Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have synthesized, cloned, and expressed the coding region for the C-terminal 159 amino acids (aa) of the human active interleukin polypeptide hormone IL-1 alpha. The sequence was assembled in stages and includes preferred Escherichia coli codons and unique restriction sites. The coding region was cloned on a multicopy plasmid vector adjacent to signals for transcription and translation that directed synthesis of 6% of total E. coli protein as IL-1 alpha. Active IL-1 alpha mutants that have a C-terminal additional eleven aa and that have N-terminal deletions of six and fourteen aa are described. Plasmids expressing beta-galactosidase fusion proteins with various parts of IL-1 alpha at their N-termini were constructed.
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PMID:Expression in Escherichia coli of synthetic human interleukin-1 alpha genes encoding the processed active protein, mutant proteins, and beta-galactosidase fusion proteins. 310 56

Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha-induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking.
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PMID:Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression. 763 22

1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) is a synthetic diether phospholipid that is competitive with phosphatidylserine binding to the regulatory domain of protein kinase C (PKC). Our previous studies indicate that the selective inhibition of tumor cell growth by ET-18-OCH3 may be due to altered signal transduction mechanisms, including the inhibition of PKC. To further define the mechanism of action of ET-18-OCH3, we have used it to study the role of PKC in regulation of the transcription factor NF-kappa B, which is activated by diverse stimuli. In the 293.27.2 human kidney cell line, as in hematopoietic cells of all lineages, NF-kappa B is stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 alpha (IL-1 alpha). The response to either TNF-alpha or IL-1 alpha is synergistically enhanced by TPA. However, the regulatory mechanisms and signal transduction systems responsible for NF-kappa B activation in response to these different stimuli have not been determined in detail. We have used ET-18-OCH3 and auranofin, which inhibit PKC by different mechanisms, to assess the role of PKC in NF-kappa B activation. ET-18-OCH3 markedly inhibits TPA-induced NF-kappa B activation, as measured by HIV long terminal repeat-directed expression of beta-galactosidase. The IC50 for inhibition by ET-18-OCH3 is approximately 2 microM, a noncytotoxic concentration. Inhibition of TPA-induced NF-kappa B activation was dependent upon preincubation with ET-18-OCH3, and the drug was active at approximately 2 mol% of total cellular phospholipid. ET-18-OCH3 did not inhibit NF-kappa B activation by either TNF-alpha or IL-1 alpha, indicating that there are multiple distinct signal transduction pathways leading to activation of NF-kappa B. We have confirmed these results using auranofin, an antirheumatic drug that is a specific PKC inhibitor interacting with the catalytic domain. Like ET-18-OCH3, auranofin blocked NF-kappa B activation by TPA but not by TNF-alpha or IL-1 alpha. Also like the ether lipid, auranofin only partially blocked the synergy exhibited by TPA and TNF-alpha. To confirm the role of NF-kappa B in this response, we measured NF-kappa B by electrophoretic mobility shift assay. Both ET-18-OCH3 and auranofin inhibited cellular induction of the active NF-kappa B complex in response to TPA but not in response to TNF-alpha.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:ET-18-OCH3 inhibits nuclear factor-kappa B activation by 12-O-tetradecanoylphorbol-13-acetate but not by tumor necrosis factor-alpha or interleukin 1 alpha. 758 18

We have previously shown that the signal peptideless cytokine interleukin 1 alpha (IL-1 alpha) may play a role as an intracellular regulator of human endothelial cell senescence (J. A. M. Maier, P. Voulalas, D. Roeder, and T. Maciag, Science 249:1570-1574, 1990). To investigate the potential intracellular function of IL-1 alpha, transformed endothelial cells were transfected with the human cDNAs that code for the two forms of IL-1 alpha, the precursor molecule IL-1(1-271) and the mature protein IL-1(113-271). The subcellular localization of the two different polypeptides was investigated directly or by using chimeric genes constructed by fusion of different fragments of the IL-1 alpha gene and the beta-galactosidase open reading frames. The IL-1(113-271) protein was cytoplasmic, while IL-1(1-271) was nuclear. The basic cluster at the NH2 terminus of IL-1, KVLKKRR, has been shown to mediate IL-1 alpha nuclear targeting. Moreover, nuclear localization of IL-1 alpha correlates with impaired cell growth and expression of some IL-1 alpha-inducible genes. These results suggest that transport of endogenous IL-1(1-271) into the nucleus is required for it to modulate endothelial cell function.
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PMID:Endogenous interleukin 1 alpha must be transported to the nucleus to exert its activity in human endothelial cells. 811 17

