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At least 721 records · Page 40Linked to original sources
PubMed2000

Genetic evidence for somatic haploidization in developing fruit bodies of Armillaria tabescens.

Armillaria spp. have vegetative hyphae with diploid uninucleate cells, but the fruit bodies of many species contain clamped dikaryotic hyphae. Earlier observations suggest that somatic haploidization takes place in developing fruit bodies. To verify this, a uninucleate diploid cell was isolated from each of the 49 mating combinations between single-spore isolates of Armillaria tabescens and they were fruited. Twenty-four isolates produced fruit bodies with at least a partially dikaryotic subhymenium. Dikaryotic hyphae were isolated from fruit-body primordia and homokaryons were obtained by micromanipulation or by protoplasting. Approximately half of the isolates proved to represent recombinant mating types in respect to parent homokaryons, and most of them contained recombinant haploid DNA, based on random-amplified microsatellite markers. The results show that the nuclei in dikaryotic hyphae found in fruit bodies result from somatic haploidization. The mechanism of haploidization remains unclear.

Basidiomycota↗
PubMed1999

Stiffening response of a cellular tensegrity model.

Living cells exhibit, as most biological tissues, a stiffening (strain-hardening) response which reflects the nonlinearity of the stress-strain relationship. Tensegrity structures have been proposed as a comprehensive model of such a cell's mechanical response. Based on a theoretical model of a 30-element tensegrity structure, we propose a quantitative analysis of its nonlinear mechanical behavior under static conditions and large deformations. This study provides theoretical foundation to the passage from large-scale tensegrity models to microscale living cells, as well as the comparison between results obtained in biological specimens of different sizes. We found two non-dimensional parameters (L*-normalized element length and T*-normalized elastic tension) which govern the mechanical response of the structure for three types of loading tested (extension, compression and shear). The linear strain-hardening is uniquely observed for extension but differed for the two other types of loading tested. The stiffening response of the theoretical model was compared and discussed with the living cells stiffening response observed by different methods (shear flow experiments, micromanipulation and magnetocytometry).

Animals↗
PubMed2002

A cellular tensegrity model to analyse the structural viscoelasticity of the cytoskeleton.

This study describes the viscoelastic properties of a refined cellular-tensegrity model composed of six rigid bars connected to a continuous network of 24 viscoelastic pre-stretched cables (Voigt bodies) in order to analyse the role of the cytoskeleton spatial rearrangement on the viscoelastic response of living adherent cells. This structural contribution was determined from the relationships between the global viscoelastic properties of the tensegrity model, i.e., normalized viscosity modulus (eta(*)), normalized elasticity modulus (E(*)), and the physical properties of the constitutive elements, i.e., their normalized length (L(*)) and normalized initial internal tension (T(*)). We used a numerical method to simulate the deformation of the structure in response to different types of loading, while varying by several orders of magnitude L(*) and T(*). The numerical results obtained reveal that eta(*) remains almost independent of changes in T(*) (eta(*) proportional, variant T(*+0.1)), whereas E(*) increases with approximately the square root of the internal tension T(*) (from E(*) proportional, variant T(*+0.3) to E(*) proportional, variant T(*+0.7)). Moreover, structural viscosity eta(*) and elasticity E(*) are both inversely proportional to the square of the size of the structure (eta(*) proportional, variant L(*-2) and E(*) proportional, variant L(*-2)). These structural properties appear consistent with cytoskeleton (CSK) mechanical properties measured experimentally by various methods which are specific to the CSK micromanipulation in living adherent cells. Present results suggest, for the first time, that the effect of structural rearrangement of CSK elements on global CSK behavior is characterized by a faster cellular mechanical response relatively to the CSK element response, which thus contributes to the solidification process observed in adherent cells. In extending to the viscoelastic properties the analysis of the mechanical response of the cellular 30-element tensegrity model, the present study contributes to the understanding of recent results on the cellular-dynamic response and allows to reunify the scattered data reported for the viscoelastic properties of living adherent cells.

