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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A system to innocuously visualize T cell lineage commitment is described. Using a "knock-in" approach, we have generated mice expressing a beta-galactosidase reporter in place of CD4; expression of beta-galactosidase in these animals appears to be an accurate and early indicator of CD4 gene transcription. We have exploited this knock-in line to trace CD4/CD8 lineage commitment in the thymus, avoiding important pitfalls of past experimental approaches. Our results argue in favor of a selective model of thymocyte commitment, demonstrating a fundamentally symmetrical process: engagement of either class of major histocompatibility complex (MHC) molecule by a differentiating CD4(+)CD8(+) cell can give rise to T cell antigen receptor (TCR)hi thymocytes of either lineage. Key findings include (a) direct demonstration of a substantial number of CD4-committed, receptor/coreceptor-mismatched cells in MHC class II- deficient mice, a critical prediction of the selective model; (b) highly efficient rescue of such "mismatched" intermediates by forced expression of CD8 in a TCR transgenic line, and an explanation of why previous experiments of this nature were less successful-a major past criticism of the selective model; (c) direct demonstration of an analogous, though smaller, population of CD8-committed mismatched intermediates in class I-deficient animals. Finally, we found no evidence of a CD4 default pathway.
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PMID:Visualization of CD4/CD8 T cell commitment. 985 18

Marek's disease is a herpesvirus (Marek's disease virus [MDV])-induced pathology of chickens characterized by paralysis and the rapid appearance of T-cell lymphomas. Lymphoblastoid cell lines (LBCLs) derived from MDV-induced tumors have served as models of MDV latency and transformation. We have recently reported the construction of mutant MDVs having a deletion (M. S. Parcells et al., J. Virol. 69:7888-7898, 1995) and an insertion (A. S. Anderson et al., J. Virol. 72:2548-2553, 1998) within the unique short region of the virus genome. These mutant MDVs retained oncogenicity, and LBCLs have been established from the mutant-induced tumors. We report the characterization of these cell lines with respect to (i) virus structure within and reactivated from the cell lines, (ii) surface antigen expression, (iii) kinetics of MDV and marker gene induction, (iv) localization and colocalization of induced MDV antigens and beta-galactosidase (beta-Gal), and (v) methylation status of the region of lacZ insertion in recombinant- and non-recombinant-derived cell lines. Our results indicate that (i) recombinant-derived cell lines contain no parental virus, (ii) the established cell lines are predominantly CD4(+) CD8(-), (iii) the percentage of Lac-expressing cells is low (1 to 3%) but increases dramatically upon 5'-iododeoxyuridine (IUdR) treatment, (iv) lacZ expression is induced with the same kinetics as several MDV lytic-phase genes (pp38, US1, gB, gI, and US10), and (v) the regulation of lacZ expression is not mediated by methylation. Furthermore, the MDV-encoded oncoprotein, Meq, could be detected in cells expressing beta-Gal and various lytic antigens but did not appear to be induced by IUdR treatment. Our results indicate that regulation of the lacZ marker gene can serve as sensitive measure of virus lytic-phase induction and the reactivation from latency.
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PMID:Recombinant Marek's disease virus (MDV)-derived lymphoblastoid cell lines: regulation of a marker gene within the context of the MDV genome. 988 41

The monoclonal antibody (mAb) ST40, specific for the immunoglobulin complementarity-determining region (CDR) 3-like loop in domain 1 of the CD4 molecule, inhibits human immunodeficiency virus type 1 (HIV-1) promoter activity and viral transcription in HIV-infected cells. To design synthetic peptides from the ST40 paratope that could mimic these biological properties, a set of 220 overlapping 12-mer peptides frameshifted by one residue, corresponding to the deduced ST40 amino acid sequence, was synthesized by the Spot method and tested for binding to recombinant soluble CD4 antigen. Several peptides that included in their sequences amino acids from the CDRs of the antibody and framework residues flanking the CDRs were found to bind soluble CD4. Eleven paratope-derived peptides (termed CM1-CM11) were synthesized in a cyclic and soluble form. All the synthetic peptides showed CD4 binding capacity with affinities ranging from 1.6 to 86.4 nM. Moreover, peptides CM2, CM6, CM7, CM9, and CM11 were able to bind a cyclic peptide corresponding to the CDR3-like loop in domain 1 of CD4 (amino acids 81-92 of CD4). Peptide CM9 from the light chain variable region of mAb ST40 and, to a lesser extent, peptides CM2 and CM11 were able to inhibit HIV-1 promoter long terminal repeat-driven beta-galactosidase gene expression in the HeLa P4 HIV-1 long terminal repeat beta-galactosidase indicator cell line infected with HIV-1. The binding of mAb ST40 to CD4 was also efficiently displaced by peptides CM2, CM9, and CM11. Our results indicate that the information gained from a systematic exploration of the antigen binding capacity of synthetic peptides from immunoglobulin variable sequences can lead to the identification of bioactive paratope-derived peptides of potential pharmacological interest.
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PMID:Synthetic peptides derived from the variable regions of an anti-CD4 monoclonal antibody bind to CD4 and inhibit HIV-1 promoter activation in virus-infected cells. 992 Sep 32

