Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

FK506, a neutral macrolide with immunosuppressive properties, was shown to selectively and rapidly inhibit the accumulation of IL-2 mRNA, as well as the mRNAs of other early (E) phase T cell activation genes such as IL-3, IL-4, GM-CSF, TNF alpha, IFN-gamma, and c-myc in activated human peripheral blood T cells. The activity of FK506, when compared to Cyclosporin A, another immunosuppressant, was 10 to 100x more potent in its ability to inhibit IL-2 mRNA synthesis. FK506 inhibited IL-2 mRNA accumulation in Con A, Con A plus PMA, Ionomycin plus PMA, anti-CD3, and anti-CD3 plus PMA activated T cells. Transcripts from other T cell gene classes such as the immediate early (IE) phase gene, c-fos, the late phase (L) genes, transferrin receptor, IL-2R alpha-chain, and TNF-beta, and the constitutive class genes glyceraldehyde-3-phosphate dehydrogenase and class I MHC HLA-B7 were not affected by FK506. The macrolide Rapamycin, which is structurally related to FK506, had no inhibitory effect on IE, E, L, or constitutive class mRNAs, but it appeared to increase the levels of the E-phase transcripts that were inhibited in FK506 treated T cells. The effect of FK506 on inducible genes in non-T and non-lymphoid human cells was studied in LPS-induced monocytes and PMA or IL-1 activated synovial fibroblasts. FK506 did not affect expression of the mRNAs for IL-1 alpha or IL-1 beta in human monocytes, or of stromelysin, collagenase, or TIMP in synovial fibroblasts. Nuclear run-off transcription studies indicate that FK506 inhibits transcription of the IL-2 gene. These studies suggest that Cyclosporin A and FK506 may effect a common early event in the T cell activation pathway.
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PMID:The immunosuppressant FK506 selectively inhibits expression of early T cell activation genes. 247 51

NK-mediated cytotoxicity is regulated by a variety of cytokines and is thought to involve perforin and granzymes. The effects of IL-2 and IL-12 on the expression and activation of cytolysis were examined in freshly isolated human NK cells. A dose-dependent increase in cytolysis of the NK-sensitive target cell, K562, and the NK-insensitive but lymphokine-activated killer (LAK) cell-sensitive target, UCLA-SO-M14, was observed after short term culture of purified human NK cells in either IL-2 or IL-12. Moreover, the two cytokines often synergized to produce augmented lytic activity. A suboptimal dose of IL-2 (60 IU/ml) combined with IL-12 (2 U/ml) could induce lytic activity equal to twice the additive effect of each cytokine alone. Northern analyses revealed time-dependent increases in mRNAs encoding for perforin and granzymes A and B following treatment with IL-2 alone or IL-2 plus IL-12. IL-2 and IL-12 also synergized for the induction of granzyme mRNAs, in that treatment with both cytokines increased mRNA levels approximately 50% above the sum of each cytokine alone, as quantitated by phosphorimage analysis, and normalized to GAPDH gene expression. However, the synergy between IL-2 and IL-12 for the induction of mRNA was less dramatic than for lytic activity. Results of experiments in which cytokine-treated cells were pulsed with actinomycin D indicated that the increased granzyme and perforin gene mRNA levels in response to IL-2, IL-12, or the combination were not due to increased transcript stability. The data suggest that low doses of IL-2 and IL-12 synergize to augment NK- and induce LAK-mediated cytotoxicity and that this increase is associated with enhanced transcription of perforin and granzyme genes in a synergistic fashion.
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PMID:IL-12 synergizes with IL-2 to induce lymphokine-activated cytotoxicity and perforin and granzyme gene expression in fresh human NK cells. 767 23