Animals↗
PubMed2001

Peptide nucleic acid-assisted topological labeling of duplex dna.

Peptide nucleic acids (PNAs) are a family of synthetic polyamide mimics of nucleic acids that offer a variety of applications. Pyrimidine bis-PNAs can be used for rational design of novel interlocked DNA nanostructures, earring labels, representing locked pseudorotaxanes or locked catenanes. These structures are created through DNA ligase-mediated catenation of duplex DNA with a circularized oligonucleotide tag at a designated DNA site. The assembly is performed via formation of the PD-loop consisting of a pair of bis-PNA openers and the probe oligonucleotide. The openers locally expose one of the two strands of duplex DNA for hybridizing the probe, whose termini are complementary to the displaced DNA strand. After hybridization, they are in juxtaposition and can subsequently be linked by DNA ligase. As a result, a true topological link forms at a precise position on the DNA double helix yielding locked, earring-like label. DNA topological labeling can be done both in solution and, for longer templates, within the agarose gel plug. Accordingly, highly localized DNA detection with rolling circle amplification of hybridization signal and effective micromanipulations with DNA duplexes become possible through precise spatial positioning of various ligands on the DNA scaffold.

DNA↗
PubMed1995

Phylogenetic relationships among karyorelictids and heterotrichs inferred from small subunit rRNA sequences: resolution at the base of the ciliate tree.

Ciliate protozoa are among the most diverse and complex cells that have been described. Ciliates are characterized by nuclear dimorphism, possessing a macronucleus and a micronucleus which share the same cytoplasm. An understanding of the evolution of ciliate diversity depends upon knowledge of their phylogeny. In this study we attempted to resolve some of the relationships at the base of the ciliate tree by determining the phylogenetic position of a sample of heterotrich and hypothesized primitive karyorelictid ciliates. Karyorelictids are considered primitive because they possess a "simple" form of nuclear dualism whereby the macronucleus does not divide once it has differentiated from a micronucleus. We micromanipulated cells of two heterotrichs, Spirostomum ambiguum and Gruberia sp., and two karyorelictids, Loxodes magnus and Tracheloraphis sp., and amplified their small subunit (SSU) rDNA using PCR. The primary structure of the SSU rDNA was determined for each species and used to infer their positions in the ciliate phylogenetic tree. The results indicate, with strong support, that the aerobic heterotrichs and the karyorelictids sampled constitute a monophyletic group. The most parsimonious interpretation of the form of nuclear dimorphism in karyorelictids is that it is derived from the general condition as found in its sister group the aerobic heterotrichs. The two anaerobic heterotrichs, Metopus contortus and Metopus palaeformis, comprise a distinct clade, so that the subclass Heterotrichia, as currently conceived, is not a monophyletic group. The complex mouth architecture which characterizes all heterotrichs must be reassessed in light of this finding.

Animals↗
PubMed1995

Mediator-induced changes in macromolecular permeability in the rat mesenteric microcirculation.

An intravital fluorescence microscopic method for measurement of changes in macromolecular permeability has been established in the mesenterial microcirculation of the rat. After exteriorization of the fat-free distal part of the ileal mesentery, a 1-hr period of stabilization was followed by the injection of FITC-labeled macromolecules. Five minutes later, histamine, leukotriene B4, or leukotriene C4 was topically applied to the tissue by means of a micromanipulator. Areas of 1 mm2 were videotaped with a SIT camera. The fluorescence intensity of these areas was measured by an analogous video image processing system and displayed as gray value histograms. The shift of the frequency of gray levels from lower to upper regions could be attributed to an increase in light intensity in the mesentery, indicating an increase in vessel wall permeability. The sites of action of histamine and leukotriene C4 were very similar. Both mediators affected mainly the larger collecting venules. In contrast, leukotriene B4 exerted its effect at postcapillary venules. Moreover, leukotriene B4-induced extravasation was inhibited by superoxide dismutase, suggesting an involvement of oxygen radicals. The studies with histamine alone and with H1- and H2-antagonists demonstrated that histamine-induced extravasation in the rat mesentery was mediated by H1-histamine receptors. The present study introduces an experimental model for the measurement of changes in macromolecular permeability, which is useful for studying mediator effects and their pharmacological inhibition in the microcirculation of the rat mesentery.

