Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “MICROMANIPULATION”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,675 records · Page 93Linked to original sources
PubMed2003

Electromagnetic actuator for generating variably oriented shear waves in MR elastography.

Magnetic resonance elastography (MRE) is a recently developed technique for determining the mechanical properties of biological tissue. In dynamic MRE, electromagnetic units (actuators) are widely used to generate shear waves in tissue. These actuators exploit the interaction between the static magnetic field B(0) and an annular coil supplied with alternating currents. Therefore, coil movements are restricted to selected orientations to B(0). Conventional actuators transfer this movement collinearly to B(0) into the tissue. In this study, an electromagnetic actuator was introduced that overcomes this limitation. It is demonstrated that different directions of mechanical excitation can be generated and monitored by MRE. Different spatial components of the propagation of the shear waves were determined using agarose phantoms. The technique allows maximum contrast for MRE images of objects with anisotropic strain components such as muscle tissue.

Anisotropy↗
PubMed2005

The utility of an erythroblast scoring system and gender-independent short tandem repeat (STR) analysis for the detection of aneuploid fetal cells in maternal blood.

OBJECTIVE: The aim of this study was to determine whether fetal nucleated red blood cells (NRBCs) could be distinguished from maternal cells in peripheral blood using an erythroblast scoring system based on the unique morphological and hemoglobin staining characteristics of this cell type. Presumptive fetal NRBCs were further analyzed for the presence of paternally inherited DNA polymorphisms to prove fetal origin. METHODS: NRBCs were isolated by density gradient separation, CD15/45 depletion, and gamma hemoglobin positive selection from peripheral blood of nine women following termination of pregnancy for trisomy 21 (n=4), 18 (n=1), 13 (n=2), and other genetic abnormalities (n=2). Candidate fetal NRBCs, based on four discrete morphological and hemoglobin staining criteria, were then subjected to fluorescent PCR (polymerase chain reaction) amplification of chromosome 21 (D21S1411, D21S11) and chromosome 18 (D18S535) short tandem repeat (STR) DNA polymorphisms. RESULTS: In all cases, candidate fetal NRBCs were accurately identified on the basis of morphologic and hemoglobin staining characteristics and confirmed to be fetal in origin based on the presence of shared and nonshared polymorphic DNA alleles when compared to DNA isolated from maternal cells. CONCLUSIONS: Using the erythroblast scoring system and subsequent analysis of inherited DNA polymorphisms, we were able to distinguish fetal NRBCs from maternal cells and prove fetal origin independent of gender. These results suggest that this novel combined approach to fetal cell isolation and genetic analysis is a promising method for noninvasive prenatal diagnostic applications.

Aneuploidy↗
PubMed2001

Preimplantation genetic diagnostic protocols for alpha- and beta-thalassaemias using multiplex fluorescent PCR.

Haemoglobinopathies including alpha- and beta-thalassaemia are the world's most common class of single gene disorder. Prenatal diagnosis (PND) for beta-thalassaemia has been proven to be an effective strategy for controlling the incidence of new cases and is widely used in several countries where the disease is common. Successful preimplantation genetic diagnosis (PGD) protocols for beta-thalassaemia have been introduced using restriction fragment length polymorphism (RFLP), single-stranded conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). However, contamination and allele dropout (ADO) remain an important concern for all of these strategies. In the present study two PGD protocols for detecting beta-thalassaemia mutations (codon 41-42 and IVSI-110) and one for alpha-thalassaemia (SEA mutation) have been designed and tested. These methods contain failsafe mechanisms to reduce the risk of misdiagnosis due to ADO or contamination and utilise multiplex fluorescent PCR (F-PCR). Interestingly, amplification efficiency and ADO were significantly affected by the choice of DNA polymerase and the freshness of the single cells used. The close similarity between the DNA sequences of beta-globin and delta-globin was also found to be an important issue that necessitated careful design of primers for the beta-globin gene.

Adult↗
PubMed2003

HLA-G positive trophoblastic cells in transcervical samples and their isolation and analysis by laser microdissection and QF-PCR.

