Showing papers by "St. Jude Children's Research Hospital published in 1996"
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TL;DR: Genetic alterations affecting p16INK4a and cyclin D1, proteins that govern phosphorylation of the retinoblastoma protein and control exit from the G1 phase of the cell cycle, are so frequent in human cancers that inactivation of this pathway may well be necessary for tumor development.
Abstract: Uncontrolled cell proliferation is the hallmark of cancer, and tumor cells have typically acquired damage to genes that directly regulate their cell cycles. Genetic alterations affecting p16(INK4a) and cyclin D1, proteins that govern phosphorylation of the retinoblastoma protein (RB) and control exit from the G1 phase of the cell cycle, are so frequent in human cancers that inactivation of this pathway may well be necessary for tumor development. Like the tumor suppressor protein p53, components of this "RB pathway," although not essential for the cell cycle per se, may participate in checkpoint functions that regulate homeostatic tissue renewal throughout life.
5,509 citations
TL;DR: The results suggest that AML1-regulated target genes are essential for definitive hematopoiesis of all lineages, and that this defect was intrinsic to the stem cells in that AMl1-/-ES cells failed to contribute to hematocerosis in chimeric animals.
1,922 citations
TL;DR: Regarding biological functions, it can be anticipated that in the very near future the phenotypes of mice deficient in the remaining STATs will be described and will thus eliminate further speculation, and it would seem less likely as time goes on that additional family members will emerge.
1,412 citations
TL;DR: Stat6-l- mice were deficient in IL-4-mediated functions including Th2 helper T-cell differentiation, expression of cell surface markers, and immunoglobulin class switching to IgE, indicating the lack of a non-redundant function in normal development.
Abstract: Signal transducers and activators of transcription (Stats) are activated by tyrosine phosphorylation in response to cytokines, and are thought to mediate many of their functional responses. Stat6 is activated in response to interleukin (IL)-4 and may contribute to various functions including mitogenesis, T-helper cell differentiation and immunoglobulin isotype switching. To evaluate the role of Stat6, we generated Stat6-null mice (Stat6 -/-) by gene disruption in embryonic stem cells. The mice were viable, indicating the lack of a non-redundant function in normal development. Although naive lymphoid cell development was normal, Stat6 -/- mice were deficient in IL-4-mediated functions including Th2 helper T-cell differentiation, expression of cell surface markers, and immunoglobulin class switching to IgE. In contrast, IL-4-mediated proliferation was only partly affected.
1,314 citations
TL;DR: All IL-12 functions tested were disrupted, including the induction of IFN-γ, mitogenesis, enhancement of natural killer cytolytic function and Thl differentiation.
Abstract: Signal transducers and activators of transcription (STATs) are activated by tyrosine phosphorylation in response to cytokines and mediate many of their functional responses. Stat4 was initially cloned as a result of its homology with Stat1 (refs 4, 5) and is widely expressed, although it is only tyrosine-phosphorylated after stimulation of T cells with interleukin (IL)-12 (refs 6,7). IL-12 is required for the T-cell-independent induction of the cytokine interferon (IFN)-gamma, a key step in the initial suppression of bacterial and parasitic infections. IL-12 is also important for the development of a Th1 response, which is critical for effective host defence against intracellular pathogens. To determine the function of Stat4 and its role in IL-12 signalling, we have produced mice that lack Stat4 by gene targeting. The mice were viable and fertile, with no detectable defects in haematopoiesis. However, all IL-12 functions tested were disrupted, including the induction of IFN-gamma, mitogenesis, enhancement of natural killer cytolytic function and Th1 differentiation.
1,181 citations
TL;DR: These findings support wider use of antigen–specific CTLs in adoptive immunotherapy and restore cellular immune responses against EBV, but also established populations of CTL precursors that could respond to in vivo or ex vivo challenge with the virus for as long as 18 months.
Abstract: Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) offers safe and effective therapy for certain viral infections and could prove useful in the eradication of tumor cells. Whether or not the infused T cells persist for extended periods, retaining their ability to expand in response to antigenic stimulation, is not known. We now report long-term detection of gene-marked Epstein-Barr virus (EBV)-specific CTLs in immunocompromised patients at risk for the development of EBV lymphoproliferative disease. Infusions of CTLs not only restored cellular immune responses against EBV, but also established populations of CTL precursors that could respond to in vivo or ex vivo challenge with the virus for as long as 18 months. Our findings support wider use of antigen-specific CTLs in adoptive immunotherapy.
