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Biomedical subjects

A Rao

Publications and source records attributed to A Rao.

At least 73 records · Page 4Linked to original sources
PubMed2000

Impaired NFAT regulation and its role in a severe combined immunodeficiency.

Severe Combined Immunodeficiency (SCID) is a primary immunodeficiency affecting T cells, B cells, or both. Whereas the clinical symptoms are uniformly dominated by recurrent infections, the molecular causes for SCID are very heterogeneous. Mutations in cell surface receptors, signal transduction molecules and transcription factors have been described, including the common gamma chain of the IL-2 (and IL-4, IL-7, IL-9 and IL-15) receptors, the kinase JAK-3, the epsilon and gamma chains of CD3, the protein tyrosine kinase ZAP-70, as well as CIITA and RFX5 involved in MHC class II gene expression. In this work we describe two infants with SCID whose T cells display a severe defect in T cell activation and cytokine transcription due to impaired activation of the transcription factor NFAT. We show that this defect in activation is not due to mutations in the NFAT proteins expressed in T cells or the phosphatase calcineurin which regulates the activation of NFAT. However, nuclear import of NFAT in response to T cell activation was severely compromised in the patients' T cells. A modest degree of nuclear translocation of NFAT was achieved in the patients' T cells when nuclear export was inhibited using lithium chloride. This low level of nuclear NFAT in the nucleus was not sufficient to compensate for the defect in cytokine production in the patients' T cells. However, elevated levels of extracellular calcium led to an increase in cytokine gene transcription by the SCID T cells, suggesting that the underlying genetic defect in the patients involved calcium influx or the initiation of calcium signalling.

Calcium↗
PubMed2000

T cell differentiation: a mechanistic view.

It has recently become clear that cytokine expression by T cells involves epigenetic changes in chromatin structure, locus accessibility and DNA methylation that occur during differentiation of naive T cells into mature effector T cells. These changes require the coordinate actions of antigen- and cytokine-induced transcription factors, chromatin remodeling complexes, histone-modifying enzymes and subset-specific transcription factors.

Animals↗
PubMed2000

Cell-type-restricted binding of the transcription factor NFAT to a distal IL-4 enhancer in vivo.

By DNase I hypersensitivity analysis, we have identified an inducible, cyclosporin A-sensitive enhancer located 3' of the interleukin-4 (IL-4) gene. The enhancer binds the Th2-specific transcription factor GATA3 in vivo but is not perceptibly influenced by the absence of a second Th2-specific factor, cMaf. The antigen-inducible transcription factor NFAT1 binds the IL-4 enhancer and the IL-4 promoter only in stimulated Th2 cells; conversely, NFAT1 binds to the interferon (IFN)-gamma promoter only in stimulated Th1 cells. Our results support a model whereby transcription factors such as NFAT1, which are nonselectively induced in antigen-stimulated T cells, gain access to cytokine regulatory regions only in the appropriate subset of differentiated T cells in vivo. This restricted access enables antigen-dependent and subset-specific transcription of cytokine genes.

3' Untranslated Regions↗
PubMed2000

Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity.

NFAT transcription factors are highly phosphorylated proteins that are regulated by the calcium-dependent phosphatase calcineurin. We show by mass spectrometry that NFAT1 is phosphorylated on fourteen conserved phosphoserine residues in its regulatory domain, thirteen of which are dephosphorylated upon stimulation. Dephosphorylation of all thirteen residues is required to mask a nuclear export signal (NES), cause full exposure of a nuclear localization signal (NLS), and promote transcriptional activity. An inducible phosphorylation site in the transactivation domain contributes to transcriptional activity. Our data suggest that dephosphorylation promotes NFAT1 activation by increasing the probability of an active conformation, in a manner analogous to that by which depolarization increases the open probability of voltage-gated ion channels. This conformational switch paradigm may explain modification-induced functional changes in other heavily phosphorylated proteins.

Animals↗
PubMed2000

Seasonal changes in the expression of neuropeptide Y and pro-opiomelanocortin mRNA in the arcuate nucleus of the ovariectomized ewe: relationship to the seasonal appetite and breeding cycles.

