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PubMed1989

A device for aeration and mixing of cell and organelle suspensions during nuclear magnetic resonance studies.

A device is described which maintains homogeneous aerobic or anaerobic cell and organelle suspensions within an NMR sample tube. Line broadening due to magnetic field inhomogeneity is reduced by elimination of gas bubbles from the area of the probe receiver coils. The linewidth of the extracellular orthophosphate resonance of a yeast suspension in 31P NMR was 0.21 ppm compared with 0.4-0.7 ppm in conventionally aerated suspensions. Recirculation of the sample results in complete mixing within 90 s of addition of aliquots of acid or alkali. The maximum rate of oxygen transfer from the gaseous to the liquid phase was approximately 600 microM min-1 when aerated with 95% oxygen/5% carbon dioxide. A 60% wet weight suspension of yeast cells was recirculated for 20 h without settling of cells occurring. A method for estimating oxygen transfer rate is described.

Candida↗
PubMed1979

Cell suspensions from rat olfactory neuroepithelium: biochemical and histochemical characterization.

Cell suspensions were generated from rat olfactory epithelium by digestion with collagenase and hyaluronidase followed by gentle mechanical disruption. These cell suspensions excluded nigrosin dye and synthesized RNA, protein and carnosine from radiolabeled precursors. Sustentacular cells, repiratory epithelial cells and olfactory neurons but not basal cells could be identified by phase-contrast microscopy. Sedimentation of these cell suspensions at unit gravity in discontinuous gradients of buffered bovine serum albumin resulted in partial separation of the various cell types as indicated by the distribution of several biochemical markers. Olfactory marker protein and carnosine synthetase activity were found in the upper gradient fractions, while carnosinase activity was present predominantly in the lower gradient fractions. Cellular localization of olfactory neuron marker protein and non-neuronal S-100 protein by immunoperoxidase staining of gradient-fractionated cells indicated that neuronal cells were only partially separated from non-neuronal cells by our fractionation techniques. Evaluation of gradient fractionated cells by histochemical staining for carbohydrates demonstrated that secretory Bowman's gland cells were quite efficiently separated from neurons. This study demonstrates the ease with which cell suspensions may be produced from the olfactory epithelium, and emphasizes the importance of utilizing both biochemical and histochemical approaches in studies of mixed populations of cells, particularly when the purity of the cell fractions is a consideration.

Animals↗
PubMed1984

Suspension culture reveals a morphogenetic property of a thyroid epithelial cell line.

It is known that freshly dissociated thyroid cell clusters form follicles in suspension culture. Thyroid epithelial cell lines, grown for many generations in vitro, fail to show colloid-containing lumina when cultured as monolayers. Several thyroid cell lines, some transformed, have been tested with respect to their ability to form extracellular lumina when transferred from monolayer to suspension culture. One cell line in particular, the T78 cell line, showed this property when cultured in suspension. Lumina formed within 3 days even in the absence of added thyrotropin (TSH). The ultrastructure of lumina within cell aggregates resembled that of the thyroid follicle in vivo. The ability to undergo morphogenesis may therefore be an intrinsic property of thyroid epithelial cells which is retained for a large number of generations in vitro and is revealed by proper culture conditions. The shift from monolayer to suspension culture may thus lead to the expression of a thyroid differentiated function such as the formation of follicle-like structures.

Animals↗
PubMed1987

Mechanical properties of rat skeletal muscle after hind limb suspension.

It is clear that there are events which occur in response to neuromuscular activity that are essential to maintain normal muscle properties. Two factors though to influence muscle contractile properties are tension and electrical activity. To study these variables the hind limbs of 10 postpubertal female Sprague-Dawley rats were prevented from supporting the weight of the body for 4 weeks, then in situ isometric properties were determined and compared with 10 age-matched control rats. The soleus (slow plantarflexor), the medial gastrocnemius (fast plantarflexor), and the tibialis anterior (fast dorsiflexor) were studied. The suspended soleus wet weight was 42% lower and the maximum isometric twitch (Pt) and tetanic (P0) tensions were 62 and 69% lower respectively, than in the control rats. The suspended medial gastrocnemius was 18% lower in wet weight and 14 and 9% lower in P0 and Pt than the controls. No differences in wet weight, P0 or Pt were observed in tibialis anterior. Mean contraction time was 22% shorter in the soleus, 9% in the medial gastrocnemius, and unchanged in the tibialis anterior following suspension. Further, the percent of P0 attained during a 330-ms tetanus at 20 Hz was 15% lower in the suspended soleus and unchanged in the other two muscles. The fatigue index (ratio of tension after 2 min of stimulation at 40 Hz for 330 ms once per second to the maximum tension developed during the test) was unaffected by suspension in the soleus and tibialis anterior but was reduced from 49% to 36% in the medial gastrocnemius. The maximum rate of shortening of all three muscles was unaffected by suspension. These results indicate that suspension of the hind limbs selectively affects the mass and force-generating capabilities of the plantarflexors, particularly the predominantly slow soleus. In contrast, the fatigability of only the fast plantarflexor was increased whereas the slow plantarflexor was unaffected. These results, considered in light of collaborative studies, suggest that the chronic force-time levels in a muscle have an important influence on tension- and speed-related properties but not necessarily the fatigability of that muscle. Further, a marked difference in the sensitivity of the contractile elements of slow and fast muscle to this influence was evident.

