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At least 847 records · Page 47Linked to original sources
PubMed1992

Effect of Pluronic F-68 on the mechanical properties of mammalian cells.

The mechanical properties of TB/C3 hybridoma cells taken from a continuous culture were measured by micromanipulation. The culture conditions were constant except for the presence or absence of Pluronic F-68 in the medium. It was found that the mean bursting membrane tension and the mean elastic area compressibility modulus of the cells were significantly greater (60% and 120%, respectively) in a medium with 0.05% (w/v) Pluronic F-68 compared to that without Pluronic. Pluronic F-68 therefore affected the strength of the membranes when the cells were exposed to it for a long period of time, i.e., in culture. The short-term effect of Pluronic F-68 on cell strength was also tested by its addition at various levels up to 0.2% (w/v) immediately before the mechanical property measurements. The resulting cell strength depended on the Pluronic concentration, but a significant short-term effect could only be detected above a threshold of 0.1% (w/v). Previous reports on the effect of Pluronic F-68 on animal cell culture are evaluated in the light of these observations.

Animals↗
PubMed1982

Inexpensive fiber optic two-dimensional densitometer for the high-resolution quantitation of autoradiogram grain densities.

The design and construction of a simple, low cost high performance fiber optic 2-dimensional microdensitometer is described. With this instrument the film to be quantitated is placed upon a back-lit frosted glass bench and scanned with an optic fiber probe attached to a transparent micromanipulator. The emerging light is transported by the fiber to the photocell of a direct reading spectrophotometer. The resulting numerical data can either be transcribed, portrayed on a recorder or entered into the memory of a microdata processor for further analysis and comparisons. The instrument was sensitive to very small differences in optical density and could resolve lines 100 but not 30 micrometers apart. It was precise, reliable and easy to build. With the use of this equipment many previously undetected, significant local brain glucose utilization differences were quantitated in groups of unrestrained rodents, maintained in several behavioral states.

Animals↗
PubMed1983

Clonal analysis of T lymphocyte response to an isolated class I disparity.

A bulk primed lymphocyte reagent generated in a class II identical class I (HLA-B) disparate sibling combination demonstrated both cytotoxic [cell mediated lympholysis (CML)] and proliferative [i.e., primed LD (lymphocyte) typing (PLT)] reactivity associated with the class I antigen, Bw62. Cells from this bulk population were plated by limiting dilution and cloned by micromanipulation. Three functional groups of clones were isolated. Some clones derived were found specifically to proliferate to and lyse cells bearing the Bw62 antigen. Based on such reactivities, these clones were analogous to the class of antigen-driven, helper cell independent cytotoxic (HITc) clones previously reported from our laboratories. Other clones responded proliferatively to stimulation by Bw62 positive cells but were not cytotoxic, thus fitting characteristics of Th, although it will be necessary to test such clones for their ability to produce Interleukin 2 (IL-2). In addition, conventional cytotoxic clones which did not proliferative to, but did lyse cells bearing the Bw62 antigen were isolated. The results were consistent with the existence of both HITc and Tc mediated cytotoxicity generated against this isolated class I disparity.

Clone Cells↗
PubMed1984

HLA-Dw/LD directed cytotoxic T cell clones.

T lymphocyte clones were derived by micromanipulation from an MLC between a stimulator and responder matched for class I but mismatched for class II HLA antigens; among the possible stimulating antigens were DR4 and Dw4. Two of the clones, when tested for cytotoxicity on a panel of DR4 positive and negative targets, appeared to recognize a determinant closely associated with Dw4, but did not lyse, with one exception, targets expressing other DR4 associated Dw specificities or DR4 negative targets. Blocking studies, using monoclonal antibodies directed against monomorphic epitopes on class I or class II molecules, revealed that the cytotoxic activity of these clones was strongly inhibited by an anti-class II (L-243) but not by an anti-class I (w6/32) monoclonal antibody. Both clones were T3+, T4+, T8-. These findings show that cytotoxic T cell clones, (directed against class II antigens), may have specificity that correlates with a T lymphocyte-defined LD/Dw determinant. The blocking experiments using monoclonal antibodies give further support to the idea that these clones recognize determinant(s) on a class II molecule. The cell surface phenotyping results are in agreement with previous reports that most anti-class II cytotoxic clones have a T4+ T8- phenotype.