Sheep experience well-documented seasonal changes in reproductive activity and voluntary food intake (VFI). Within the hypothalamus, neurones that express neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) have been implicated in the regulation of reproduction and appetite. In this study, we aimed to determine the extent to which the expression of these two neuronal systems is linked to the seasonal reproductive cycle and/or the seasonal appetite cycle. VFI in our sheep reaches a nadir in August with no difference occurring between December and February. We examined the brains of ovariectomized (OVX) female sheep (n=5-7) that were killed during the breeding season (February) or during the early or late nonbreeding season (August and December, respectively). The brains of these animals were perfused with paraformaldehyde and processed for in situ hybridization histochemistry, using ribonucleotide probes labelled with 35S. The number of NPY and POMC cells and the number of silver grains per cell were counted using an image analysis system. For NPY, the number of cells counted in the arcuate nucleus/median eminence region and the number of silver grains per cell was significantly lower in animals killed during August than in animals killed in February or December. The number of grains per cell over NPY cells was also significantly lower in animals killed during August. For POMC, the number of cells was lower in February than in August and December. Similarly, the number of grains per cell for POMC were lower in February than in August and December. VFI was significantly lower in animals during August than at other times of the year. We conclude that in OVX ewes: (i) NPY gene expression is lower at the time of the year when VFI is reduced and (ii) POMC gene expression is greater at the time of the nonbreeding season than during the breeding season. Because these results were obtained in OVX animals, the changes appear to be independent of alterations in the secretion and/or action of ovarian steroids. Thus, the activity of NPY neurones appears to relate to changes in appetite whereas changes in POMC expression may be relevant to the seasonal breeding cycle.

Animals↗
PubMed2000

Uninterrupted moderately accelerated radiotherapy in the treatment of unresectable/advanced head and neck cancer: one institution's experience and a comparative review.

Conventional radiotherapy alone in treatment of unresectable or locally advanced head and neck cancer has poor results. To improve outcome without significant increase in acute and late morbidity, we began a moderately accelerated hyperfractionation radiation therapy protocol without breaks for treatment of unresectable/advanced head and neck malignancies. From August 1984 to June 1995, 48 patients with unresectable or advanced carcinoma of the head and neck were treated using a protocol of accelerated hyperfractionation radiation therapy at Kaiser Permanente Medical Center, Los Angeles. Patients were treated twice a day using 150 cGy per fraction, 4 days per week, to a final dose of 60 Gy. Two patients were excluded from this analysis because they did not complete treatment. With a median follow-up of 33 months, 31 (67%) patients have had disease recurrence, 30 (65%) of whom had a locoregional component to their failures. At the last follow-up, 12 patients (26%) were alive with no evidence of disease, 30 patients had died of disease, and 4 had died of intercurrent disease without recurrence. Nine (19%) patients required treatment interruptions averaging 8 days in duration. This accelerated regimen resulted in outcomes similar to those with conventional radiotherapy, most likely because of a conservative total dose. Further refinement of fractionation schedules with potential incorporation of chemotherapy must be investigated.

Adult↗
PubMed2000

Long-term alterations in adiposity affect the expression of melanin-concentrating hormone and enkephalin but not proopiomelanocortin in the hypothalamus of ovariectomized ewes.