IL-2 is the major mitogenic cytokine for mature human T cells. This growth factor has been shown previously to induce the expression of a number of genes, including structural proteins, proto-oncogenes, and metabolic enzymes. Multiple mechanisms, including increases in mRNA stability, protein synthesis, and new transcriptional initiation, have been studied to determine how IL-2 induces such a wide variety of genes. The following studies show that a release of transcriptional attenuation is important in IL-2-induced gene expression. A thymic blast cell system was developed and used to demonstrate that IL-2-deprived cells have a marked attenuation of transcription in the 3' ends of the pim-1 and c-myb genes. IL-2 stimulation removes this attenuation and leads to read-through transcription. This effect is gene-specific, as demonstrated by the fact that GAPDH is not attenuated in unstimulated cells. The IL-2-mediated relief of attenuation occurs within 1 h of IL-2 stimulation and is insensitive to the translation inhibitor cycloheximide, suggesting that new protein synthesis is not necessary. Further, the effect is insensitive to the immunosuppressant cyclosporin A, but is sensitive to rapamycin and the tyrosine kinase inhibitor genistein. These studies demonstrate that release of transcription attenuation is a mechanism used to induce gene expression in response to IL-2 treatment.
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PMID:The effect of IL-2 treatment on transcriptional attenuation in proto-oncogenes pim-1 and c-myb in human thymic blast cells. 875 12

T-cell activation is the key event in the development of acute allograft rejection and precedes clinically apparent organ damage. We have performed competitive RT-PCR to quantify the intragraft gene expression for T-cell associated cytokines (IL-2, IL-4, IL-7, IL-15), CTLA4 and cytotoxic lymphocyte specific molecules to test their potential as rejection markers and to further elucidate mechanisms involved in graft rejection. RNA was isolated from snap-frozen portions of core biopsies obtained for the evaluation of graft dysfunction in 34 adults and 8 children. Reverse transcription derived cDNA was coamplified with a known amount of a competitor (a mutated target gene fragment) and normalized for the house keeping gene GAPDH. IL-2, the principal T-cell growth factor and IL-4 were not detectable in any biopsy at the time of histologically apparent rejection. Transcripts of the novel cytokine IL-15 were found in all dysfunctioning grafts and in two donor kidneys prior to reperfusion. CTLA-4, expressed in activated T-cells after costimulation by CD28 was uniformly present post transplantation, but not in the two donor kidneys. Transcripts for IL-7 (p < 0.001), IL-15 (p < 0.0005), CTLA4 (p = 0.04), granzyme B (p < 0.00015) and perforin (p < 0.0003) showed a significant correlation to acute rejection episodes. Heightened gene expression declined rapidly after initiation of rejection treatment. Fas-ligand mRNA gene expression was upregulated in both acute and chronic rejections. While this study shows that competitive RT-PCR is a reliable diagnostic tool to detect acute rejection in renal core biopsies, a future challenge will be to identify molecular markers of evolving rejections utilizing RT-PCR in sequential samples of fine needle aspirations, urine and blood.
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PMID:The intragraft gene activation of markers reflecting T-cell-activation and -cytotoxicity analyzed by quantitative RT-PCR in renal transplantation. 883 47

Spontaneous tumor regression, which is observed clinically and histologically in some primary melanomas, occurs in the absence of any effective therapy. It is probably immunologically mediated, because regressing melanomas are infiltrated with larger numbers of activated T cells, primarily CD4+, than nonregressing melanomas. To investigate the hypothesis that spontaneous regression of melanomas is caused by T-cell cytokine production, cytokine mRNA expression in 20 primary melanomas was examined using a noncompetitive, quantitative reverse-transcriptase polymerase chain reaction method. DNA standards were used to generate known numbers of molecules in each sample. Results were standardized to the internal control, glyceraldehyde-3-phosphate dehydrogenase. mRNA for CD35, lymphotoxin (TNF-beta), and IL-2 were significantly elevated in the ten regressing melanomas compared to the ten nonregressing melanomas. IFN-gamma mRNA was also elevated in regressing melanomas but failed to reach statistical significance. The Th2 cytokines IL-10 and IL-13 did not show differences in the regressing melanomas compared to nonregressing melanomas; neither did the pro-inflammatory cytokines IL-1alpha, IL-1beta, IL-6, IL-8, and TNF-alpha, nor the growth factors, bFGF and TGF-beta or GM-CSF. This study shows an association between Th1 cytokines and spontaneously regressing melanomas. Although we have not shown that these cytokines cause regression, these findings support our hypothesis that activated CD4+ T cells may mediate melanoma regression by secretion of Th1 cytokines.
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PMID:T helper 1 cytokine mRNA is increased in spontaneously regressing primary melanomas. 918 21

