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A Rao

Publications and source records attributed to A Rao.

At least 163 records · Page 9Linked to original sources
PubMed1995

Immunosuppressive drugs prevent a rapid dephosphorylation of transcription factor NFAT1 in stimulated immune cells.

The immunosuppressive drugs cyclosporin A and FK506 interfere with the inducible transcription of cytokine genes in T cells and in other immune cells, in part by preventing the activation of NF-AT (nuclear factor of activated T cells). We show that transcription factor NFAT1 in T cells is rapidly dephosphorylated on stimulation, that dephosphorylation occurs before translocation of NFAT1 into the cell nucleus, and that dephosphorylation increases the affinity of NFAT1 for its specific sites in DNA. Cyclosporin A prevents the dephosphorylation and the nuclear translocation of NFAT1 in T cells, B cells, macrophages, and mast cells, delineating at least one mechanism that contributes to the profound immunosuppressive effects of this compound.

Animals↗
PubMed1995

An ion pair in class II major histocompatibility complex heterodimers critical for surface expression and peptide presentation.

In this report we demonstrate that the ion pair Arg-80 alpha and Asp-57 beta, located in the peptide-binding site of nearly all class II major histocompatibility complex (MHC) proteins, is important for surface expression and function of the murine class II heterodimer I-Ad. Charge reversal at either of these two residues by site-directed mutagenesis generated mutant class II molecules that failed to appear at the cell surface. This defect in surface expression was partially reversed when the invariant chain was present or when the mutants were paired with the corresponding charge-reversed variant of the opposite chain. Surprisingly, surface expression was restored when cells expressing the single-site mutants were cultured at reduced temperature. In addition, the substitution of Asp-57 beta with residues found in alleles of class II molecules associated with diabetes resulted in heterodimers that were inefficiently expressed at the cell surface and presented foreign peptide poorly. Together, these results demonstrate that the formation of a salt-bridge between Arg-80 alpha and Asp-57 beta is required for efficient surface expression of class II MHC molecules, therefore representing an important step in the assembly and transport of functional class II heterodimers to the cell surface.

Animals↗
PubMed1995

Phosphorylation of the transcription factor NFATp inhibits its DNA binding activity in cyclosporin A-treated human B and T cells.

Cyclosporin A (CsA) exerts its immunosuppressive effect by inhibiting the activity of nuclear factor of activated T cells (NFAT), thus preventing transcriptional induction of several cytokine genes. This effect is thought to be largely mediated through inactivation of the phosphatase calcineurin, which in turn inhibits translocation of an NFAT component to the nucleus. Here we report that CsA treatment of Raji B and Jurkat T cell lines yields a phosphorylated form of NFATp that is inhibited in DNA-binding and in its ability to form an NFAT complex with Fos and Jun. Immunoblot analyses and metabolic labeling with [32P]orthophosphate show that CsA alters NFATp migration on SDS-polyacrylamide gel electrophoresis by increasing its phosphorylation level without affecting subcellular distribution. Dephosphorylation by in vitro treatment with calcineurin or alkaline phosphatase restores NFATp DNA binding activity and its ability to reconstitute an NFAT complex with Fos and Jun proteins. These data point to a new mechanism for CsA-sensitive regulation of NFATp in which dephosphorylation is critical for DNA binding.

Alkaline Phosphatase↗
PubMed1995

Activation and expression of the nuclear factors of activated T cells, NFATp and NFATc, in human natural killer cells: regulation upon CD16 ligand binding.