Animals↗
PubMed2002

Laser Doppler flowmetry mapping of cerebrocortical microflow: characteristics and limitations.

The aim of this study was to quantitatively analyze the amount of methodological noise and the spatial and temporal variability of laser Doppler flowmetry (LDF) signals mapping cerebrocortical microflow. In an experimental setup with latex beads, the methodological LDF-signal variability was determined (coefficient of variation or CV(method)). The biological variability of the LDF signals was measured in animal experiments using 10 anesthetized rabbits. One stationary reference probe was used to assess temporal heterogeneity (CV(temp)) and a micromanipulator-driven scanning probe was used to assess spatial heterogeneity (CV(spat)) in a cortical area of 3.5 x 4.5 mm with 252 measurement points. CO(2) tests were used to modulate cerebrovascular resistance. CV(method) was found to be 4.94 +/- 1.7. The CV(temp) for the LDF-velocity signal was assessed to be 13.93 +/- 5.9 during normocapnia. Scanning of the brain surface with the scanning probe revealed a CV(spat) for LDF velocity of 65.0 +/- 16.2 during normocapnia. CO(2) modulation (hypocapnia --> normocapnia --> hypercapnia) of the cerebral resistance did not show a significant change in temporal heterogeneity (10.84 +/- 3.1 --> 13.93 +/- 5.9 --> 14.82 +/- 3.9), whereas spatial heterogeneity decreased significantly (81.31 +/- 12.0 --> 65.0 +/- 16.2 --> 54.04 +/- 21.8). Although the spatial and temporal variability of LDF signals evoked by cerebrocortical microflow is in the same range as with other methods and in other organs, LDF cerebrocortical mapping is restricted by the large temporal and spatial heterogeneity of the cerebrocortical vasculature. The definitions of sample volume, scanning step width, probe to brain surface distance, and average time per scanning point are critical concerning reliable LDF cerebrocortical mapping techniques.

Animals↗
PubMed2006

Handmade somatic cell cloning in cattle.

Apart from the biological and ethical problems, technical difficulties also hamper the improvement and widespread application of somatic cell nuclear transfer (NT). Recently introduced zona-free procedures may offer a solution for the latter problem. The most radical approach of these techniques is the so-called handmade cloning (HMC). It does not require micromanipulators because the manipulations required for both enucleation and nucleus transfer are performed by hand. The HMC technique includes manual bisection of zona-free oocytes, selection of cytoplasts by staining, and the simultaneous fusion of the somatic cell with two cytoplasts to produce a cloned embryo. HMC is a rapid and efficient technique that suits large-scale NT programs. It requires less expertise and time than traditional NT methods and the cost of equipment is significantly less. Production efficiency is high and embryo quality, in terms of pregnancy rates and live births, is not compromised. Although HMC has been developed particularly for bovine NT, the technique is applicable to other species. The method may become a useful tool for both experimental and commercial somatic cell cloning because it allows for standardization of procedures and provides the possibility of automation.

Animals↗
PubMed1990

In vitro survival of cells derived from isodiploid uniparental half embryos of the mouse after aggregation with normal embryos.

Fertilized mouse eggs, heterozygous for two allozymes of glucose phosphate isomerase (GPI) were bisected, and the resulting half eggs were diploidized with cytochalasin B. After separate aggregation with normal embryos carrying a third allozyme of GPI, the resulting chimaeras were kept in culture up to 10 days. The majority grew out on the culture dish during this period. By GPI analysis, 7.7% of the embryos were found to be chimaeric. Both types of uniparental cells, from gynogenetic and from androgenetic half eggs, were capable of surviving in chimaeras in vitro. These results are comparable with published data obtained by using uniparental embryos generated by micromanipulation.