Animals↗
PubMed1991

Blood-brain barrier permeability is not altered by allograft or xenograft fetal neural cell suspension grafts.

Alterations in blood-brain barrier (BBB) function after brain grafting seem dependent on the donor phenotype and possibly on the grafting technique. Intracerebral blood grafts of nonneural tissue permanently disrupt the host BBB, while fetal neural block grafts probably do not. Cell suspensions, an alternative technique in brain grafting, disrupt the extracellular matrix of the graft. Fetal cell suspension allografts appear to form a functional BBB. We confirm and extend this finding to include fetal neural xenografts. Allograft and xenograft fetal neural cell suspensions were intracerebrally injected, and the BBB was examined using intravenous horseradish peroxidase (HRP). Neither graft type showed disruption of the BBB at the graft site from 2 weeks to more than 6 months after grafting. Vascular supply was prominent at all time points. Xenograft survival was improved with cyclosporine, yet cyclosporine did not affect BBB permeability. Cyclosporine did not interfere with repair of the BBB after simple brain trauma was induced by a control injection of saline. We conclude that fetal allograft and xenograft neural cell suspensions rapidly form and maintain a BBB impermeable to intravenous HRP.

Acetylcholinesterase↗
PubMed1984

Skeletal muscle fibers in suspension: a new approach to the study of oxidative and glycolytic metabolism in differentiated muscle.

A preparation of suspended fibers from m. flexor digitorum brevis of the rat was characterized with respect to morphological features, and its relevance for the study of muscular metabolism investigated. The activities of oxidative (palmitate and pyruvate oxidation) and glycolytic (lactate formation) pathways were enhanced in myofiber suspensions when compared to intact whole muscle. The rate of glycolysis was stimulated about two-fold by insulin in both the myofiber suspensions and intact muscle. Parameters of oxidative metabolism responded similarly to metabolic effectors in the myofiber suspensions and in intact muscle. It is concluded that the myofiber suspensions have distinct advantages over intact muscle for biochemical and pharmacological studies.

Animals↗
PubMed1987

Mechanical, morphological and biochemical adaptations of bone and muscle to hindlimb suspension and exercise.

The influences of weightbearing forces on the structural remodeling, matrix biochemistry, and mechanical characteristics of the rat tibia and femur and surrounding musculature were examined by means of a hindlimb suspension protocol and highly intensive treadmill running. Female, young adult, Sprague-Dawley rats were designated as either normal control, sedentary suspended, or exercise suspended rats. For 4 weeks, sedentary suspended rats were deprived of hindlimb-to-ground contact forces, while the exercise suspended rats experienced hindlimb ground reaction forces only during daily intensive treadmill training sessions. The suspension produced generalized atrophy of hindlimb skeletal muscles, with greater atrophy occurring in predominantly slow-twitch extensors and adductors, as compared with the mixed fiber-type extensors and flexors. Region-specific cortical thinning and endosteal resorption in tibial and femoral diaphyses occurred in conjunction with decrements in bone mechanical properties. Tibial and femoral regional remodeling was related to both the absence of cyclic bending strains due to normal weightbearing forces and the decrease in forces applied to bone by antigravity muscles. To a moderate extent, the superimposed strenuous running counteracted muscular atrophy during the suspension, particularly in the predominantly slow-twitch extensor and adductor muscles. The exercise did not, however, mitigate changes in bone mechanical properties and cross-sectional morphologies, and in some cases exacerbated the changes. Suspension with or without exercise did not alter the normal concentrations of collagen, phosphorus, and calcium in either tibia or femur.

