Transcriptional regulation. Modification by nuclear export?
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Binding, Competitive↗
Biomedical subjects
A Rao
Publications and source records attributed to A Rao.
Central administration of leptin to ovariectomized ewes inhibits food intake without affecting the secretion of hormones from the pituitary gland: evidence for a dissociation of effects on appetite and neuroendocrine function.
We have studied the effect of leptin on food intake and neuroendocrine function in ovariectomized ewes. Groups (n = 5) received intracerebroventricular infusions of either vehicle or leptin (20 microg/h) for 3 days and were blood sampled over 6 h on days -1, 2, and for 3 h on day 3 relative to the onset of the infusion. The animals were then killed to measure hypothalamic neuropeptide Y expression by in situ hybridization. Plasma samples were assayed for metabolic parameters and pituitary hormones. Food intake was reduced by leptin, but did not change in controls. Leptin treatment elevated plasma lactate and nonesterified fatty acids, but did not affect glucose or insulin levels, indicating a state of negative energy balance that was met by the mobilization of body stores. Pulse analysis showed that the secretion of LH and GH was not affected by leptin treatment, nor were the mean plasma concentrations of FSH, PRL, or cortisol. Expression of messenger RNA for neuropeptide Y in the arcuate nucleus was reduced by the infusion of leptin, primarily due to reduced expression per cell rather than a reduction in the number of cells observed. Thus, the action of leptin to inhibit food intake is dissociated from neuroendocrine function. These results suggest that the metabolic effects of leptin are mediated via neuronal systems that possess leptin receptors rather than via endocrine effects.
Animals↗
Evidence for differential regulation of multiple transcripts of the gonadotropin releasing hormone receptor in the ovine pituitary gland; effect of estrogen.
The number of pituitary gonadotropin-releasing hormone receptors (GnRH-R) varies across the estrous cycle. We report that there is variable expression of the differently-sized GnRH-R transcripts in cyclic ewes and in an experimental model. During the follicular phase of the cycle, and compared to the luteal phase, there was increased expression of the 1.5, 2.3 and 3.7 kilobase (kb) transcripts with no change in the levels of the 5.6 or the 1.2 kb transcripts. Steady state levels of mRNA for luteinising hormone beta and common alpha subunit were also increased in the follicular phase of the cycle. In hypothalamo-pituitary disconnected ovariectomised ewes given pulsatile GnRH replacement, injection of estrogen increased the 1.5, 2.3 and 3.7 kb, while the levels of the 5.6 and 1.2 kb transcripts were not altered. We conclude that the differential regulation of GnRH-R mRNA occurs through a direct effect of E on the pituitary.
Animals↗
Two-site interaction of nuclear factor of activated T cells with activated calcineurin.
Transcription factors belonging to the nuclear factor of activated T cells (NFAT) family regulate the expression of cytokine genes and other inducible genes during the immune response. The functions of NFAT proteins are directly controlled by the calcium- and calmodulin-dependent phosphatase calcineurin. Here we show that the binding of calcineurin to NFAT is substantially increased when calcineurin is activated with calmodulin and calcium. FK506.FKBP12 drug-immunophilin complexes inhibited the interaction of NFAT with activated calcineurin much more effectively than they inhibited the interaction with inactive calcineurin, suggesting that part of the interaction with activated calcineurin involved the enzyme active site. We have previously shown that NFAT is targeted to inactive calcineurin at a region distinct from the calcineurin active site (Aramburu, J., Garcia-Cozar, F. J., Raghavan, A., Okamura, H., Rao, A., and Hogan, P. G. (1998) Mol. Cell 1, 627-637); this region is also involved in NFAT binding to activated calcineurin, since binding is inhibited by an NFAT peptide spanning the calcineurin docking site on NFAT. The interacting surfaces are located on the catalytic domain of the calcineurin A chain and on an 86-amino acid fragment of the NFAT regulatory domain. NFAT binding to the calcineurin catalytic domain was inhibited by the calcineurin autoinhibitory domain and the RII substrate peptide, which bind in the calcineurin active site, as well as by the NFAT docking site peptide, which binds to a region of calcineurin distinct from the active site. We propose that, in resting cells, NFAT is targeted to a region of the calcineurin catalytic domain that does not overlap the calcineurin active site. Upon cell activation, displacement of the autoinhibitory domain by calmodulin binding allows NFAT to bind additionally to the calcineurin active site, thus positioning NFAT for immediate dephosphorylation at functional phosphoserine residues.