We have developed a ruminant model to study long-term alterations in adiposity on the expression of appetite-regulating peptides in the hypothalamus. In this model endocrine and metabolic status are fully defined as well as body composition. The current study sought to define the effects of altered adiposity on the expression of genes for neuropeptide Y (NPY), POMC, enkephalin (ENK), and melanin-concentrating hormone (MCH). Ovariectomized ewes with high (60 +/- 1 kg) (FAT) or low (37 +/- 3 kg) body weights (THIN) were blood sampled every 10 min for 8 h to determine metabolic and endocrine status. The animals were then killed and the brains perfused for in situ hybridization. Body composition analysis was performed on the carcass using dual energy x-ray absorptiometry; this indicated that the FAT animals were 36 +/- 1% fat, whereas the THIN animals were 15 +/- 2% fat. The LH interpulse interval was lower and mean GH concentrations were higher in the THIN animals; cortisol and TSH levels were not different between the two groups but free T4 and free T3 levels were lower; the FT3:FT4 ratio was higher in THIN ewes. Levels of insulin, lactate, and nonesterified fatty acids were lower in the THIN group, and plasma glucose and urea concentrations were similar in THIN and FAT animals. Levels of gene expression of NPY and MCH were higher in THIN ewes. POMC expression was similar in the two groups. In the THIN animals, ENK expression was lower in the paraventricular and ventromedial nuclei but higher in the periventricular region. In conclusion, we have shown that alterations in adiposity influence the expression of appetite-regulating peptides in the absence of ovarian steroids. The appetite stimulators, NPY and MCH, appear to be involved in the metabolic response to altered adiposity, whereas ENK in the periventricular region may be linked to the secretion of GH and possibly LH. Our results suggest that altered expression of appetite- regulating peptides can be linked with the endocrine and metabolic adaptations that occur with long-term changes in adiposity.

Adipose Tissue↗
PubMed2000

Molecular aspects of T-cell differentiation.

Differentiated T helper 1 (Th1) and T helper 2 (Th2) T-cells show striking differences in their patterns of cytokine expression. This process is initiated by stimulation with antigen and the cytokines IL-12 and IL-4, respectively, and requires antigen-induced transcription factors such as NFAT and cytokine-induced transcription factors such as STAT4, induced by IL-12, and STAT6, induced by IL-4. This results in induction and maintained expression of subset-specific transcription factors including T-bet in Th1 cells and GATA3 in Th2 cells, which are involved in ensuring the commitment of T-cells to Th1 or Th2 lineages. Here we review the signalling pathways and transcription factors that mediate T-cell differentiation, and describe the epigenetic changes in chromatin structure, locus accessibility and DNA methylation that are known to accompany this process.

Bacterial Infections↗
PubMed1999

Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A.

The flow of information from calcium-mobilizing receptors to nuclear factor of activated T cells (NFAT)-dependent genes is critically dependent on interaction between the phosphatase calcineurin and the transcription factor NFAT. A high-affinity calcineurin-binding peptide was selected from combinatorial peptide libraries based on the calcineurin docking motif of NFAT. This peptide potently inhibited NFAT activation and NFAT-dependent expression of endogenous cytokine genes in T cells, without affecting the expression of other cytokines that require calcineurin but not NFAT. Substitution of the optimized peptide sequence into the natural calcineurin docking site increased the calcineurin responsiveness of NFAT. Compounds that interfere selectively with the calcineurin-NFAT interaction without affecting calcineurin phosphatase activity may be useful as therapeutic agents that are less toxic than current drugs.

Amino Acid Sequence↗
PubMed1999

NFAT5, a constitutively nuclear NFAT protein that does not cooperate with Fos and Jun.

NFAT transcription factors are related to NF-kappaB/Rel proteins and form cooperative complexes with Fos and Jun on DNA. We have identified an NFAT-related protein, NFAT5, which differs from the conventional NFAT proteins NFAT1-4 in its structure, DNA binding, and regulation. NFAT5 contains a NFAT-like Rel homology domain, conserves the DNA contact residues of NFAT1-4, and binds DNA sequences similar to those found in the regulatory regions of well-characterized NFAT-dependent genes. However, it lacks the majority of Fos/Jun contact residues and does not bind cooperatively with Fos and Jun to DNA. Unlike NFAT1-4, whose nuclear import is tightly regulated by calcineurin-mediated dephosphorylation, NFAT5 is a constitutively nuclear phosphoprotein regardless of calcineurin activation. These features suggest that unlike the conventional NFAT proteins, NFAT1-4, which activate gene transcription by integrating inputs from calcium/calcineurin and protein kinase C/mitogen-activated protein kinase signaling pathways, NFAT5 participates in as-yet-unidentified signaling pathways in diverse immune and nonimmune cells.

Amino Acid Sequence↗