Antibodies, Monoclonal↗
PubMed1984

Cellular basis of anti-SB response.

Cloning of cells allosensitized in vitro against SB1, SB2, and SB3 antigens was performed by micromanipulation. One hundred and twenty-six clones were tested for both proliferative and cytolytic responses; 14 proliferative, noncytotoxic clones and one clone which demonstrated both proliferative and cytotoxic reactivity specific for SB antigens were obtained. The proliferative noncytotoxic clones tested and the clone with both cytotoxic and proliferative activity were all able to produce IL-2-like activity upon specific antigen stimulation in vitro and were positive for OKT3, OKT4 but negative for OKT8. The proliferative clones fit the characteristics of helper T cell (Th) clones while the clone with both cytotoxic and proliferative reactivities is analogous to the class of antigen-driven, helper cell-independent cytotoxic (HITc) clones. No cytolytic nonproliferative SB specific clones were detected. The prevalent induction of Th clones strongly suggests that the biological function of SB antigens is similar to other class II antigens of HLA. The existence of an SB specific HITc clone demonstrates that a determinant on an SB molecule can induce both proliferative and cytolytic responses.

Antibodies, Monoclonal↗
PubMed1986

Allospecific proliferative human T-cell clones acquire the cytotoxic effector function after three months in culture, in IL-2 conditioned medium.

Allostimulated T lymphocytes were cloned by micromanipulation and expanded in IL-2 conditioned medium. Three T3+,T4+,T8-, clones called BJ1, BJ4, and BJ37, were extensively studied. The BJ1 cells were able to proliferate and kill the specific target. The BJ4 and BJ37 cells were able to proliferate with the specific restimulator but could not kill even in lectin-dependent cell-mediated cytotoxic assay; however, they acquired the specific cytolytic activity in the 6-day culture when fresh irradiated autologous peripheral blood mononuclear cells as feeder cells were added to the specific irradiated Epstein-Barr virus transformed cell line, in the presence of recombinant IL-2. This observation strongly suggested that the culture conditions could be involved in the differentiation of proliferative clones into cytotoxic T lymphocyte (CTL) clones, by the lymphokines, either present in the IL-2 conditioned medium or secreted by the mixed allogeneic irradiated feeder cells. Moreover, it was shown that the acquisition of the cytolytic function could be blocked by the monoclonal antibody LeoA1, previously described and which recognized the TLiSA1 structure involved in the CTL differentiation.

Antibodies, Monoclonal↗
PubMed1992

Positional changes of the mandibular condyle assessed by three-dimensional computed tomography.

This investigation was designed to test the validity and reliability of three-dimensional computed tomography (3-D CT) for quantification of positional changes of the condyle in a laboratory model. The model consisted of a mounted dried human skull and a mandibular condyle attached to a micromanipulator. Controlled changes in condylar position were made and the condyle/fossa was imaged. Positional changes were measured by triangulation methods based on specific 3-D CT landmarks. The data were analyzed using descriptive statistics, analyses of variance to evaluate the sources of variability, and linear contrasts to evaluate the differences between observed and expected values. The results indicated that selection of appropriate anatomic landmarks for assessment of movement influences technique accuracy. The data also indicate that 3-D CT is most accurate in detecting inferior condylar movements. Lateral and posterior movements were assessed with less accuracy than the inferior positional changes. The clinical significance of these differences has yet to be determined.

Analysis of Variance↗
PubMed1988

CD4 cytotoxic T lymphocyte differentiation.