849 citations
TL;DR: It is demonstrated that enhanced gene transfer into mammalian target cells is due to direct binding of retroviral particles to sequences within the fibronectin molecule.
Abstract: Hematopoietic cells are important targets for genetic modification with retroviral vectors. Attempts at human gene therapy of stem cells have achieved limited success partly because of low gene transfer efficiency. Chymotryptic fragments of the extracellular matrix molecule fibronectin used during infection have been shown to increase transduction of human hematopoietic progenitor cells. Here, we demonstrate that this enhanced gene transfer into mammalian target cells is due to direct binding of retroviral particles to sequences within the fibronectin molecule. Transduction of mammalian cells, including murine long-term repopulating hematopoietic cells, is greatly enhanced when cells are adherent to chimeric fragments containing these retroviral binding sequences. In addition, colocalization of retrovirus and target cells on fibronectin peptides allows targeted transduction of specific cell types by exploiting unique ligand/receptor interactions.
587 citations
TL;DR: The results are consistent with the interpretation that CD8+ T cell-mediated control of this persistent gammaherpesvirus is progressively lost in the absence of the CD4- T cell subset, which parallels what may be happening in AIDS patients who develop Kaposi's sarcoma and various Epstein Barr virus associated disease processes.
Abstract: A unique experimental model has been developed for dissecting the integrity of CD8+ T cell-mediated immunity to a persistent gammaherpesvirus under conditions of CD4+ T cell deficiency. Respiratory challenge of major histocompatibility complex class II -/- and +/+ C57BL/6J mice with the murine gammaherpesvirus 68 (MHV-68) leads to productive infection of both lung and adrenal epithelial cells. Virus titers peak within 5-10 d, and are no longer detected after day 15. Persistent, latent infection is established concurrently in splenic and lymph node B cells, with higher numbers of MHV-68+ lymphocytes being found in all lymphoid sites analyzed from the +/+ mice concurrent with the massive, but transient splenomegaly that occurred only in this group. From day 17, however, the numbers of infected B lymphocytes were consistently higher in the -/- group, while the frequency of this population diminished progressively in the +/+ controls. Infectious MHV-68 was again detected in the respiratory tract and the adrenals of the -/- (but not the +/+) mice from day 22 after infection. The titers in these sites rose progressively, with the majority of the -/- mice dying between days 120 and 133. Even so, some CD8+ effectors were still functioning as late as 100 d after infection. Depletion of CD8+ T cells at this stage led to higher virus titers in the -/- lung, and to the development of wasting in some of the -/- mice. Elimination of the CD8+ T cells from the +/+ group (day 80) increased the numbers of MHV-68+ cells in the spleen, but did not reactivate the infection in the respiratory tract. The results are consistent with the interpretation that CD8+ T cell-mediated control of this persistent gammaherpesvirus is progressively lost in the absence of the CD4+ T cell subset. This parallels what may be happening in AIDS patients who develop Kaposi's sarcoma and various Epstein Barr virus associated disease processes.
517 citations
Journal Article•
TL;DR: The apparent coordinated coexpression of the CYP3A gene family and P-glycoprotein in the LS180 cells suggests that for common orally administered drugs, P- glycoprotein may play an important role in net drug absorption and drug/drug interactions of shared CYP 3A4/P-gly Coprotein substrates.
Abstract: Xenobiotics frequently induce proteins involved in their detoxification. Because many drugs that are metabolized by human cytochromes P450 (CYP) 3A4 and 3A5 are also transported by the drug efflux pump P-glycoprotein, we determined whether expression of these proteins was altered by a variety of drugs in a cell line derived from a human colon adenocarcinoma, LS180/WT, and its adriamycin-resistant subline, LS180/AD50. P-glycoprotein and CYP3A4 were constitutively expressed in both LS180/AD50 and LS180/WT cells, and both proteins were up-regulated after treatment with many drugs, including rifampicin, phenobarbital, clotrimazole, reserpine, and isosafrole. However, there were some exceptions because P-glycoprotein was up-regulated by midazolam and nifedipine, whereas CYP3A4 was not. CYP3A5, which is also constitutively expressed in these cells, remained unchanged with most drug treatments but was up-regulated by reserpine and clotrimazole. The apparent coordinated coexpression of the CYP3A gene family and P-glycoprotein in the LS180 cells suggests that for common orally administered drugs, P-glycoprotein may play an important role in net drug absorption and drug/drug interactions of shared CYP3A4/P-glycoprotein substrates.
