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A Rao

Publications and source records attributed to A Rao.

At least 109 records · Page 6Linked to original sources
PubMed1998

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↗
PubMed1998

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↗
PubMed1998

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↗
PubMed1998

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↗
PubMed1998

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↗
PubMed1998

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↗
PubMed1998

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↗
PubMed1998

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↗
PubMed1998

Proopiomelanocortin mRNA levels in ovine hypothalamus are not reduced at the time of the preovulatory luteinising hormone surge.

A reduction in inhibition (disinhibition) of gonadotropin-releasing hormone (GnRH) secretion by endogenous opioid systems in the hypothalamus is thought to be permissive of the preovulatory surge of GnRH and luteinising hormone (LH). In rats, proopiomelanocortin (POMC) mRNA levels in the arcuate nucleus are reduced at the time of the LH surge, and this is thought to be part of the neural mechanism of oestrogen positive feedback. There are no studies of POMC mRNA levels at the time of the LH surge in other species. POMC mRNA levels were measured by in-situ hybridization using a 35S-labelled cRNA probe and computer-assisted grain counting in the arcuate nucleus of ovary-intact ewes (n=4) killed on day 10 of the luteal phase or 24 or 48 h into the follicular phase (experiment 1), and ewes killed on day 10 of the luteal phase or during the preovulatory LH surge (experiment 2). Grain counts per cell, the proportion of cellular area covered by grains and the number of labelled cells per section were not significantly different (P > 0.1) between animals killed in the luteal phase and animals killed during the follicular phase or during the LH surge. We conclude that in sheep, POMC mRNA levels are not reduced at the time of the preovulatory LH surge, and reduced POMC gene transcription does not appear to be part of the neural mechanism of oestrogen positive feedback in this species.

Animals↗
PubMed1998

Synthesis, structure and in vitro anti-human immunodeficiency virus activity of novel 3-methyl-1H,3H-thiazolo[3,4-a]benzimidazoles.

A series of novel 1-aryl-3-methyl-1H,3H-thiazolo[3,4-a]benzimidazoles, TBZ analogues, were synthesized and investigated as anti-human immunodeficiency virus (HIV) agents in order to study the effects of structural modifications on antiviral activity and cytotoxicity. They were proved to inhibit significantly HIV-1 replication in vitro without showing inhibitory activity on HIV-2 or simian immunodeficiency virus. Their potency was influenced by the presence of suitable substituents in the phenyl ring at C-1 as well as by their stereochemical characteristics. In fact, the most active compound of the series was the trans-1-(2,6-difluorophenyl)-3-methyl-1H,3H-thiazolo[3,4- a]benzimidazole, in which the butterfly-like conformation is stabilized by two intramolecular hydrogen bonds between the fluorine atoms and H-1 and H-3. This was made possible by the trans arrangement of C-1 and C-3 substituents, as shown by X-ray and NMR analysis.

Anti-HIV Agents↗
PubMed1998

Neonatal immunization against gonadotropin-releasing hormone (GnRH) results in diminished GnRH secretion in adulthood.

The effects of neonatal immunization against GnRH were studied in sheep after they had reached adulthood (3-4 yr) and the antibody titers had fallen to undetectable levels. The immunized animals had small gonads, and the females did not have large follicles (>3 mm) or corpora lutea in their ovaries. Compared with controls, the immunized animals had low or nondetectable levels of LH and FSH in peripheral plasma, and the immunized animals generally failed to respond to a single i.v. GnRH challenge. After ovariectomy, the control ewes, but not the immunized ewes, showed an elevation in plasma LH and FSH levels. The sampling of hypophysial portal blood, with a newly described method, showed that the secretion of GnRH was reduced in the immunized animals, but the amount of GnRH in the median eminence was similar in the control and immunized ewes. The pituitary content of LH and FSH was reduced in the immunized ewes as was messenger RNA for the gonadotropin subunits and the GnRH receptor. These data indicate that neonatal immunization does not affect the synthesis of GnRH in adulthood but reduces the secretion of GnRH, causing long-term sterility in these animals.

Animals↗
PubMed1997

IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation.

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit IkappaB. A large multiprotein complex, the IkappaB kinase (IKK) signalsome, was purified from HeLa cells and found to contain a cytokine-inducible IkappaB kinase activity that phosphorylates IkappaB-alpha and IkappaB-beta. Two components of the IKK signalsome, IKK-1 and IKK-2, were identified as closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 had pronounced effects on RelA nuclear translocation and NF-kappaB-dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-kappaB signaling pathway.

Cell Cycle Proteins↗
PubMed1997

A genetic animal model of human neocortical heterotopia associated with seizures.

Malformations of the human neocortex are commonly associated with developmental delays, mental retardation, and epilepsy. This study describes a novel neurologically mutant rat exhibiting a forebrain anomaly resembling the human neuronal migration disorder of double cortex. This mutant displays a telencephalic internal structural heterotopia (tish) that is inherited in an autosomal recessive manner. The bilateral heterotopia is prominent below the frontal and parietal neocortices but is rarely observed in temporal neocortex. Neurons in the heterotopia exhibit neocortical-like morphologies and send typical projections to subcortical sites; however, characteristic lamination and radial orientation are disturbed in the heterotopia. The period of neurogenesis during which cells in the heterotopia are generated is the same as in the normotopic neocortex; however, the cells in the heterotopia exhibit a "rim-to-core" neurogenetic pattern rather than the characteristic "inside-out" pattern observed in normotopic neocortex. Similar to the human syndrome of double cortex, some of the animals with the tish phenotype exhibit spontaneous recurrent electrographic and behavioral seizures. The tish rat is a unique neurological mutant that shares several features with a human cortical malformation associated with epilepsy. On the basis of its regional connectivity, histological composition, and period of neurogenesis, the heterotopic region in the tish rat is neocortical in nature. This neurological mutant represents a novel model system for investigating mechanisms of aberrant neocortical development and is likely to provide insights into the cellular and molecular events contributing to seizure development in dysplastic neocortex.

Animals↗