OBJECTIVE: To assess the frequency of cytotrophoblastic cells in endocervical samples collected by lavage at early stages of gestation using a specific anti-HLA-G McAb (G233). From a set of four selected samples, cells identified by immunostaining were collected by laser microdissection and then tested by quantitative fluorescent polymerase chain reaction (QF-PCR) for the presence of paternally derived DNA markers, in order to establish their fetal origin. METHODS: Syncytial fragments and cytotrophoblastic cells from 23 transcervical samples were identified by immunostaining with McAb G233 reacting against HLA-G antigen and with antibodies against cytokeratin. Slides from the same samples were also tested by fluorescent in situ hybridization (FISH), while selected samples were analysed by QF-PCR. Slides from four samples retrieved from mothers with male fetuses were immunolabelled and then cytotrophoblastic cells, syncytial fragments and maternal epithelial cells were collected by laser microdissection and tested by QF-PCR. RESULTS: All endocervical samples retrieved from mothers with male fetuses were found to contain some cells with chromosome Y-specific signals when tested by FISH. Using McAb anti- HLA-G, cytotrophoblastic cellular elements were detected in about 50% of the samples. From four samples, cellular elements identified by immunostaining as cytotrophoblast or syncytial fragments were collected by laser microdissection and shown to be of fetal origin when tested by QF-PCR for the presence of fetal DNA markers. CONCLUSIONS: These results confirm that, during an early phase of gestation, fetal cells are released in the lower uterine cavity and that they can be isolated and analysed for prenatal diagnosis of single gene defects and aneuploidies.

Adult↗
PubMed2003

A simple and sensitive erythroblast scoring system to identify fetal cells in maternal blood.

OBJECTIVES: Nucleated red blood cells (NRBCs) of fetal origin appear to have distinguishable characteristics from that of maternal NRBCs in both nuclear morphology and properties of hemoglobin staining. However, these differences have yet to be quantified. Our aim was to develop an erythroblast scoring system using four distinct phenotypic parameters (nuclear roundness, nuclear morphology, gamma hemoglobin staining intensity, and peripheral brightness of the stained cytoplasm) to address this issue. METHODS: NRBCs were isolated from four maternal blood samples by density gradient separation, CD15/45 depletion, and gamma hemoglobin positive selection after elective termination of a trisomy 21 male fetus (47,XY,+21). All cells were deposited onto microscope slides and every NRBC was analyzed according to the scoring system. Each of the four individual parameters was given a value from 0 to 3 points and a combined score was obtained for each cell (range 0-12). Fluorescence in situ hybridization (FISH) using X- and Y-specific probes was performed to determine, on the basis of interphase karyotype, whether the cell was maternal or fetal. RESULTS: The majority of maternal NRBCs were found to have a combined score of 6 or less (103/117) and the majority of fetal NRBCs were found to have a score of 7 or greater (43/53). The proportion of cells that were identified correctly as fetal increased with each ascending category of combined score. For example, 5.7% of NRBCs with a combined score of 5 points or less were found to be fetal, whereas 19.2% of NRBCs with a combined score of 6 points were fetal. At combined scores of 11 and 12 points, 100% of NRBCs were found to be fetal. CONCLUSION: Fetal NRBCs have characteristic morphology and a gamma hemoglobin staining appearance that makes them distinguishable from maternal NRBCs. The scoring system presented here is a simple and sensitive method to distinguish fetal NRBCs from adult cells in maternal blood. This system may have clinical utility for noninvasive prenatal diagnosis as well as applications for basic research into the developmental biology of NRBCs. In addition, these defined parameters may serve as computational classifiers for the automated detection of fetal cells in maternal blood.

Adult↗
PubMed1993

Ultrathin-layer zone electrophoresis of lactate dehydrogenase isoenzymes in microdissected liver samples.

A highly sensitive electrophoretic separation of lactate dehydrogenase isoenzymes in 150-microns ultrathin-layer polyacrylamide gels is described. By means of the incorporation of miniature-sized wells into the gel and by executing all the analytical steps under paraffin oil, this technique allows the exact and sharp separation of the lactate dehydrogenase isoenzymes in nanogram-sized microdissected liver tissue samples. With this method the heterogeneous distribution pattern of lactate dehydrogenase isoenzymes in the liver acinus of various mammals could be demonstrated, and a new interpretation of their functional roles is offered.

Animals↗
PubMed1994

Microanalysis of cholesterol, phospholipid, and medium- and long-chain fatty acids in biologic materials.

We have developed a procedure for the microanalysis of the cholesterol and phospholipid concentrations and the fatty acid (including medium-chain fatty acids) composition of total lipid and phospholipid. One-milligram lipid samples were extracted from jejunal brush border membranes isolated from rats fed a diet containing 14% medium-chain triglycerides. The samples were analyzed by gas chromatography using an autosampler. We were able to identify 17 fatty acids in the total lipid and in the phospholipid fractions. The cholesterol and phospholipid contents of the membranes were 270 +/- 21 and 298 +/- 35 nmol.mg protein-1, respectively; thus the cholesterol/phospholipid ratio was 0.92 +/- 0.05, a value typically reported for the rat. Medium-chain fatty acids were not detected in the brush border membranes. Therefore, to demonstrate that medium-chain fatty acids were not lost during analysis, we analyzed the rat diet, and brush border membrane samples to which medium-chain triglycerides had been added. Recovery of medium-chain fatty acids from brush border membrane samples was 95.7 +/- 2.6%. The percentage of medium-chain fatty acids detected in the diet was not significantly different from the percentage when the diet was prepared. In summary, our procedure enabled a complete lipid analysis of very small biologic specimens.

