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PubMed2006

Oxidative stress by biphenyl metabolites induces inhibition of bacterial cell separation.

Cell-cell separation of a polychlorinated biphenyl (PCB)-degrading bacterium Comamonas testosteroni TK102 was monitored by flow cytometry. When monohydroxy metabolites of biphenyl (BP) (2-hydroxybiphenyl and 3-hydroxybiphenyl) were added to the culture, cell-cell separation of strain TK102 was inhibited at stationary phase. This inhibition was reproduced on non-PCB degrading bacteria such as Pseudomonas putida PpY101 and Escherichia coli MV1184, but was not observed on Pseudomonas aeruginosa PAO1. An opportunistic pathogen, P. aeruginosa PAO1, produces exopolysaccharide, which is known to scavenge damaging chemicals such as reactive oxygen species (ROS). The higher level of ROS and lipid peroxidants were detected in the cells treated by monohydroxybiphenyls. Fat-soluble vitamin E, which is a lipid radical scavenger, maintained bacterial cell separation during monohydroxybiphenyls treatment. Our results demonstrated that intracellular oxidative stress played an important role in the inhibition of bacterial cell separation during BP metabolism. This study shows that metabolites of environmental pollutants, such as monohydroxylated BP, inhibit bacterial cell separation by oxidative stress.

Antioxidants↗

PubMed1992

Large-scale cell separation by centrifugal elutriation.

Countercurrent elutriation is a powerful method for fast and gentle large-scale isolation of cells according to size and density. Current limitations of this approach are the suboptimal selectivity that is obtained and the fact that the total number of cells that can be separated per run is restricted. We describe a new separation chamber, which was calculated according to the hydrodynamic boundaries of the counterflow separation. In comparison to the chamber commonly in use, a fivefold higher cell number can be separated with this chamber at an increased separation selectivity. Furthermore the use of this chamber, which fits to the standard Beckman equipment, avoids large sampling volumes.

Cell Line↗

PubMed1996

Heart-focused and general illness fears in relation to parental medical history and separation experiences.

Disease fears, such as excessive heart-focused anxiety (HFA), are quite common, and yet their origin is only poorly understood. Explanatory models of HFA have emphasized observational learning, parental cardiac disease, and the effects of separation experiences as key ethological factors. The purpose of this study was: (a) to provide descriptive information on the prevalence of HFA in an unselect sample of younger adults; and (b) to investigate the relation of HFA and general illness fears to parental medical history and different types of separation experiences. 421 undergraduate students completed the Cardiac Anxiety Questionnaire (CAQ), Illness Attitude Scales, Parental Medical History Questionnaire, and Separation Anxiety and Experience Questionnaire. Approximately 2-3% of our sample reported excessive HFA, and both illness attitudes and parental cardiac disease predicted 23% of total CAQ variance. Subjects with high CAQ scores reported more parental cardiac and other medical problems than low HFA subjects. Although high and low HFA subjects did not differ in terms of number of personal intimate relationships that ended in separation, subjects with separated parents reported more HFA than persons with nonseparated parents. We discuss the relevance of these findings for our understanding of HFA.

Adolescent↗

PubMed1987

Separation distress in infant rhesus monkeys: effects of diazepam and Ro 15-1788.

Disruption of the primate mother-infant attachment bond is a naturally occurring stressor that results in marked behavioral, physiological, and endocrine activation. We studied the effect that altering benzodiazepine systems has on the behavioral and endocrine response of infant rhesus monkeys (1-27 weeks of age) to brief separation from their mothers. In the first experiment, the benzodiazepine agonist diazepam (0.1 and 1.0 mg/kg) significantly increased locomotion and social behavior and decreased inactivity and distress vocalizations in infant monkeys undergoing separation. In the second experiment, the benzodiazepine antagonist Ro 15-1788 (5 and 10 mg/kg) had no significant effects on the infants' separation response. In the third experiment, administration of diazepam 1.0 mg/kg was followed by administration of Ro 15-1788 10 mg/kg in infants undergoing separation. Ro 15-1788 blocked the decreases both in inactivity and in plasma ACTH and cortisol concentrations caused by diazepam, suggesting that these effects are mediated through benzodiazepine receptors. These data support the hypothesis that in primates, endogenous benzodiazepine systems modulate the behavioral and endocrine response to the naturally occurring stress of separation.

