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Stellate cells of aortic intima: II. Arborization of intimal cells in culture.

The present study analyzed effects of different cAMP-elevators on cell morphology in primary culture of human intimal and medial cells from grossly normal and atherosclerotic areas. In primary culture of human aortic cells adenylate cyclase activator forskolin and other cAMP elevators induced arborization of cells, i.e. they reversibly changed the shape of cells. This resulted in the formation of thin branching processes and in the concentration of cytoplasm around the nucleus. In the culture, the shape of the arborized cells resembled that of stellate ones detected in the aortic intima in situ. The arborization of cells was accompanied by destruction of myofilaments. Due to cAMP elevators' effect, most of the arborized cells were exhibited in the cultures isolated from the elastic-hyperplastic layer of the intima. The number of arborized cells was significantly less in the cultures isolated from the musculo-elastic layer and still lesser in those isolated from media. We failed to reveal any significant difference in the number of arborized cells cultured from fatty streaks, atherosclerotic plaques and grossly normal aortic areas. Obtained results suggest that the previously revealed polymorphism of human aortic intimal cells may be accounted for by the cell shape transformations underlined by the mechanism similar to that of arborization in vitro.

Actins↗

Mitogens for glial cells: a comparison of the response of cultured astrocytes, oligodendrocytes and Schwann cells.

We have identified two growth factors for cultured rat astrocytes: fibroblast growth factor, a peptide derived from either whole bovine brain, or pituitaries, and a growth factor in extracts of bovine pituitary which was previously identified as a Schwann cell mitogen. Oligodendrocytes in primary cultures derived from neonatal rat central nervous system divide only rarely if at all. These growth factors did not stimulate primary oligodendrocytes to divide. Occasionally cells found in suspension in long-term cultures of the central nervous system were enriched for cells which were identified as oligodendrocytes by the presence of galactocerebroside on their surface and myelin basic protein in their cytoplasm. When provided with a monolayer of irradiated 3T3 cells, these oligodendrocytes were able to spread out and extend elaborate branched processes typical of oligodendrocytes in the primary cultures. Unlike their counterparts in the primary cultures, these suspension-derived oligodendrocytes are capable of cell division as demonstrated by the uptake of [3H]thymidine and autoradiography.

Animals↗

Ontogeny of enkephalin- and VIP-containing neurons in dissociated cultures of embryonic mouse spinal cord and dorsal root ganglia.

The ontogeny of vasoactive intestinal polypeptide (VIP), and Met-enkephalin in primary cultures of spinal cord/dorsal root ganglia from 12-day mouse embryos was examined by radioimmunoassay and immunohistochemistry. Met-enkephalin levels rose from less than 5 to 700 pg/culture over 26 days and were half maximal by day 16-18 in culture. VIP levels rose from less than 1 to 30 pg/culture over the same period, but were already half maximal by day 9. Met-enkephalin immunoreactivity was localized in multipolar medium sized neurons while VIP immunoreactivity was visualized both in neurons with extensively branched processes and in bipolar cells some of which appeared to be dorsal root ganglion cells. Tetrodotoxin (TTX)-sensitive spontaneous release of both peptides developed in parallel with the ability to stimulate peptide release with elevated potassium. Factors affecting the ontogeny of neuropeptide expression in, and release from, spinal cord neurons can now be examined in vitro in a strictly defined neurochemical environment.

Animals↗

Cell attachment and neurite stability in NG108-15 cells: effects of 5'-deoxy, 5'-methyl thioadenosine (MTA) compared with laminin, kinase inhibitor H-7, and Mn2+ ions.

Methylation inhibitors such as 5'-deoxy, 5'-methyl thioadenosine (MTA) have been shown to exert an intriguing spectrum of effects upon neural cells: inhibiting responses to nerve growth factor in PC12 cells; stimulating outgrowth of rapid-onset neurites in NG108-15 cells; inhibiting the resorption and remodelling of these rapid-onset neurites; and inducing fibroblasts to extend long, multipolar, branched processes. To learn whether the apparently diverse effects of these agents might reflect some common underlying cellular sites of action, we examined simpler, short-term effects of MTA upon cell attachment and stability of rapid-onset neurites in NG108-15 cells, and compared the effects with those produced by the kinase inhibitor H-7, substratum-bound laminin, or Mn2+ ions. MTA was shown to selectively enhance the response of cells and their neurites in attaching to their substrata, without inducing other 'adhesive' responses such as cell spreading or motility. The data suggest that MTA activates or increases the effectiveness of 'attachment receptors', and that this at least partially explains its neurite-promoting effects. While it is still premature to propose a common mechanism of action for MTA and related agents, all of their known effects thus appear to involve modulating responses generated at cell-surface receptors. A further clue is that kinase C activity appears to be critical, since cells pretreated with phorbol esters for 24 h (which down-regulated levels of kinase C) selectively failed to extend neurites in response to MTA, but responded normally to laminin and Mn2+ ions.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Polyamines promote neurite elongation of cultured rat hippocampal neurons.