507 citations
TL;DR: Results suggest that NFAT1 exerts a negative regulatory influence on the immune response, and in an in vivo model of allergic inflammation, the accumulation of eosinophils and levels of serum immunoglobulin E were increased.
Abstract: 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.
374 citations
TL;DR: Non-Hodgkin's lymphomas are the third most common group of cancers in children and adolescents in the United States, accounting for approximately 13 percent of newly diagnosed cancers in this age group.
Abstract: Lymphomas are the third most common group of cancers in children and adolescents in the United States, accounting for approximately 13 percent of newly diagnosed cancers in this age group1 Non-Hodgkin's lymphomas represent approximately 60 percent of these diagnoses, and Hodgkin's disease accounts for the remainder2,3 Approximately 500 cases of childhood non-Hodgkin's lymphoma occur annually in the United States4 Non-Hodgkin's lymphomas are categorized as low, intermediate, or high grade on the basis of their clinical aggressiveness Low- and intermediate-grade tumors predominate in adults, whereas more than 90 percent of children with non-Hodgkin's lymphoma have high-grade tumors This difference is
TL;DR: In this article, the isolation of a human RNA polymerase II complex containing a subset of the basal transcription factors and the human homologues of the yeast SRB proteins was reported.
Abstract: WE report here the isolation of a human RNA polymerase II complex containing a subset of the basal transcription factors and the human homologues of the yeast SRB (for suppressors of RNA polymerase B) proteins1–3. The complex contains transcriptional coactivators and increases the activation of transcription. In addition, some components of the RNA polymerase II complex participate in DNA repair.
TL;DR: These findings suggest that Trβ controls the maturation of auditory function but not morphogenesis of the cochlea, and identify Trβ as an essential transcription factor for auditory development and indicate that distinct Tr genes serve certain unique functions.
Abstract: Congenital thyroid disorders are often associated with profound deafness, indicating a requirement for thyroid hormone (T3) and its receptors in the development of hearing. Two T3 receptor genes, Tr alpha and Tr beta are differentially expressed, although in overlapping patterns, during development. Thus, the extent to which they mediate unique or redundant functions is unclear. We demonstrate that Tr beta-deficient (Thrb-/-) mice exhibit a permanent deficit in auditory function across a wide range of frequencies, although they show no other overt neurological defects. The auditory-evoked brainstem response (ABR) in Thrb-/- mice, although greatly diminished, displayed normal waveforms, which suggested that the primary defect resides in the cochlea. Although hypothyroidism causes cochlear malformation, there was no evidence of this in Thrb-/- mice. These findings suggest that Tr beta controls the maturation of auditory function but not morphogenesis of the cochlea. Thrb-/- mice provide a model for the human endocrine disorder of resistance to thyroid hormone (RTH), which is typically associated with dominant mutations in Tr beta. However, deafness is generally absent in RTH, indicating that dominant and recessive mutations in Tr beta have different consequences on the auditory system. Our results identify Tr beta as an essential transcription factor for auditory development and indicate that distinct Tr genes serve certain unique functions.
TL;DR: The overall risk of second malignancies after Ewing's sarcoma is similar to that associated with treatment for other childhood cancers, and the radiation dose-dependency of secondary sarcomas justifies modification in therapy to reduce radiation doses.