The putative factors that couple the signal transduction from surface receptors to the activation of cytokine synthesis in natural killer (NK) cells have not been elucidated. We report here that the nuclear factor of activated T cells (NFATp), a cyclosporin A (CsA)-sensitive factor that regulates the transcription of several cytokines, mediates CD16-induced activation of cytokine genes in human NK cells. CD16 (Fc gamma RIIIA)-induced expression of cytokine mRNA in NK cells occurs via a CsA-sensitive and Ca(2+)-dependent mechanism. Stimulation of NK cells with CD16 ligands induces NFAT-like DNA binding activity in the nuclear extracts from these cells, as detected in electrophoretic mobility shift assays. This occurs with fast kinetics after stimulation, via a CsA-sensitive and Ca(2+)-dependent mechanism that does not require de novo protein synthesis. NK cell NFAT is present in the cytosol of nonstimulated cells, migrates to the nucleus upon stimulation, and can associate with AP-1. Two distinct molecules, NFATp and NFATc, have been reported to mediate NFAT activity. The results of supershift assays using NFATp- and NFATc- specific antibodies indicate that NK cell activation early after CD16 ligand binding involves primarily, if not exclusively, NFATp, and Western blot analysis shows that this has the same electrophoretic mobility (approximately 120 kD) as that of T lymphocytes. NK cells do not express NFATc constitutively, but NFATc mRNA accumulation is induced in these cells within 2 h of stimulation with CD16 ligands. However, supershift assays using the available mAb recognizing the T cell NFATc revealed no detectable NFATc protein in nuclear and cytoplasmic extracts from CD16- or phorbol ester-stimulated cells at any time tested, up to 4 h. These results provide the first direct evidence that both CsA-sensitive transcription factors, NFATp and NFATc, are expressed in human NK cells, and that their activation and/or expression can be regulated in primary cells by a single stimulus, that, in the case of CD16 in NK cells, results in early activation of NFATp and subsequently induced expression of NFATc mRNA.

Animals↗
PubMed1995

Only one of the two DNA-bound orientations of AP-1 found in solution cooperates with NFATp.

BACKGROUND: The transcription factor AP-1 activates the expression of numerous genes in response to mitogenic stimuli. AP-1 regulates gene expression both through solitary binding to independent recognition sites and, in cooperation with various heterologous transcription factors, through targeting to composite response elements. The two subunits that make up the AP-1 heterodimer, Fos and Jun, possess identical residues at positions that make sequence-specific contacts to DNA. This degeneracy leaves the protein with no apparent way of orienting itself uniquely on DNA by differentially recognizing its two non-identical half-sites. Here, we have analyzed the orientation of the AP-1 basic-leucine-zipper (bZip) domain on a cognate site, both alone and in the cooperative complex formed together with the 'nuclear factor of activated T cells' (NFATp). RESULTS: The results of affinity cleaving experiments demonstrate that, in solution, the AP-1 bZip binds DNA as a mixture of two orientational isomers. However, in the cooperative complex formed with NFATp on a composite response element, the AP-1 bZip adopts a single orientation, with Jun and Fos bound to the NFATp-proximal and NFATp-distal half-sites, respectively. Protein cross-linking experiments demonstrate that protein-protein contacts are responsible for this 'orientational locking'. CONCLUSIONS: Our results demonstrate that, through protein-protein interactions, one protein can force another to adopt a single DNA-bound orientation. Thus, cooperative interactions between adjacent regulatory proteins can influence not only the energetics of their interactions with DNA, but also their precise geometric and stereochemical arrangement. Because orientational isomers present markedly different structures to the transcriptional apparatus, it seems likely that orientation will exert an effect on the ability to activate transcription.

Amino Acid Sequence↗
PubMed1995

Effects of Celastrus paniculatus on passive avoidance performance and biogenic amine turnover in albino rats.

The effects of an indigenous drug, Celastrus oil, extracted from the seeds of Celastrus paniculatus on learning and memory in a two compartment passive avoidance task was studied in albino rats. The effects on the contents of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in the brain and on the levels of their metabolites both in the brain and urine were also assessed. Significant improvement was observed in the retention ability of the drug treated rats compared with the saline administered controls. The contents of NE, DA and 5-HT and their metabolites in the brain were significantly decreased in the drug treated group. The urinary metabolite levels were also significantly decreased except for total 3-methoxy-4-hydroxyphenyl glycol. These data indicate that Celastrus oil causes an overall decrease in the turnover of all the three central monoamines and implicate the involvement of these aminergic systems in the learning and memory process.

