Morphoregulatory Effects Research Articles

Mouse Acetylcholinesterase Enhances Neurite Outgrowth of Rat R28 Cells Through Interaction With Laminin-1

The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine, but can also exert ‘non-classical’, morpho-regulatory effects on developing neurons such as stimulation of neurite outgrowth. Here, we investigated the role of AChE binding to laminin-1 on the regulation of neurite outgrowth by using cell culture, immunocytochemistry, and molecular biological approaches. To explore the role of AChE, we examined fiber growth of cells overexpressing different forms of AChE, and/or during their growth on laminin-1. A significant increase of neuritic growth as compared with controls was observed for neurons over-expressing AChE. Accordingly, addition of globular AChE to the medium increased total length of neurites. Co-transfection with PRIMA, a membrane anchor of AChE, led to an increase in fiber length similar to AChE overexpressing cells. Transfection with an AChE mutant that leads to the retention of AChE within cells had no stimulatory effect on neurite length. Noticeably, the longest neurites were produced by neurons overexpressing AChE and growing on laminin-1, suggesting that the AChE/laminin interaction is involved in regulating neurite outgrowth. Our findings demonstrate that binding of AChE to laminin-1 alters AChE activity and leads to increased neurite growth in culture. A possible mechanism of the AChE effect on neurite outgrowth is proposed due to the interaction of AChE with laminin-1.

In vitro culture of Hypericum rumeliacum Boiss. and production of phenolics and flavonoids

The Balkan endemic species, Hypericum rumeliacum, Guttiferae was introduced in vitro for the first time with the aim to study the type of morphogenetic response to plant growth regulators and ability to produce phenolics and flavonoid compounds. The morphoregulatory effect of 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphtaleneacetic acid (NAA), 6-benzyladenine (BA) and combination of BA with NAA in Murashige–Skoog's basal medium on leaf lamina, internode stem segment, stem node and root cuttings was studied. Histological analysis of the structures regenerated from the primary explants proved the presence of both, embryoids and meristemoids. The node explants cultivated on BA-supplemented medium were the most favourable for regeneration through meristemoids. Therefore a double-stage culture approach, allowing an effective multiplication of large quantities of plant shoots in vitro along with maintenance of the biosynthetic capacity of the culture was developed. It comprised one subculture of three-nodal stem explants derived from the stock shoot cultures on MS medium supplemented with 0.2 mg/l BA followed by subculture of the induced multiple shoots on cytokinin-free MS medium. Determination of the total phenolics and flavonoids showed that the decrease of the levels of these secondary metabolites is transitional, as the exclusionof BA from the medium resulted in an increase of their total content.

Morphoregulation by acetylcholinesterase in fibroblasts and astrocytes

Acetylcholinesterase (AChE) terminates neurotransmission at cholinergic synapses by hydrolysing acetylcholine, but also has non-enzymatic morphoregulatory effects on neurons such as stimulation of neurite outgrowth. It is widely expressed outside the nervous system, but its function in non-neuronal cells is unclear. Here we have investigated the distribution and function of AChE in fibroblasts and astrocytes. We show that these cells express high levels of AChE protein that co-migrates with recombinant AChE but contains little catalytic activity. Fibroblasts express transcripts encoding the synaptic AChE-T isoform and its membrane anchoring peptide PRiMA-I. AChE is strikingly distributed in arcs, rings and patches at the leading edge of spreading and migrating fibroblasts and astrocytes, close to the cell-substratum interface, and in neuronal growth cones. During in vivo healing of mouse skin, AChE becomes highly expressed in re-epithelialising epidermal keratinocytes 1 day after wounding. AChE appears to be functionally important for polarised cell migration, since an AChE antibody reduces substratum adhesion of fibroblasts, and slows wound healing in vitro as effectively as a beta1-integrin antibody. Moreover, elevation of AChE expression increases fibroblast wound healing independently of catalytic activity. Interestingly, AChE surface patches precisely co-localise with amyloid precursor protein and the extracellular matrix protein perlecan, but not focal adhesions or alpha-dystroglycan, and contain a high concentration of tyrosine phosphorylated proteins in spreading cells. These findings suggest that cell surface AChE, possibly in a novel signalling complex containing APP and perlecan, contributes to a generalised mechanism for polarised membrane protrusion and migration in all adherent cells.