Abstract: BACKGROUNDAn excess risk of second malignancies has been reported in survivors of Ewing's sarcoma. We examined a multiinstitutional data base to reevaluate the risk among survivors of Ewing's sarcoma and to identify possible causal factors.METHODSInformation was derived from a data base that included 266 survivors of Ewing's sarcoma. Cumulative incidence rates of second malignancies were calculated. Contributions of clinical features, type and dose of chemotherapy, and cumulative radiation dose to the risk of second malignancies were evaluated.RESULTSAfter a median follow-up duration of 9.5 years (range, 3.0 to 30), 16 patients have developed second malignancies, which included 10 sarcomas (five osteosarcomas, three fibrosarcomas, and two malignant fibrous histiocytomas) and six other malignancies (acute myeloblastic leukemia, acute lymphoblastic leukemia, meningioma, bronchioalveolar carcinoma, basal cell carcinoma, and carcinoma-in-situ of the cervix). The median latency to the diagnosis of the second m...
Journal Article•
TL;DR: Using mutation specific PCR-RFLP analysis, the TPMT*3 allele was detected in genomic DNA from approximately 75 percent of unrelated white subjects with heterozygous phenotypes, indicating that TPMt*3 is the most prevalent mutant allele associated with T PMT-deficiency in Caucasians.
Abstract: The autosomal recessive trait of thiopurine S-methytransferase (TPMT) deficiency is associated with severe hematopoietic toxicity when patients are treated with standard doses of mercaptopurine, azathioprine, or thioguanine To define the molecular mechanism of this genetic polymorphism, we cloned and characterized the cDNA of a TPMT-deficient patient, which revealed a novel mutant allele (TPMT*3) containing two nucleotide transitions (G460-->A and A719-->G) producing amino acid changes at codons 154 (Ala-->Thr) and 240 (Tyr--> Cys), differing from the rare mutant TPMT allele we previously identified (ie, TPMT*2 with only G238-->C) Site-directed mutagenesis and heterologous expression established that either TPMT*3 mutation alone leads to a reduction in catalytic activity (G460-->A, ninefold reduction; A719-->G, 14-fold reduction), while the presence of both mutations leads to complete loss of activity Using mutation specific PCR-RFLP analysis, the TPMT*3 allele was detected in genomic DNA from approximately 75 percent of unrelated white subjects with heterozygous phenotypes, indicating that TPMT*3 is the most prevalent mutant allele associated with TPMT-deficiency in Caucasians
TL;DR: The broad substrate specificity of FabZ coupled with the inactivity of FabA toward a long chain unsaturated β-hydroxyacyl-ACP provides a biochemical explanation for the phenotypes of cells with genetically altered levels of the two dehydratases.
Journal Article•
TL;DR: RNA-based polymerase chain reaction analysis revealed identical NPM-MLF1 mRNA fusions in each of the three t(3;5)-positive cases of AML examined, indicating that NPM trafficking signals direct MLF1 to an inappropriate cellular compartment in myeloid leukemia cells.
Abstract: A t(3;5)(q25.1;q34) chromosomal translocation associated with myelodysplastic syndrome and acute myeloid leukemia (AML) was found to rearrange part of the nucleophosmin (NPM) gene on chromosome 5 with sequences from a novel gene on chromosome 3. Chimeric transcripts expressed by these cells contain 5' NPM coding sequences fused in-frame to those of the new gene, which we named myelodysplasia/myeloid leukemia factor 1 (MLF1). RNA-based polymerase chain reaction analysis revealed identical NPM-MLF1 mRNA fusions in each of the three t(3;5)-positive cases of AML examined. The predicted MLF1 amino acid sequence lacked homology to previously characterized proteins and did not contain known functional motifs. Normal MLF1 transcripts were expressed in a variety of tissues, most abundantly in testis, ovary, skeletal muscle, heart, kidney and colon. Anti-MLF1 antibodies detected the wild-type 31 kDa protein in K562 and HEL erythroleukemia cell lines, but not in HL-60, U937 or KG-1 myeloid leukemia lines. By contrast, t(3;5)-positive leukemia cells expressed a 54 kDa NPM-MLF1 protein, but not normal MLF1. Immunostaining experiments indicated that MLF1 is normally located in the cytoplasm, whereas NPM-MLF1 is targeted to the nucleus, with highest levels in the nucleolus. The nuclear/nucleolar localization of NPM-MLF1 mirrors that of NPM, indicating that NPM trafficking signals direct MLF1 to an inappropriate cellular compartment in myeloid leukemia cells.