Renal cell carcinoma (RCC) is a solid tumour of the kidney and is the most common renal neoplasm. Despite the presence of tumour infiltrating lymphocytes (TIL) in RCC, these tumours continue to progress in vivo suggesting a poor host immune response to the tumour, and the suppression of TIL effector function. Cytokines are key molecules that modulate the function of T cells. The possibility is investigated that the local production of cytokines in RCC contributes to immunosuppression of TIL. The expression of pro-inflammatory (IFN-gamma/IL-2) and immunosuppressive (IL-10/TGF-beta) cytokine mRNA transcripts was determined in RCC, normal kidney and peripheral blood of RCC patients using a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) with cytokine-specific primers. Following Southern blot hybridization of the PCR products with internal radiolabelled oligonucleotide probes, cytokine transcript levels were measured by densitometry and expressed relative to the glyceraldehyde-3-phosphate dehydrogenase densitometry score. With the exception of IL-10, there were no differences in expression of cytokine mRNA transcripts between the peripheral blood of patients and normal healthy individuals. It was found that TGF-beta transcripts were well represented in normal kidney and RCC. In contrast, the expression of IFN-gamma transcripts, while low in the majority of samples, was significantly increased in RCC when compared to normal kidney (P=0.05). The IL-2 and IL-10 transcripts showed a more variable expression in normal kidney and RCC, with no significant differences in expression between the sample groups. The data demonstrating pro-inflammatory and immunosuppressive cytokine expression in RCC do not support a prominent immunosuppressive cytokine profile in these tumours.
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PMID:Expression of cytokine mRNA transcripts in renal cell carcinoma. 972 77

Recently, a novel technique for "real time" quantitative Reverse Transcriptase-PCR which measures PCR-product accumulation during the exponential phase of the PCR reaction using a dual-labelled fluorogenic probe, has been developed. This method allows direct detection of PCR-product formation by measuring the increase in fluorescent emission continuously during the PCR reaction. Here we present data validating this PCR-method for the quantification of murine cytokines and other factors playing a role in immune regulation (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-13, IL-15, IFN-gammaTNF-alphaTGF-beta and iNOS). For each substance of interest, a set of primers and internal probe was designed, which specifically amplify the target cDNA, not co-amplifying contaminating genomic DNA. Furthermore, a corresponding reference plasmid cDNA clone was constructed, allowing direct quantification. Additionally, normalization to the housekeeping genes beta-actin or GAPDH was performed. The assay is very sensitive and accurate. It is a "closed-tube" PCR reaction, avoiding time-consuming and hazardous post-PCR manipulations and decreasing the potential risk of PCR contamination.
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PMID:Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR. 1032 70