Amino Acid Sequence↗
Nuclear factor of activated T cells (NFAT)-dependent transactivation regulated by the coactivators p300/CREB-binding protein (CBP).
p300 and cAMP response element-binding protein (CREB)-binding protein (CBP) are members of a family of coactivators involved in the regulation of transcription and chromatin. We show that transcription factors of the nuclear factor of activated T cells (NFAT) family bind p300/CBP and recruit histone acetyltransferase activity from T cell nuclear extracts. The NH2-terminal transactivation domain of NFAT1 and the phospho-CREB- and E1A-binding sites of p300/CBP are involved in the interaction. The viral oncoprotein E1A inhibits NFAT-dependent transactivation in a p300-dependent manner. Recruitment of the coactivators p300/CBP by the transactivation domains of NFAT proteins is likely to play a critical role in NFAT-dependent gene expression during the immune response.
Acetyltransferases↗
Subcortical connections of normotopic and heterotopic neurons in sensory and motor cortices of the tish mutant rat.
Orthograde and retrograde tracers were used to examine subcortical connections of neurons in the neurological mutant tish rat. This animal exhibits bilateral heterotopia similar to those observed in epileptic humans with subcortical band heterotopia. Terminal varicosities were labeled in the striatum, thalamus, brainstem, and spinal cord following injections of the anterograde tracer biotinylated dextran amine (BDA) into the heterotopic cortex. The general topography of corticothalamic projections was evaluated by injecting the retrograde tracer Fluoro-Gold (FG) into ventral thalamic nuclei. Retrograde labeling of small-to-medium sized neurons was observed in layer VI of topographically restricted portions of the normotopic cortex. Similar appearing cells were labeled in the neighboring portions of the underlying heterotopia; however, these neurons did not display characteristic lamination or radial orientation. Thalamocortical terminals labeled by injecting BDA into the ventroposterolateral nucleus (VPL) were observed primarily in layer IV of the medial aspect of the normotopic somatosensory cortex. In contrast, a radial column of terminals was present in the underlying heterotopia. Typical barrel labeling was found in the lateral aspect of the normotopic somatosensory cortex after injecting the ventroposteromedial nucleus (VPM), whereas more diffuse patches of labeling were observed in the underlying heterotopia. Heterotopic neurons in the tish cortex, thus, exhibit characteristic features of subcortical connectivity. Both normotopic and heterotopic neurons in the tish brain project to appropriate subcortical sites and establish bidirectional topographic connections with the thalamus. These results suggest that primary sensory-motor information is represented in a parallel manner in the normotopic and heterotopic cortices of the tish rat.
Afferent Pathways↗
Paracrine interactions within the pituitary gland.
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Adrenocorticotropic Hormone↗
Regulation of allergic inflammation and eosinophil recruitment in mice lacking the transcription factor NFAT1: role of interleukin-4 (IL-4) and IL-5.
Transcription factors of the NFAT (nuclear factor of activated T cells) family regulate the expression of many genes encoding immunoregulatory cytokines and cell surface proteins during the immune response. The NFAT protein NFAT1 (NFATp) is expressed and functional in T cells, B cells, mast cells, and natural killer cells. Here we report a detailed analysis of the enhanced eosinophil responses of NFAT1-deficient mice, observed in an in vivo model of allergic inflammation. In addition to the pleural eosinophilia described previously, NFAT1-/- mice that have been sensitized with antigen display a significant increase, relative to wild-type mice, in the numbers of eosinophils in bone marrow and peripheral blood. After restimulation with antigen in vitro, antigen-responsive T cells from the draining lymph nodes of NFAT1-/- mice show increased expression of mRNA encoding the Th2 cytokines interleukin-4 (IL-4), IL-5, and IL-13. Consistent with this finding, there is a pronounced increase in the levels of IL-5 and IL-13 in the pleural cavities of sensitized NFAT1-/- mice after allergen challenge in vivo. Furthermore, development of eosinophilia depends on overexpression of IL-4 and IL-5, because it is strongly inhibited by administration of neutralizing antibodies to either of these cytokines. These results indicate that NFAT1-deficient mice are prone to develop a classically allergic phenotype characterized by eosinophilia and increased production of Th2 cytokines. Thus, the presence of NFAT1 might inhibit the allergic response, perhaps by interfering with the development of Th2 immune responses, and the lack or dysfunction of NFAT1 could potentially underlie certain cases of atopic disease.
Animals↗
Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA.