Animals↗
PubMed1994

Colorimetric micromethod for protein determination with erythrosin B.

Reaction of erythrosin B with proteins results in a stable, highly colored chromophore with an absorbance maximum at 545 nm. This is the basis for a new quantitative determination method of proteins in solution. The assay can be performed at room temperature, but is faster and more sensitive at 90-95 degrees C. The erythrosin assay is characterized by (i) stable dye-protein color, (ii) high sensitivity (2-14 micrograms/ml protein), (iii) short reaction time (1.5-2 min at 90-95 degrees C), (iv) good reproducibility, (v) limited interference by common reagents, and (vi) low protein-to-protein variability. Thus, the erythrosin assay can be useful for routine analytical purposes and may overcome some of the limitations of other currently employed assays.

Artifacts↗
PubMed1995

Microtiter plate-based determination of multiple concentration-inhibition relationships.

The microtiter plate reader has been used to obtain data on the inhibition of aldehyde dehydrogenase over a wide range of inhibitor concentrations. Multiple reactions can be run simultaneously and assayed rapidly with repetitive determinations of enzyme activity using this technique. The microplate reader allows for the efficient generation of concentration-inhibition curves of excellent precision, using only small amounts of material under uniform experimental conditions. The microplate reader can be very useful for pharmacologic studies which make use of spectrophotometric assays.

Aldehyde Dehydrogenase↗
PubMed1994

Preparative in situ hybridization: selection of chromosome region-specific libraries on mitotic chromosomes.

We have developed preparative in situ hybridization (Prep-ISH) of complex DNA populations to mitotic chromosomes as a means of generating chromosome region-specific DNA subpopulations. Prep-ISH is a combination of two cytogenetic techniques: in situ hybridization of DNA molecules to mitotic chromosomes and chromosome microdissection. Here, we present test cases demonstrating the feasibility of this approach on mouse and human genomes, using single nuclei, single chromosomes, or single chromosomal subregions to assess sensitivity, specificity, and representation of the Prep-ISH technique. Prep-ISH has a number of applications in studies of gene expression and genome organization, including efficient cytogenetic sorting of tissue-specific cDNAs and genomic DNA libraries. In addition, Prep-ISH is likely to dramatically reduce the number of candidate genes to aid in gene discovery efforts and to improve efficiency of developing transcription maps and YAC and cosmid contigs through defined cytogenetic regions.

Animals↗
PubMed1994

Microdissected double-minute DNA detects variable patterns of chromosomal localizations and multiple abundantly expressed transcripts in normal and leukemic cells.

Double-minute (dm) chromosomes are cytogenetically resolvable DNA amplification-mediating acentric extrachromosomal structures that are commonly seen in primary tumors, tumor cell lines, and drug-resistant cells grown in vitro. Selective isolation of dm DNAs with standard molecular biological techniques is difficult, and thus, detailed studies to elucidate their structure, site of chromosomal origin, and chromosomal reintegration patterns have been limited. In those instances in which a gene has been localized on dms, characterization of the remainder of the DNA, which far exceeds the size of the gene identified, has remained inconclusive, dms seen in the acute myeloid leukemia cell line HL-60 have been shown to harbor the c-myc protooncogene. In this paper, we report the successful isolation of the dm-specific DNAs from these cells by the microdissection/polymerase chain reaction technique and demonstrate that the dm DNAs derived from a single discrete normal chromosome segment 8q24.1-q24.2 reintegrate at various specific locations in the leukemic cells. The microdissected dm DNA detects multiple abundantly expressed transcripts distinct from c-myc mRNA on Northern blots. By devising a "transcript selection" strategy, we cloned the partial genomic sequence of a gene from the microdissected DNA that encodes two of these RNAs. This strategy will be generally applicable for rapid cloning of unknown amplified genes harbored on dms. With DNA from 20 microdissected dms, we constructed a genomic library of about 20,000 recombinant microclones with an average insert size of about 450 bp.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1999

Single molecule force spectroscopy of spectrin repeats: low unfolding forces in helix bundles.