The expression of wild-type human amyloid precursor protein affects the dendritic phenotype of neocortical pyramidal neurons in transgenic mice

The current study addresses the morphoregulatory effects of human amyloid precursor protein expression on neocortical pyramidal cells in vivo. For this purpose, a transgenic mouse line was used that expresses wild-type human amyloid precursor protein (APP) at levels similar to endogenous mouse APP [introduced by Lamb, B.T., Sisodia, S.S., Lawler, A.M., Slunt, H.H., Kitt, C.A., Kearns, W.G., Pearson, P.L., Price, D.L., Gearhart, J.D., 1993. Introduction and expression of the 400 kilobase amyloid precursor protein gene in transgenic mice. Nat. Genet. 5, 22–30]. This strain does not develop Alzheimer's disease-related pathology which allowed to study effects of APP or APP cleavage products but excluded the influence of amyloid deposits. Commissural projecting pyramidal neurons of layers II/III within the primary somatosensory cortex were retrogradely labelled by injection of biotinylated dextran amine into the corpus callosum. In transgenic mice, computer-aided morphometric analysis revealed an increase in the surface area of proximal and intermediate basal dendritic segments resulting from an enlarged diameter. On the other hand, the length of the same segments was reduced. Both basal and apical dendrites were characterized by a higher dendritic density within the proximal and intermediate fields. Although the total spatial extension of basal and apical dendrites remained unchanged, a moderate withdrawal of arbors is suggested. The results implicate a physiological function for APP in regulatory mechanisms of neuronal morphogenesis.

Prohexadione‐Ca induces resistance to fireblight and other diseases1

Prohexadione‐Ca is primarily used for the control of shoot growth in pome and other fruit trees. During its development, treated apple and pear trees were found to be significantly less affected by fireblight (Erwinia amylovora) and other pathogens, although prohexadione‐Ca is inactive as a bactericide or fungicide. Prohexadione is a structural mimic of 2‐oxoglutaric acid, and the distinct dioxygenases involved in gibberellin biosynthesis that require this compound as a cosubstrate are blocked. As a result, less growth‐active gibberellins are formed and treated plants remain compact. 2‐Oxoglutaric acid‐dependent dioxygenases are also involved in flavonoid metabolism. In shoots of apples and pears, prohexadione‐Ca causes considerable changes in the formation of flavonoids and their phenolic precursors by inhibiting flavanone 3‐hydroxylase. Convincing evidence is available that prohexadione‐Ca triggers pathogen resistance primarily by inducing the formation of 3‐deoxyflavonoids, in particular luteoforol, with phytoalexin‐like properties. Morphoregulatory effects caused by prohexadione‐Ca are only of secondary relevance. The simultaneous control of excessive shoot growth and shoot infections by fireblight is seen as a major advantage of using prohexadione‐Ca in pome fruit trees.

Prohexadione-Ca in Fruit Trees: Modes of Action of a Multifunctional Bioregulator

APOGEE and REGALIS have recently been introduced in a number of countries for use in pome and other fruit trees. These products contain 27.5% and 10% of prohexadione-Ca (ProCa), respectively. As a result of inhibiting excessive vegetative growth, less summer and dormant pruning is required, the ratio between vegetative growth and fruit formation is improved, and crop protection is facilitated due to the reduction of tree row volume and a more open canopy. Additionally, a lowered incidence of diseases such as fire blight and scab is observed, which is not due to a direct bactericidal or fungicidal effect of the compound. Further, the compound may reduce fruit drop early in the season. Prohexadione is a structural mimic of 2-oxoglutaric and ascorbic acid. Therefore, distinct dioxygenases are blocked, which require these compounds as a co-substrate. Such enzymes catalyze late steps in gibberellin biosynthesis. After treatment with ProCa, less growth-active gibberellins are formed and treated plants remain more compact. ProCa also affects ACC oxidase, another dioxygenase. The resulting reduction of ethylene formation, in addition to the availability of more assimilates for fruit growth, is most likely the cause of reduced fruit drop. 2-Oxoglutaric acid-dependent dioxygenases are also involved in the metabolism of flavonoids and their phenolic precursors: In shoots of apples and pears, ProCa causes considerable changes by inhibiting flavanone 3-hydroxylase. Convincing evidence is now available that ProCa triggers pathogen resistance by inducing the formation of 3-deoxyflavonoids, in particular luteoforol, with phytoalexin-like properties. Morphoregulatory effects caused by ProCa are only of secondary relevance for the reduction of disease incidence.

