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Biomedical subjects

A Rao

Publications and source records attributed to A Rao.

At least 145 records · Page 8Linked to original sources
PubMed1997

Effects of vasopressin and elimination of corticotropin-releasing hormone-target cells on pro-opiomelanocortin mRNA levels and adrenocorticotropin secretion in ovine anterior pituitary cells.

Although arginine-vasopressin (AVP) is reported to produce greater ACTH biosynthetic and secretary responses than does corticotropin-releasing hormone (CRH) in sheep anterior pituitary cells, neither factor appears to increase pro-opiomelanocortin (POMC) mRNA levels, as does CRH in the cells of some other species. Since only a fraction of cells that express POMC mRNA may be able to respond to AVP, the aim of this study was to further delineate the regulation of POMC mRNA in ovine anterior pituitary corticotrophs, as a whole and in functional subpopulations of corticotrophs. We measured the effects of AVP, CRH or activation of protein kinase C by phorbol myristate acetate (PMA) in cultured cells. We compared responses in intact populations with those of cultures from which CRH-target cells were pharmacologically eliminated. Dissociated adult ovine anterior pituitary cells were cultured overnight, treated with either vehicle (intact) or a CRH-toxin conjugate that specifically eliminates CRH-target cells (CRH-target-depleted), washed, returned to culture and subsequently challenged with vehicle, AVP (100 nM), CRH (10 nM) or PMA (1 microM) for 5 h. The media were assayed for ACTH by RIA and the cells for POMC mRNA by Northern blot analysis. In intact populations, AVP and CRH increased ACTH secretion from 6.5 +/- 1.2 to 216 +/- 22 and 81 +/- 14 ng/well respectively, but only AVP caused an increase in steady-state POMC mRNA levels (+48 +/- 10%). Direct activation of protein kinase C with PMA mimicked the effect of AVP on ACTH secretion (318 +/- 16 ng/well), but did not alter POMC mRNA levels. In CRH-target-depleted populations, control ACTH secretion (11 +/- 3 ng/well) and POMC mRNA (+69 +/- 7%) were elevated, compared with intact populations. AVP (55 +/- 8 ng/well) and PMA (120 +/- 17 ng/ well), but not CRH, increased ACTH secretion; POMC mRNA was not significantly elevated by any of the treatments. Taken together, these data provide further support for the notion of dissociation between secretion of ACTH and expression of POMC mRNA, and demonstrate that AVP increases steady-state POMC mRNA levels in ovine anterior pituitary cells. The data are also consistent with the concept that complex interactions, possibly including those between cells, influence ACTH secretion and steady-state POMC mRNA levels.

Adrenocorticotropic Hormone↗
PubMed1997

Anti-HIV agents. V. Synthesis, structure and in vitro anti-HIV activity of novel 1H,3H-naphtho[2',3':4,5]imidazo[1,2-c]thiazoles.

A series of 1H,3H-naphtho[2',3':4,5]imidazo[1,2-c]thiazoles, TBZ analogues, was prepared in order to explore the influence on anti-HIV activity of the spatial requirements of the hydrophobic moiety fused to imidazothiazole system. In spite of a butterfly-like conformation evidenced by X-ray analysis of compound 4g, the results of in vitro screening suggest that the replacement of the benzene fused ring by the naphthyl moiety negatively influences the activity of analogous thiazolobenzimidazole derivatives, some of which are potent non-nucleoside HIV-1 RT inhibitors.

Anti-HIV Agents↗
PubMed1996

Normal function of the transcription factor NFAT1 in wasted mice. Chromosome localization of NFAT1 gene.

NFAT1 (NFATp), a cytosolic component of the nuclear factor of activated T cells (NFAT), is encoded by a single gene which was mapped to mouse chromosome 2 in the vicinity of the wasted (wst) locus. Although wasted mice display a severe immune disorder, they express normal levels of NFAT1 protein. The NFAT1 protein in wasted mice is properly regulated and possesses comparable DNA binding activity as that in their littermate controls. Therefore, the wasted phenotype is not due to a defect in the expression or early regulation of the NFAT1 protein.

Animals↗
PubMed1996

Interaction of calcineurin with a domain of the transcription factor NFAT1 that controls nuclear import.