Transfer of genes by injection of plasmid DNA into skeletal muscle has a wide variety of applications ranging from treatment of neuromuscular disorders to genetic vaccination. We examined each component involved in the intramuscular injection of plasmid DNA in terms of the induction of inflammatory responses. The insertion of a needle and the injection of a relatively large volume of saline caused very little muscle damage except in rare cases. In contrast, barium chloride-induced regeneration of muscle, injection of lipopolysaccharide, plasmid backbone or plasmid expressing a neo-antigen (beta-galactosidase) all generated widespread inflammation of injected muscle, with mononuclear infiltrate, comprised largely of macrophages and with both CD4+ and CD8+ T lymphocytes, present. Such inflammation may hamper clinical application of this technology and may encourage undesirable immune responses in gene therapy trials. Inflammation was not greatly reduced by CD4- or CD8-depleting antibodies, suggesting this initial inflammation did not involve T cells, but methylation of plasmid DNA before injection substantially lessened the inflammatory response and resulted in longer term expression of the transgene.
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PMID:Inflammatory responses following direct injection of plasmid DNA into skeletal muscle. 993 Mar 31

This study characterized gene transfer into both normal and injured adult rat dorsal spinal cord using first (E1-/E3-) or second (E1-/E2A125/E3-, temperature-sensitive; ts) generation of replication-defective adenoviral (Ad) vectors. A novel immunosuppressive regimen aimed at blocking CD4/CD45 lymphocytic receptors was tested for improving transgene persistence. In addition, the effect of gene transfer on nociception was also evaluated. Seven days after treatment, numerous LacZ-positive cells were observed after transfection with either viral vector. By 21 days after transfection, beta-galactosidase staining was reduced and suggestive of ongoing cytopathology in both Ad-treated groups, despite the fact that the immunogenicity of LacZ/Adts appeared less when compared with that elicited by the LacZ/Ad vector. In contrast, immunosuppressed animals showed a significant (P < or = 0.05) increase in the number of LacZ-positive cells not displaying cytopathology. In these animals, a concomitant reduction in numbers of macrophages/microglia and CD4 and CD8 lymphocytes was observed. Only animals that received LacZ/Adts and immunosuppression showed transgene expression after 60 days. Similar results were observed in animals in which the L4-L5 dorsal roots were lesioned before transfection. Gene transfer into the dorsal spinal cord did not affect nociception, independent of the adenovirus vector. These results indicate that immune blockade of the CD4/CD45 lymphocytic receptors enhanced transgene stability in adult animals with normal or injured spinal cords and that persistent transgene expression in the spinal cord does not interfere with normal neural function.
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PMID:Adenoviral gene transfer into the normal and injured spinal cord: enhanced transgene stability by combined administration of temperature-sensitive virus and transient immune blockade. 1002 40

The potential use as vaccine delivery system of Salmonella typhimurium strains harboring defined mutations in the sseC (HH104) and sseD (MvP101) genes, which encode putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2, was evaluated and compared with that of the well-characterized aroA mutant strain SL7207 by using beta-galactosidase (beta-Gal) as a model antigen. When orally administered to immune-competent or gamma interferon-deficient (IFN-gamma-/-) BALB/c mice, both mutants were found to be highly attenuated (50% lethal dose, >10(9) bacteria). Both strains were also able to efficiently colonize and persist in Peyer's patches. Immunization with HH104 and MvP101 triggered beta-Gal-specific serum and mucosal antibody responses equivalent to or stronger than those observed in SL7207-immunized mice. Although immunoglobulin G2 (IgG2) serum antibodies were dominant in all groups, IgG1 was also significantly increased in mice vaccinated with MvP101 and SL7207. Comparable beta-Gal-specific IgA and IgG antibodies were detected in intestinal lavages from mice immunized with the different strains. Antigen-specific CD4(+) T-helper cells were generated after vaccination with all vaccine prototypes; however, responses were significantly more efficient when HH104 and MvP101 were used (P < 0.05). Significantly higher levels of IFN-gamma were produced by restimulated spleen cells from mice immunized with HH104 than from those vaccinated with the MvP101 or SL7207 derivatives (P </= 0.05). Interestingly, the three strains induced major histocompatibility complex class I-restricted CD8(+) cytotoxic T cells against beta-Gal; however, cytotoxic T-lymphocyte responses were significantly stronger after immunization with HH104 (P < 0.05). These novel S. typhimurium attenuated strains constitute promising delivery systems for vaccine antigens. The qualitative differences observed in the obtained responses with different carriers may be useful for those applications in which a targeted immunomodulation is required.
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PMID:Pathogenicity island 2 mutants of Salmonella typhimurium are efficient carriers for heterologous antigens and enable modulation of immune responses. 1002 48

