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)

Chemical conjugates of recombinant soluble CD4 (sCD4) with toxins, or with antibodies that activate cytotoxic T cells, can be used to direct selective killing of human immunodeficiency virus (HIV)-infected cells. This approach takes advantage of the ability of sCD4 to bind with high affinity to gp120, the envelope protein of HIV-1, which is expressed on actively infected cells. However, conjugation of sCD4 via reagents that target amino groups may reduce its affinity for gp120, since at least one such group is important for gp120 binding. Here, we describe a novel cross-linking reagent which enables the conjugation of sCD4 via its carbohydrate moieties rather than its free amino groups. This heterobifunctional reagent, 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), combines a nucleophilic hydrazide with an electrophilic maleimide, thereby allowing coupling of carbohydrate-derived aldehydes to free thiols. We describe conditions by which MPBH is coupled selectively to the sialic acid residues of sCD4, and exemplify the use of MPBH by conjugating sCD4 to hemoglobin and to beta-galactosidase. We show that, whereas conjugation of sCD4 via amino groups markedly reduces its gp120 binding affinity, conjugation via the carbohydrate chains using MPBH does not affect binding. Moreover, we demonstrate the ability of a sCD4-MPBH-fluorescein conjugate to label HIV-infected human CEM cells selectively. These results indicate that, by targeting its carbohydrate moieties, sCD4 can be cross-linked to other molecules without compromising its function. The approach described here can be useful for glycoproteins in which amino groups, but not carbohydrates, are important for function. More generally, this approach can be considered for use in cross-linking glycoconjugates to compounds which either contain thiols, or to which thiols can be added.
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PMID:Conjugation of soluble CD4 without loss of biological activity via a novel carbohydrate-directed cross-linking reagent. 163 20

The multiple drug-resistant human lymphoblastic leukemic cell, CEM/VLB100, in which P-glycoprotein (P-170) is overexpressed, has a lowered content of lysosomal enzymes, such as N-acetylglucosaminidase and beta-galactosidase, and the relative rates of secretion of these enzymes are significantly greater than those of its drug-sensitive counterpart, CEM. The ability of CEM/VLB100 cells to accumulate [3H]vinblastine ([3H]-VLB) is also greatly reduced. Multiple drug-resistant cells whose mode of resistance is not associated with P-170 do not have reduced enzyme content, and their rate of secretion is the same as that of their drug-sensitive parents. Linkage of drug and enzyme elimination is suggested by the observation that verapamil inhibits both the efflux of [3H]VLB and the secretion of lysosomal enzymes in CEM/VLB100 cells; the content of both [3H]VLB and enzyme increases in these cells when chronically exposed to verapamil. Further, both secretion of N-acetylglucosaminidase and efflux of [3H]VLB by CEM/VLB100 cells are enhanced by the addition of NaCl to the suspending, sucrose-containing medium. When cells have taken up [3H]VLB and are then fractionated by means of a Percoll centrifugation gradient, the distribution of drug among the various populations of vesicles is similar to that of N-acetylglucosaminidase. Losses of both enzyme and drug take place from these vesicular populations to varying degrees, when CEM/VLB100 cells are induced to secrete. It is proposed that, in a multiple drug-resistant cell such as CEM/VLB100, the presence of P-170 in the plasma membrane may, in some indirect manner, lead to increased exocytosis of lysosomal enzyme, ultimately resulting in a significant depletion of enzyme. Further, a toxic, cationic drug such as vinblastine, accumulating in lysosomes and acidic vesicles, is also eliminated from the cell by exocytosis. This pathway may supplement the known, major mode of efflux directly involving P-170.
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PMID:Secretion of lysosomal enzymes by drug-sensitive and multiple drug-resistant cells. 167 22

The sequences encoding the p24 core protein of human immunodeficiency virus type 1 were identified in a cDNA library made from infected CEM cells. The nucleotide sequence of the DNA coding for p24 was shown to be very similar but not identical to the sequences of lymphadenopathy virus and human T-cell leukaemia virus type IIIb. These sequences were expressed in Escherichia coli at the amino terminus of beta-galactosidase and used to screen a panel of monoclonal antibodies raised against virus-expressed p24. Regions containing the epitopes of five of the monoclonal antibodies were located using a series of amino- and carboxy-terminal deletion mutants of the recombinant p24 protein.
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PMID:The cloning and expression in Escherichia coli of sequences coding for p24, the core protein of human immunodeficiency virus, and the use of the recombinant protein in characterizing a panel of monoclonal antibodies against the viral p24 protein. 247 10

