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At least 379 records · Page 21Linked to original sources
PubMed1999

Regulators of G protein signaling exhibit distinct patterns of gene expression and target G protein specificity in human lymphocytes.

The newly recognized regulators of G protein signaling (RGS) attenuate heterotrimeric G protein signaling pathways. We have cloned an IL-2-induced gene from human T cells, cytokine-responsive gene 1, which encodes a member of the RGS family, RGS16. The RGS16 protein binds Gialpha and Gqalpha proteins present in T cells, and inhibits Gi- and Gq-mediated signaling pathways. By comparison, the mitogen-induced RGS2 inhibits Gq but not Gi signaling. Moreover, the two RGS genes exhibit marked differences in expression patterns. The IL-2-induced expression of the RGS16 gene in T cells is suppressed by elevated cAMP, whereas the RGS2 gene shows a reciprocal pattern of regulation by these stimuli. Because the mitogen and cytokine receptors that trigger expression of RGS2 and RGS16 in T cells do not activate heterotrimeric G proteins, these RGS proteins and the G proteins that they regulate may play a heretofore unrecognized role in T cell functional responses to Ag and cytokine activation.

Cells, Cultured↗
PubMed1998

CXCR4 and CCR5 expression delineates targets for HIV-1 disruption of T cell differentiation.

HIV-1 disease is often associated with CD4+ T lymphopenia as well as quantitative reductions in naive CD8+ T cells and cytopenias involving nonlymphoid hemopoietic lineages. Studies in HIV-1-infected humans as well as in animal models of lenti-virus disease indicate that these effects may be secondary to infection and destruction of multilineage and lineage-restricted hemopoietic progenitor cells. To define the stages of T cell differentiation that might be susceptible to HIV-1, we performed flow cytometric analysis of the surface expression of CXCR4 and CCR5 on T cells and their progenitors from fetal tissue, cord blood, SCID-hu Thy/Liv mice, and adult peripheral blood. We found that CXCR4 is expressed at low levels on hemopoietic progenitors in the bone marrow, is highly expressed on immature (CD3-CD4+CD8-) T cell progenitors in the thymus, and then is down-regulated during thymocyte differentiation. As thymocytes leave the thymus and enter the peripheral circulation, the expression of CXCR4 is again up-regulated. In contrast, CCR5 is undetectable on most hemopoietic progenitors in the bone marrow and on intrathymic T progenitor cells. It is up-regulated when thymocytes coexpress CD4 and CD8, then down-regulated either in the thymus (CD4+ cells) or during exit from the thymus (CD8+ cells). These results indicate that discrete, lineage-related populations of T cell progenitors may vary widely in their potential to respond to chemokines and to be infected by HIV-1, and that T lymphoid differentiation is particularly vulnerable to CXCR4-using viruses.

Adult↗
PubMed2000

In vitro generation of regulatory CD8+ T cells similar to those found in mice with anterior chamber-associated immune deviation.

PURPOSE: When injected intravenously into naive mice, peritoneal exudate cells (PECs) incubated with ovalbumin (OVA) in the presence of transforming growth factor (TGF)-beta2 induce immune deviation similar to that evoked by injection of OVA into the anterior chamber of the eye. Intraocular antigen injection elicits two distinct populations of regulatory T cells that impair delayed hypersensitivity (DH) by two different mechanisms: a CD4+ T cell that suppresses the induction of DH (afferent) and a CD8+ T cell that inhibits DH expression. In an effort to understand the origin and mechanism of action of these regulatory cells, CD8+ T cells from OVA-specific T cell receptor (Tcr) transgenic mice (OT-1) were used. METHODS: CD8+ T cells were harvested from Tcr transgenic OT-1 mice whose Tcr recognize an OVA peptide in the context of the class I major histocompatibility complex molecule Kb. These cells were stimulated in vitro with OVA-pulsed PECs exposed (or not) to TGF-beta2, then analyzed for their capacity to proliferate, to secrete various cytokines, to lyse OVA-expressing target cells, and to regulate bystander T cells in vitro and in vivo. RESULTS: When OVA-pulsed PECs were used in vitro as stimulators, responding OT-1 T cells proliferated and preferentially secreted interferon (IFN)-gamma, interleukin (IL)-2, and tumor necrosis factor (TNF)-alpha, rather than IL-4 and IL-10. When the stimulator PECs were pretreated with TGF-beta2 and then pulsed with OVA, responding OT-1 T cells proliferated even more swiftly, but they secreted significantly less IFN-gamma, IL-2, and TNF-alpha, and no IL-4 or IL-10. OT-1 T cells, which constitutively display cytotoxicity toward OVA-expressing target cells, lost this activity when stimulated with OVA-pulsed, TGF-beta2-pretreated PECs. Moreover, OT-1 T cells stimulated in this manner displayed the capacity to inhibit proliferation of OVA-primed T cells exposed to OVA in vitro and to suppress in vivo the expression of OVA-triggered DH. CONCLUSIONS: OVA-pulsed PECs, pretreated with TGF-beta2, coerce naive OVA-specific CD8+ T cells to become efferent regulators of DH similar to the regulatory T cells evoked by intraocular injection of OVA.