In vitro allostimulated CD4+ human lymphocytes were cloned by micromanipulation and expanded for a short time in IL-2 conditioned medium. In the present study we observed that proliferative noncytotoxic cloned cells were able to acquire the specific cytolytic activity under some modification of the cloned cells restimulation cycle. We demonstrated that rIFN-alpha and -gamma are the agents responsible for the acquisition of specific lytic activity.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1984

A quantitative analysis of the spatial organization of the vestibulo-ocular reflexes in lateral- and frontal-eyed animals--I. Orientation of semicircular canals and extraocular muscles.

The spatial relationship between extraocular muscles and semicircular canals was evaluated in cat (frontal-eyed animal) and rabbit (lateral-eyed animal). Semicircular canal orientations in the rabbit were determined by a principal components analysis of data points obtained from the exposed osseous canals using a three-axis micromanipulator. Canal orientations were presented in terms of unit sensitivity vectors. Orientation of extraocular muscles in rabbits and cats was derived from measurements of the insertion and origin of each muscle with respect to a reference point on the skull and a calculated estimate of the center of the eye. Muscle orientations were presented in terms of unit action vectors. Semicircular canal planes of the rabbit labyrinth were not orthogonal, having deviations up to 14 degrees. Pairs of antagonistically acting vertical semicircular canals, left (right) anterior-right (left) posterior deviated from coplanarity by 16 degrees, while the deviation for the horizontal canals was 9 degrees. In both animals, muscles of an antagonistic pair were coplanar to within 8 degrees, with the exception of the oblique muscles in the rabbit for which the deviation was 19 degrees. The three pairs of antagonistic muscles were almost orthogonal to each other, the maximum deviation between any of the pairs being 8 degrees in the cat and 18 degrees in the rabbit. Comparing extraocular muscle planes and semicircular canal planes reveals that they are roughly aligned. However, there were slight but consistent differences between a given semicircular canal plane and the planes of the muscles to which this canal is connected by the classical three-neuron-arc (principal vestibulo-ocular reflex circuits).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Comparison of isolated outer hair cells from five mammalian species.

Live outer hair cells were isolated from guinea pig, chinchilla, rat, mouse, and gerbil. The organ of Corti from selected turns of the cochlea was briefly incubated with collagenase and outer hair cells were separated from the tissue by micromanipulation under microscopic observation. Morphological criteria for cell viability were: cylindrical cell shape without swelling or distortion of the membrane; location of the nucleus in its normal position near the base of the cell; cytoplasm devoid of Brownian motion and granulation. Both yield and quality (as judged by these morphological criteria) of isolated hair cells varied with the species and the turn from which the isolation was attempted. Consistently high yields and cells of good morphology were obtained from guinea pigs and chinchillas. Fewer cells were obtained from rats and mice, and their quality was less consistent. Gerbils gave the poorest yield and quality of outer hair cells. In all species, the preparation was more successful from the apical than from the basal turn. The length of apical hair cells varied almost 4-fold from 60 to 80 microns in guinea pig and chinchilla to 20 to 40 microns in the other species, while their diameter only varied 1.5-fold from 7 (mouse) to 10 microns (chinchilla). Outer hair cells could be maintained in vitro in good condition for several hours. Typical early signs of degeneration were increased Brownian motion and granulation in the cytoplasm, upward movement of the nucleus, or distortion of cell shape. Degeneration was always accompanied by a shortening along the long axis.

Animals↗
PubMed1993

Contribution of the anterior inferior cerebellar artery to cochlear blood flow in guinea pig: a model-based analysis.

