Plasticity of the neoplastic phenotype in vivo is regulated by epigenetic factors

Age of host and transplantation-site microenvironment influence the tumorigenic potential of neoplastically transformed liver epithelial cells. Tumorigenic BAG2-GN6TF rat liver epithelial cells consistently form tumors at ectopic sites, but differentially express tumorigenicity or hepatocytic differentiation in the liver depending on host age and route of cell transplantation into the liver. Direct inoculation into host livers concentrates tumor cells locally, resulting in undifferentiated tumors near the transplantation site in both young (3-month-old) and old (18-month-old) rats. Transplantation-site tumors regress within 1 month in the livers of young rats, but grow progressively in old rats. However, inoculation of cells into the spleen distributes transplanted cells individually throughout the liver, resulting in hepatocytic differentiation by tumor cells with concomitant suppression of their tumorigenicity in young rats. When transplanted into livers of old rats by splenic inoculation, or when young hepatic-transplant recipients are allowed to age, hepatocytic progeny of BAG2-GN6TF cells proliferate to form foci, suggesting that the liver microenvironment of old rats incompletely regulates the proliferation and differentiation of tumor cell-derived hepatocytes. Upon removal from the liver, BAG2-GN6TF-derived hepatocytes revert to an undifferentiated, aggressively tumorigenic phenotype. We posit that the spectrum between normal differentiation and malignant potential of these cells reflects the dynamic interaction of the specific transformation-related genotype of the cells and the characteristics of the tissue microenvironment at the transplantation site. Changes in the tissue milieu, such as those that accompany normal aging, may determine the ability of a genetically aberrant cell to produce a tumor.

Abscisic acid-induced stomatal closure mediated by cyclic ADP-ribose

Abscisic acid (ABA) is a plant hormone involved in the response of plants to reduced water availability. Reduction of guard cell turgor by ABA diminishes the aperture of the stomatal pore and thereby contributes to the ability of the plant to conserve water during periods of drought. Previous work has demonstrated that cytosolic Ca2+ is involved in the signal transduction pathway that mediates the reduction in guard cell turgor elicited by ABA. Here we report that ABA uses a Ca2+-mobilization pathway that involves cyclic adenosine 5′-diphosphoribose (cADPR). Microinjection of cADPR into guard cells caused reductions in turgor that were preceded by increases in the concentration of free Ca2+ in the cytosol. Patch clamp measurements of isolated guard cell vacuoles revealed the presence of a cADPR-elicited Ca2+-selective current that was inhibited at cytosolic Ca2+ ≥ 600 nM. Furthermore, microinjection of the cADPR antagonist 8-NH2-cADPR caused a reduction in the rate of turgor loss in response to ABA in 54% of cells tested, and nicotinamide, an antagonist of cADPR production, elicited a dose-dependent block of ABA-induced stomatal closure. Our data provide definitive evidence for a physiological role for cADPR and illustrate one mechanism of stimulus-specific Ca2+ mobilization in higher plants. Taken together with other recent data [Wu, Y., Kuzma, J., Marechal, E., Graeff, R., Lee, H. C., Foster, R. & Chua, N.-H. (1997) Science 278, 2126–2130], these results establish cADPR as a key player in ABA signal transduction pathways in plants.

A model of long-term memory storage in the cerebellar cortex: A possible role for plasticity at parallel fiber synapses onto stellate/basket interneurons

By evoking changes in climbing fiber activity, movement errors are thought to modify synapses from parallel fibers onto Purkinje cells (pf*Pkj) so as to improve subsequent motor performance. Theoretical arguments suggest there is an intrinsic tradeoff, however, between motor adaptation and long-term storage. Assuming a baseline rate of motor errors is always present, then repeated performance of any learned movement will generate a series of climbing fiber-mediated corrections. By reshuffling the synaptic weights responsible for any given movement, such corrections will degrade the memories for other learned movements stored in overlapping sets of synapses. The present paper shows that long-term storage can be accomplished by a second site of plasticity at synapses from parallel fibers onto stellate/basket interneurons (pf*St/Bk). Plasticity at pf*St/Bk synapses can be insulated from ongoing fluctuations in climbing fiber activity by assuming that changes in pf*St/Bk synapses occur only after changes in pf*Pkj synapses have built up to a threshold level. Although climbing fiber-dependent plasticity at pf*Pkj synapses allows for the exploration of novel motor strategies in response to changing environmental conditions, plasticity at pf*St/Bk synapses transfers successful strategies to stable long-term storage. To quantify this hypothesis, both sites of plasticity are incorporated into a dynamical model of the cerebellar cortex and its interactions with the inferior olive. When used to simulate idealized motor conditioning trials, the model predicts that plasticity develops first at pf*Pkj synapses, but with additional training is transferred to pf*St/Bk synapses for long-term storage.