The nuclear factor of activated T cells (NFAT) and the AP-1 heterodimer, Fos-Jun, cooperatively bind a composite DNA site and synergistically activate the expression of many immune-response genes. A 2.7-A-resolution crystal structure of the DNA-binding domains of NFAT, Fos and Jun, in a quaternary complex with a DNA fragment containing the distal antigen-receptor response element from the interleukin-2 gene promoter, shows an extended interface between NFAT and AP-1, facilitated by the bending of Fos and DNA. The tight association of the three proteins on DNA creates a continuous groove for the recognition of 15 base pairs.
Amino Acid Sequence↗
Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture.
To determine their roles in the assembly of glutamatergic postsynaptic sites, we studied the distributions of NMDA- and AMPA-type glutamate receptors; the NMDA receptor-interacting proteins alpha-actinin-2, PSD-95, and chapsyn; and the PSD-95-associated protein GKAP during the development of hippocampal neurons in culture. NMDA receptors first formed nonsynaptic proximal dendrite shaft clusters within 2-5 d. AMPA receptors were diffuse at this stage and began to cluster on spines at 9-10 d. NMDA receptor clusters remained partially nonsynaptic and mainly distinct from AMPA receptor clusters until after 3 weeks in culture, when the two began to colocalize at spiny synaptic sites. Thus, the localization of NMDA and AMPA receptors must be regulated by different mechanisms. alpha-Actinin-2 colocalized with the NMDA receptor only at spiny synaptic clusters, but not at shaft nonsynaptic or synaptic clusters, suggesting a modulatory role in the anchoring of NMDA receptor at spines. PSD-95, chapsyn, and GKAP were present at some, but not all, nonsynaptic NMDA receptor clusters during the first 2 weeks, indicating that none is essential for NMDA receptor cluster formation. When NMDA receptor clusters became synaptic, PSD-95 and GKAP were always present, consistent with an essential function in synaptic localization of NMDA receptors. Furthermore, PSD-95 and GKAP clustered opposite presynaptic terminals several days before either NMDA or AMPA receptors clustered at these presumptive postsynaptic sites. These results suggest that synapse development proceeds by formation of a postsynaptic scaffold containing PSD-95 and GKAP in concert with presynaptic vesicle clustering, followed by regulated attachment of glutamate receptor subtypes to this scaffold.
Actinin↗
Differential regional expression and ultrastructural localization of alpha-actinin-2, a putative NMDA receptor-anchoring protein, in rat brain.
Fast chemical neurotransmission is dependent on ionotropic receptors that are concentrated and immobilized at specific postsynaptic sites. The mechanisms of receptor clustering and anchoring in neuronal synapses are poorly understood but presumably involve molecular linkage of membrane receptor proteins to the postsynaptic cytoskeleton. Recently the actin-binding protein alpha-actinin-2 was shown to bind directly to the NMDA receptor subunits NR1 and NR2B (), suggesting that alpha-actinin-2 may function to attach NMDA receptors to the actin cytoskeleton. Here we show that alpha-actinin-2 is localized specifically in glutamatergic synapses in cultured hippocampal neurons. By immunogold electron microscopy, alpha-actinin-2 is concentrated over the postsynaptic density (PSD) of numerous asymmetric synapses where it colocalizes with NR1 immunoreactivity. Thus alpha-actinin-2 is appropriately positioned at the ultrastructural level to function as a postsynaptic-anchoring protein for NMDA receptors. alpha-Actinin-2 is not, however, exclusively found at the PSD; immunogold labeling was also associated with filaments and the spine apparatus of dendritic spines and with microtubules in dendritic shafts. alpha-Actinin-2 showed marked differential regional expression in rat brain. For instance, the protein is expressed at much higher levels in dentate gyrus than in area CA1 of the hippocampus. This differential regional expression implies that glutamatergic synapses in various parts of the brain differ with respect to their alpha-actinin-2 content and thus, potentially, the extent of possible interaction between alpha-actinin-2 and the NMDA receptor.
Actinin↗
Tacrolimus enhances the immunosuppressive effect of cyclophosphamide but not that of leflunomide or mycophenolate mofetil in a model of discordant liver xenotransplantation.
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Animals↗
Postsynaptically silent synapses in single neuron cultures.
We have used the synapses that isolated hippocampal cells in culture form onto themselves (autapses) to determine if some synapses lack functional AMPA receptors (AMPARs). A comparison of the synaptic variability of the AMPAR- and NMDAR-mediated evoked responses, as well as of miniature synaptic responses, indicates that a population of events exists that only contains an NMDAR component. Spillover of glutamate from adjacent synapses cannot explain these results because in single cell cultures all synaptic events mediated by AMPARs should be detected. Immunocytochemical analysis of these cultures clearly reveals a population of synapses with puncta for NR1 (NMDAR) but not for GluR1 (AMPAR). These results provide strong anatomical and physiological evidence for the existence of postsynaptically silent synapses.