Animals↗
PubMed1992

The physical and mechanical effects of suspension-induced osteopenia on mouse long bones.

The present investigation addresses the extent of tail-suspension effects on the long bones of mice. The effects are explored in both sexes, in both forelimb and hindlimb bones, and in both diaphyseal and metaphyseal/epiphyseal bones. Two weeks of suspension provided unloading of the femora and tibiae and an altered loading of the humeri. Whole-bone effects included lower mass (approximately 10%) and length (approximately 4%) in the bones of suspended mice compared to controls. The geometric and material properties of the femora were considered along the entire length of the diaphysis and in the metaphysis/epiphysis portions as a unit. Geometric effects included lower cross-sectional cortical area (16%), cortical thickness (25%) and moment of inertia (21%) in the femora of suspended mice; these differences were observed in both distal and proximal portions of the femur diaphysis. The relative amount of bone comprising the middle 8 mm of the diaphysis was greater (3%) in the control mice than in the suspended mice. Significant mass differences between the group in the metaphysis/epiphysis were not observed. Material effects included lower %ash (approximately 2%) in the femora and tibiae as well as in the humeri of suspended mice compared to controls. With respect to the measured physical and material properties, suspension produced similar bone responses in male and female mice. The effects of suspension are manifested largely through geometric rather than through material changes.

Animals↗
PubMed1980

Susceptibility of adherent versus suspension target cells derived from adherent tissue culture lines to cell-mediated cytotoxicity in rapid 51Cr-release assays.

Preparation of target cells from tissue culture lines which grow adherent to tissue culture vessels is often desirable for tests of cell-mediated cytotoxicity (CMC). In the present study we used cells derived from adherent tissue culture lines to compare the merits of suspension vs. adherent target cells in short-term 51Cr-release assays. Cytotoxic activity of murine spleen cells sensitized in vitro against allogeneic spleen cells or syngeneic sarcoma cells was tested with fibroblast or sarcoma target cells. In parallel tests, aliquots of tissue culture lines were detached and used as either suspension or adherent target cells in CMC assays, matching the concentrations of suspension and adherent target cells. In both allogeneic and syngeneic combinations adherent target cells released less 51Cr spontaneously and were more susceptible to CMC than their suspension counterparts.

Animals↗
PubMed1980

A simple method for the isolation of platelet-free lymphocyte suspensions from rat whole blood.

A simple technique for obtaining platelet-free lymphocyte suspensions from rat whole blood for use in complement-dependent microcytotoxicity assays is presented. Rat lymphocytes were purified by filtering buffy coat preparations containing thrombin over nylon fiber columns prior to Ficoll-Hypaque centrifugation. The addition of thrombin to buffy coat preparations significantly (P < 0.001) enhanced platelet retention on the nylon fiber columns. This procedure yielded lymphocyte suspensions which had a 98% mean reduction in platelet contamination compared to lymphocyte preparations which were purified using only Ficoll-Hypaque centrifugation. The thrombin-nylon fiber filtration method produced lymphocyte suspensions containing an average of 98% lymphocytes, and the total yield of lymphocytes isolated from 2 ml of blood averaged 2.85 X 10(6) cells. The sensitivity of complement-dependent microcytotoxicity assays was increased by the use of the thrombin-nylon fiber-filtered platelet-free lymphocyte preparations compared to the use of platelet-contaminated lymphocyte suspensions.

Animals↗
PubMed1984

Characterization of biological response modifier release by human monocytes cultured in suspension in serum-free medium.

Human monocytes have been shown to be critical immunoregulatory cells for a variety of frequently measured in vitro human immune functions and are secretors of potent biologic response modifiers ( BRMs ). These BRMs , such as interferon (IFN), colony stimulating factor (CSF) and prostaglandin E (PGE), may play important roles in the host immune response to cancer. A new approach for culturing circulating peripheral blood monocytes has been developed to retain the native characteristics of these cells, avoiding possible alteration or activation by adherence. The ability of elutriator-purified human monocytes to secrete IFN and PGE was examined under conditions of suspension culture as well as after adherence, and no difference in secretion of these BRMs was noted. In contrast, Teflon-cultured monocytes demonstrated a significantly enhanced CSF release over culturing in polystyrene plates. A new serum-free medium has also been developed, and monocytes cultured in vitro in this medium showed a 5-fold increase in IFN release, and up to a 72% increase in CSF release when compared to optimal standard culture medium (containing 10% AB serum) and an increased stimulation index for PGE release. By these procedures, hundreds of millions of highly purified human monocytes can be sterilely isolated in suspension and cultured in suspension in serum-free medium with retention of BRM-releasing capabilities. This system should permit more detailed molecular studies of monocytes (in which serum can be an impediment) and may also facilitate clinical therapy studies involving the in vivo transfer of monocytes activated in suspension in vitro.

