute leukemia (AL), as well as on CD34+ cells purified from the blood of 8 patients with agnogenic myeloid metaplasia (AMM). We used 3 x 10(6) BM cells from MM patients (for testing 6 different culture conditions), 10(6) BM cells from AL (2 culture conditions) and 4 x 10(3) CD34+ cells from AMM patients. In most patients, 20 good quality metaphases per slide were easily analyzed, showing that a 10-40 times reduction of the number of cells used for cytogenetics allows a reliable karyotypic analysis in hematological malignancies.

Bone Marrow↗

Changes of spontaneous miniature excitatory postsynaptic currents in rat hippocampal pyramidal cells induced by aniracetam.

Spontaneous miniature excitatory postsynaptic currents (mEPSCs) in rat hippocampal pyramidal neurones in slices (CA1 region) were recorded at 35-37 degrees C using the whole-cell patch-clamp technique before and after addition of aniracetam (1 mM) to determine how a partial blockade of desensitization alters the relationship between the amplitude (A) and kinetics of mEPSCs, and to evaluate the factors that determine their variability. The rise time (taur) and the time constant of decay of mEPSCs (taud) are essentially amplitude independent in control conditions, but become clearly amplitude dependent in the presence of aniracetam. The slopes of the best fitting lines to taud:A and taur:A data pairs were (+/- SD; ms/pA; n = 5): (1) (control) 0.07 +/- 0.02 and 0.008 +/- 0.003; (2) (aniracetam) 0.40 +/- 0.19 and 0.22 +/- 0.22. The amplitude-dependent prolongation of taud is explained by the concentration dependence of two related processes, the buffering of glutamate molecules by AMPA receptor channels, and the occupancy of the double-bound activatable states. A slower deactivation makes an amplitude-independent contribution. Desensitization reduces the amplitude dependence of taud by minimizing repeated openings of alpha-amino-3-hydroxy-methyl-isoxazole (AMPA) receptor channels. A greater amplitude dependence of taur probably involves both pre- and postsynaptic factors. The variability of A and taud values did not change significantly, but the factors underlying the variability of taud values were much affected. The greater amplitude dependence and the greater scatter about the best fitting lines to taud:A data pairs are approximately balanced by the greater mean values. The greater scatter of taud about the best fitting lines probably occurs because the saturation of AMPA receptors is not the same at different synapses with different numbers of AMPA receptors.

Animals↗

Effect of cyclothiazide on spontaneous miniature excitatory postsynaptic currents in rat hippocampal pyramidal cells.

Spontaneous miniature excitatory postsynaptic currents (mEPSCs) were recorded from the CA1 region of slices using the whole-cell patch-clamp technique. Cyclothiazide (0.1 mM), a complete blocker of desensitization of (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) channels, was applied to determine the changes in amplitude and kinetics of mEPSCs occurring with complete suppression of desensitization. The amplitude of mEPSC (A) was not affected significantly by cyclothiazide, but both the rise (taur) and the decay time (taud) were consistently increased (from 2.3 to 6.5 ms and from 9.9 to 22.2 ms respectively). The amplitude dependence of both taud and taur became much greater, but there was no upward shift of the best-fitting lines. The slopes of the control best-fitting lines were (+/-SD; ms/pA; n=5) 0.39+/-0.05 for taud:A and 0.12+/-0.07 for taur:A, but, in the presence of cyclothiazide, the corresponding slopes were much steeper (2.1+/-0.60 and 0.68+/-0. 21; holding potential was -50 mV and temperature 32 degreesC). These changes, which were slow to develop, suggest that cyclothiazide blocks AMPA receptor channel desensitization, whilst having no effect on the closing rate of AMPA channels. Judging by the extent of change, the speed of diffusion of glutamate in the synaptic cleft is probably similar to that in water. In conclusion, this study provides evidence that: (1) under control conditions, desensitization of AMPA channels plays a major role in shaping the time course of synaptic currents in CA1; and (2) cyclothiazide prolongs their time course solely by abolishing desensitization.

Animals↗

Examining the timing of miniature endplate potential releases at the frog and mouse neuromuscular junctions for deviations from Poisson expectations.

The intervals between miniature endplate potentials (MEPPs) were measured at frog and mouse neuromuscular junctions in several different solutions. Data sets with monotonic trends in MEPP frequency were discarded. The remaining data sets had between 283 and 5024 MEPPs. The fit to the exponential distribution, which describes a Poisson process, was tested with Sherman's statistic. If the intervals are not distributed exponentially, this statistic indicates whether they deviate because they are more concentrated or more diffuse. In 6 of 20 data sets from the frog and in 3 of 7 data sets from the mouse the intervals between MEPPs were not distributed exponentially. Some of these were clustered, while others were diffuse. In one frog data set release was periodic. In all data sets releases appeared to be independent, judging from the integrated power spectra. One data set may show fractal behavior. Calculations of the approximate entropy gave no indication of an underlying regularity, so there is no evidence for a chaotic process. The lack of fit to the exponential distribution due to either concentrated or diffuse interval distributions is mimicked by a model in which release is Poisson, but with a mean rate that randomly shifts to a different level.

Action Potentials↗

An efficient in situ hybridization protocol for multiple tissue sections and probes on miniaturized slides.