Morphologic and neurotoxic effects of ethanol vary with timing of exposure in vitro

Results of investigations with animal models of fetal alcohol syndrome (FAS) seem to indicate that neuronal vulnerability to ethanol-induced cell death may be correlated with specific developmental events. In the present study, we sought to test this observation in a cell culture model of neuronal development in which morphogenesis as well as survival could be assessed. Using embryonic rat hippocampal pyramidal neurons in primary cultures, we compared the sensitivity of neurons to ethanol added, at 400 mg/dl, to the medium at different times relative to the development of axons and dendrites. Quantitative morphometric analysis was performed by using phase contrast at 12 h (0.5 day) and 24 h (1 day), or fluorescence microscopy after microtubule-associated protein-2 (MAP2) immunostaining at 6 and 14 days. Survival was assessed by counting the number of neurons per unit area of the substrate at 14 days. Addition of ethanol 1 day after plating, when most neurons had developed an axon, had no effect on survival up to 14 days in vitro, but resulted in significantly shorter, less branched dendrites than observed when ethanol was added 2 h after plating. Despite the shorter duration of ethanol exposure, the addition of ethanol on day 6, after rapid growth of dendrites and synapses had begun, resulted in loss of all but about one third of the neurons by 14 days. This supports the suggestion that increased neuronal vulnerability to the morphoregulatory effects of ethanol is correlated with the establishment of polarity, but that the sensitivity of neurons to the cytotoxic effects of ethanol occurs later, when dendrites and synapses are rapidly forming.

Lysophosphatidic acid antagonizes the morphoregulatory effects of the luteinizing hormone on luteal cells: possible role of small Rho-G-proteins.

Lysophosphatidic acid (LPA) is a biologically active phospholipid recently introduced as a new marker for ovarian cancer. Because high concentrations of LPA have also been found in the follicular fluid from healthy subjects, one can presume that this biological mediator may have relevance for normal ovarian physiology as well. We have reported earlier that luteal cells possess specific binding sites for LPA. Using these cells as a model, we show now that LPA is able to modulate the morphological cell shape changes induced by LH in that it inhibits the formation of stellate processes induced by LH. This morphoregulatory effect of LPA is mimicked by cytotoxic necrotizing factor 1, a bacterial toxin known to activate small G-proteins from the Rho family. On the other hand, C3-exotransferase that acts mainly through the inhibition of Rho A mimics the effects of LH. Furthermore, we report here that the morphoregulatory effects of LPA are accompanied by the translocation of Rho proteins from the cytosol to cell membrane, an effect generally considered to be an indicator for the activation of Rho-GTPases. During the development and rescue of the corpus luteum, major morphoregulatory effects are exerted by LH that appear to be modulated by LPA via an activation of Rho proteins.

Morphoregulatory effect of plant growth regulators on Hypericum perforatum L. Seedlings

AbstractThe morphogenetic response of Hypericum perforatum seedlings to different auxin and cytokinin concentrations was studied. A stimulation of the concentration‐dependent rooting ability was observed under the influence of indole‐3‐acetic acid and indole‐3‐butyric acid. Rooting was not enhanced by the effects of 2,4‐dichlorophenoxyacetic acid and 1‐naphtaleneacetic acid. Differentiated roots were isolated and cultured in liquid media with the same combination of growth‐promoting auxins. Chromosome counts in root tip cells after long‐term cultivation indicated a high degree of chromosomal instability. Multiple shoot formation occurred under the influence of 6‐benzylaminopurine and kinetin. Adenine and 6‐(γ,γ‐dimethylallylamino)‐purine did not stimulate shoot differentiation. No differences in the morphogenetic response to auxins and cytokinis were detected between diploid and tetrapoloid plants.

Modulation of root growth and organogenesis in thidiazuron-treated ginseng (Panax quinquefolium L.)

The morphoregulatory effect of thidiazuron (TDZ) applications to ginseng (Panax quinquefolium L.) seedlings and 3-year-old plants was determined. Applications of TDZ (0.22 and 2.20 ppm), either as foliar sprays or soil drench to greenhouse-grown seedlings over 18 weeks (2 weeks after sowing to 20 weeks when plants were harvested) induced similar responses, in particular, increased stem length and diameter, and shoot and root weight (economic yield). Single foliar applications of TDZ at 62.5 and 125 ppm to 3-year-old field-grown ginseng plants 3 months prior to harvest increased root biomass (economic yield) by 19 to 23%. Roots of TDZ-treated seedlings and 3-year-old field-grown plants developed thickened secondary roots on the upper part of the taproot. The root-like structure of these secondary roots was confirmed by histology. In addition, TDZ treatments induced adventitious buds on the shoulder of 3-year-old roots. These buds developed into shoots to give multi-stem plants following a period of dormancy, which was overcome with GA3 (gibberellic acid) treatment prior to planting.