TL;DR: E extent of tumor resection, as estimated by the neurosurgeon, does not correlate with outcome but that extent of residual tumor does correlate with prognosis in certain children (those who are > 3 years old, with no tumor dissemination).
Abstract: We reviewed the data of children with high-stage primitive neuroectodermal tumors (medulloblastomas) who were treated on Children's Cancer Group-921 protocol to evaluate the correlation between tumor resection and prognosis. Patients enrolled in the study had either tumors that were operatively categorized to be Chang tumor stage 3b or 4, postoperative residual tumors > 1.5 cm2, or evidence of tumor dissemination (Chang metastasis Stages [M Stages] 1-4) at diagnosis. Resections were analyzed in two ways, as follows: 1) by the extent of resection (percent of the tumor that was removed), as estimated by the treating neurosurgeon; and 2) by the extent of residual tumor (how much of the tumor was left), as estimated from postoperative scans. Two hundred and three children were enrolled in the study with institutional diagnoses of primitive neuroectodermal tumors-medulloblastomas; diagnoses were confirmed by central neuropathological review in 188 patients. Progression-free survival (PFS) at 5 years was 54% (standard error, 5%). As in previous Children's Cancer Group studies, age and M stage correlated with survival; PFS was significantly lower in children 1.5 to 3.0 years old at diagnosis and in those with any evidence of tumor dissemination (M Stage 1-4). On univariate analysis, neither extent of resection nor extent of residual tumor correlated with PFS. However, adjusting for other factors, extent of residual tumor was important; PFS was 20% (standard error, 14%) better at 5 years in children with no dissemination (M Stage 0) who had 3 years old with no tumor dissemination (M Stage 0) and with 3 years old, with no tumor dissemination). In contrast to age and M stage, the major factors associated with outcome, residual tumor is an important variable in outcome, one that neurosurgeons can control.
TL;DR: The t(12;21) fusion protein dominantly interferes with AML-1B-dependent transcription, suggesting that the inhibition of expression of AML -1 genes is critical for B-cell leukemogenesis.
Abstract: The t(12;21) translocation is present in up to 30% of childhood B-cell acute lymphoblastic and fuses a potential dimerization motif from the ets-related factor TEL to the N terminus of AML1. The t(12;21) translocation encodes a 93-kDa fusion protein that localizes to a high-salt- and detergent-resistant nuclear compartment. This protein binds the enhancer core motif, TGTGGT, and interacts with the AML-1-binding protein, core-binding factor beta. Although TEL/AML-1B retains the C-terminal domain of AML-1B that is required for transactivation of the T-cell receptor beta enhancer, it fails to activate transcription but rather inhibits the basal activity of this enhancer. TEL/AML-1B efficiently interferes with AML-1B dependent transactivation of the T-cell receptor beta enhancer, and coexpression of wild-type TEL does not reverse this inhibition. The N-terminal TEL helix-loop-helix domain is essential for TEL/AML-1B-mediated repression. Thus, the t(12;21) fusion protein dominantly interferes with AML-1B-dependent transcription, suggesting that the inhibition of expression of AML-1 genes is critical for B-cell leukemogenesis.
TL;DR: A new hemagglutinin (HA) subtype, H15, was proposed in this paper, where the amino acid sequence data was evaluated when determining the HA subtypes of influenza A viruses.
TL;DR: In this paper, the authors identified a full-length cDNA clone in the dbEST data base, of which the predicted amino acid sequence has extensive homology to other mammalian and bacterial neuraminidases, including the F(Y)RIP domain and Asp-boxes.