The effects of spermine, spermidine and putrescine on the survival and morphology of hippocampal neurons obtained from rat embryos were investigated using low cell density culture. The number of surviving neurons gradually decreased in the control cultures, while addition of spermine significantly promoted neuronal survival. The survival-promoting effect of spermine was seen maximally at a concentration of 10(-8) M. On the other hand, spermidine and putrescine showed no significant effect on neuronal survival. The axon length of neurons cultured in the presence of spermine was significantly longer than that in the medium with no added spermine, indicating that spermine promotes process elongation. The concentration-effect curve for spermine was bell-shaped with the maximum effect at a concentration of 10(-8) M. However, the number of processes per soma, the elongation of dendrites and the process branching in axons were not significantly affected by the presence of spermine. Addition of spermidine and putrescine also promoted the axon elongation of cultured neurons, without affecting the branching number. The effects of spermidine and putrescine on neuronal growth were very similar to that of spermine in terms of the effective concentrations and the patterns of morphological changes. Since spermine specifically promoted neuronal survival while three polyamines affected the neuronal morphology in a similar manner, it is possible that the effects of polyamines on neuronal survival and neurite elongation are mediated by different mechanisms.

Animals↗

The CNS-PNS transitional zone of the rat. Morphometric studies at cranial and spinal levels.

The transitional zone is that length of rootlet containing both central and peripheral nervous tissue. The CNS-PNS interface may be defined as the basal lamina covering the intricately interwoven layer of astrocyte processes which forms the CNS surface and which is pierced by axons passing between the CNS and PNS. Study of transitional zone development defines morphologically the growth, relative movement and interaction of central and peripheral nervous tissues as they establish their mutually exclusive territories on either side of the CNS-PNS boundary, and helps to explain the wide variations in the form of the mature transitional zone. Nerve rootlets at first consist of bundles of bare axons. These become segregated by matrices of fine Schwann cell processes peripherally and of astrocyte processes centrally. The latter may prevent Schwann cell invasion of the CNS. Astrocyte processes branch profusely and come to form the principal central nervous tissue component of the transitional zone. Developmental changes in the transitional zone vary markedly between nerves, reflecting differences in its final morphology. Widespread relative movements and migration of CNS and PNS tissues take place during development, so that the central-peripheral interface changes shape and position, commonly oscillating along the proximodistal axis of the rootlet. For example, developing cervical ventral rootlets contain a transient central tissue projection, while that of lumbar ventral rootlets and to a lesser extent that of cervical dorsal rootlets alternately increase and decrease in length. In the developing cochlear nerve, a central tissue projection is present before birth, but regresses somewhat before a marked outgrowth of central nervous tissue along the nerve takes place, which reaches into the modiolus during the first week postnatum. During development, some astrocytic tissue may even break off and migrate distally into the root, giving rise to one or more glial islands within it. During the period immediately preceding birth, Schwann cells come to be present in very large numbers in that part of the rootlet immediately distal to the CNS-PNS interface, the proximal rootlet segment. Here they form prominent sleeves or clusters of closely packed cells which intertwine with and encapsulate one another on the rootlet surface. Such Schwann cell overcrowding in the proximal rootlet segment could result in part from distal overgrowth of the rapidly expanding CNS around axon bundles, which might strip the Schwann cells distally off the bundle segments so engulfed.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

A simple model for the statistics of events in idiotypic networks.