The nuclear import of the nuclear factor of activated T cells (NFAT)-family transcription factors is initiated by the protein phosphatase calcineurin. Here we identify a regulatory region of NFAT1, N terminal to the DNA-binding domain, that controls nuclear import of NFAT1. The regulatory region of NFAT1 binds directly to calcineurin, is a substrate for calcineurin in vitro, and shows regulated subcellular localization identical to that of full-length NFAT1. The corresponding region of NFATc likewise binds calcineurin, suggesting that the efficient activation of NFAT1 and NFATc by calcineurin reflects a specific targeting of the phosphatase to these proteins. The presence in other NFAT-family transcription factors of several sequence motifs from the regulatory region of NFAT1, including its probable nuclear localization sequence, indicates that a conserved protein domain may control nuclear import of all NFAT proteins.

Amino Acid Sequence↗
PubMed1996

Mechanisms of transactivation by nuclear factor of activated T cells-1.

Nuclear factor of activated T cells-family proteins (NFAT1/NFATp, NFATc, NFAT3, and NFAT4/NFATx/NFATc3) play a key role in the transcription of cytokine genes and other genes during the immune response. We have defined the mechanisms of transactivation by NFAT1. NFAT1 possesses two transactivation domains whose sequences are not conserved in the other NFAT-family proteins, and a conserved DNA-binding domain that mediates the recruitment of cooperating nuclear transcription factors even when it is expressed in the absence of other regions of the protein. The activity of the NH2-terminal transactivation domain is modulated by an adjacent regulatory region that contains several conserved sequence motifs represented only in the NFAT family. Our results emphasize the multiple levels at which NFAT-dependent transactivation is regulated, and predict significant differences in the architecture of cooperative transcription complexes containing different NFAT-family proteins.

Binding Sites↗
PubMed1996

An enhanced immune response in mice lacking the transcription factor NFAT1.

Transcription factors of the NFAT family are thought to play a major role in regulating the expression of cytokine genes and other inducible genes during the immune response. The role of NFAT1 was investigated by targeted disruption of the NFAT1 gene. Unexpectedly, cells from NFAT1 -/- mice showed increased primary responses to Leishmania major and mounted increased secondary responses to ovalbumin in vitro. In an in vivo model of allergic inflammation, the accumulation of eosinophils and levels of serum immunoglobulin E were increased in NFAT1 -/- mice. These results suggest that NFAT1 exerts a negative regulatory influence on the immune response.

Amino Acid Sequence↗
PubMed1996

Calcineurin binds the transcription factor NFAT1 and reversibly regulates its activity.

NFAT1 (previously termed NFATp) is a cytoplasmic transcription factor involved in the induction of cytokine genes. We have previously shown that the dephosphorylation of NFAT1, accompanied by its nuclear translocation and increased DNA binding activity, is regulated by calcium- and calcineurin-dependent mechanisms, as each of these hallmarks of NFAT1 activation is elicited by ionomycin and blocked by the immunosuppressive drugs cyclosporin A and FK506 (Shaw, K.T.-Y., Ho, A.M., Raghavan, A., Kim, J., Jain, J., Park, J., Sharma, S., Rao, A., and Hogan, P.G. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11205-11209). Here we show that the activation state of NFAT1 in T cells is remarkably sensitive to the level of calcineurin activity. Addition of cyclosporin A, even in the presence of ongoing ionomycin stimulation, results in rephosphorylation of NFAT1, its reappearance in the cytoplasm, and a return of its DNA binding activity to low levels. Similar effects are observed upon removal of ionomycin or addition of EGTA. We also demonstrate a direct interaction between calcineurin and NFAT1 that is consistent with a direct enzyme-substrate relation between these two proteins and that may underlie the sensitivity of NFAT1 activation to the level of calcineurin activity. The NFAT1-calcineurin interaction, which involves an N-terminal region of NFAT1 conserved in other NFAT family proteins, may provide a target for the design of novel immunosuppressive drugs.

Amino Acid Sequence↗
PubMed1996

Molecular variants of epidermal growth factor in malignant astrocytoma.