Animals↗
PubMed1995

Transfection of TCR alpha-chains into suppressor and T helper cell hybridomas. Production of suppressor factors with predicted antigen specificity.

Conditioned medium from Ag-specific suppressor T cell hybridomas contains soluble factors (TsF) that modulate immune responses in an Ag-specific manner. We previously generated a series of TCR-alpha- and TCR-beta- expression variants from a 4-hydroxy-3-nitrophenyl acetyl (NP)-specific inducer suppressor T cell hybridoma and demonstrated that loss of TCR alpha-chain mRNA, but not TCR-beta chain mRNA, was accompanied by concomitant loss of suppressor bioactivity. Suppressor factor bioactivity was restored by expression of TCR alpha-chain cDNA, suggesting that the TCR alpha-chain plays a critical role in Ag-specific suppressor cell function. We have now transfected TCR alpha-chain from a Th cell clone specific for arsanylated peptides plus I-Ad into a TCR-alpha- derivative of an NP-specific inducer suppressor T cell hybridoma. The transfectants expressed a new hybrid TCR-alpha beta complex and produced soluble factors that suppressed azobenzenearsonate hapten (ABA) but not NP delayed-type hypersensitivity responses. These supernatants mediated suppression of the induction, but not the effector phase of the delayed-type hypersensitivity reaction. In reciprocal experiments we transfected a TCR alpha-chain from an NP-specific suppressor T cell hybridoma into a TCR-alpha- hybridoma derived from the ABA-specific Th cell hybridoma. The NP-specific TCR alpha-chain was expressed in the Th cell hybridoma, but the supernatant from this transfectant did not suppress DTH responses to either NP or ABA. However, the latter supernatants, when combined with cell lysates derived from a TCR-alpha- Ts hybridoma, specifically suppress NP DTH responses. These data are consistent with the interpretation that TCR alpha-chain imparts Ag specificity to the suppressor molecule and a second, yet undefined, component produced by the Ts hybridoma controls the immunoregulatory bioactivity.

Animals↗
PubMed1995

A similar DNA-binding motif in NFAT family proteins and the Rel homology region.

The cyclosporin-sensitive factor NFATp cooperates with Fos and Jun family proteins to regulate transcription of the interleukin 2 gene in activated T cells. We have defined a 187-amino-acid fragment of NFATp, located centrally within the protein sequence, as the minimal region required for DNA binding and for complex formation with Fos and Jun. The sequence of this region of NFATp shows a low degree of similarity to the Rel homology region. One specific short sequence in NFATp (RAHYETEG), located near the NH2 terminus of the DNA-binding domain, resembles a highly conserved sequence (RFRYxCEG) that is located near the NH2 terminus of the Rel homology region and that has been implicated in DNA binding by Rel family proteins. Mutational analysis demonstrates that the residues in this sequence that are identical in NFATp and Rel family proteins contribute to DNA binding by NFATp. Further, mutation of the threonine residue in this sequence to cysteine, as in Rel proteins, confers on NFATp a sensitivity to sulfhydryl modification similar to that of Rel family proteins. The results suggest that NFATp and Rel family proteins bind to DNA using similar structural motifs.

Amino Acid Sequence↗
PubMed1995

NFATp, a cyclosporin-sensitive transcription factor implicated in cytokine gene induction.

The transcription factor NFATp (nuclear factor of activated T cells, preexisting) is likely to regulate the cyclosporin-sensitive transcription of cytokine genes and other activation-associated genes during the immune response. NFATp is the first identified member of a new family of transcription factors, whose DNA binding domains show a weak sequence similarity to those of the Rel (NF-kappa B) family of proteins. NFATp is expressed in several types of immune cells as a cytosolic protein that translocates to the nucleus following activation. The nuclear translocation is regulated by calcium and calcineurin and inhibited by cyclosporin A and FK506. In the nucleus, NFATp cooperates with Fos-Jun dimers and possibly with other transcription factors at composite elements in the regulatory regions of cytokine genes. The potential roles of NFATp and a related family member, NFATc, in cytokine gene transcription are discussed.

Amino Acid Sequence↗
PubMed1995

Circulating immune complexes in intracranial neoplasms.