Total ribonucleic acid (RNA) isolated from a continuous canine macrophage cell line (DH82) was used in reverse transcription polymerase chain reactions (RT-PCR) for the detection of transcripts of interleukin (IL)-8, -12, and tumour necrosis factor-alpha (TNF). Three different methods of RNA isolation (standard guanidinium-thiocyanate method with and without application of RNA matrix, and boiling) were used and compared in regard to RT-PCR results. The most suitable method was used to establish RT-PCR amplification of mRNA transcripts of IL-2, -10, and interferon-gamma (IFN) in RNA isolated from canine peripheral blood leukocytes. Integrity of RNA isolates was ensured by amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or beta-actin, IL-8, -12, and TNF were amplified from RNA isolated by various methods. Use of guanidinium-thiocyanate with and without RNA matrix gave the most consistent results. Boiling as a mean of RNA isolation was quick and easy, but the RT-PCR results were extremely variable and multiple smaller bands were observed in the agarose gel in some preparations. IL-2, -10 and IFN transcripts were amplified from RNA isolated with guanidinium-thiocyanate from leukocytes stimulated with concanavalin A. DNase-treatment of RNA isolates was necessary to assure the destruction of genomic DNA and to avoid amplification of genomic sequences. This was especially a problem when using primers for GAPDH, beta-actin, IL-12, and TNF. Lack of DNase-treatment may lead to false positive results. This may be especially a problem when amplification of so-called house-keeping genes is used as internal control for RNA integrity. These findings demonstrated that isolation of total RNA with guanidinium-thiocyanate followed by DNase-treatment gave reliable and consistent results for detection of cytokine transcripts by RT-PCR in a canine macrophage cell line and canine peripheral blood leukocytes.
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PMID:RT-PCR amplification of various canine cytokines and so-called house-keeping genes in a species-specific macrophage cell line (DH82) and canine peripheral blood leukocytes. 1041 64

Further characterization of the canine immune system will greatly benefit from the availability of tools to detect canine cytokines. Our interest concerns the study on the role of cytokines in canine visceral leishmaniasis. For this purpose, we have designed specific primers using previously published sequences for the detection of canine IL-2, IFN-gamma and IL10 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). For IL-4, we have cloned and sequenced this cytokine gene, and developed canine-specific primers. To control for sample-to-sample variation in the quantity of mRNA and variation in the RT and PCR reactions, the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a housekeeping gene, were determined in parallel. Primers to amplify G3PDH were designed from consensus sequences obtained from the Genbank database. The mRNA levels of the cytokines mentioned here were detected from ConA-stimulated peripheral mononuclear cells derived from Leishmania-infected dogs. A different pattern of cytokine production among infected animals was found.
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PMID:Detection of canine cytokine gene expression by reverse transcription-polymerase chain reaction. 1050 99

Eye muscle (EM) and retroorbital fat tissue are two major sites of involvement in thyroid-associated ophthalmopathy (TAO). Lymphocytic infiltration in these tissues is a prominent histological feature of TAO. We have investigated the cytokine gene profiles in EM and orbital fat (OF) tissues from patients with TAO. Total RNA was isolated from EM tissue of 14 patients and from OF tissues of 29 patients with TAO. Cytokine gene expression was assessed by RT-PCR using paired primers for interferon gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-10, CD4, CD8, and glyceraldehyde-3-phosphate dehydrogenase. IFNgamma, TNFalpha, IL-1beta, and IL-6 messenger RNA (mRNA) were mainly detected in EM tissue, whereas IL-4 and IL-10 mRNA were detected in only one patient. On the other hand, in OF tissue, IL-4 and IL-10 mRNA were detected in 24% and 38% of the patients, respectively, and IFNgamma, IL-1beta, and IL-6 mRNA were less often detected compared with EM tissue. The enlargement of EM tissue as assessed by computed tomography correlated significantly with TNFalpha mRNA expression in EM tissue. The orbital volume was positively correlated with IL-6 mRNA expression and negatively correlated with IL-4 mRNA and IL-10 mRNA expression in OF tissue. These results suggest that T helper (Th) 1-like cytokines predominate in EM tissue in most patients and that the predominant cytokine profile in OF tissue varies from patient to patient. Both Th1-like and Th2-like immune responses may play roles in the development of two components of ophthalmopathy.
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PMID:Cytokine profiles in eye muscle tissue and orbital fat tissue from patients with thyroid-associated ophthalmopathy. 1072 61


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