Modifications of the glutathione (GSH) intracellular level have been implicated in the regulation of human immunodeficiency virus (HIV) transcription and expression. In regard to this hypothesis, we have investigated the effects of valproic acid (VPA) on HIV replication. Indeed, it has been recently reported that VPA inhibits the human red blood cell glutathione reductase. In the supernatant of a CEM-SS T-lymphocytic cell line infected with the LAI strain of HIV-1, we observed an increase, in a dose-dependent fashion, of the reverse transcriptase activity after treatment of cells with VPA. VPA also induced HIV expression in the chronically infected monocytic U1 cell line which constitutively expresses low levels of virus, enhanced the HIV-long terminal repeat (LTR)-directed expression of beta-galactosidase in transiently transfected Jurkat T-cells, and potentiated the PMA effect on the LTR transactivation. GSH assays showed that VPA treatment led to a decrease in the intracellular level of this thiol compound in U937 (U1 parent-cell line) and in Jurkat T-cells. Work to understand the molecular mechanism of VPA-induced HIV transcription and expression are now in progress. VPA seems to be an adequate molecule to study the implications of a GSH decrease in the stimulation of HIV replication. However, a modification of the intracellular balance between reduced and oxidized glutathione, rather than a simple reduction of the intracellular glutathione level, could be of importance in the regulation of HIV replication and we are now testing this hypothesis. Finally, these findings already suggest that VPA, which is an anticonvulsive drug frequently prescribed for the management of various seizure disorders, should not be recommended for treatment of epilepsy or other related illnesses in HIV-positive individuals.
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PMID:Valproic acid reduces the intracellular level of glutathione and stimulates human immunodeficiency virus. 751 59

Cell-surface sugar receptors may participate in interactions of lymphoid cells that influence their adhesive properties and proliferation. Their expression on cells of the pre-B line BLIN-I, the B-lymphoblastoid line Croco II, the myeloma line RPMI 8226 and the T-lymphoblastoid line CCRF-CEM was monitored with a panel of 14 types of chemically glycosylated E. coli beta-galactosidase at a non-saturating ligand concentration. Quantitative differences were determined for the capacity of the different cell types to bind constituents of the carbohydrate part of glycoconjugates. They were corroborated by analyses of binding for lactose-, beta-N-acetylgalactosamine-, beta-N-acetylglucosamine- and fucose-exposing neoglycoenzymes up to saturation levels. Values of dissociation constants of the tetrameric enzyme were in the range of 3-300 nM. Several types of sugar receptor led to carbohydrate-inhibitable adhesion of cells to 6 types of nitrocellulose-immobilized neoglycoprotein, their effectiveness being most obvious for the myeloma cells. Analyses of the carbohydrate-ligand-mediated adhesion of the other cell types revealed a comparatively decreased response. Only a few carbohydrates among the 7 types tested were effective in reducing cell adhesion to a far more complex ligand-bearing matrix than immobilized neoglycoproteins, namely bone-marrow stromal cell layers: sialic acid and N-acetylgalactosamine for B-lymphoblastoid cells and rhamnose for pre-B cells. These cellular interactions may encompass sugar receptors on the stromal cells and other types of molecular recognition in addition to the detected activities on the lymphoid cells.
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PMID:Adhesion of human lymphoid cell lines to immobilized carbohydrates and to bone-marrow stromal cell layers by surface sugar receptors. 839 77

Sodium valproate (VPA), a simple branched-chain fatty acid that has anticonvulsant activity and is used in the treatment of many forms of epilepsy, has been reported to stimulate human immunodeficiency virus (HIV) type 1 replication in acutely infected CEM and chronically infected U1 cells (Chemico-Biological Interactions 1994;91:111-121). When attempting to reproduce and extend these findings, we confirmed that VPA is able to stimulate HIV-1(IIIB) replication in acutely infected CEM and C8166 T lymphocytic cell lines and chronically infected ACH-2 and U937/IIIB/LAI cells in a concentration-dependent manner. The stimulatory effect of VPA on HIV replication in CEM cells was not increased by pretreatment of the cells with VPA for 24 hr before infection. However, we could not detect any stimulatory effect of VPA on HIV-1(IIIB) replication in acutely infected peripheral blood mononuclear cells (PBMCs), MT-4, MT-2, HUT-78, and MOLT-4 (clone 8) cells and in chronically infected HUT-78/IIIB/LAI cells. The stimulatory effect by VPA under certain conditions (see above) may be ascribed to an enhanced HIV transcription, as VPA was found to enhance the HIV long terminal repeat (LTR)-directed expression of beta-galactosidase in transiently transfected HLtat, P4, and COS7 cells. VPA did not enhance beta-galactoside expression mediated by the cytomegalovirus (CMV) promoter. VPA did not affect HIV-induced syncytium formation. Nor had VPA any direct inactivating effect on HIV.
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PMID:Cell type-dependent effect of sodium valproate on human immunodeficiency virus type 1 replication in vitro. 900 4