The levels of circulating immune complexes (CIC) were assayed in the sera of 109 patients with intracranial space occupying lesions. The CIC levels were significantly increased in all the brain tumours. After treatment, the CIC levels were still significantly increased when compared to the controls but showed no change when compared to their respective pre-operative values. Further, no change was observed in the CIC levels between the malignant and benign tumour case. Moreover, in brain tumours, 90% of the CIC precipitate consisted of IgG. However, the CIC levels fail to prognosticate the process of the disease in these patients.

Adolescent↗
PubMed1995

Anacardoside from the seeds of Semecarpus anacardium.

From the seeds of Semecarpus anacardium, a new phenolic glucoside, anacardoside, was isolated, and its structure and configuration were elucidated by a combination of NMR techniques as 1-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyloxy-3-hydro xy-5-methylbenzene.

Carbohydrate Sequence↗
PubMed1995

Transcriptional regulation of the IL-2 gene.

The past two years have seen significant advances in our understanding of IL-2 gene transcription. Many of the relevant transcription factors have been identified, the intracellular mechanisms regulating their functions are being elucidated, and the multiple roles of calcineurin are beginning to be appreciated.

Base Sequence↗
PubMed1995

Molecular identification of viruses in sudden infant death associated with myocarditis and pericarditis.

A subset of infants dying suddenly and unexpectedly have myocarditis with or without pericarditis found at autopsy. To address whether viruses known to cause infantile myocarditis and pericarditis might be present in such infants, we examined myocardium, liver and skeletal muscle for the presence of genomic sequences from adenovirus, cytomegalovirus, enterovirus and echovirus 22/23 in infants enrolled in a comprehensive evaluation protocol. We studied eight infants who died suddenly and unexpectedly with histologic evidence of myocarditis and/or pericarditis detected at postmortem examination. One infant with myocarditis and pericarditis had adenovirus genome detected in the myocardium. In an additional infant with severe pericarditis alone, enterovirus genome was detected in the liver. Although echovirus 22/23 has been associated with myopericarditis in young infants, no previous studies have used molecular methods to search for the genomic sequences of these viruses in clinical samples. No echovirus 22/23 genome was detected in the patients reported here. The significance of enterovirus and adenovirus genome in the tissues of two patients dying suddenly and unexpectedly remains speculative but raises the possibility that pathogenic viruses may cause little or no clinical symptoms and yet be contributory to sudden death in young infants.

Adenoviridae↗
PubMed1995

Prohormones in adrenal venous effluent in patients with primary hyperaldosteronism.

Aldosterone-producing adenoma (APA) and idiopathic hyperplasia (IHA) are two main causes of primary hyperaldosteronism, which differ in the modes of treatment. Some of the prohormones, such as 18-hydroxycorticosterone, are elevated in adenomas. 19-Nor-deoxycorticosterone (19-nor-DOC), produced in the kidney, has been shown to be excreted in excess in patients with APA. Deoxycorticosterone is a known precursor of aldosterone and 19-nor-DOC. This study is designed to evaluate the levels of prohormones in adrenal venous effluent in eight patients, three with APA, of which two were confirmed by surgical pathology and four with IHA, and one patient with primary adrenal hyperplasia. 19-OH-DOC, a precursor of 19-nor-DOC, was found to be the main prohormone in adrenal venous effluent in patients with both APA and IHA. 19-Oic-deoxycorticosterone and 19-nor-DOC were also detected, but in smaller quantities. 19-OH-DOC appears to be the main prohormone in adrenal venous effluent for the biosynthesis of 19-nor-DOC.

Adenoma↗
PubMed1995

The current status of hepatic transplantation at the University of Pittsburgh.