Theory for normal and impaired experience-dependent plasticity in neocortex of adult rats

We model experience-dependent plasticity in the cortical representation of whiskers (the barrel cortex) in normal adult rats, and in adult rats that were prenatally exposed to alcohol. Prenatal exposure to alcohol (PAE) caused marked deficits in experience-dependent plasticity in a cortical barrel-column. Cortical plasticity was induced by trimming all whiskers on one side of the face except two. This manipulation produces high activity from the intact whiskers that contrasts with low activity from the cut whiskers while avoiding any nerve damage. By a computational model, we show that the evolution of neuronal responses in a single barrel-column after this sensory bias is consistent with the synaptic modifications that follow the rules of the Bienenstock, Cooper, and Munro (BCM) theory. The BCM theory postulates that a neuron possesses a moving synaptic modification threshold, θM, that dictates whether the neuron's activity at any given instant will lead to strengthening or weakening of its input synapses. The current value of θM changes proportionally to the square of the neuron's activity averaged over some recent past. In the model of alcohol impaired cortex, the effective θM has been set to a level unattainable by the depressed levels of cortical activity leading to “impaired” synaptic plasticity that is consistent with experimental findings. Based on experimental and computational results, we discuss how elevated θM may be related to (i) reduced levels of neurotransmitters modulating plasticity, (ii) abnormally low expression of N-methyl-d-aspartate receptors (NMDARs), and (iii) the membrane translocation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in adult rat cortex subjected to prenatal alcohol exposure.

Plasticity of the cochleotopic (frequency) map in specialized and nonspecialized auditory cortices

Auditory conditioning (associative learning) causes reorganization of the cochleotopic (frequency) maps of the primary auditory cortex (AI) and the inferior colliculus. Focal electric stimulation of the AI also evokes basically the same cortical and collicular reorganization as that caused by conditioning. Therefore, part of the neural mechanism for the plasticity of the central auditory system caused by conditioning can be explored by focal electric stimulation of the AI. The reorganization is due to shifts in best frequencies (BFs) together with shifts in frequency-tuning curves of single neurons. In the AI of the Mongolian gerbil (Meriones unguiculatus) and the posterior division of the AI of the mustached bat (Pteronotus parnellii), focal electric stimulation evokes BF shifts of cortical auditory neurons located within a 0.7-mm distance along the frequency axis. The amount and direction of BF shift differ depending on the relationship in BF between stimulated and recorded neurons, and between the gerbil and mustached bat. Comparison in BF shift between different mammalian species and between different cortical areas of a single species indicates that BF shift toward the BF of electrically stimulated cortical neurons (centripetal BF shift) is common in the AI, whereas BF shift away from the BF of electrically stimulated cortical neurons (centrifugal BF shift) is special. Therefore, we propose a hypothesis that reorganization, and accordingly organization, of cortical auditory areas caused by associative learning can be quite different between specialized and nonspecialized (ordinary) areas of the auditory cortex.

Spontaneous corticospinal axonal plasticity and functional recovery after adult central nervous system injury

Although it is believed that little recovery occurs after adult mammalian spinal cord injury, in fact significant spontaneous functional improvement commonly occurs after spinal cord injury in humans. To investigate potential mechanisms underlying spontaneous recovery, lesions of defined components of the corticospinal motor pathway were made in adult rats in the rostral cervical spinal cord or caudal medulla. Following complete lesions of the dorsal corticospinal motor pathway, which contains more than 95% of all corticospinal axons, spontaneous sprouting from the ventral corticospinal tract occurred onto medial motoneuron pools in the cervical spinal cord; this sprouting was paralleled by functional recovery. Combined lesions of both dorsal and ventral corticospinal tract components eliminated sprouting and functional recovery. In addition, functional recovery was also abolished if dorsal corticospinal tract lesions were followed 5 weeks later by ventral corticospinal tract lesions. We found extensive spontaneous structural plasticity as a mechanism correlating with functional recovery in motor systems in the adult central nervous system. Experimental enhancement of spontaneous plasticity may be useful to promote further recovery after adult central nervous system injury.

Cyclic AMP Increases Cell Surface Expression of Functional Na,K-ATPase Units in Mammalian Cortical Collecting Duct Principal Cells

Cyclic AMP (cAMP) stimulates the transport of Na+ and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCDc14 collecting duct cells. db-cAMP (10−3 M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of 86Rb+ uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20°C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca2+ chelator bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.

Further Studies of the Role of Cyclic β-Glucans in Symbiosis. An ndvC Mutant of Bradyrhizobium japonicum Synthesizes Cyclodecakis-(1→3)-β-Glucosyl1

The cyclic β-(1→3),β-(1→6)-d-glucan synthesis locus of Bradyrhizobium japonicum is composed of at least two genes, ndvB and ndvC. Mutation in either gene affects glucan synthesis, as well as the ability of the bacterium to establish a successful symbiotic interaction with the legume host soybean (Glycine max). B. japonicum strain AB-14 (ndvB::Tn5) does not synthesize β-glucans, and strain AB-1 (ndvC::Tn5) synthesizes a cyclic β-glucan lacking β-(1→6)-glycosidic bonds. We determined that the structure of the glucan synthesized by strain AB-1 is cyclodecakis-(1→3)-β-d-glucosyl, a cyclic β-(1→3)-linked decasaccharide in which one of the residues is substituted in the 6 position with β-laminaribiose. Cyclodecakis-(1→3)-β-d-glucosyl did not suppress the fungal β-glucan-induced plant defense response in soybean cotyledons and had much lower affinity for the putative membrane receptor protein than cyclic β-(1→3),β-(1→6)-glucans produced by wild-type B. japonicum. This is consistent with the hypothesis presented previously that the wild-type cyclic β-glucans may function as suppressors of a host defense response.