A simple random graph model of idiotypic networks is introduced: this model allows (1) to evaluate the stability of the network dynamics' fixed points, and (2) to compute the statistics of events triggered in response to the arrival of new molecules (metadynamics) using a dynamic mean-field approximation based on the theory of branching processes. It is shown that (1) the network dynamics is unlikely to have many stable fixed points in a strict sense, but that (2) the reorganizations which the network undergoes owing to the metadynamics are always subcritical if plausible figures are injected into the model. In other words the distance between successive (unstable or weakly stable) fixed points is relatively small, so that the overall behavior is stable.

Animals↗

Nerve cells immunoreactive for vasoactive intestinal polypeptide in dissociation cultures of rat hypothalamus and midbrain.

Immunocytochemistry using the unlabeled antibody (PAP) technique reveals the presence of 0.01-0.3% neurons reacting with an antiserum against vasoactive intestinal polypeptide (VIP) in dissociation cultures of hypothalamus and midbrain. The cultures are obtained from 18-day-old rat fetuses and maintained for 22 and 29 days in vitro. Most of the neurons appear well differentiated giving off branched processes with pronounced varicosities. These can often be seen to contact perikarya as well as processes of unstained neurons.

Animals↗

Human cerebral cortical cell lines from patients with unilateral megalencephaly and Rasmussen's encephalitis.

Continuous cerebral cortical cell lines have been developed from two patients, an 11-month-old with unilateral megalencephaly and a seven-year-old with Rasmussen's encephalitis, designated HCN-1 and HCN-2, respectively. The two cell lines stain for neuronal markers such as neurofilament and neuron-specific enolase but not for non-neuronal markers such as glial fibrillary acidic protein and S-100 protein. In the presence of appropriate growth factors, the cells extend long, branched processes resembling neurons. Differentiation of HCN-1 cells can be induced with nerve growth factor, dibutyryl cyclic AMP and isobutylmethylxanthine, while for HCN-2 cells nerve growth factor, isobutylmethylxanthine and the phorbol ester 12-O-tetradecaoylphorbol-13-acetate are most effective. Immunohistochemical staining of both differentiated cell lines reveals intense staining for GABA, glutamate, somatostatin, cholecystokinin-8 and methionine enkephalin. Two human cortical neuronal cell lines have been developed which represent neuronal precursors. These cell lines propagate in culture and are capable of differentiating upon the addition of a variety of growth factors and chemical agents. These cell lines should prove to be useful models for the study of in vitro neuronal processes.

Blotting, Western↗

Electrophysiological properties of paraventricular magnocellular neurons in rat brain slices: modulation of IA by angiotensin II.

Whole-cell patch-clamp recordings obtained from magnocellular neurons of the hypothalamic paraventricular nucleus in brain slice preparations of adult Sprague-Dawley rats have been utilized to examine three outward potassium conductances and the ionic mechanisms through which angiotensin II exerts its neurotransmitter actions within this region. Lucifer Yellow fills showed that neurons from which we recorded had large ovoid cell bodies 11-17 microns wide and 22-35 microns long, as well as 1-3 minimally branched processes, anatomical features in accordance with those previously described for magnocellular neuroendocrine neurons. These neurons had an average resting membrane potential of -58.3 +/- 0.9 (mean +/- S.E.M.) mV, spike amplitude of 92.8 +/- 1.4 mV, and input resistance of 788.9 +/- 50.4 M omega. Most of these cells displayed irregular or continuous spontaneous activity with a mean frequency of 2.44 +/- 0.33 Hz. Voltage-clamp recordings revealed three outward potassium currents; (1) a delayed outward current (IK), (2) a Ca(2+)-dependent outward current (IK(Ca)) and (3) a transient outward current (IA). These currents were classified according to their voltage dependence, inactivation, Ca2+ dependence and pharmacology. The IK was activated by depolarization beyond -40 mV and its amplitude consistently increased with depolarizing steps. The membrane conductance underlying this current was 27.3 +/- 3.8 nS for depolarization to +50 mV. In medium containing 2 mM Ca2+, depolarization to above -20 mV evoked a slowly-activating IK(Ca) which showed minimal inactivation. This current was suppressed in Ca(2+)-free/Co2+ medium and its membrane conductance was also smaller (19.4 +/- 3.5 nS at +50 mV) than that of IK. The IA demonstrated both fast activation and inactivation and was evoked only if depolarizing pulse steps were preceded by conditioning hyperpolarization. The activation threshold was approximately -65 mV and IA amplitude increased in non-linear fashion as test voltage steps became more positive. The 90% maximum of IA conductance was 15.7 +/- 1.1 nS, and was observed at membrane potentials around -15 mV. The reversal potentials of these currents were in accordance with the K+ equilibrium potential. Tetra-ethylammonium reversibly inhibited both the peak and steady-state currents of the IK, while 4-aminopyridine suppressed the IA. Replacement of 2 mM Ca2+ with 2 mM Co2+ in our bath solution or addition of Co2+ into Ca(2+)-free medium reduced the magnitude of IA, revealing the existence of a Co(2+)-sensitive IA. Bath administration of 10(-7) M angiotensin was without significant effect on IK, but resulted in a statistically significant reduction in IA (-31.0 +/- 4.1%) in 12 of 14 paraventricular nucleus cells tested, effects which were not observed following pretreatment with the AT1 receptor antagonist losartan. We conclude that in paraventricular nucleus magnocellular cells, like other CNS neurons, at least three sets of potassium channels contribute to the outward current evoked by depolarization. Our data also demonstrate ionic mechanisms through which angiotensin may act at AT1 receptors to influence the excitability of hypothalamic neuroendocrine cells.