Selective enhancement of axonal branching of cultured dentate gyrus neurons by neurotrophic factors.

Epileptic seizures in the mature nervous system are associated with axonal sprouting of the hippocampal dentate granule cells and pathological synapse formation. The molecular basis of this morphological rearrangement is obscure. Since epileptic seizures induce the transcriptional activation of genes encoding diverse neurotrophic and growth factors in the dentate granule cells and their targets, morphoregulatory effects of these proteins may contribute to this morphological rearrangement. To determine whether neurotrophins or growth factors exert morphoregulatory effects on dentate gyrus neurons, quite homogeneous preparations of these neurons from postnatal rats were established in primary culture at low density in defined media. Dendrites were distinguished from axons by phase contrast appearance together with microtubule-associated protein-2 immunocytochemistry. Multiple factors enhanced branching of axons but not dendrites of these neurons. The rank order of effectiveness was: basic fibroblast growth factor > brain-derived growth factor > neurotrophin-4 > neurotrophin-3; nerve growth factor was ineffective. No additives of synergistic effects were detected. These results are consistent with the idea that activity-driven expression of these genes contributes to the axonal sprouting and pathological synapse formation evident in diverse forms of epilepsy.

Specification of the effect of chelating complex of iron ions in androgenesisin vitro by means of cation-free minimal medium

The addition of Na2EDTA to a minimal medium (agar, sucrose) deprived of cations produces no morphoregulatory effect. This effect is induced only by the addition of iron. Traces of iron present in the agar are sufficient for the development of globular embryoid into a complete plant. The traces of iron in distilled water and sucrose (p. a.) are insufficient for the morphoregulation. The marked difference between the necessary limiting amount of chelate and necessary limiting amount of iron in non-modified media is explained by the presence of other cations, which saturate and thereby inactivate a greater part of Na2EDTA. It is recommended to decrease the amount of iron and leave the commonly used amount of chelate in the prepared media. Simultaneously with optimal morphoregulatory effect in androgenesis and somatic embryogenesis, vitality of tissue culture will be lengthened and ageing as well as necrosis of the cultures be decreased.

In vitro regulation of androgenesis by iron ions and chelate: A common property of two androgenic species (Nicotiana tabacum L. andDatura innoxia Mill.)

The morphoregulatory effect of FeEDTA chelating complex was verified in an androgenically more productive cultivar of tobacco White Burley and inDatura innoxia Mill. The presence of EDTA in Nitsch (1969) medium (N medium) was sufficient for the conversion of isolated embryoids and embryoids in anthers into complete plants. The androgenic development was most rapid in N medium with FeEDTA. In N medium with EDTA, the cultures developed more slowly and remained vital for a long time. In the medium without EDTA, new embryoids developed continuously. The embryoids arose and developed also in closed anthers. All embryoids which arose in anthers cultured on N medium were capable of forming complete plants. The threshold morphoregulatory concentration of FeEDTA was 40 μM FeEDTA per 1 litre of medium for embryoids in anthers and 30 μM FeEDTA per 1 litre for isolated embryoids. The dark brown colour of the anthers of tobacco andD. innoxia was due to the presence of iron (FeEDTA) in the culture medium. The morphoregulatory effect of EDTA in N medium is explained by the formation of a complex with iron present in the form of pollution of chemicals and agar. This amount of iron in complex with EDTA is sufficient for the conversion of embryoids into plants, but it is insufficient for the browning of anthers.

Effect of Minimization of Culture Medium on the Process of Androgenesis in vitro (Nicotiana tabacum L. var. White Burley, Datura innoxia Mill.)

Summary The medium consisting of agar and sucrose is sufficient for the development of ernbryoids into globular stage, whereas for the development of complete plants it is necessary to add chelated iron. The highest androgenic embryogenesis occurred on AS medium (0.8% sugar, 2% sucrose). The specifirity of culture medium is not decisive for the ini tiation of androgenesis. The initiation of androgenic embryogenesis is assumed to be an autonomous function of extirpated anthers. A continual androgenic development of pollen microspores into globular embryoids takes place on both minimal medium without FeEDTA and complete medium without FeEDTA. Isolated embryoids transferred to the medium consisting of agar, sucrose and FeEDTA markedly change their morphology but do not develop into complete plants, whereas in a complete medium, the complete plants are formed. Sucrose concentration of 0.5- -l% in complete medium does not influence the transformation of isolated embryoicls into green plantlets. In minimal medium, the wall of cultured anther can replace nutritionally the complete medium, but cannot replace the morphoregulatory effect of FeEDTA. The nutritional capacity of the another wall is limited.