Recent studies have suggested that the signal peptide-less cytokine, interleukin (IL)-1 alpha, may play a role as an intracellular regulator of human endothelial cell proliferation in vitro (Garfinkel, S., Haines, D. S., Brown, S., Wessendorf, J., Gillespie, D. H., and Maciag, T. (1992) J. Biol. Chem. 267, 24375-24378). In order to determine the intracellular locale of the IL-1 alpha precursor, we fused the open reading frame of the IL-1 alpha precursor to the reporter gene beta-galactosidase (Gal) and studied the cellular distribution of the chimera in NIH 3T3 cells after transfection. Immunological and enzymatic analysis demonstrated that the IL-1 alpha:beta-Gal fusion protein was associated with the nucleus. To further define the region responsible for this activity, we ligated the mature form of IL-1 alpha (IL-1 alpha 113-271) and the IL-1 alpha precursor domain (IL-1 alpha 1-112) to beta-Gal. Analysis of the intracellular distribution of these chimeric polypeptides following transfection demonstrated a differential distribution of IL-1 alpha 1-112:beta-Gal in the nucleus and IL-1 alpha 113-271:beta-Gal in the cytosol. Because the IL-1 alpha precursor domain contains a sequence that resembles a nuclear translocation signal (KVLKKRRL, residues 79-86), we prepared an IL-1 alpha precursor point mutant in which Lys82 was replaced by Glu. Transfection of NIH 3T3 cells with the IL-1 alpha precursor point mutant (IL-1 alpha 1-271 Glu82:beta-Gal) resulted in a significant reduction in the ability of the IL-1 alpha precursor to associate with the nucleus and similar data were obtained as a result of Lys82 mutagenesis in the IL-1 alpha precursor domain (IL-1 alpha 1-112 Glu82:beta-Gal). These data suggest that the IL-1 alpha precursor contains a functional nuclear localization sequence within the structure of the precursor domain and Lys82 is critical for its function.
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PMID:Identification of a nuclear localization sequence within the structure of the human interleukin-1 alpha precursor. 840 68

In this study, an in vitro model has been developed to examine the interactions of macrophages with ultrahigh molecular-weight polyethylene (UHMWPE) and high-density polyethylene (HDPE) particles. Polyethylene particles are the major constituent of the material debris formed as a result of orthopedic implant wear. However, the study of polyethylene particle interactions with cells has been limited. UHMWPE (18-20 microns) and HDPE (4-10 microns) were suspended in soluble collagen type I and subsequently solidified on glass coverslips. The particle chemistry was characterized by Fourier transform infra-red spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). Mouse cell line macrophages (IC-21) were established on the collagen-particle substrata and maintained for up to 24 h. The response of the cells to the particles was examined by light and transmission electron microscopy (LM and TEM), as well as by scanning electron microscopy (SEM), and compared to cells on control collagen surfaces without particles. Histological analysis of the samples revealed that the macrophages surrounded larger particles (18-20 microns) and the cells appeared to be attached to the surface of the particles, and the smaller particles (4-10 microns) had been phagocytosed within 2 h. Inflammatory cytokines (TNF-alpha, IL-1 alpha, IL-1 beta, and IL-6), lysosomal enzymes (beta-galactosidase and hexosaminidase), and prostaglandin E2 were released into the medium, and IL-1 alpha, IL-1 beta, PGE2, beta-galactosidase, and hexosaminidase levels were significantly increased over collagen control values. The results demonstrate active phagochemotaxis by macrophages for wear particulates and validate this model as a means of studying the specific in vitro interactions of polyethylene with cells.
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PMID:Macrophage phagocytosis of polyethylene particulate in vitro. 942 95