Adoptive Transfer↗
PubMed2002

Pag, a putative tumor suppressor, interacts with the Myc Box II domain of c-Myc and selectively alters its biological function and target gene expression.

The highly conserved Myc Box II (MBII) domain of c-Myc is critically important for transformation and transcriptional regulation. A yeast two-hybrid screen identified Pag as a MBII-interacting protein. Pag, a member of the peroxiredoxin family, has been reported previously to bind to and inhibit the cytostatic properties of the c-Abl oncoprotein. We now show that Pag promotes increased cell size and confers a proapoptotic phenotype, two hallmark features of ectopic c-Myc overexpression. Pag and c-Myc also confer resistance to oxidative stress, a previously unrecognized property of the latter protein. In contrast, Pag inhibits tumorigenesis by c-Myc-overexpressing fibroblasts and causes a broad but selective loss of c-Myc target gene regulation. Pag is therefore an MBII-interacting protein that can either mimic or enhance some of the c-Myc properties while at the same inhibiting others. These features, along with the previously identified interaction with c-Abl, provide support for the idea that Pag functions as a tumor suppressor.

3T3 Cells↗
PubMed1998

Target-specific expression of presynaptic mossy fiber plasticity.

Mossy fiber synaptic transmission at hippocampal CA3 pyramidal cells and interneurons was compared in rat brain slices to determine whether mossy terminals are functionally equivalent. Tetanic stimulation of mossy fibers induced long-term potentiation in pyramidal neurons but was either without effect or it induced depression at synapses onto interneurons. Unlike transmission onto pyramidal neurons, transmission onto interneurons was not potentiated after adenosine 3',5'-monophosphate (cAMP) activation. Furthermore, metabotropic glutamate receptor depression of transmission onto interneurons did not involve cAMP-dependent pathways. Thus, synaptic terminals arising from a common afferent pathway do not function as a single compartment but are specialized, depending on their postsynaptic target.

Afferent Pathways↗
PubMed1995

Thermostability of a nuclear-targeted luciferase expressed in mammalian cells. Destabilizing influence of the intranuclear microenvironment.

Protein denaturation and aggregation are most likely the cause for the noxious effects of heat shock. There are some indications that the nucleus is one of the most sensitive cellular compartments. To test the possibility that the intranuclear microenvironment might be detrimental to the heat stability of proteins, we compared the in situ thermal stability of a reporter protein localized in the nucleus or in the cytoplasm. A recombinant firefly (Photynus pyralis) luciferase carrying a point mutation in the C-terminal domain remains in the cytoplasm (cyt-luciferase). A nuclear localization sequence was fused to the N-terminal domain of cyt-luciferase; the resulting nuc-luciferase was efficiently targeted to the cell nucleus. In both cases, decreased luciferase activity and solubility were found in lysates from heat-shocked cells. These characteristics were taken as an indication of thermal denaturation in situ. The heat-inactivated luciferases were partially reactivated during recovery after stress, indicating the capacity of both the cytoplasmic and nuclear compartments to reassemble proteins from an aggregated state. Although both the nuc- and the cyt-luciferases were heat inactivated at similar rates in vitro, nuc-luciferase was more susceptible to thermal denaturation in situ compared to cyt-luciferase. This observation suggests that the microenvironment of an intracellular compartment may modulate the thermal stability of proteins. The local concentration might be one element of this microenvironment affecting the heat-stability of proteins. In cells made thermotolerant by a priming shock, the thermal inactivation of the recombinant luciferases occurred at a slower rate during a second challenging stress. However, this decreased thermal sensitivity was less pronounced for the nuc-luciferase (threefold) than for the cyt-luciferase (sevenfold). The nuclear luciferase might become a useful tool to investigate the action of molecular chaperones in the nucleus.