Animals↗
Long-range transcriptional regulation of cytokine gene expression.
Most studies on the control of cytokine gene expression have involved the functional analysis of proximal promoters. Recent work has identified distal elements that mediate long-range cytokine gene regulation and has implicated chromatin reorganization in regulation of cytokine gene loci. These studies have begun to elucidate the basis for cell-specificity and high-level expression of cytokine genes.
Animals↗
Clinical intestinal transplantation: new perspectives and immunologic considerations.
BACKGROUND: Although tacrolimus-based immunosuppression has made intestinal transplantation feasible, the risk of the requisite chronic high-dose treatment has inhibited the widespread use of these procedures. We have examined our 1990-1997 experience to determine whether immunomodulatory strategies to improve outlook could be added to drug treatment. STUDY DESIGN: Ninety-eight consecutive patients (59 children, 39 adults) with a panoply of indications received 104 allografts under tacrolimus-based immunosuppression: intestine only (n = 37); liver and intestine (n = 50); or multivisceral (n = 17). Of the last 42 patients, 20 received unmodified adjunct donor bone marrow cells; the other 22 were contemporaneous control patients. RESULTS: With a mean followup of 32 +/- 26 months (range, 1-86 months), 12 recipients (3 intestine only, 9 composite grafts) are alive with good nutrition beyond the 5-year milestone. Forty-seven (48%) of the total group survive bearing grafts that provide full (91%) or partial (9%) nutrition. Actuarial patient survival at 1 and 5 years (72% and 48%, respectively) was similar with isolated intestinal and composite graft recipients, but the loss rate of grafts from rejection was highest with intestine alone. The best results were in patients between 2 and 18 years of age (68% at 5 years). Adjunct bone marrow did not significantly affect the incidence of graft rejection, B-cell lymphoma, or the rate or severity of graft-versus-host disease. CONCLUSIONS: These results demonstrate that longterm rehabilitation similar to that with the other kinds of organ allografts is achievable with all three kinds of intestinal transplant procedures, that the morbidity and mortality is still too high for their widespread application, and that the liver is significantly but marginally protective of concomitantly engrafted intestine. Although none of the endpoints were markedly altered by donor leukocyte augmentation (and chimerism) with bone marrow, establishment of the safety of this adjunct procedure opens the way to further immune modulation strategies that can be added to the augmentation protocol.
Actuarial Analysis↗
Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation.
Differentiating cells undergo programmed alterations in their patterns of gene expression, which are often regulated by structural changes in chromatin. Here we demonstrate that T cell differentiation results in long-range changes in the chromatin structure of effector cytokine genes, which persist in resting Th1 and Th2 cells in the absence of further stimulation. Differentiation of naive T helper cells into mature Th2 cells is associated with chromatin remodeling of the IL-4 and IL-13 genes, whereas differentiation into Th1 cells evokes remodeling of the IFNgamma but not IL-4 or IL-13 genes. IL-4 locus remodeling is accompanied by demethylation and requires both antigen stimulation and STAT6 activation. We propose that chromatin remodeling of cytokine gene loci is functionally associated with productive T cell differentiation and may explain the coordinate regulation of Th2 cytokine genes.
Animals↗
Selective inhibition of NFAT activation by a peptide spanning the calcineurin targeting site of NFAT.
NFAT transcription factors play a key role in the immune response. The activation of NFAT proteins is controlled by calcineurin, the calmodulin-dependent phosphatase that is inhibited by the immunosuppressive drugs cyclosporin A and FK506. Here we identify a short conserved sequence in NFAT proteins that targets calcineurin to NFAT. Mutation of a single residue in this sequence impairs the calcineurin-mediated dephosphorylation and nuclear translocation of NFAT1. Peptides spanning the region inhibit the ability of calcineurin to bind to and dephosphorylate NFAT proteins, without affecting the phosphatase activity of calcineurin against other substrates. When expressed intracellularly, a corresponding peptide inhibits NFAT dephosphorylation, nuclear translocation, and NFAT-mediated expression in response to stimulation. Thus, disruption of the enzyme-substrate docking interaction that directs calcineurin to NFAT can effectively block NFAT-dependent functions.
Amino Acid Sequence↗
Sampling the universe of gene expression.
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Animals↗