Blood Physiological Phenomena↗
PubMed1984

Efficacy of 2.5% Pimafucin suspension in the treatment of denture stomatitis.

This double-blind clinical trial was carried out in 38 patients to compare the efficacy of 2.5% Pimafucin suspension with a placebo in the treatment of denture stomatitis. The diagnosis of Candida infection was confirmed by the making of cultures and microscopic examination. Data were analyzed from 32 patients, of whom 18 received Pimafucin suspension and 14 a placebo suspension. Pimafucin suspension-treated patients showed a significant reduction of Candida infection in terms of culture, culture growth, and number of colonies within the first week of therapy. There was no statistical evidence of relapse during the 4-week period after therapy was stopped. The group receiving a placebo failed to show any statistically significant improvement with respect to the mentioned parameters. Erythema of oral mucosa showed significant improvement in both groups. No side effects were found.

Adult↗
PubMed1982

Regulation of tyrosine hydroxylase activity in retinal cell suspensions: effects of potassium and 8-bromo cyclic AMP.

Tyrosine hydroxylase (TH) contained in dopamine (DA) neurons of rat retina is activated in vivo as a consequence of photic stimulation. Experiments were conducted to test the effects of changes of membrane potential and of cyclic AMP-dependent protein phosphorylation on TH activity of these retinal neurons. Retinas were dissociated into suspensions of apparently viable cells to test the direct effects of pharmacological manipulations on TH activity in the absence of trans-synaptic influences. To test the effects of changes of membrane potential on TH activity we examined the effects of a depolarizing agent, potassium. Incubation of cell suspensions in Ringer's solution containing a depolarizing concentration of K+ (52 mM) resulted in a significant increase of TH activity, suggesting that membrane depolarization may trigger a series of molecular events that leads to TH activation. Incubation of cell suspensions in the presence of 8-bromo cyclic AMP, a cyclic AMP analog that is known to activate cyclic AMP-dependent processes following extra-cellular application, resulted in a significant activation of TH that was comparable to that produced in vitro by cyclic AMP-dependent protein phosphorylation. These data support the hypothesis that membrane potential plays a role in the regulation of TH activity, and indicate that cyclic AMP-dependent phosphorylation can activate retinal TH in situ. The apparent viability of the retinal cells in suspension suggests that this preparation may be useful for studying synaptic regulatory mechanisms.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Effects of a short term hindlimb suspension on central and peripheral norepinephrine turnover in rats.

The loss of appropriate cardiovascular reflexes which contributes to the cardiovascular deconditioning observed after an exposure to actual or simulated microgravity (in man or animals) is well known, but the mechanisms responsible remain unclear. This protocol, a 2.5 h hindlimb suspension in rats, was undertaken to study the early adaptation of the sympathetic neurons involved in arterial pressure regulation: we determined central norepinephrine (NE) turnover in the brainstem catecholaminergic cell groups responsible for the central cardiovascular regulation i.e. A1, A2 (rostral and caudal), A5 and A6 cell groups and peripheral NE turnover in target organs (heart and kidneys). The NE turnover in suspended rats significantly decreased in rostral A2 (48% p < 0.001), caudal A2 (52% p < 0.001) and A5 (40% p < 0.05) cell groups while it was unchanged in A1 and A6 cell groups compared with rats attached to the suspension device but maintained in the horizontal position. The short term hindlimb suspension did not alter the NE turnover in cardiac atria and ventricles or in kidneys nor did it alter the blood variables studied (hematocrit, osmolality, plasma sodium, potassium, proteins and renin concentration). We concluded that a 2.5 h hindlimb suspension reduced noradrenergic neuron activity in A2 and A5 cell groups involved in the central control of arterial pressure and particularly in the baroreceptor reflex mechanisms. This duration was probably not sufficient to modify the NE turnover in the two peripheral organs studied.

Animals↗
PubMed1988

The modified forced-swim test in rats: influence of rope- or straw-suspension on climbing behavior.