Adrenocorticotropic Hormone↗

PubMed1983

Separation of lymphocyte subsets by expanding velocity sedimentation of E-rosettes at unit gravity.

An expanding velocity sedimentation procedure at unit gravity has been used to separate human lymphocyte subsets on the basis of E-rosette formation. It is shown that the method allows gentle yet efficient separation of T and non-T lymphocytes in a continuous gradient of colloidal silica (Percoll). Less efficient separation was obtained in gradients consisting of polymerized sucrose (Ficoll 70). The technique offers a special advantage in the possibility of separating lymphocytes binding differing numbers of indicator red cells. The method should be generally adaptable to various rosetting techniques, including systems where the indicator cells are attached with weak bonds. As judged by vital staining, the lymphocytes are in excellent condition after the separation.

Animals↗

PubMed1986

Optimized density gradient separation of leukocyte fractions from whole blood by adjustment of osmolarity.

Some of the compounds used for density gradient separation of blood cells have high osmolarities at the concentrations needed to create the required specific densities. Several mixed media use a combination of hyperosmolar shrinkage and red cell aggregation to improve cell separation. Due to the characteristics of Percoll density gradient medium the density and osmolarity of the gradient can be controlled separately. In the present study, Percoll gradients were used to determine the buoyant densities of different human blood cells at the osmolarities 300 mosM, 350 mosM and 400 mosM. Cell volumes were measured at the same osmolarities using a Coulter counter with channelyzer. As expected, the cell buoyant densities increased and the cell volumes decreased at the higher osmolarities used. There were, however, quantitative differences between the cells with respect to the effects of an increased osmolarity, making a 350 mosM density gradient the most effective in separating mononuclear leukocytes from polymorphonuclear leukocytes. A 400 mosM gradient offered the best possibilities to separate red blood cells from polymorphonuclear leukocytes. A one-step centrifugation procedure, based on these principles, is presented. This procedure makes possible the simultaneous purification of mononuclear leukocytes and polymorphonuclear leukocytes, suitable for functional assays.

Cell Separation↗

PubMed1988

A comparative investigation of three methods of separation of CD4 and CD8 cells from human peripheral blood cells.

A comparison was made of the phenotypic purities and functional efficiencies of CD4 and CD8 cell subsets, separated by three methods. CD4 and CD8 monoclonal antibodies were used for positive and negative selection of T cells by (a) panning, (b) red cell rosetting, and (c) complement mediated killing techniques. The viability of the CD4 and CD8 cell subsets, separated by any of the three methods and assessed by the dye exclusion method, was greater than 95%. An inverse relationship was found between the yields and purities of the CD4 and CD8 cell subset. The most efficient separation of the T cells was achieved by positive selection using the rosetting method; 95.3 +/- 1.8% of the CD4 subset reacted with antibodies to CD4 and 1.4 +/- 0.5% with antibodies to CD8 antigens. The separated CD8 subset showed 90.7 +/- 0.4% reacting with CD8 and 3.5 +/- 1.3% with CD4 antibodies. This separation was slightly better than that achieved by the panning method. However, the positive methods of selection gave lower yields than the negative selection methods. The five CD4 and five CD8 cell subsets were assessed functionally by stimulating with a mitogen (phytohaemagglutinin), a bacterial antigen (purified protein derivative) and a viral antigen (herpes simplex virus). The results suggest that the uptake of [3H]thymidine is generally most efficient with the positively selected rosetting CD4 and CD8 cell subset.

Antibodies, Monoclonal↗

PubMed1992

High gradient magnetic separation of cells on the basis of expression levels of cell surface antigens.

The possibility of separating cells on the basis of levels of antigen expression was explored in a model system using fixed erythrocytes and high gradient magnetic separation (HGMS). Fixed human erythrocytes were labelled to varying degrees with tetrameric monoclonal antibody complexes specific for both dextran and glycophorin A-M. The cells were then mixed and incubated with dextran iron particles prior to magnetic separation. The small size of the dextran iron particles (less than 0.2 microns) resulted in quantitative magnetic labelling of cells as shown using fluoresceinated anti-dextran antibodies and flow cytometry. The relationships between the initial percentage of labelled cells, cell recovery, non-specific entrapment of unlabelled cells, the purity of the removed fraction, the degree of antigen expression and separation conditions (flow rate and field strength) were determined and used to establish separation conditions that allowed recovery of cells that differ only in the degree of antibody labelling.