Spectrin repeats fold into triple helical coiled-coils comprising approximately 106 amino acid residues. Using an AFM-related technique we measured the force required to mechanically unfold these repeats to be 25 to 35 pN. Under tension, individual spectrin repeats unfold independently and in an all-or-none process. The dependence of the unfolding forces on the pulling speed reveals that the corresponding unfolding potential is shallow with an estimated width of 1.5 nm. When the unfolded polypeptide strand is relaxed, several domains refold within less than a second. The unfolding forces of the alpha-helical spectrin domains are five to ten times lower than those found in domains with beta-fold, like immunoglobulin or fibronectin Ill domains, where the tertiary structure is stabilized by hydrogen bonds between adjacent strands. This shows that the forces stabilizing the coiled-coil lead to a mechanically much weaker structure than multiple hydrogen-bonded beta-sheets.

Animals↗
PubMed1993

A micropipette which allows in situ perfusion of arterioles and capillaries.

In order to investigate capillary physiology, a glass micropipette system was developed that allowed in situ perfusion of microvessels as well as rapid changes of perfusion solutions. Theta tube (WPI, Inc.; 1.5-mm o.d. glass stock capillary tubing which is divided into two hemicylindrical sides by a central glass septum) was pulled to a smaller diameter of approx 300-600 microns and inserted into the shank of a sharpened cannulating micropipette tip constructed from large-bore glass stock (1.6 mm i.d.). The resulting dead volume between the end of the Theta supply tube and the tip of the outer cannulating tip was approximately 90 nl. The perfusate was driven in a circuit from a pressurized feed reservoir down one side of the Theta supply tube pipette and back through the second side into a reservoir maintained at a lower pressure. The pressure gradient between the two reservoirs established a high-volume flow rate and subsequently a short perfusate transit time from the feed to the collection reservoir. The average pressure in the two reservoirs determined the pressure which drove the perfusate from the cannulating tip. At normal pressures and flows, the time required to change perfusion fluid composition at the pipette tip was less than 1 min, and discharge hematocrit of a red blood cell suspension was indistinguishable from the hematocrit measured in the feed reservoir.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Direct measurement of retinal microvascular pressures in the live, anesthetized cat.

Microvascular pressures in retinal circulation were measured in living, anesthetized cat by the servonull micropuncture technique. Of the total vascular pressure drop 32% was in the segment proximal to the large retinal arteries when intraocular pressure was set at 10 mm Hg and decreased to 27% when intraocular pressure was increased to 20 mm Hg. The pressure in the retinal veins was substantially (> 7 mm Hg) greater than intraocular pressure at both values of intraocular pressure. There was negligible pressure drop within venous segment; however, the pressure drop from the retinal veins to the systemic veins was large and cannot be explained on the basis of a venous waterfall within the retina.

Animals↗
PubMed1994

Two phases of pseudopod protrusion in tumor cells revealed by a micropipette.

Pseudopod protrusion at the leading edge is a characteristic of migrating tumor cells as they traverse vascular subendothelial basement membranes to establish metastases. A micropipette system has been developed to study the dynamics of pseudopod protrusion in individual human melanoma cells in response to type IV collagen, a component of basement membranes. Soluble type IV collagen stimulates chemotaxis of A2058 melanoma cells through a G protein-coupled receptor, and induces an early burst of intracellular calcium (Savarese et al., 1992, J. Biol. Chem. 267, 21928-21935). A micropipette filled with type IV collagen solution (100 micrograms/ml) was positioned so that the tip was adjacent to a cell suspended in Dulbeceo's modified Eagle's medium. Within 10 min, tumor cells generated a pseudopod which entered the micropipette with an average velocity of 0.24 micron/min and proceeded to lengthen for 40 min. Pseudopods from individual cells ranged from 7.5-10 microns at this time and were characterized by an irregular shape which did not fill the lumen of the micropipette. Pretreatment of cells with pertussis toxin (0.5 microgram/ml), which inhibits cell migration by approximately 90% (but not the calcium burst), blocked formation of the irregular, extended pseudopod, while allowing a much smaller outpouching, or bleb, to form. Lengths of such blebs from individual PT-treated cells reached a plateau at approximately 20 min and ranged from 2.2-4.0 microns at the end point. Treatment of cells with bis-(amino-phenoxy)ethane tetraacetic acid (75 microM), an intracellular Ca2+ chelator, blocked initial bleb formation and prevented extension. From these observations we hypothesize that tumor cell pseudopod protrusion induced by soluble type IV collagen takes place in distinct, separable phases: an initial convex, symmetrical outpouching, caused by localized Ca(2+)-activated actin depolymerization and osmotic flux, followed by an extension with an irregular shape, which requires G protein-mediated actin polymerization.

Calcium↗