Angiotensin I↗

The trochlear nucleus of the frog Rana ridibunda: localization, morphology and ultrastructure of identified motoneurons.

The organization of the trochlear nucleus (N IV) was investigated in the frog Rana ridibunda. Retrograde tracing with horseradish peroxidase and biotinylated dextran amines resulted in labeling on the contralateral N IV of motoneurons with diverse morphologies and in direct caudal continuation with the oculomotor nucleus. Their dendritic arborizations extend profusely in the ipsilateral tegmentum and reach the oculomotor nucleus, the fasciculus longitudinalis medialis and also small processes branch towards the ventricle. Occasionally, one to three cells are labeled in the ipsilateral N IV, whereas mesencephalic trigeminal cells that would send their peripheral branch in the IVth nerve are never observed. The course of the labeled trochlear axons varies depending on the localization within the N IV of their cells of origin and different points of decussation are present above the midbrain ventricle. The ultrastructural analysis of identified trochlear motoneurons shows numerous axo-somatic synaptic contacts and six types of terminals with variable morphologies have been observed. Among them, a peculiar type of axon terminal forms mixed junctions with synaptic specializations and gap junctions together in the membrane interfaces that could represent the simultaneous presence of a chemical as well as an electrical component. The present data give more insights into the organization of the N IV and demonstrate that, although the organization of the trochlear nucleus is highly conservative in gnathostome vertebrates, it shows specific features for each species studied, as demonstrated for amphibians.

Animals↗

The ability to re-express polysialylated NCAM in soleus muscle after denervation is reduced in aged rats compared to young adult rats.

The neural cell-adhesion molecule, NCAM, contains an unusual homopolymer of sialic acid units, polysialic acid. This carbohydrate seems to be involved in neurite outgrowth, bundling and branching, processes which are important during reinnervation. In aged rats, reinnervation of denervated muscle fibres is incomplete. In this study, age-related changes in the degree of polysialylation of NCAM re-expressed after denervation were examined using a monoclonal antibody recognizing polysialic acid and a polyclonal antibody recognizing NCAM. The results show that, after denervation, the degree of polysialylation on NCAM was clearly reduced in rat soleus muscle of aged, compared to young, adult rats. This age-related change in expression of polysialic acid probably influences the reinnervation process in aged muscle.

Animals↗

Calbindin D-28K and NADPH-diaphorase activity are localized in different populations of periglomerular cells in the rat olfactory bulb.

Calbindin D-28k (CaBP) immunocytochemistry and NADPH-diaphorase (ND) histochemistry have been combined in the rat olfactory bulb by successive incubations of the same sections. The outer strata showed a similar neuronal staining pattern for both markers with positive periglomerular neurons (although the CaBP-stained periglomerular cells were six-fold more abundant than the ND-active ones) and larger neurons scattered in the glomerular and external plexiform layers. Both populations of periglomerular cells were distinct but they did not show specific morphological characteristics nor a predominant distribution around ND-positive and negative glomeruli. The colocalization study demonstrates that the larger ND and CaBP-stained juxtaglomerular cells, identified according to their size, location and processes branching patterns as two types of short axon cells (superficial short-axon and Van Gehuchten Cells) were also independent populations.