Antibodies, Monoclonal↗

PubMed1996

Production of IL-8 and IL-4 by positively and negatively selected CD4+ and CD8+ human T cells following a four-step cell separation method including magnetic cell sorting (MACS).

Highly enriched CD4+ and CD8+ human T cells were obtained from peripheral blood using a relatively simple and inexpensive method consisting of four steps: separation of mononuclear cells on Lymphoprep, removal of adherent monocytes by incubation in plastic petri dishes, removal of B cells, NK cells and further depletion of nonadherent monocytes by panning with anti-CD19, -CD16, -CD14, -CD11b and -CD33 mAb, and separation of CD4+ and CD8+ T lymphocytes by magnetic cell sorting (MACS). Cell culture for up to 48 h showed preservation of function by both positively and negatively selected cells as determined by production of IL-8. Although the cell separation procedure had no effect on interleukin-2 receptor (IL-2R, CD25) expression, it induced production of IL-4 by both T cell subsets selected positively, implying cell activation by ligation of CD4 and CD8 molecules. Irrespective of the mode of separation, CD8+ T cells produced more IL-4, a cytokine which is associated with a Th2-type cytokine profile of CD4+ T cells. We conclude that our method for separating T cells into their CD4+ and CD8+ subsets results in high cell purities with preservation of function, as determined by cytokine generation. If enriched cells are to be used for functional studies we recommend isolation by negative selection which has less effect on cell function. The relevance of the finding that CD8+ T cells can be an important source of IL-4 remains to be elucidated.

CD4-Positive T-Lymphocytes↗

PubMed1984

Cerebrospinal fluid measures of neurotransmitter changes associated with pharmacological alteration of the despair response to social separation in rhesus monkeys.

Social separation is a risk factor for major depressions that can be modeled in nonhuman primates. Changes in central monoamine neurotransmission are also likely to be involved in major depression. This study examined the relationship between separation-induced depressive-like behavior and central monoamine neurotransmitter changes in rhesus monkeys. Measures of cerebrospinal fluid (CSF) norepinephrine (NE), 5-hydroxyindoleacetic acid (5HIAA), and homovanillic acid (HVA) were used to assess the neurobiological impact of social separation and drug treatments alone or in combination. alpha-Methyl-p-tyrosine exacerbated, and fusaric acid ameliorated, the depressive-like response to separation. Probenecid-induced accumulations of HVA and 5HIAA reflected changes in behavior, but were not consistently affected by drug treatment. In contrast, pretreatment CSF NE was comparatively stable across repeated sampling, and drug-induced changes in this measure were correlated with changes in behavior. Low CSF NE, whether drug-induced or naturally occurring, was associated with a more severe depressive-like response to separation.

Animals↗

PubMed1987

Separation and identification of bovine lymphocyte populations.

Various methods for separation of lymphocyte populations have been modified and adapted for use in isolating and identifying bovine lymphocytes. Ficoll diatrizoate (F-D) with a specific density of 1.084 was found to be superior to those with densities of 1.080 and 1.077 which were developed originally for the mouse and human mononuclear cells, respectively. F-D with a density of 1.084 attained a lymphocyte (absolute number) recovery rate of 92% whereas those with densities of 1.080 and 1.077 yielded 81% and 71% recovery rate of lymphocytes, respectively. Subsequent separation of T lymphocytes was achieved best by nylon wool column whereas separation of B lymphocytes was attained best by complement-mediated depletion of T lymphocytes with the T lymphocyte specific monoclonal antibody (MAb), BLT-1. The former yielded 95 +/- 3% T lymphocytes with 47 +/- 9% recovery rate, and the latter gave 96 +/- 3% B lymphocytes with 71 +/- 9% recovery rate. In comparison, direct panning of F-D gradient separated mononuclear cells with goat anti-bovine IgG coated plates yielded 80% B lymphocytes with 31% recovery rate and indirect panning of MAb BLT-1 treated F-D gradient-separated mononuclear cells with goat anti-mouse IgG coated plates yielded 89% T lymphocytes with 35% recovery rate.