Base Sequence↗
PubMed1997

Mutant molecular motors disrupt neural circuits in Drosophila.

A dominant negative mutation, Glued1, that codes for a component of the dynactin complex, disrupted the axonal anatomy of leg sensory neurons in Drosophila. To examine neuron structure in mutant animals, a P[Gal4] enhancer trap targeted expression of lacZ to the sensory neurons and thereby labeled neurons in the femoral chordotonal organ and their axons within the central nervous system. When these sensory axons were examined in the Glued1 mutant specimens, they were observed to arborize abnormally. This anatomical disruption of the sensory axons was associated with a corresponding disruption in a reflex. Normally, the tibial extensor motor neurons were excited when the femoral-tibial joint was flexed, but this resistance reflex was nearly absent in mutant animals. We used the P[Gal4] insertion strains to target expression of tetanus toxin light chain to these sensory neurons in wild-type animals and showed that this blocked the resistance reflex and produced a phenocopy of the Glued result. We conclude that disruption of the dynein-dynactin complex disrupts sensory axon path finding during metamorphosis, and this in turn disrupts synaptic connectivity.

Animals↗
PubMed1998

Neuronal basic helix-loop-helix proteins (NEX, neuroD, NDRF): spatiotemporal expression and targeted disruption of the NEX gene in transgenic mice.

Basic helix-loop-helix (bHLH) genes have emerged as important regulators of neuronal determination and differentiation in vertebrates. Three putative neuronal differentiation factors [NEX for neuronal helix-loop-helix protein-1 (mammalian atonal homolog-2), neuroD (beta-2), and NDRF for neuroD-related factor (neuroD2)] are highly homologous to each other in the bHLH region and comprise a new bHLH subfamily. To study the role of NEX, the first bHLH protein identified in this group, we have disrupted the NEX gene by homologous recombination. NEX-deficient mice have no obvious developmental defect, and CNS neurons appear fully differentiated. To investigate further whether the absence of NEX is compensated for by neuroD and NDRF, we compared the spatiotemporal expression of all three genes. We demonstrate, by in situ hybridization, that the transcription patterns of NEX, neuroD, and NDRF genes are highly overlapping in the developing CNS of normal rats between embryonic day 12 and adult stages but are not strictly identical. The most prominent transcription of each gene marks the dorsal neuroepithelium of the telencephalon in early development and is sustained in the adult neocortex, hippocampus, and cerebellum. In general, neuroD provides the earliest marker of neuronal differentiation in any given region compared with NDRF or NEX. Whereas a few CNS regions are specific for neuroD, no region was detected in which solely NEX or NDRF is expressed. This suggests that the function of the mutant NEX gene in neuronal differentiation is compensated for by neuroD and NDRF and that, in analogy with myogenic bHLH proteins, neuronal differentiation factors are at least in part equivalent in function.

Aging↗
PubMed1998

Insertion specificity and trans-activation of IS801.

The transposable element IS801, isolated from plasmid pMMC7105 of Pseudomonas syringae pv. phaseolicola, transposes in Escherichia coli to plasmid targets, expressing a relatively relaxed target specificity. The target sequences are tetramers with homology with the left terminus (GAAC) of the transposing unit, the alternative targets being GAAC, GGAC, CAAG, and CGAC. In the areas flanking IS801 in 13 different locations, no similarities other than the target tetramer were observed. The transposase is physically and functionally separable from the transposing unit since transposition of constructs carrying marker genes occurs with the transposase expressed in trans. The IS801 transposase shows amino acid sequence homology to the transposases of the E. coli elements IS91 and IS1294. These transposases contain conserved amino acid motifs found in the replicases of certain plasmids that replicate as rolling circles.