Abstract: Neuraminidases (sialidases) have an essential role in the removal of terminal sialic acid residues from sialoglycoconjugates and are distributed widely in nature. The human lysosomal enzyme occurs in complex with beta-galactosidase and protective protein/cathepsin A (PPCA), and is deficient in two genetic disorders: sialidosis, caused by a structural defect in the neuraminidase gene, and galactosialidosis, in which the loss of neuraminidase activity is secondary to a deficiency of PPCA. We identified a full-length cDNA clone in the dbEST data base, of which the predicted amino acid sequence has extensive homology to other mammalian and bacterial neuraminidases, including the F(Y)RIP domain and "Asp-boxes." In situ hybridization localized the human neuraminidase gene to chromosome band 6p21, a region known to contain the HLA locus. Transient expression of the cDNA in deficient human fibroblasts showed that the enzyme is compartmentalized in lysosomes and restored neuraminidase activity in a PPCA-dependent manner. The authenticity of the cDNA was verified by the identification of three independent mutations in the open reading frame of the mRNA from clinically distinct sialidosis patients. Coexpression of the mutant cDNAs with PPCA failed to generate neuraminidase activity, confirming the inactivating effect of the mutations. These results establish the molecular basis of sialidosis in these patients, and clearly identify the cDNA-encoded protein as lysosomal neuraminidase.
TL;DR: It is concluded that Pgp-mediated transport is a critical element influencing the CYP3A inductive response.
Abstract: The P-glycoprotein (Pgp) efflux pump can influence the hepatocellular concentration of xenobiotics that are modulators and substrates of cytochrome P4503A (CYP3A). We tested the hypothesis that Pgp is a determinant of drug-inducible expression of CYP3A. The magnitude of CYP3A induction by rifampicin was compared in the human parental colon carcinoma cell line LS 180/WT (wild type) and in two derivative clones overexpressing the human multidrug resistance gene MDR1 (also designated PGY1) because of either drug selection (LS 180/ADR) or transfection with MDRI cDNA (LS 180/MDR). In both MDR1 cDNA-overexpressing clones, rifampicin induction of CYP3A mRNA and protein was decreased and required greater rifampicin concentrations compared with parental cells. The role of Pgp in regulation of CYP3A expression in vivo was analyzed in mice carrying a targeted disruption of the mdr1a mouse gene. Oral treatment with increasing doses of rifampicin resulted in elevated drug levels in the livers of mdr1a (-/-) mice compared with mdr1a (+/+) mice at all doses. Consistent with the enhanced accumulation of rifampicin in mdr1a (-/-) mice, lower doses of rifampicin were required for induction of CYP3A proteins, and the magnitude of CYP3A induction was greater at all doses of rifampicin in mdr1a (-/-) mice compared with mdr1a (+/+) mice. We conclude that Pgp-mediated transport is a critical element influencing the CYP3A inductive response.
TL;DR: The results demonstrate the natural existence of potentially dominantly suppressive variants of Stat5 and implicate the carboxyl domain of Stats in transcriptional regulation and functions related to dephosphorylation and Surprisingly, the tyrosine phosphorylation of the car Box5 forms is considerably more stable than that of the wild-type proteins.
Abstract: Stat5 was initially identified as a prolactin-induced member of the signal transducer and activator of transcription (Stat) family in sheep. However, Stat5 is also activated in the response to a variety of cytokines. In mice, and possibly in other species, there exist two Stat5 genes (Stat5a and Stat5b) that encode proteins of 92 and 94 kDa that are 95% identical. In the studies described here, we demonstrate that naturally occurring carboxyl-truncated, variant Stat5 proteins of 77 and 80 kDa exist and that these proteins are inducibly tyrosine phosphorylated in the response to several cytokines and form heterodimers with the full-length, wild-type proteins. Using expression constructs encoding truncated forms, we demonstrate that the truncated forms can be tyrosine phosphorylated and bind DNA. Surprisingly, the tyrosine phosphorylation of the carboxyl-truncated forms is considerably more stable than that of the wild-type proteins. Overexpression of a carboxyl-truncated Stat5a in cells resulted in the specific inhibition of the transcriptional activation by interleukin-3 of the genes for oncostatin M (Osm) and the cytokine-inducible, SH2 domain-containing gene (Cis), both of which have been shown to be normally regulated by Stat5. Although Stat5 dominantly suppressed the induction of these genes, no effects on cell proliferation were observed. Together, the results demonstrate the natural existence of potentially dominantly suppressive variants of Stat5 and implicate the carboxyl domain of Stats in transcriptional regulation and functions related to dephosphorylation.
TL;DR: The studies presented here demonstrate that this region is also necessary and sufficient for the activation of Stat5A and Stat5B, and can disrupt Stat5 DNA binding activity, consistent with a role of Y-343 as a site of recruitment to the receptor.