Animals↗

PubMed1992

Simultaneous separation and purification of mononuclear and polymorphonuclear cells from the peripheral blood of cats.

Peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMNC) play important roles in immunodeficiency diseases and AIDS-like syndromes in cats caused by feline leukemia virus (FeLV) and presumably also when caused by feline immunodeficiency virus (FIV). For comparative or functional studies it is advantageous or necessary to obtain these cells as separate entities from the same sample of an animal. Therefore, we analyzed the technical parameters of obtaining, separating and purifying these cells simultaneously from several blood samples of several cats. Flow cytometric studies with outbred cats indicated that of various cell separation/purification methods, e.g. from lysed whole blood, by Histopaque 1.077 or 1.119 single-density, or by 1.077/1.119 double-density centrifugation, the 1.077/1.119 double-density centrifugation of diluted whole blood is the most consistent, practical and effective method of yielding both highly purified PBMC and PMNC as separate entities from the same sample. The interface between plasma and 1.077 contained an average 86% PBMC vs. 14% PMNC, and the interface between 1.077 and 1.119 an average of 2% PBMC vs. 98% PMNC. Lymphocytes separated by this method had an average CD4/CD8 T-cell ratio of 2.0. These data indicate that Histopaque 1.077/1.119 double-density gradient allows purification and physical separation of lymphocytes and phagocytes from a blood sample, enabling the investigator to examine both cell types from the same sample simultaneously.

Animals↗

PubMed1992

Further analyses of human kidney cell populations separated on the Space Shuttle.

Cultured human embryonic kidney cells were separated into electrophoretic subpopulations in laboratory experiments and in two separation experiments on the STS-8 (Challenger) Space Shuttle flight using the mid-deck Continuous Flow Electrophoretic Separator (CFES). Populations of cells from each fraction were cultured for the lifetime of the cells, and supernatant medium was withdrawn and replaced at 4-day intervals. Withdrawn medium was frozen at -120 degrees C for subsequent analysis. Enzyme assays, antibodies and gel electrophoresis were used as analytical tools for the detection and quantitation of plasminogen activators in these samples. These assays of frozen culture supernatant fluids confirmed the electrophoretic separation of plasminogen-activator producing cells from non-producing cells, the isolation of cells capable of sustained production, and the separation of cells that produce different plasminogen activators from one another.

Cell Line↗

PubMed2005

A comparison of BoviPure and Percoll on bull sperm separation protocols for IVF.

In the present study, we have examined the effect of density gradient preparations BoviPure and Percoll on bull sperm separation and the in vitro fertilization (IVF) and culture (IVC) results. Frozen/thawed semen from five simmental bulls were pooled. Sperm quality parameters such as sperm motility, concentration, membrane activity (HOS assay), membrane integrity (SYBR-14/PI assay) and acrosomal status (EthD-1/FITC-PSA assay) were evaluated before and after sperm processing for IVF using BoviPure and Percoll density gradient separations. The results of the evaluated parameters before sperm processing were: motility 50%, concentration 82.33x10(6)spz/mL, membrane activity 39.05%, membrane integrity 42.97% and the acrosomal status 46.90% of the live spermatozoa with intact acrosomes. After sperm processing with BoviPure and Percoll the motility was 66.67 and 64.17%, the concentration was 25.50x10(6) and 27.67x10(6)spz/mL, the membrane activity was 53.78 and 56.58%, the membrane integrity was 70.85 and 68.76% of and the acrosomal status was 74.16 and 67.46% of the live spermatozoa with intact acrosomes, respectively. Percentages were referred to the total number of spermatozoa. There were significant differences (P<0.05) between the evaluated parameters before and after sperm processing for both separation protocols. We found no significant differences (P>0.05) regarding sperm evaluation parameters between the protocols. A total of 492 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA in six replicates. The cleavage (D2) and blastocysts (D7) rate were significantly higher (P<0.05) for the BoviPure group compared to the Percoll group: 75.80 and 28.21%; 61.58 and 20.83%, respectively. However, the number of hatched blastocysts (D10) did not differ significantly between sperm separation protocols (P>0.05). Our results indicate that both protocols gave suitable sperm for IVF. This finding and the similarity between those two density gradient preparations suggests that BoviPure is a good alternative for sperm separation in bovine IVF.