Base Sequence↗
PubMed2000

The AF-1 and AF-2 activating domains of retinoic acid receptor-alpha (RARalpha) and their phosphorylation are differentially involved in parietal endodermal differentiation of F9 cells and retinoid-induced expression of target genes.

Retinoic acid (RA) induces the differentiation of F9 cells cultured as monolayers into primitive endodermal-like cells, whereas a combination of RA and cAMP leads to parietal endodermal differentiation. In RA receptor alpha-null F9 cells (RARalpha-/- cells), RA still efficiently triggers RARgamma-mediated primitive endodermal differentiation, but parietal endodermal differentiation is markedly delayed. To investigate the role of RARalpha1 activation functions AF-1 and AF-2 and of their phosphorylation sites during RA- and cAMP-induced parietal differentiation, cell lines reexpressing WT or mutated RARalpha1 were established in RARalpha-/- cells. We have found that the protein kinase A (PKA) phosphorylation site and the AF-2AD core (helix 12) of RARalpha1 are required for efficient parietal endodermal differentiation, whereas the AF-1 proline-directed kinase phosphorylation site is dispensible. Interestingly, deletion of the AF-1 activating domain (the A/B region), but not of the AF-2AD core, generates a dominant negative mutant that abrogates primitive endodermal differentiation when expressed in RARalpha-/- cells. We also show that the RARalpha AF-1 and AF-2 activation functions, but not their phosphorylation sites, are involved in the induction of RA-responsive genes in a differential promoter context-dependent manner.

Animals↗
PubMed1999

Cytolytic activity of mitogen activated old and young mouse spleen cells against tumor target cells expressing high or low levels of Fas antigen.

Sensitivity of Fas expressing tumor cells (high levels in Hut78 & Jurkat; low levels in P815) toward the cytotoxic Con-A (5 microg/ml) activated spleen cells from young (12 to 16 week old males) and old (2 year old males) mice were studied. The spleen cells from young mice activated for a day showed high levels of cytotoxic activity against Hut78 and Jurkat cell lines but not against P815 cells. The cytotoxic activity against P815 cells were detected in the spleen cells from old but not young mice following a longer period of Con-A activation (three days). Comparable levels of cytotoxic activity against Hut78 and Jurkat cells were observed in the spleen cells from both young and old mice following three days of activation. Treatment of Hut78 cells with anti-Fas antibody affected the tumor cells become resistant against the cytotoxic activity of the spleen cells from young mice in a dose dependent manner however P815 cells were not affect by the anti-Fas antibody treatment. These results show that there are differences in the sensitivity of target tumor cells toward Con-A induced cytotoxic spleen cells from young and old mouse. Mitogen-induced cytotoxic lymphocytes from young mouse spleen appear to kill targets through mechanisms involving Fas antigen, specially, in early stage (1 day) of activation. Old mouse spleen cells generated high levels of cytotoxic cells in later phase (3 days), which appear to kill through Fas-unrelated mechanisms.

Age Factors↗
PubMed1995

Expression and targeting of Syrian hamster prion protein induced by heat shock in transgenic Drosophila melanogaster.

To evaluate the fruit fly as a model for studying neurodegenerative diseases caused by prions, transgenic flies were generated by introducing the Syrian hamster prion protein (SHaPrP) gene into the Drosophila melanogaster germ line by P element-mediated transformation. Nine transgenic lines were isolated; induction of transgenes that had been placed under the control of the Drosophila heat shock promoter, hsp 70, resulted in the synthesis of full-length SHaPrP. The relative molecular weight of the recombinant protein was lower than that of authentic SHaPrP due to incomplete processing of Asn-linked CHOs. To determine the cellular localization of SHaPrP, Drosophila Schneider line 2 cells were transfected with the same constructs used for fly transformation. Heat shock induced SHaPrP was anchored to the surface of S2 cells by a glycolipid, demonstrating that the carboxy-terminal glycolipidation signal of SHaPrP is recognized by this evolutionarily distant host. When SHaPrP was synthesized in transgenic flies constitutively by subjecting them to heat pulses continuously, no difference in their lifespans compared with controls was detected. Furthermore, expression of SHaPrP for 20 days did not produce protease resistant SHaPrP, which is the major and possibly only component of the infectious prion. In contrast to transgenic mice overexpressing SHaPrP, which develop a profound neuromyopathy, no disease phenotype was associated with expression of SHaPrP over the entire lifespan of transgenic flies.