The present study has measured EGF levels in primary brain tumor tissues. EGF levels were measured by specific radioimmunoassay (RIA) and further analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) followed by RIA and radioreceptor binding. The levels of EGF-like immunoreactivity (EGF-LI) in astrocytoma-IV tumors were fourfold greater than those in normal brain tissues. In astrocytoma-II and astrocytoma-III tumors, however, levels of EGF-LI were not different from those in normal brain. HPLC analysis of extracts from normal brain tissue and astrocytoma-II showed one peak of EGF-LI that coeluted with standard human EGF (retention time 22 min). Interestingly, EGF-LI in extracts of astrocytoma-IV tumors eluted in two distinct peaks with retention times of 24 and 26 min (Astro-A and Astro-B, respectively). Materials in both Astro-A and Astro-B peaks reduced the specific binding of [125I]hEGF to EGF receptors in human placental membranes. These studies demonstrate elevated levels of EGF-LI in malignant astrocytoma, but not in benign tumors. Furthermore, two different EGF-like molecules that are different from native EGF are present in malignant astrocytoma.

Adolescent↗
PubMed1996

Immunophenotypic characterization of tumor infiltrating lymphocytes and peripheral blood lymphocytes isolated from melanomatous and non-melanomatous Sinclair miniature swine.

Selectively-bred Sinclair miniature swine exhibit a high incidence of congenital malignant melanoma which either proves fatal (10-15% of tumor-bearing piglets) or spontaneously regresses with a biphasic immunological phenomenon (85-90%) and no recurrence of malignancy. Mononuclear leukocytes were isolated from cutaneous melanomas and peripheral blood specimens collected from melanomatous (tumor-bearing) Sinclair swine during second-phase regression, and from peripheral blood specimens collected from non-melanomatous (tumor-free) Sinclair swine and control Hanford swine. Leukocyte identities were determined with single- and dual-parameter indirect immunofluorescence assays via flow cytometry. Assays for the specific surface antigens CD45, CD2, CD4, CD8, CD1, MHC class II, and N1 were employed to develop immunophenotypic profiles within the gated lymphocyte clusters from each TIL and PBL suspension. Significantly more CD8+ T-lymphocytes were identified in TIL suspensions than in peripheral blood leukocyte (PBL) suspensions (P < and = 0.05), regardless of breed or tumor status. Conversely, PBL suspensions contained significantly higher percentages of CD4+ T-lymphocytes than the levels found in TIL suspensions (P < and = 0.05). Virtually all TIL were MHC class II+, whereas the percentages of PBL expressing this antigen were markedly lower (P < and = 0.05). The percentages of T-lymphocytes co-expressing CD4 and CD8, a normal subset unique to swine, were generally consistent in all TIL and PBL suspensions examined. The results of this study have firmly established the immunophenotypic identities of cells associated with the second-phase regression phenomenon of this melanoma and have identified specific variations in the leukocyte profiles of the respective TIL and PBL suspensions.

Animals↗
PubMed1996

Some practical biological phantoms for calibrating multifrequency electrical impedance tomography.

Three groups of materials have been assessed with a Hewlett-Packard 4284A impedance analyser and Sheffield Mark 1 electrical impedance tomography (EIT) system for suitability for calibration of multifrequency EIT systems. They were required to be easy to use, stable over several hours, and have complex impedance similar to biological tissue. The groups were: (i) inorganic materials including barium titanate, polystyrene microspheres and fumed silica, all in aqueous suspension; these had phase angles below 1 degrees and so were unsuitable. (ii) Cucumber in KCl solution. Cucumber cortex had a phase angle of 40 degrees at a centre frequency of 50 kHz. Contrast between the cucumber and bathing solution could be selected by varying the KCl concentration. (iii) Polyurethane sponge immersed in packed red cells. The phase angle of packed cells was about 25 degrees at 1 MHz. Sponge resistivities and permittivities when immersed in packed cells were 5-20% higher than the bathing solution itself, for densities of 2-6.2% w/v. Both the biological materials appear suitable for the intended purpose; system (iii) is inherently more stable, and has capacitance in both bathing medium and test object. If an initial accuracy of greater than about +/- 15% is required, direct measurement with an impedance analyser is recommended prior to imaging studies.

Electric Impedance↗
PubMed1996

Imaging of physiologically evoked responses by electrical impedance tomography with cortical electrodes in the anaesthetized rabbit.