Animals↗

PubMed2006

Magnetic separation of CD14+ cells using antibody binding with protein A expressed on bacterial magnetic particles for generating dendritic cells.

Herein the potential of a highly efficient cell separation system using bacterial magnetic particles expressing protein A (protein A-BacMPs) was demonstrated. Protein A was expressed on BacMPs using the transmembrane proteins Mms13 and MagA as anchor molecules. The evaluations of the numbers of bound antibody molecules and binding capabilities of the protein A-BacMPs using Mms13 indicated that the antibodies were efficiently introduced into protein A-BacMPs using Mms13 in comparison to MagA. In addition, the recovery ratio of the target cells on the magnetic cell separation system was enhanced by using protein A-BacMPs with Mms13. Using positive selection against peripheral blood mononuclear cells, the CD14(+) cells were separated at a purity of more than 99% by protein A-BacMPs using Mms13. Furthermore, in the evaluation of the influence of protein A-BacMPs on the separated cells, the CD14(+) cells separated using protein A-BacMPs and were successfully differentiated into dendritic cells.

Bacterial Proteins↗

PubMed2005

Lateral separation of colloids or cells by dielectrophoresis augmented by AC electroosmosis.

Colloidal particles and biological cells are patterned and separated laterally adjacent to a micropatterned electrode array by applying AC electric fields that are principally oriented normally to the electrode array. This is demonstrated for yeast cells, red blood cells, and colloidal polystyrene particles of different sizes and zeta-potentials. The separation mechanism is observed experimentally to depend on the applied field frequency and voltage. At high frequencies, particles position themselves in a manner that is consistent with dielectrophoresis, while at low frequencies, the positioning is explained in terms of a strong coupling between gravity, the vertical component of the dielectrophoretic force, and the Stokes drag on particles induced by AC electroosmotic flow. Compared to high frequency dielectrophoretic separations, the low frequency separations are faster and require lower applied voltages. Furthermore, the AC electroosmosis coupling with dielectrophoresis may enable cell separations that are not feasible based on dielectrophoresis alone.

Cell Membrane↗

PubMed2000

NGF expression in the developing rat brain: effects of maternal separation.

A number of studies have shown that mothering style in rodents can produce neuroendocrine, neurochemical and behavioural changes in the adult, although the basic mechanisms initiating this cascade of events still need to be investigated. Long term changes in neuronal function might be due to alterations in the expression of neurotrophins which have been shown to promote neuronal survival, differentiation and function during development, such as Nerve Growth Factor (NGF). NGF is essential for proper development of sympathetic and neural crest-derived sensory neurons of the peripheral nervous system as well as of central cholinergic neurons. In previous studies, using a maternal separation paradigm, we have shown that NGF expression is increased in the dentate gyrus and the hilus of the hippocampus as a result of brief (45 min) maternal separations. In the present study neonatal rats were separated for longer periods of time (up to 3 h) and at different ages during development (9 and 16 days postnatally). Results indicate that the effects of maternal separation on NGF expression are stronger with longer separations and are not restricted to the hippocampal region but can be seen also in other brain areas. Overall these results indicate that external factors, such as the presence/absence of the mother, can modify neurotrophic factor's availability in the brain, thus indicating NGF as a potential player in environmentally-mediated brain plasticity during development.

Animals↗

PubMed1979

Artificial insemination homologous with oligospermic semen separated on albumin columns.

Semen samples obtained from 27 infertile men were separated on human serum albumin (HSA) columns prior to artificial homologous insemination (AIH). The columns contained either a single 7.5% HSA layer or two 17.5% and 7.5% HSA layers. Separated specimens were free of seminal debris, had significantly improved motility and progressive drive, and had a decreased percentage of abnormal forms. The total sperm count was also significantly decreased after separation, especially with the two-layer technique. No conceptions occurred in 21 couples when two-layer separation was used, but four pregnancies in 12 couples resulted from AIH with semen separated on the single-layer column. The mean total count of motile sperm obtained for AIH was 5 million with the two-layer and 31 million with the one-layer technique.

Adult↗