Animals↗
PubMed1996

Use of Epstein-Barr virus nuclear antigen-1 in targeted therapy of EBV-associated neoplasia.

To target expression of toxic genes to Epstein-Barr virus (EBV)-associated tumor cells, we have developed an EBV-driven enzyme prodrug system (EDEPS) that takes advantage of the trans-activating properties of EBNA1, a latent protein expressed in all EBV-containing cells, to direct expression of cytosine deaminase (CD) at high levels in those cells only. Plasmids were constructed in which the CD gene or a luciferase reporter gene were cloned downstream of the herpes simplex virus thymidine kinase (tk) promoter and the family of repeats (FR) sequence from the oriP region of EBV. Analysis of luciferase activity after transient transfection into a panel of EBV-negative or -positive human cell lines showed that the presence of the FR element enhanced transcription from the tk promoter in all EBV-positive cell lines, whereas transcription from tk was repressed in all EBV-negative cell lines, including B, T, and fibroblast cell lines. In clonogenicity assays following transfection with the CD vector, the presence of 5-fluorocytosine (5-FC) in the culture medium completely abolished cell growth in EBV-positive cell lines, but did not affect the growth of EBV-negative cell lines. This vector system should have wide applicability in that it allows targeted expression of any gene of interest to tumors that carry EBV, irrespective of the role EBV plays in their pathogenesis.

Antigens, Viral↗
PubMed2002

Tumor-targeted Salmonella expressing cytosine deaminase as an anticancer agent.

The study was designed to evaluate whether TAPET-CD, an attenuated strain of Salmonella typhimurium expressing Escherichia coli cytosine deaminase (CD), was capable of converting nontoxic 5-fluorocytosine (5-FC) to the active antitumor agent 5-fluorouracil (5-FU). The antitumor effect of TAPET-CD plus 5-FC against subcutaneously implanted colon tumors was also evaluated. TAPET-CD was given to tumor-bearing mice by a single bolus intravenous administration followed with 5-FC by intraperitoneal administration. TAPET-CD accumulated in tumors at levels 1000-fold higher than that in normal tissues and high levels of 5-FU were detected in tumors in mice treated with both TAPET-CD and 5-FC. No 5-FU could be detected in normal tissues. Inhibition of tumor growth was observed in mice treated with either TAPET-CD alone or TAPET-CD in combination with 5-FC (TAPET-CD/5-FC), but not with 5-FC alone. TAPET-CD/5-FC inhibited tumor growth by 88%-96%, compared to TAPET-CD alone, which inhibited tumor growth by 38%-79%. These data suggest that tumor-targeting Salmonella could be used to deliver prodrug-converting enzyme selectively to tumors and produced anti-tumor effects when the corresponding prodrug was also given.

Animals↗
PubMed1999

Ectopic substance P-immunoreactive boutons are preferentially presynaptic to neurokinin-1 receptor immunoreactive dendrites in the spinal white matter of transgenic mice.

A recent immunocytochemical study has shown that substance P (SP) preferentially innervates targets expressing the neurokinin-1 receptor (NK-1r) in the superficial spinal dorsal horn of the rat. Based on these findings, we decided to further investigate the relationship between SP and the NK-1r in a transgenic mouse model in which SP fibres are ectopically located. Double-labelling immunocytochemistry at both the light and electron microscopic levels was performed to study the association between SP and the NK-1r in the spinal white matter of both control and transgenic mice. Light microscopy revealed NK-1r-immunoreactive (IR) dendrites in the white matter of the dorsolateral funiculus in both control and transgenic mice. In transgenic mice, but not in controls, SP-IR fibres were observed in close proximity to the NK-1r-IR dendrites in the white matter. At the ultrastructural level, SP-IR boutons were apposed to NK-1r-IR dendrites in the dorsolateral funiculus of transgenic mice, and a synapse was frequently observed as well. These results indicate that, even in conditions in which SP fibres are ectopically located, they still preferentially innervate targets expressing the NK-1r.