γ-Aminobutyric acid type A receptors modulate cAMP-mediated long-term potentiation and long-term depression at monosynaptic CA3–CA1 synapses

cAMP induces a protein-synthesis-dependent late phase of long-term potentiation (LTP) at CA3–CA1 synapses in acute hippocampal slices. Herein we report cAMP-mediated LTP and long-term depression (LTD) at monosynaptic CA3–CA1 cell pairs in organotypic hippocampal slice cultures. After bath application of the membrane-permeable cAMP analog adenosine 3′,5′-cyclic monophosphorothioate, Sp isomer (Sp-cAMPS), synaptic transmission was enhanced for at least 2 h. Consistent with previous findings, the late phase of LTP requires activation of cAMP-dependent protein kinase A and protein synthesis. There is also an early phase of LTP induced by cAMP; the early phase depends on protein kinase A but, in contrast to the later phase, does not require protein synthesis. In addition, the cAMP-induced LTP is associated with a reduction of paired-pulse facilitation, suggesting that presynaptic modification may be involved. Furthermore, we found that Sp-cAMPS induced LTD in slices pretreated with picrotoxin, a γ-aminobutyric acid type A (GABAA) receptor antagonist. This form of LTD depends on protein synthesis and protein phosphatase(s) and is accompanied by an increased ratio of failed synaptic transmission. These results suggest that GABAA receptors can modulate the effect of cAMP on synaptic transmission and thus determine the direction of synaptic plasticity.

Growth factors regulate phototransduction in retinal rods by modulating cyclic nucleotide-gated channels through dephosphorylation of a specific tyrosine residue

Illumination of vertebrate rod photoreceptors leads to a decrease in the cytoplasmic cGMP concentration and closure of cyclic nucleotide-gated (CNG) channels. Except for Ca2+, which plays a negative feedback role in adaptation, and 11-cis-retinal, supplied by the retinal pigment epithelium, all of the biochemical machinery of phototransduction is thought to be contained within rod outer segments without involvement of extrinsic regulatory molecules. Here we show that insulin-like growth factor-I (IGF-I), a paracrine factor released from the retinal pigment epithelium, alters phototransduction by rapidly increasing the cGMP sensitivity of CNG channels. The IGF-I-signaling pathway ultimately involves a protein tyrosine phosphatase that catalyzes dephosphorylation of a specific residue in the α-subunit of the rod CNG channel protein. IGF-I conjointly accelerates the kinetics and increases the amplitude of the light response, distinct from events that accompany adaptation. These effects of IGF-I could result from the enhancement of the cGMP sensitivity of CNG channels. Hence, in addition to long-term control of development and survival of rods, growth factors regulate phototransduction in the short term by modulating CNG channels.

Developmental plasticity of CNS microglia

Microglia arise from CD45+ bone marrow precursors that colonize the fetal brain and play a key role in central nervous system inflammatory conditions. We report that parenchymal microglia are uncommitted myeloid progenitors of immature dendritic cells and macrophages by several criteria, including surface expression of “empty” class II MHC protein and their cysteine protease (cathepsin) profile. Microglia express receptors for stem cell factor and can be skewed toward more dendritic cell or macrophage-like profiles in response to the lineage growth factors granulocyte/macrophage colony-stimulating factor or macrophage colony-stimulating factor. Thus, in contrast to other organs, where terminally differentiated populations of resident dendritic cells and/or macrophages outnumber colonizing precursors, the majority of microglia within the brain remain in an undifferentiated state.

T cell activation up-regulates cyclic nucleotide phosphodiesterases 8A1 and 7A3

Agents that increase intracellular cAMP inhibit the activation and function of T cells and can lead to cell death. Recently, it has been postulated that cAMP inhibits T cell function in large part by acting as a brake on the T cell receptor and costimulatory receptor pathways. Therefore, for full activation of the T cell to occur, this inhibitory influence must be removed. One likely mechanism for accomplishing this is by up-regulation and/or activation of specific cyclic nucleotide phosphodiesterases (PDEs), and such a mechanism for one phosphodiesterase, PDE7A1, has been reported. In this paper, we extend this mechanism to another isozyme variant of the same PDE family, PDE7A3. We also report the full-length sequence of human PDE8A1 and show that it also is induced in response to a combination of T cell receptor and costimulatory receptor pathway activation. However, the time course for induction of PDE8A1 is slower than that of PDE7A1. The basal level measured and, therefore, the apparent fold induction of PDE7A1 mRNA and protein depend in large part on the method of isolation of the T cells. On the other hand, regardless of the isolation method, the basal levels of PDE7A3 and PDE8A1 are very low and fold activation is much higher. Constitutively expressed PDE8A1 and PDE7A3 also have been isolated from a human T cell line, Hut78.