Animals↗
PubMed1995

Endoscopy under operating microscope. A new device.

Existing technical conditions make it difficult for a single surgeon to simultaneously observe the endoscope and the observation field of the operating microscope. Video endoscopy is not adapted, because it respects neither the constraints of microsurgery (the surgeon has to stop observing the binoculars of the microscope to watch the video screen) nor the micromanipulations necessary for the specialties using micro endoscopy. A new device is described that closely joins endoscope and microscope. This coupling device allows the surgeon to permanently control and alternate the images according to his choice. The surgeon is able to observe in the binoculars the images transmitted by the endoscope and/or the operating microscope. This device respects the requirements of the microscope and, thanks to its optical capabilities, the endoscopic image is improved. The professions requiring double observation will improve their ability in diagnosis and therapy.

Endoscopes↗
PubMed1996

Microinjection technique for pharmacological evaluation of microvascular permeability in human skin.

A new technique which combines skin microinjection of minute amounts of drugs (0.5 microliter) and measurement of capillary permeability by intravital fluorescence videomicroscopy and densitometry is introduced. Glass micropipettes with a tip diameter of 7-9 microns are inserted by a micromanipulator into the stratum papillare containing the capillary loops and used for microinjection by microsyringe and special dispenser. Transcapillary diffusion of sodium fluorescein applied by intravenous bolus injection is visualised by fluorescence videomicroscopy and stored on videotape. Perivascular fluorescent light intensity (FLI) is measured in arbitrary units (AU) by videodensitometry around the site of microinjection during playback of the videotapes. The method was tested by microinjection of 0.5 microliter histamine (1% solution) at the distal tibial plateau. Mean FLI values representing microvascular permeability were 2186 AU 10 min after microinjection of histamine, 420 AU after physiologic saline and 1228 AU after histamine combined with oral intake of 20 mg cetirizine. Cetirizine significantly reduced (P < 0.01) the increased permeability induced by histamine. However, the mean values after injection of the H1-blocker were still significantly enhanced (P < 0.01) when compared to the mean values observed after injection of physiologic saline solution, which provoked only minor microtrauma. The technique offers new scope for pharmacological testing in man.

Adult↗
PubMed1990

Microdissection of banded human chromosomes.

Physical dissection of metaphase chromosomes is the most straightforward approach for the isolation of DNA sequences from specific chromosome regions. However, conventional microdissection techniques are too crude and inefficient for analysis of the human genome. Here we describe a technique for the precise dissection of single bands from GTG-banded chromosomes. Cells from normal amniotic fluid cell cultures are harvested by the pipette method. Microdissection is performed on an inverted microscope (magnification 1250X) with the help of extended siliconized glass needles and an electronically controlled micromanipulator. Enzymatic amplification of the dissected DNA allows the construction of band-specific DNA libraries from as few as 20 dissected chromosome fragments.

Cells, Cultured↗
PubMed1989

Telomeric sequences derived from laser-microdissected polytene chromosomes.

Telomeric fragments from salivary gland squashes of Drosophila melanogaster Oregon R. were produced by a new microdissection technique, UV laser microbeam dissection. Microdissection, an essential step in microcloning procedures, is usually performed using micromanipulators and microneedles. Recently it has been shown that microdissection can be improved to very high precision if a laser coupled into a microscope is used. A laser microbeam, generated by an excimer pumped dye laser, allows chromosomes to be cut into slices of less than 0.5 micron. Here it is shown, that single copy DNA probes prepared from Drosophila chromosomes by laser microdissection and microcloning relocalize to the chromosomal regions from which they are derived. The combination of laser technique and microcloning provides an advantageous approach for rapid genetic analysis with potential for the study of genetic diseases and genome mapping.

Animals↗
PubMed1979

Internuclear control of DNA synthesis in exconjugant cells of Paramecium caudatum.