The purpose of this study was to determine if electrical impedance tomography (EIT) could be used to image impedance changes of several per cent over tens of seconds, known to occur during evoked activity of the cerebral cortex. A ring of 16 electrodes was placed on the exposed superior surface of the brain of anaesthetized rabbits. EIT images were acquired every 15 s using a Sheffield Mark 1 EIT system. During periods of 2.5-3 min of intense photic stimulation of both eyes or electrical stimulation of a forepaw, reproducible impedance decreases of 4.5 +/- 2.7% and 2.7 +/- 2.4% (mean +/- SD) respectively occurred in appropriate cortical areas, with a time course similar to the period of stimulation. They were accompanied by adjacent smaller impedance increases. The decreases are probably due to increased blood flow and temperature; the cause of the adjacent increases may be a shadowing artefact of the reconstruction algorithm or due to physiological shrinkage of the extracellular space. This demonstrated, for the first time, that such small changes may be imaged under optimal conditions. These results are encouraging to the prospect that EIT may eventually be suitable for imaging evoked responses or epilepsy in human subjects.

Anesthesia↗
PubMed1996

Direct actions of the luteinizing hormone-releasing hormone agonist, deslorelin, on anterior pituitary contents of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), LH and FSH subunit messenger ribonucleic acid, and plasma concentrations of LH and FSH in castrated male cattle.

The objective in this study was to characterize direct effects of the LHRH agonist, deslorelin, on anterior pituitary gland function in male cattle in the absence of gonadal feedback. Castrated bulls (steers), 30 mo old, were allocated to four groups: group 1, control, no treatment (n = 8); group 2, five deslorelin implants (approximately 250 micrograms total deslorelin/day) for 42 days (n = 8); group 3, control+ LHRH (50 micrograms i.m.) at weekly intervals (n = 3); group 4, five deslorelin implants+LHRH as for group 3 (n = 3). Plasma LH was similar (p > 0.05) for steers in groups 1 and 2 on Day 0 and lower (p < 0.05) for steers in group 2 on Day 4, and continued to decrease to Day 41 (group 1, 1.71 +/- 0.20 ng/ml [mean +/- SEM]; group 2, 0.38 +/- 0.03 ng/ml [p < 0.001]). Mean plasma concentrations of FSH were similar (p > 0.05) for steers in groups 1 and 2 on Day 0 and lower (p < 0.05) for steers in group 2 on Day 7, and declined to Day 41 (group 1, 43.5 +/- 3.9 ng/ml; group 2, 17.5 +/- 1.5 ng/ml [p < 0.001]). Steers in group 3 showed increases in plasma LH after injection of LHRH on all occasions, while steers in group 4 did not show increases in plasma LH from Day 14 onward. Mean relative pituitary contents (arbitrary units) of LH beta- and FSH beta-subunit mRNAs were reduced on Day 42 in steers treated with deslorelin (LH beta: groups 1 and 3, 1.56 +/- 0.27; groups 2 and 4, 0.08 +/- 0.01 [p < 0.001]; FSH beta: groups 1 and 3, 1.01 +/- 0.08; groups 2 and 4, 0.34 +/- 0.07 [p < 0.001]). However, alpha-subunit mRNA was similar for control steers and steers treated with deslorelin (groups 1 and 3, 1.00 +/- 0.11; groups 2 and 4, 0.86 +/- 0.12 [p > 0.1]). Pituitary content of LH, but not FSH, was reduced in steers treated with deslorelin. In summary, steers treated with deslorelin showed desensitization to natural LHRH, and this was associated with reduced pituitary contents of LH and FSH beta-subunit mRNAs, a reduction in pituitary content of LH, and decreases in plasma concentrations of LH and FSH. This demonstrated, for the first time, a direct action of LHRH agonist on LH and FSH beta-subunit gene expression in cattle, independent of gonadal feedback. Also, there was a differential effect of treatment with deslorelin on gonadotropin alpha- and beta-subunit mRNA contents in the anterior pituitary.

Animals↗
PubMed1996

Tumor necrosis factor alpha gene regulation in activated T cells involves ATF-2/Jun and NFATp.