Animals↗
PubMed1994

Participation of target Fas protein in apoptosis pathway induced by CD4+ Th1 and CD8+ cytotoxic T cells.

The results presented here provide evidence that the presence of Fas protein in target cells is essential to permit cytotoxicity (resulting in apoptosis) mediated by cloned CD4+ Th1 cells. Using mitogen-activated B cells as targets, antigen-dependent lysis by CD4+ Th1 effectors was observed with MRL/MpJ+ but not with MRL/MpJ-lpr targets. The congenic MRL/MpJ-lpr strain is defective in Fas expression. Target cells from various lymphoid tissues of C3H.MRL-lpr mice were also resistant to the lectin-dependent cytotoxicity of Th1 effectors, whereas C3H/HeJ targets were sensitive. Moreover, a rapid DNA fragmentation prior to 51Cr release was induced only in C3H/HeJ targets. Thus, cytotoxicity induced by Th1 effectors correlates with target Fas expression. In contrast to Th1 effectors, CD8+ cytotoxic T lymphocytes (CTLs) killed C3H.MRL-lpr targets. When cytotoxicity was assayed in the presence of EGTA and MgCl2, which chelates extracellular Ca2+ [(Ca2+)ext], only C3H.MRL-lpr targets became resistant to CD8+ CTLs. This (Ca2+)ext-independent cytotoxicity of both Th1 and CD8+ effectors could be inhibited with unlabeled C3H/HeJ thymocytes or with a transfectoma carrying a murine Fas-human mu gene construct. In comparison, C3H.MRL-lpr thymocytes and the nontransfected parental cell line were poor inhibitors. Our study demonstrates that CD4+ Th1 cells and CD8+ CTLs differ in their (Ca2+)ext-dependent cytotoxicity but share a (Ca2+)ext-independent cytotoxicity that requires participation of Fas molecules for cytotoxic signal transduction leading to target apoptosis.

Animals↗
PubMed1999

NF kappa B activity and target gene expression in the rat brain after one and two exposures to ionizing radiation.

The central nervous system injury that can result after radiotherapy has been suggested to involve induced gene expression and cytokine production. We have previously shown that irradiation of primary cultures of rat astrocytes results in the activation of NF kappa B. To determine whether such an effect also occurs in vivo, NF kappa B activity was analyzed in the cerebral cortex of the rat brain after whole body irradiation. After a single dose of 15 Gy, NF kappa B activity was increased by 2 h postirradiation, returning to unirradiated levels by 8 hours. The increase was dose-dependent beginning at 2 Gy and continuing to at least 22.5 Gy. NF kappa B activity in the irradiated cortex was not accompanied by I kappa B alpha degradation. When 7.5 Gy was delivered 24 h before the 15 Gy, the increase in NF kappa B activity after 15 Gy was significantly reduced. These results suggest that an initial exposure to radiation induced a refractory period in the brain during which the susceptibility of NF kappa B to activation by subsequent irradiation was significantly reduced. This period of reduced sensitivity to radiation was also apparent for the induction of the NF kappa B-regulated cytokines IL-1 beta, IL-6, and TNF alpha.

Animals↗
PubMedDate not supplied

The Fugu rubripes tyrosinase gene promoter targets transgene expression to pigment cells in the mouse.

The regulation of the mouse tyrosinase gene expression is controlled by a highly conserved element at -100 bp, the M-box, and an enhancer at -12 kb. In most vertebrates, the length of intergenic sequences makes it difficult to analyze the whole gene and the complete regulatory region. We took advantage of the compact Fugu genome to identify regulatory regions involved in pigment cell-specific expression. We isolated the Fugu tyrosinase gene, and identified putative cis-acting regulatory elements within the promoter. We then asked whether the Fugu promoter sequence functions in mouse pigment cells. We showed that E11.5 transgenic embryos bearing 6 kb or 3 kb of Fugu tyrosinase 5' sequence fused to the reporter gene lacZ revealed melanoblast and RPE-specific expression. This is the first evidence that the tyrosinase promoter is active at midgestation in melanoblasts, long before the onset of pigmentation.

Amino Acid Sequence↗