An exconjugant cell of Paramecium caudatum has two kinds of macronuclei, fragmented prezygotic macronuclei and postzygotic new macronuclei (anlagen). Although the DNA synthesis in the fragmented prezygotic macronucleus continues until the third cell cycle after conjugation, selective suppression of the DNA synthesis in the prezygotic macronucleus takes place at the fourth cell cycle. The inhibition of DNA synthesis in prezygotic fragmented macronuclei is due to the presence of a postzygotic macronucleus (anlage) in the same cytoplasm because the inhibition does not occur when the postzygotic macronucleus (anlage) is removed by micromanipulation during the third or fourth cell cycle. Well-developed postzygotic macronuclei (anlagen) with full ability to divide have the ability to depress the DNA synthesis of prezygotic macronuclear fragments. The suppression of DNA synthesis in prezygotic macronuclear fragments seems to be irreversible. Competition for the limited amount of DNA precursors also plays an important role in the onset of the selective suppression of the DNA synthesis.

Animals↗
PubMed1978

Identification of the small nuclear RNAs associated with the mitotic chromosomes of Amoeba proteus.

Amebas contain 7 electrophoretically distinct species of small nuclear RNAs (snRNAs), some of which are known to associate in a striking manner with mitotic chromosomes. These RNAs can be divided into 2 classes, one consisting of 4 snRNA species that shuttle in a non-random way between nucleus and cytoplasm during interphase and one consisting of 3 snRNA species that do not leave the nucleus at all during interphase. In the work reported here we sought to determine which class is associated with mitotic chromosomes. Through a series of micromanipulative procedures we arranged for the shuttling snRNAs to be the only radioactive molecules in the cell. Such cells were allowed to enter mitosis, whereupon they were fixed and subjected to autoradiography. In those cells no radioactive snRNAs were found associated with mitotic chromosomes. It is concluded, therefore, that those snRNAs that do associate with mitotic chromosomes must be one or more of the non-shuttling species.--In the Discussion, how the non-shuttling snRNAs may function in cell activities is considered.

Amoeba↗
PubMed1993

Self-diploidization in Saccharomyces cerevisiae kar2 heterokaryons.

Zygotes isolated by micromanipulation from crosses of Saccharomyces cerevisiae strains, one of which carries a kar mutation, give rise most frequently to cytoductant colonies showing the nuclear constitution of either one of the two haploid parental strains. In crosses of kar2-1 strains to wild-type, about 10% of the cytoductants of both mating types are homozygous autodiploids. There is evidence indicating that self-diploidization occurs by fusion between sibling nuclei in the heterokaryotic zygote. Here we describe this phenomenon and propose to take advantage of it for the construction of genotypically-defined diploids able to mate, and of polyploid strains, which are useful tools in genetic and cytological studies.

Crosses, Genetic↗
PubMed1994

Gene mapping from a bovine 1;29 DNA library prepared with chromosome microdissection.

Bovine gene mapping is progressing rapidly using syntenic group mapping based on somatic cell hybrids and linkage, and to a lesser extent on in situ hybridization. Single chromosome DNA libraries are a logical next step, and this was, therefore, the aim of our laboratory. Since we have access to several cattle with t(1;29) and this chromosome is readily distinguishable, we chose this as our first target--recognizing that we would not produce a "single" chromosome library in the strict sense because two autosomes are represented. We utilized an inverted microscope and a micromanipulator fitted with glass instruments pulled specifically to dissect off approximately 100 t(1;29) chromosomes per microdrop. A glass chamber made to accommodate a hanging drop was used to extract the DNA under a dissecting microscope. The DNA was then cleaved with EcoRI and inserted in lambda gtwes arms. Host cells were then infected with these phage and positive clones obtained. The first clone, isolated from this library by hybridization with a human collagen 6A1 cDNA, was mapped by in situ hybridization to bovine Chromosome some (Chr) 1q12-q14, near the centromere. The second clone, an anonymous DNA fragment (D1S11), was mapped to 1q43-q46, near the terminal end.

Animals↗