The human tumor necrosis factor alpha (TNF-alpha) gene is one of the earliest genes expressed upon the activation of a T or B cell through its antigen receptor. Previous experiments have demonstrated that in stimulated T cells, a TNF-alpha promoter element, kappa 3, which binds NFATp, is required for the cyclosporin A-sensitive transcriptional activation of the gene. Here, we demonstrate that a cyclic AMP response element (CRE), which lies immediately upstream of the kappa 3 site, is also required for induction of TNF-alpha gene transcription in T cells stimulated by calcium ionophore or T-cell receptor ligands. The CRE binds ATF-2 and Jun proteins in association with NFATp bound to kappa 3. These proteins bind noncooperatively in vitro; however, the transcriptional activity of the CRE/kappa 3 composite site is dramatically higher than the activity of the kappa 3 site alone, indicating that the two sites cooperate in vivo. This study is the first demonstration of a role for ATF-2 in TNF-alpha gene transcription and of a functional interaction between ATF-2/Jun and NFATp. This novel pairing of NFATp with ATF-2/Jun may account for the specific and immediate pattern of TNF-alpha gene transcription in stimulated T cells.

Activating Transcription Factor 2↗
PubMed1996

T-cell receptor stimulation elicits an early phase of activation and a later phase of deactivation of the transcription factor NFAT1.

We show here that NFAT1 is rapidly activated, then slowly deactivated, by stimulation of T cells through their antigen receptor. Within minutes of T-cell receptor stimulation, NFAT1 is dephosphorylated, translocates from the cytoplasm into the nucleus, and shows an increase in its ability to bind to DNA. These changes are dependent on calcium mobilization and calcineurin activation, since they are also elicited by ionomycin and are blocked by the immunosuppressive drug cyclosporin A. After several hours of T-cell receptor stimulation, the majority of the NFAT1 in the cell reverts to its original phosphorylated form, reappears in the cytoplasm, and again displays a low affinity for DNA. Deactivation of NFAT1 is facilitated by phorbol 12-myristate 13-acetate and inhibitors of capacitative calcium entry and most likely reflects the slow return of intracellular free calcium concentrations towards resting levels. Our results suggest that calcineurin-dependent signalling pathways mediate the early activation of NFAT1, while phorbol 12-myristate 13-acetate-dependent feedback pathways contribute to the late deactivation. Persistent NFAT-dependent cytokine gene transcription in activated T cells may be mediated by other NFAT family proteins in addition to NFAT1 during the immune response.

Animals↗
PubMed1996

Recombinant NFAT1 (NFATp) is regulated by calcineurin in T cells and mediates transcription of several cytokine genes.

Transcription factors of the NFAT family play a key role in the transcription of cytokine genes and other genes during the immune response. We have identified two new isoforms of the transcription factor NFAT1 (previously termed NFATp) that are the predominant isoforms expressed in murine and human T cells. When expressed in Jurkat T cells, recombinant NFAT1 is regulated, as expected, by the calmodulin-dependent phosphatase calcineurin, and its function is inhibited by the immunosuppressive agent cyclosporin A (CsA). Transactivation by recombinant NFAT1 in Jurkat T cells requires dual stimulation with ionomycin and phorbol 12-myristate 13-acetate; this activity is potentiated by coexpression of constitutively active calcineurin and is inhibited by CsA. Immunocytochemical analysis indicates that recombinant NFAT1 localizes in the cytoplasm of transiently transfected T cells and translocates into the nucleus in a CsA-sensitive manner following ionomycin stimulation. When expressed in COS cells, however, NFAT1 is capable of transactivation, but it is not regulated correctly: its subcellular localization and transcriptional function are not affected by stimulation of the COS cells with ionomycin and phorbol 12-myristate 13-acetate. Recombinant NFAT1 can mediate transcription of the interleukin-2, interleukin-4, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor promoters in T cells, suggesting that NFAT1 contributes to the CsA-sensitive transcription of these genes during the immune response.

Amino Acid Sequence↗
PubMed1996

Synthesis and antitumor activity evaluation of Delta2-1,2,4-oxadiazoline derivatives.

4-Adamantyl-5-aryl-3-phenyl-Delta2-1,2,4-oxadiazolines were synthesized and evaluated in vitro as antitumor agents against 60 human tumor cell lines. Some derivatives exhibit both cell selectivity and tumor growth inhibition activity. In particular, 2,6-dichlorophenyl derivative (2q), the most active of the series, possesses significant activity on 26 cell lines at 10(-5) M.

Antineoplastic Agents↗