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Two types of three-dimensional structures formed by epithelial cell aggregates from the rat ventral prostate in collagen gel culture. The amount of activated charcoal for absorbing serum.

Cells taken from the rat ventral prostate and cultured formed a tubular structure inside the collagen gel in a medium containing activated charcoal-absorbed serum after a 14-day incubation. This might suggest that the active substances of serum could induce isolated epithelial cells to form such a spherical or tubular structure, depending on the amount of activated charcoal used for the absorption of serum.

Absorption↗

Structure and function of C-CAM1: effects of the cytoplasmic domain on cell aggregation.

C-CAMs are epithelial cell-adhesion molecules of the immunoglobulin supergene family with sequences highly homologous to carcinoembryonic antigen (CEA). C-CAMs and their human homologues, biliary glycoproteins, are unique among the CEA-family proteins in that they have cytoplasmic domains. Furthermore, alternative splicing generates C-CAM isoforms with different cytoplasmic domains, suggesting that the cytoplasmic domains of C-CAM may play important roles in regulating the function or functions of C-CAM. By using both sense and antisense approaches, we have shown that C-CAM1 is a tumour suppressor in prostate carcinogenesis. This observation raises the possibility that the cytoplasmic domain of C-CAM1 may be involved in signal transduction or interaction with cytoskeletal elements to elicit the tumour suppressor function. The cytoplasmic domain of C-CAM1 contains several potential phosphorylation sites, including putative consensus sequences for cyclic AMP-dependent kinase and tyrosine kinase. One of the potential tyrosine phosphorylation sites is located within the antigen-receptor homology (ARH) domain. The ARH domain of the membrane-bound IgM molecule is necessary for signal transduction in B-cells. These structural features suggest that the cytoplasmic domain of C-CAM1 may be important for signal transduction. To test this possibility, we generated several site-directed C-CAM1 mutants and tested their ability to support adhesion and their abilities to be phosphorylated in vivo. Results from these studies revealed that Tyr-488 is phosphorylated in vivo. However, replacing this tyrosine with phenylalanine did not significantly compromise its adhesion function. Similarly, Ser and Thr residues are phosphorylated in vivo, but deletion of the potential cyclic AMP-dependent kinase site did not significantly reduce the adhesion function. These results suggest that the kinase phosphorylation sites in the cytoplasmic domain of C-CAM1 are not required for the adhesion function. However, these phosphorylation sites are probably involved in the regulation of C-CAM-mediated signal transduction. Thus, there are probably distinct structural requirements for the adhesion and the signal transduction functions of C-CAM. Incidentally, a C-CAM1 deletion mutant containing a 10-amino-acid cytoplasmic domain was able to support adhesion activity. This is in contrast to our previous finding that a C-CAM isoform, C-CAM3, with a 6-amino-acid cytoplasmic domain could not support cell adhesion. This result indicates that the extra four amino acids, which are absent in C-CAM3 and contain a potential Ser/Thr phosphorylation site, are important for the adhesion function.

Adenosine Triphosphatases↗

In vitro culture and labeling of neural cell aggregates followed by transplantation.

Septal, cortical, or whole brain fetal (E15-17) cells were dissociated and cultured in serum-supplemented Dulbecco minimum essential medium under rotating culture conditions. Preincubation and exposure to cytosine arabinoside was utilized to produce "neuron-rich" cultures. Fluorescent latex microbeads were added to cultures at seeding time or early after aggregate formation. All cell types were found to incorporate the fluorescent beads, although apparently not to the same extent. Two- to five-day-old aggregates tended to attach and grow neurites after their transfer to poly-l-lysine- or Matrigel-coated dishes under stationary conditions. Early aggregates transplanted to the hippocampus of adult rats developed into identifiable grafts, with fluorescent-labeled cells. We conclude that "young" neural cell aggregates maintain their ability to undergo two basic phenomena for cellular interaction, i.e., attachment and neuritic growth. Floating aggregates may provide a convenient cellular condition whenever culturing of neural cells is to be used before grafting them into a host animal.

Animals↗

[Action of Rhodobrum roserm Limpr on changes of red cell aggregation and yield-shear stress in dogs with acute experimental coronary occlusion].

Following acute occlusion of the left anterior descending coronary artery in dogs, significant increases were observed in the red cell aggregation index, yield-shear stress and red cell electrophoretic time in blood drained from ischemic area. When transfusion was performed with Rhodobrum roseum solution from the right femoral vein, significant rises of the above-said items were observed.

Animals↗

Cholinergic neurons of fetal rat telencephalon in aggregating cell culture respond to NGF as well as to protein kinase C-activating tumor promoters.