We modified Porsolt's forced-swim test by suspending ropes or straws above the water in order to investigate a possible relationship between immobility and perceived escape responses from water. In this modified test, it was demonstrated clearly that rats reduced their duration of immobility and attempted to climb up the suspended ropes or straws. Most rats which had remained immobile during a 5-min test period in the forced-swim test, exhibited such climbing responses within 5-10 min of rope-suspension. Despite the suspension of ropes, however, some rats showed immobile postures and did not respond to the rope. On the other hand, straws were used in order to produce sliding and prevent climbing when the animals attempted to climb. There were no differences in immobility during either rope- or straw-suspension. It seems that the climbing behavior displayed by forced-swimming rats is due to a "pseudo-escape" effect produced by the suspension of an object above the water. The present findings were interpreted as further evidence for the notion that immobility in forced-swimming rats does not necessarily imply "behavioral despair," but rather an emotional reaction to an inescapable stressor.

Animals↗
PubMed1989

Gas transfer in isolated lungs perfused with red cell suspension or hemoglobin solution.

Rapid mixing experiments have shown that the reaction between oxygen and hemoglobin is faster in hemoglobin solutions than in red cell suspensions. In this study we tested whether this observation can also be made in the lung. Excised rabbit lungs were perfused either with washed human red cell suspensions or with hemoglobin solutions, each with 50 g hemoglobin/L, and steady-state diffusing capacities (DLO2) for oxygen elimination measured. Mean settings were a temperature of 29.5 degrees C of the inflowing and outflowing perfusate of the lung, a total ventilation of 1.7 L.min-1, and a perfusion rate of 116 ml.min-1. Under those conditions resulted a DLO2 with hemoglobin solution of 0.68 +/- 0.18 ml.min-1.mm Hg-1, and a significantly lower value of 0.50 +/- 0.06 ml.min-1.mm Hg-1 with red cell suspension (P less than 0.01). An extraerythrocytic diffusing resistance, formed by a plasma layer and/or arising from a dynamic diffusion boundary layer, which is also known as unstirred layer, could explain the lower value with red cell suspensions.

Animals↗
PubMed1988

The effect of fish oil consumption on platelet aggregation responses in washed human platelet suspensions.

Although the biochemical mechanisms relating to the inhibitory action of eicosapentaenoic acid on platelet function have previously been investigated in washed platelet suspensions, there is little existing evidence in washed platelet suspensions to indicate that dietary fish oil consumption reduces platelet aggregation responses. In the present work, eight healthy male volunteers consumed 20 fish oil capsules (MaxEPA) per day for a period of six weeks followed by a six week "dry-out" period during which no fish oil was consumed. Washed platelet suspensions were prepared and aggregation responses to collagen, platelet activating factor (PAF), and thrombin were assessed initially at week 0 and subsequently at 3, 6, 9, and 12 weeks of the experimental period. Three weeks of fish oil consumption did not produce any marked alterations in aggregation responses; however, six weeks of dietary fish oil supplementation resulted in decreased maximum aggregation responses to collagen and PAF by 50.1% and 27.2%, respectively. These results provide evidence, in washed platelet suspensions, that dietary fish oil consumption significantly diminishes platelet responses to collagen and PAF. The significance of these observations remains to be explored and interpreted.

Adult↗
PubMed1991

Transvaginal needle suspension procedures for recurrent stress incontinence.

The results of transvaginal needle suspension in 36 patients with stress urinary incontinence who failed previous operation were reviewed. Eighteen patients underwent a Stamey endoscopic needle suspension, and a Peyrera-Raz cystourethropexy was performed on the remainder. Recurrent stress incontinence was confirmed by history, physical examination, and urodynamic studies. Cure rates of 78 percent for the Stamey procedure and 83 percent for the Raz operation were achieved. Excluding patients with voiding dysfunction or urge incontinence postoperatively due to bladder instability, the failure rates were 6 percent and 11 percent, respectively. Analysis of the results according to the preoperative maximal urethral pressures demonstrated that low-pressure urethras if accompanied with urethrovesical hypermobility have no impact on the results if the anatomic abnormality was corrected adequately by the needle suspension. Thus, the transvaginal needle suspension was found to be highly effective in patients with low-pressure urethras, who failed previous operation, but had persistent bladder neck hypermobility, and suggests that this method of surgical repair should be the first choice in this selected group.

Female↗