Tacrolimus is a more potent and satisfactory immunosuppressant than CyA for combination therapy with prednisone. In randomized trials comparing the 2 drugs, the ability of tacrolimus to rescue intractably rejecting grafts on the competing CyA arm allowed equalization of patient and graft survival on both arms when the intent-to-treat analytic methodology was applied. The ability of tacrolimus to systematically rescue the treatment failures of CyA suggested, as a matter of common sense, that it is the preferred baseline drug for hepatic transplantation. This conclusion was supported by analysis of secondary end points, including the ability to prevent rejection. Hepatic-intestinal, multivisceral and isolated intestinal transplantation became feasible on a practical basis only after the advent of tacrolimus. Nevertheless, better management strategies must be devised before intestinal transplantation, alone or with other abdominal viscera, will meet its potential. One such strategy is based on the discovery of the presence of previously unsuspected, low-level donor leukocyte chimerism in long-surviving allograft recipients. We believe that this chimerism is the essential explanation for the feasibility of organ transplantation and a link to the acquired neonatal tolerance demonstrated by Billingham, Brent and Medawar (32). The hematolymphopoietic chimerism in organ recipients explains why weaning to a drug-free state in selected long-term survivors is frequently feasible and particularly if the allograft is a liver. Weaning should never be attempted without a stepwise protocol and careful monitoring of graft function. Recognition of the natural chimerism that develops after whole organ transplantation has led to efforts to augment it with perioperative donor BM infusion. This procedure has been shown to be free of significant complications (including GVHD) in all kinds of whole organ recipients, including those given intestine. The prospects of clinical xenotransplantation must be evaluated in the same context of chimerism as that delineated for allotransplantation with the discovery of spontaneous chimerism. Before addressing chimerism-related questions in xenotransplantation, the additional barrier of the complement activation syndromes that cause hyperacute rejection will have to be surmounted. Although measures to effectively transplant xenografts have so far eluded us, the availability of the more potent drug, tacrolimus, and recognition of the seminal basis of allograft (or xenograft) acceptance via chimerism has inserted an element of reality into the largely wishful thinking that has been evident in discussions about the future of xenotransplantation.

Adolescent↗
PubMed1994

The role of NFATp in cyclosporin A-sensitive tumor necrosis factor-alpha gene transcription.

The tumor necrosis factor-alpha (TNF alpha) gene is an immediate early gene in activated T cells, in that it is rapidly induced without a requirement for protein synthesis. Maximal induction of TNF alpha mRNA can be induced by treatment of T cells with calcium ionophores alone, via a calcineurin-dependent process that is blocked by cyclosporin A. We have previously identified a promoter element, kappa 3, that is required for calcium-stimulated, cyclosporin A-sensitive induction of the TNF alpha gene in activated T cells. Here, we demonstrate that the kappa 3 binding factor contains NFATp, a cyclosporin-sensitive DNA-binding protein required for interleukin-2 gene transcription. NFATp binds to two sites within the kappa 3 element, and occupancy of both sites is required for TNF alpha gene induction. Thus, although the kappa 3 element has little sequence similarity to other NFATp-binding sites, it appears to function as a cyclosporin-sensitive promoter element in T cells by virtue of its ability to bind NFATp. The involvement of NFATp in transcriptional activation of both the interleukin-2 and TNF alpha genes suggests that this factor plays an important role in the coordinate induction of multiple cytokine genes, starting at the earliest stages of T cell activation.

Base Sequence↗
PubMed1994

Expression of the transcription factor NFATp in a neuronal cell line and in the murine nervous system.

The transcription factor NFATp, which has a central role in cytokine gene induction in T cells, was previously thought to be expressed only in T cells and other immune cells. Here we report that NFATp is also expressed in the rat PC12 pheochromocytoma cell line and in the murine nervous system. Antisera against T cell NFATp recognize proteins of similar apparent molecular weight in T cells, PC12 cells, and murine olfactory bulb. The immunoreactive proteins from PC12 cells and from olfactory bulb bind to an NFAT site of the murine interleukin-2 promoter with the same specificity as T cell NFATp. Immunohistochemical staining localizes NFATp to neurons, specifically to olfactory receptor neurons and to their terminals in the olfactory bulb. The presence of NFATp in the nervous system suggests that it has a role in the transcription of specific neuronal genes in response to increases in cytosolic calcium.

Adrenal Gland Neoplasms↗