Serum-free aggregating cell cultures of fetal rat telencephalon treated with the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) showed a dose-dependent, persistent stimulation of the enzymes choline acetyltransferase (ChAT), glutamic acid decarboxylase and glutamine synthetase. After elimination of the proliferating cells by treatment of the cultures with Ara-C (0.4 microM) only the cholinergic marker enzyme, ChAT, could be stimulated by tumor promoters. The non-promoting phorbol ester, 4 alpha-phorbol 12,13-didecanoate proved to be inactive in these cultures, whereas the potent non-phorbol tumor promoter, mezerein, produced an even greater stimulatory effect than PMA. Since PMA and mezerein are potent and specific activators of protein kinase C, the present results suggest a role for this second messenger in the development of cholinergic telencephalon neurons. Stimulation of ChAT required prolonged exposure (48 h) of the cultures to PMA and the responsiveness of the cholinergic neurons to the tumor promoters decreased with progressive cellular maturation. The cholinergic telencephalon neurons showed the same pattern of responsiveness for tumor promoters as for nerve growth factor (NGF). However, the combined treatment with NGF and either PMA or mezerein produced an additive stimulatory effect, suggesting somewhat different mechanisms of action.

Animals↗

Molecular rearrangement of lactose plasmid DNA associated with high-frequency transfer and cell aggregation in Lactococcus lactis 712.

High-frequency conjugation of the lactose plasmid pLP712 is associated with a constitutive cell aggregation phenotype and is facilitated by cointegration with a sex factor. Analysis of 23 independently derived enlarged lactose plasmids revealed that the sex factor DNA present in cointegrates varied in size. This suggested that more than simple cointegration with a sex factor plasmid was involved. Further analysis led to the discovery of a chromosomally located sex factor that could excise and be lost or exist as labile plasmid DNA. Cointegration with this sex factor was shown to be promoted by transposition of a copy of ISSI present on the lactose plasmid, and models are presented to account for the complex and variable structures of the resulting enlarged lactose plasmids.

Base Sequence↗

Native associations of early hematopoietic stem cells and stromal cells isolated in bone marrow cell aggregates.

In suspensions of murine bone marrow, many stromal cells are tightly entwined with hematopoietic cells. These cellular aggregations appear to exist normally within the marrow. Previous studies showed that lymphocytes and stem cells adhered to stromal cells via vascular cell adhesion molecule 1 (VCAM1). Injection of anti-VCAM1 antibody into mice disrupts the aggregates, showing the importance of VCAM1 in the adhesion between stromal cells and hematopoietic cells in vivo. Early hematopoietic stem cells were shown to be enriched in aggregates by using a limiting-dilution culture assay. Myeloid progenitors responsive to WEHI-3CM in combination with stem cell factor (c-kit ligand) and B220- B-cell progenitors responsive to insulin-like growth factor-1 in combination with interleukin-7 are not enriched. We propose a scheme of stromal cell-hematopoietic cell interactions based on the cell types selectively retained within the aggregates. The existence of these aggregates as native elements of bone marrow organization presents a novel means to study in vivo stem cell-stromal cell interaction.

Animals↗

Flow-resistant red blood cell aggregation in morbid obesity.

OBJECTIVE: Enhanced red blood cell (RBC) aggregation has an adverse effect on microcirculatory blood flow and tissue oxygenation. It has been previously shown that obesity is associated with increased RBC aggregation. The objectives of the present study were to further characterize obesity-related RBC aggregation and to examine whether the enhanced aggregation is a plasma- or cellular-dependent process. METHODS: Obese (body mass index (BMI)=40+/-6.3 kg/m2, n=22) and nonobese (BMI=24+/-3.4 kg/m2, n=18) individuals were evaluated for inflammation markers and aggregation parameters. Aggregation parameters were derived from the distribution of RBC population into aggregate sizes, and from the variation of the distribution as a function of flow-derived shear stress, using a cell flow properties analyzer. To differentiate plasmatic from cellular factors, we determined the aggregation in the presence of autologous plasma or dextran-500 kDa and calculated the plasma factor (PF) in the obese group. PF ranges from 0 to 1. When the PF=1, the aggregation is all due to plasmatic factors, when PF=0, the altered aggregation depends entirely on cellular factors, whereas 0<PF<1 reflects the joint contribution of cellular and plasmatic factors. RESULTS: Obese subjects had relatively larger aggregates that were more resistant to dispersion by flow. The calculated PF in the obese group was 0.9, indicating a pronounced contribution of plasma to RBC aggregation in obesity. DISCUSSION: Our results suggest that obese individuals present pathological plasma-dependent RBC aggregation, which is probably triggered by plasma macromolecules associated with the inflammatory response. These findings impact the future attempts to develop strategies aimed at attenuation of the enhanced RBC aggregation in obese individuals.

Adult↗

Effect of specific membrane perturbations on alpha 1-adrenergic and muscarinic-cholinergic signal transduction in rat parotid cell aggregates.

Alpha 1-adrenergic and muscarinic-cholinergic stimulated IP3 production and calcium mobilization are inhibited by treatment of parotid cell aggregates with methanol, hydrogen peroxide and saponin. Only methanol exerts an effect on binding to receptors. In most cases a close correspondence exists between inhibition of alpha 1-adrenergic and muscarini-cholinergic responses as well as inhibition of IP3 production and calcium mobilization. G-protein dependent signal transduction, therefore appears to be quite sensitive to plasma membrane perturbation and membrane active agents may provide useful tools for elucidation of transduction mechanisms and their regulation.