Abstract: The cytoplasmic domain of the erythropoietin receptor (EpoR) contains a membrane-distal region that is dispensable for mitogenesis but is required for the recruitment and tyrosine phosphorylation of a variety of signaling proteins. The membrane-proximal region of 96 amino acids is necessary and sufficient for mitogenesis as well as Jak2 activation, induction of c-fos, c-myc, cis, the T-cell receptor gamma locus (TCR-gamma), and c-pim-1. The studies presented here demonstrate that this region is also necessary and sufficient for the activation of Stat5A and Stat5B. The membrane-proximal domain contains a single tyrosine, Y-343, which when mutated eliminates the ability of the receptor to couple Epo binding to the activation of Stat5. Furthermore, peptide competitions demonstrate that this site, when phosphorylated, can disrupt Stat5 DNA binding activity, consistent with a role of Y-343 as a site of recruitment to the receptor. Cells expressing the truncated, Y343F mutant (a mutant with a Y-to-F alteration at position 343) proliferate in response to Epo in a manner comparable to that of the controls. However, in these cells, Epo stimulation does not induce the appearance of transcripts for cis, TCR-gamma, or c-fos, suggesting a role for Stat5 in their regulation.
TL;DR: Ongoing studies aim to better define the influence of TPMT activity on thiopurine efficacy, to identify additional mutant alleles and determine their frequency in different ethnic groups, to elucidate the mechanism(s) for loss of function of mutant proteins, to identified potential endogenous substrates and to define the molecular mechanisms of T PMT regulation.
Abstract: Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurines such as mercaptopurine and thioguanine. TPMT activity exhibits genetic polymorphism, with about 1 in 300 inheriting TPMT-deficiency as an autosomal recessive trait. If treated with standard dosages of thiopurines, TPMT-
TL;DR: Analysis of the individual enzymes and the pattern of intermediate accumulation in the reconstituted system identified initiation of fatty acid synthesis by β-ketoacyl-ACP synthase III (fabH) and enoyl- ACP reductase (fabI) in the elongation cycle as two steps attenuated by long chain acyl-ACP.
TL;DR: Defects in the mu heavy-chain gene are a cause of agammaglobulinemia in humans, which implies that an intact membrane-bound mu chain is essential for B-cell development.
Abstract: Background Most patients with congenital hypogammaglobulinemia and absent B cells are males with X-linked agammaglobulinemia, which is caused by mutations in the gene for Bruton's tyrosine kinase (Btk); however, there are females with a similar disorder who do not have mutations in this gene. We studied two families with autosomal recessive defects in B-cell development and patients with presumed X-linked agammaglobulinemia who did not have mutations in Btk. Methods A series of candidate genes that encode proteins involved in B-cell signal-transduction pathways were analyzed by linkage studies and mutation screening. Results Four different mutations were identified in the mu heavy-chain gene on chromosome 14. In one family, there was a homozygous 75-to-100-kb deletion that included D-region genes, J-region genes, and the mu constant-region gene. In a second family, there was a homozygous base-pair substitution in the alternative splice site of the mu heavy-chain gene. This mutation would inhibit productio...
TL;DR: It is suggested that direct physical interactions between RAD52 and RPA are essential for homologous recombination in mammalian cells.
TL;DR: Coexpression of a T–cell chemokine and T-cell growth factor potentiates antitumor responses in vivo, suggesting a general strategy to improve cancer immunotherapy.
Abstract: The probability of producing a specific antitumor response should be increased by multiplying the number of T lymphocytes that encounter the malignant cells. We tested this prediction in a murine model, using a recently discovered T-cell chemokine, lymphotactin (Lptn). This chemokine increased tumor cell infiltration with CD4+ lymphocytes but generated little antitumor activity. Coexpression of the T-cell growth factor interleukin-2, however, greatly expanded the T lymphocytes attracted by Lptn, affording protection from the growth of established tumor in a CD4+ and CD8+ T cell-dependent manner. Lesser synergy was seen with GM-CSF. Hence coexpression of a T-cell chemokine and T-cell growth factor potentiates antitumor responses in vivo, suggesting a general strategy to improve cancer immunotherapy.