Animals↗

Degradation and mineralization of Direct Blue 71 in a circulating upflow reactor by UV/TiO2 process and employing a new method in kinetic study.

Direct Blue 71 (C(40)H(23)N(7)Na(4)O(13)S(4)), an azo dye with a high worldwide consumption and providing toxic effluents, can be highly degraded using TiO(2) catalyst suspension and irradiation with a UV-C lamp in a circulating upflow photo-reactor with no dead zone. An initial concentration of 50 mgL(-1) of dye, within the range of typical concentration in textile wastewaters, was used. The influence of catalyst concentration, pH and temperature were investigated. The results showed that degradation of this dye can be conducted in the both processes of only UV irradiation and UV/TiO(2); but with the aim of mineralization, the later process provides significantly better results. Accordingly, a degradation of more than 97% of dye was achieved by applying the optimal operational parameters with 40 mgL(-1) of catalyst, natural pH and 45 degrees C, during 120 min irradiation. A removal of about 50% of COD could also be obtained at the same time. In kinetic investigations, the effect of catalyst particles' turbidity was taken into account and the rate of degradation of the dye, under mild conditions, was expressed as the sum of the rates of individual photolysis and photocatalysis process branches, with mainly influence of the bulk hydroxyl radicals.

Azo Compounds↗

Evolutionary dynamics of invasion and escape.

Whenever life wants to invade a new habitat or escape from a lethal selection pressure, some mutations may be necessary to yield sustainable replication. We imagine situations like (i) a parasite infecting a new host, (ii) a species trying to invade a new ecological niche, (iii) cancer cells escaping from chemotherapy, (iv) viruses or microbes evading anti-microbial therapy, and also (v) the repeated attempts of combinatorial chemistry in the very beginning of life to produce self-replicating molecules. All such seemingly unrelated situations have a common structure in terms of Darwinian dynamics: a replicator with a basic reproductive ratio less than one attempts to find some mutations that allow indefinite survival. We develop a general theory, based on multitype branching processes, to describe the evolutionary dynamics of invasion and escape.

Animals↗

Sampling distribution for microsatellites amplified by PCR: mean field approximation and its applications to genotyping.

Due to microsatellite mutations during PCR, stutter patterns may appear in the final PCR product, which hinder us from accurate genotyping microsatellite markers. The existing methods for microsatellite stutter pattern deconvolution required large amount of data. A mathematical model for microsatellite mutations during PCR and an estimation method based on mean field approximation for branching processes have recently been developed. In this paper, we study the asymptotic behaviors for mean field approximation when experiments are started from a large number of molecules, and we derive an upper bound for the approximation error when experiments are started from a finite number of molecules. Based on the theories of mean field approximation and Bayesian statistics, we develop a novel method for microsatellite stutter pattern deconvolution.

Gene Amplification↗

Perturbation avalanches and criticality in gene regulatory networks.

Boolean networks are simplified models of gene regulatory networks. We derive an approximation of the size distribution of perturbation avalanches in Boolean networks based on known results in the theory of branching processes. We show numerically that the approximation works well for different kinds of Boolean networks. It has been suggested that gene regulatory networks may be dynamically critical. To study this, as an application of the presented theory we present a novel method for estimating an order parameter from microarray data. According to the available data and our method, we find that gene regulatory networks appear to be stable and reside near the phase transition between order and chaos.

Animals↗

Epithelial tissue architecture protects against cancer.

We consider the design of colon crypts from the point of view of minimizing the likelihood of generation of cancerous mutations. A stochastic mathematical model (a finite branching process) is developed and fully analyzed. It is found that depending on the mutation rates, different designs are evolutionarily advantageous. If the mutation rates associated with stem cells are a lot higher than the mutation rates of daughter cells, then few stem cells per crypt is the evolutionarily optimal strategy. If the mutation rates of stem cells are of the same order of magnitude or lower than those for daughter cells, then having as many stem cells per crypt as possible is the desirable design. We also found that the optimal evolutionary strategy may work very well to protect the organism from cancer in the young age, but the same strategy becomes detrimental as the organism ages. It pushes the onset of cancer back in time, but it results in an elevated cancer initiation rates as the organism gets older. Our model quantifies the idea that cancer and aging are the two sides of one coin.

Adenomatous Polyposis Coli Protein↗