To facilitate detection of gene activity in tissue sections we combined common protocols of in situ hybridization on tissue sections (TSISH) with the technique of whole-mount in situ hybridization (WMISH). Miniature glass slides for mounting tissue sections were cut from regular microscope slides and handled for in situ hybridization in laboratory-made 2-ml containers (baskets) similar to those originally used for WMISH on Drosophila embryos. A salient point of the method is the use of airtight reaction vessels placed in a dry thermostat for critical hybridization steps as this facilitates reproducible and stringent hybridization conditions which are difficult to achieve on tissue sections otherwise. The practicability of the method is illustrated on consecutive serial frozen sections of murine neonatal cerebellum hybridized for math1 and neuroD, two developmentally regulated genes with distinct expression patterns. For both genes excellent spatial resolution and a highly dynamic range of signal intensity was obtained. The approach enables simple processing of multiple probes, allows the efficient and economic use of small tissue samples and is amenable to automation.

Animals↗

Genome-wide distribution and potential regulatory functions of AtATE, a novel family of miniature inverted-repeat transposable elements in Arabidopsis thaliana.

A study of transgenic promoter::beta-glucuronidase lines showed that the promoters of the two Arabidopsis ARGININE DECARBOXYLASE paralogues, ADC1 and ADC2, exhibited extremely different patterns of activity. One major feature of the promoter of ADC1 was the presence of a novel transposable element, which was shown to possess all of the characteristics of Miniature Inverted-repeat Transposable Elements (MITEs), and to be present in 26 full-length copies and 1617 partial copies and fragments distributed throughout the Arabidopsis genome. TRANSFAC analysis showed that this transposable element possesses a significant number of transcription-factor binding motifs. A bioinformatics approach based on a suffix-tree compilation was used to obtain an exhaustive description of exact copy numbers and positions of the element in the Arabidopsis genome. The distribution among the chromosomes was non-random, and a significant number of copies were found in regions flanking genes. Full-length copies of the transposable element were detected in the immediate vicinity of 22 genes, either upstream or downstream.

Arabidopsis↗

Zaba: a novel miniature transposable element present in genomes of legume plants.

A novel family of miniature transposable elements, named Zaba, was identified in pea (Pisum sativum) and subsequently also in other legume species using computer analysis of their DNA sequences. Zaba elements are 141-190 bp long, generate 10-bp target site duplications, and their terminal inverted repeats make up most of the sequence. Zaba elements thus resemble class 3 foldback transposons. The elements are only moderately repetitive in pea (tens to hundreds copies per haploid genome), but they are present in up to thousands of copies in the genomes of several Medicago and Vicia species. More detailed analysis of the elements from pea, including isolation of new sequences from a genomic library, revealed that a fraction of these elements are truncated, and that their last transposition probably did not occur recently. A search for Zaba sequences in EST databases showed that at least some elements are transcribed, most probably due to their association with genic regions.

Base Sequence↗

How have the alpine dwarf plants in Yakushima been miniaturized? A comparative study of two alpine dwarf species in Yakushima, Blechnum niponicum (Blechnaceae) and Lysimachia japonica (Primulaceae).

Many plant species are miniaturized in the alpine region in Yakushima, Japan. To examine how these alpine dwarf plants are different from their related lowland ones of the same species, we analyzed two phylogenetically distinct species cytologically, genetically and morphologically: one is a fern species, Blechnum niponicum, and the other is an angiosperm species, Lysimachia japonica. The analysis shows that the alpine dwarf and the lowland plants in each of these species do not differ in chromosome number or genetic constitution. The organ-level comparison between the alpine dwarf and lowland plants of B. niponicum shows that the fertile leaf size correlates closely with the sterile one. By contrast, the flower size does not correlate with the leaf size in L. japonica. At the cell level, the leaf size of the alpine dwarf plants of B. niponicum consists of a smaller number of epidermal cells than that of the lowland plants of this species. On the other hand, the smaller leaf size of the alpine dwarf plants of L. japonica depends on both the smaller number and the smaller size of the epidermal cells. We conclude that plant dwarfism in Yakushima shows variation at both the organ and cell levels.

Ferns↗

Mobilization and evolutionary history of miniature inverted-repeat transposable elements (MITEs) in Beta vulgaris L.

We have identified three families of miniature inverted-repeat transposable elements (VulMITEs) in the genome of sugar beet (Beta vulgaris L.), evidently derived from a member of the Vulmar family of mariner transposons. While VulMITEs I are typical stowaway-like MITEs, VulMITEs II and VulMITEs III are rearranged stowaway elements of increased size. The integration of divergent moderately and highly repetitive sequences into VulMITEs II and, in particular in VulMITEs III, respectively, shows that amplification of repetitive DNA by MITEs contribute to the increase of genome size with possible implications for plant genome evolution. Fluorescent in-situ hybridization (FISH), for the first time visualizing stowaway MITE distribution on plant chromosomes, revealed a dispersed localization of VulMITEs along all B. vulgaris chromosomes. Analysis of the flanking sequences identified a dispersed repeat as target site for the integration of the stowaway element VulMITE I. Recent transposition of VulMITE I, which most likely occurred during the domestication of cultivated beets, was concluded from insertional polymorphisms between different B. vulgaris cultivars and species.

Base Sequence↗

Tuareg, a novel miniature-inverted repeat family of pearl millet (Pennisetum glaucum) related to the PIF superfamily of maize.

Miniature-inverted repeat transposable elements (MITEs) are abundantly repeated in plant genomes and are especially found in genic regions where they could contribute regulatory elements for gene expression. We describe with molecular and cytological tools the first MITE family reported in pearl millet: Tuareg. It was initially detected in the pearl millet ortholog of Teosinte-branched1, an important developmental gene involved in the domestication of maize. The Tuareg family was amplified recently in the pearl millet genome and elements were found more abundant in wild than in domesticated plants. We found that they shared similarity in their terminal repeats with the previously described mPIF MITEs and that they are also present in other Pennisetum species, in maize and more distantly related grasses. The Tuareg family may be part of MITEs activated by PIF-like transposases and it could have been mobile since pearl millet domestication.

Base Sequence↗