Adrenergic alpha-1 Receptor Antagonists↗

Morphological and functional correlates of synchronous beating between embryonic heart cell aggregates and layers.

We have examined correlations between morphological and functional evidence of cell coupling between aggregates of beating embryonic heart cells and underlying layers. Synchronously beating aggregate-layer pairs were compared with asynchronous pairs. Intracellular microelectrode studies demonstrated that asynchronously beating aggregate-layers could not be induced to beat synchronously by electrical stimulation of the aggregate, whereas 86% of synchronous instances showed propagation of stimulating current pulses from aggregate to layer. By freeze fracture we have found significant differences both in the number and in the total area of gap junctions between the aggregate-layer interfaces of synchronous and asynchronous preparations. The data suggest that synchronous beating is a reliable functional indication of effective ionic coupling, and requires a certain area and number of gap junction/cell.

Action Potentials↗

High and low molecular weight heparins do not modify red blood cell aggregability in vitro.

The usefulness of heparins as anticoagulants has been demonstrated, but their effects on hemorheological parameters, such as erythrocyte aggregability, are under discussion. If the heparins had adverse effect on erythrocyte aggregability, its use would be especially undesirable in patients with pathologies involving red blood cell hyperaggregability as is the case of cardiac disease. In the present study we examine the in vitro effect of unfractionated and fractionated heparins on red blood cell aggregability. The results show that heparins do not increase this rheological parameter but show a slight tendency to lower it.

Dose-Response Relationship, Drug↗

Negative regulation by dexamethasone of the potentiation of neuromedin C-induced growth hormone and prolactin release by estradiol in anterior pituitary cell aggregates.

The bombesin-like peptide neuromedin C (NMC) stimulated the release of growth hormone (GH) and prolactin (PRL) from adult male rat anterior pituitary cell aggregates cultured in serum-free medium. This effect, particularly on GH release, was significantly increased when the cells were cultured in the presence of estradiol (E2). The GH response to NMC was not affected by 4 nM dexamethasone (Dex) but was enhanced in the presence of 80 nM Dex. However, the strong stimulatory action of E2 on the GH response to NMC was completely abolished by 4 nM Dex. In contrast, the PRL response to NMC was reduced by 4 nM Dex and this to a proportionally similar extent in the presence or absence of E2. The present data show that glucocorticoids can be both positive and negative regulators of the same response system.

Animals↗

Evaluation of red blood cell aggregation in diabetes by computerized image analysis.

Red blood cell (RBC) aggregation has been widely studied and its importance is well established in the rheology of microcirculation. RBC aggregation is a major factor responsible for the flow properties of blood. Increased RBC aggregation has been observed in several pathological states. Therefore, the measurement of erythrocyte aggregation is rheologically important for quantifying flow abnormality in pathological conditions. Normal RBC under low flow or at rest form rouleaux aggregates, while abnormal RBC aggregation may lead to the formation of irregular aggregate structures, which may be induced by cell-associated factors (reduced membrane sialic acid levels) but also by extracellular factors. The main objective of the present investigation was to study RBC aggregate morphology in diabetic patients, using direct microscopic observation and numerical processing of recorded digitized images. Blood samples were obtained from 20 diabetic patients and from 15 normal control subjects. The aggregate morphology was quantified by the so-called Aggregate Shape Parameter (ASP) defined as the ratio of the aggregate projected area to its square perimeter. ASP appeared significantly higher (p < 10(-5)) in diabetic patients (0.65 +/- 0.18) than in normal controls (0.28 +/- 0.15). This rheo-optical method based on the theoretical model for rouleaux aggregates provides a useful reference for measuring deviations of RBC aggregate morphology. Increased aggregation of RBC resulting from a decreased sialylation of glycophorins may be an important factor in the development of vascular diseases and in the microcirculation impairment.

Adult↗

Phase resetting of embryonic chick atrial heart cell aggregates. Experiment and theory.

The influence of brief duration current pulses on the spontaneous electrical activity of embryonic chick atrial heart cell aggregates was investigated experimentally and theoretically. A pulse could either delay or advance the time of the action potential subsequent to the pulse depending upon the time in the control cycle at which it was applied. The perturbed cycle length throughout the transition from delay to advance was a continuous function of the time of the pulse for small pulse amplitudes, but was discontinuous for larger pulse amplitudes. Similar results were obtained using a model of the ionic currents which underlie spontaneous activity in these preparations. The primary ion current components which contribute to phase resetting are the fast inward sodium ion current, INa, and the primary, potassium ion repolarization current, IX1. The origin of the discontinuity in phase resetting of the model can be elucidated by a detailed examination of the current-voltage trajectories in the region of the phase response curve where the discontinuity occurs.

Action Potentials↗