This study was performed to determine the contribution of the anterior inferior cerebellar artery (AICA) to cochlear blood flow (CBF) in guinea pig. The AICA and the basilar-vertebral arterial complex in twelve animals was exposed through the basal portion of the skull. The cochlea was ventrally approached and the CBF of the apical area monitored with laser Doppler flowmetry. A specially designed microclamp was held in a micromanipulator and used to obstruct the AICA. When the AICA was clamped, CBF decreased to approximately 60% of baseline (BL) (not to 'biological zero'), followed by a gradual increase. When the clamp was released, CBF quickly increased to more than 160% BL and then slowly declined to baseline. To quantify the contribution of AICA to CBF, we formulated an electrical analog model of the cochlear vessel system. With this model, AICA contribution to CBF and the relationship among blood pressure, blood flow, and vascular resistance or vascular conductance in the cochlea can be explored. Results in the present study indicate that the AICA contributes only about 45% of CBF to the cochlea; 55% of CBF must come from other supplying vessels. Contrary to previous reports, CBF response to AICA clamping did not exhibit a stable or constant decrease but showed time-dependent dynamic changes. In addition, the cochlear vascular system showed a marked autoregulatory response, instead of a passive response, to the perfusion pressure change. AICA clamping is, therefore, not a suitable model for investigation of ischemia effects in the guinea pig cochlea, but it is a useful approach to study autoregulation and the myogenic mechanism of the cochlear vascular system.

Animals↗
PubMed1996

Cryopreservation of mammalian embryos and oocytes: recent advances.

The cryopreservation of embryos of most domestic species has become a routine procedure in embryo transfer, and recently, advances have been made in the cold storage of mammalian oocytes. The ability to sustain viable oocytes and embryos from mammalian species at low temperature for prolonged periods of time has important implications to basic and applied biotechnology. Recent advances in the study of physico-chemical behaviour of different cryoprotectants, use of various macromolecule additives in cryoprotective solutions and isolation and use of proteins of plant and animal origin with antifreeze activity offers many new options for cryopreservation of oocytes and embryos of animal and human origin. At the same time rapidly developing methods of oocyte/embryo manipulation such as in vitro embryo production, embryo splitting, embryo biopsying for gene and sex determination, embryo cloning and the isolation of individual blastomers, create new challenges in cryopreservation. Very recent advances in the cryopreservation of mammalian oocytes, in vivo- and in vitro-derived embryos, and micromanipulated embryos are reviewed in this manuscript.

Journal Article↗
PubMed1989

Formation and modulation of chemical connections: evoked acetylcholine release from growth cones and neurites of specific identified neurons.

The ability to release acetylcholine (ACh) from cultured neurons of Helisoma was assessed by micromanipulating ACh-sensitive somata into contact with presynaptic neurons. ACh release was reliably detected from neurites and growth cones of cholinergic neuron B5, but not neuron B19, as early as 3 s after contact with novel target neurons. The rapid onset of transmission correlates with the ability of neuron B5, but not neuron B19, to indiscriminately form chemical connections and may be related to the specificity of synaptogenesis. The neuropeptide FMRFamide reduces ACh release at early chemical connections. The rapid onset of functional transmission and the ability of FMRFamide to modulate chemical transmission at this early chemical connection suggest that neuron B5 acquires its presynaptic apparatus through an intrinsic program independently of target contact.

Acetylcholine↗
PubMed1991

Target contact regulates the calcium responsiveness of the secretory machinery during synaptogenesis.

Neuron B19 of Helisoma is selective in synaptogenesis. Presynaptic mechanisms underlying this selectivity were tested. Acetylcholine-sensitive assay cells were micromanipulated into contact with B19 somata to assess its secretory state. Prior to appropriate muscle target contact, spontaneous synaptic currents were detected; however, action potential-evoked release of neurotransmitter was detected only following hours of muscle contact. Photolysis of a calcium cage, DM-nitrophen, accelerated the frequency of synaptic currents in muscle-contacted, but not novel neuron-contacted, B19 somata. These studies demonstrate that contact with appropriate target muscle enhances the responsiveness of this neuron's secretory machinery to internal calcium levels, thereby imparting the presynaptic cell with the ability to couple action potentials with neurotransmitter release.

Acetates↗
PubMed1993

PCR amplification using a single cell allows the detection of the mtDNA lesion associated with Leber's hereditary optic neuropathy.

The development of the polymerase chain reaction (PCR), which routinely can amplify specific target sequences more than one billion-fold, has made it possible to produce readily detectable amounts of DNA from a few copies of very rare sequences. We have begun a study of mitochondrial myopathies with the purpose of developing a diagnostic test using PCR to amplify appropriate mitochondrial DNA (mtDNA) target sequences from small amounts of sample. We have developed a 15-min procedure for recovering mtDNA which can be amplified by PCR to detectable levels, from as little as 30 microliters of blood or 5 microliters of amniotic fluid. We have microscopically selected HL60 cells, and have found that 28 cycles of PCR allows the detection of mitochondrial targets from a single cell. Using micromanipulation techniques, we utilized this approach to analyze mtDNA from a single cell isolated from an 8-cell stage mouse blastocyst. Finally, a single cell cultured from a patient with Leber's hereditary optic neuropathy, a mitochondrial myopathy, provided sufficient mtDNA for detection of the single base substitution that leads to loss of a restriction endonuclease recognition site for SfaNI and generation of a site for MaeIII.

Animals↗
PubMed1995

New models of lymphoma in transgenic mice.

We are in a 'golden age' of mouse genetics. Genetic engineering and micromanipulation techniques now allow not only the overexpression or misexpression of transgenes randomly inserted into the mouse genome, but also the directed homologous replacement of normal genes with transgenes coding for null or mutant alleles (gene 'knockouts'). Both of these approaches have been used in the past 1-2 years to illuminate the contributions of novel oncogenes and of anti-oncogenes to the molecular basis of lymphomagenesis.

Animals↗
PubMed1995

Molecular single-cell analysis of Hodgkin and Reed-Sternberg cells.

In Hodgkin's disease, the malignant Hodgkin and Reed-Sternberg (HRS) cells are present in very small numbers in the diseased tissue, thus making molecular analysis of these cells very difficult. Using micromanipulation and single-cell polymerase chain reaction (PCR), rearranged immunoglobulin genes can be amplified and sequenced from single HRS cells. Oligonucleotides chosen from the variable (V)-gene sequences identified in the HRS cells can be used as specific markers for the tumour clone. This technique will allow one to search for members of the tumour clone in various compartments of the patient's body, to follow disease progression during therapy, and to analyse stem-cell populations for contamination by tumour cells before autologous bone-marrow transplantation.

Animals↗
PubMed2005

Development of water buffalo (Bubalus bubalis) embryos from in vitro matured oocytes reconstructed with fetal skin fibroblast cells as donor nuclei.

The present study was carried out to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to find out the developmental competence of embryos following transfer of these nuclei to in vitro matured enucleated buffalo oocytes. Skin cells were isolated from 1 to 2-month-old fetuses obtained from slaughterhouse, by enzymatic digestion (0.5% w/v trypsin +0.05% w/v collagenase in Dulbecco's PBS) for 15-20 min. The cells were washed 4 times with Dulbecco's PBS and then once with RPMI-1640+10% FBS by centrifugation at 600 x g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 2-3 days. Cumulus-oocyte complexes (COCs) collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 5 microg/ml FSH-P + 10 microg/ml LH+10% FBS) for 20-22 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body with 10-15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electro fused and incubated in the activation medium (TCM-199 + 8 microg/ml cytochalasin-B+10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199+10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 48 h. The cleaved embryos were then co-cultured with buffalo oviduct cells in embryo development media (EDM). Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electro fused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to 2-cell stage, 3 (3.8%) reached to 4-cell stage and 3 (3.8%) reached to 8-cell stage. In the synchronized group, out of 62 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40 (65%) were electrofused, activated and cultured, out of which 4 (10%) developed to 2-cell stage, 3 (7.50%) to 4-cell stage, 2 (5.0%) to early morula stage and 1 (2.50%) to blastocysts stage. These results suggest that buffalo fetal skin fibroblasts could be used as donor nuclei for the production of buffalo embryos after nuclear transfer to enucleated in vitro matured buffalo oocytes.

Animals↗