The retroviral nucleocapsid (NC) protein contains highly conserved amino acid sequences (-Cys-X2-Cys-X4-His-X4-Cys-) designated retroviral (CCHC) Zn2+ fingers. The NC protein of murine leukemia viruses contains one NC Zn2+ finger and mutants that were competent in metal binding (CCCC and CCHH) packaged wild-type levels of full-length viral RNA but were not infectious. These studies were extended to human immunodeficiency virus type 1 (HIV-1), a virus with two NC Zn2+ fingers. Viruses with combinations of CCHC, CCCC, and CCHH Zn2+ fingers in each position of HIV-1 NC were characterized. Mutant particles contained the normal complement of processed viral proteins. Four mutants packaged roughly wild-type levels of genomic RNA, whereas the remaining mutants packaged reduced levels. Virions with mutated C-terminal position NC fingers were replication competent. One interesting mutant, containing a CCCC Zn2+ finger in the N-terminal position of NC, packaged wild-type levels of viral RNA and showed approximately 5% wild-type levels of infectivity when examined in CD4-expressing HeLa cells containing an HIV-1 LTR/beta-galactosidase construct. However, this particular mutant was replication defective in H9 cells; all other mutants were replication defective over the 8-week course of the assay. Two long terminal repeat viral DNA species could be detected in the CCCC mutant but not in any of the other replication-defective mutants. These studies show that the N-terminal Zn2+ finger position is more sensitive to alterations than the C-terminal position with respect to replication. Additionally, the retroviral (CCHC) NC Zn2+ finger is required for early infection processes. The evolutionary pressure to maintain CCHC NC Zn2+ fingers depends mainly on its function in infection processes, in addition to its function in genome packaging.
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PMID:Strict conservation of the retroviral nucleocapsid protein zinc finger is strongly influenced by its role in viral infection processes: characterization of HIV-1 particles containing mutant nucleocapsid zinc-coordinating sequences. 1008 30

We recently established an effective immune T-cell-mediated graft-versus-leukemia (GVL) murine model system in which complete tumor remissions were achievable even in advanced metastasized cancer. We now describe that this T-cell-mediated therapy is dependent on host macrophages expressing the lymphocyte adhesion molecule sialoadhesin (Sn). Depletion of Kupffer cells in tumor-bearing mice during adoptive immunotherapy (ADI) or the treatment of these animals with anti-Sn monoclonal antibodies led to complete or partial inhibition of the immune T-cell-mediated therapeutic effect. Furthermore, Sn+ host macrophages in livers formed clusters during ADI with donor CD8 T cells. To test for a possible antigen presentation function of these macrophages, we used as an in vitro model the antigen beta-galactosidase for which a dominant major histocompatibility complex (MHC) class I Ld-restricted peptide epitope is known to be recognized by specific CD8 cytotoxic T lymphocytes (CTL). We demonstrate that purified Sn+ macrophages can process exogenous beta-galactosidase and stimulate MHC class I peptide-restricted CTL responses. Thus, Sn+ macrophages, which are significantly increased in the liver after ADI, may process tumor-derived proteins via the MHC class I pathway as well as via the MHC class II pathway, as shown previously, and present respective peptide epitopes to CD8 as well as to CD4 immune T cells, respectively. The synergistic interactions observed before between immune CD4 and CD8 T cells during ADI could thus occur in the observed clusters with Sn+ host macrophages.
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PMID:Sialoadhesin-positive host macrophages play an essential role in graft-versus-leukemia reactivity in mice. 1036 Nov 36

In contrast to adherent cells, cells growing in suspension and particularly hematopoietic cells, are notoriously difficult to transfect in vitro using nonviral approaches. In the present study, the effect of cell adhesion on gene transfer efficacy was investigated by allowing hematopoietic cells to bind to an adherent cell monolayer (ACM) before being subjected to cationic liposome-mediated DNA transfer. Human CD34 and T CD4 cell lines were cultivated on an ACM constituted of murine fibroblast NIH3T3 cells and transfected with a plasmid carrying the beta-galactosidase gene. X-gal staining showed that up to 27% of the cells expressed the transgene. In contrast, less than 0.1% of these cells were positively transfected in suspension. This adhesion-assisted lipofection (AAL) procedure was also successfully tested on blood lymphocytes, since it resulted in up to 30% of transfected human primary T lymphocytes. Flow cytometry analysis performed on T lymphocyte subsets revealed that 8 and 9%, respectively, of CD4 and CD8 cells could be transfected with a plasmid carrying the green fluorescent protein gene. Other adherent cells, such as MS5 murine stromal cells or HeLa epithelial cells, were also a compatible matrix for AAL. Moreover, the pCMV beta plasmid was present in similar amounts in the nuclei of TF1 cells transfected in suspension or with the AAL procedure. These data raise the possibility that cell matrix/hematopoietic cell interactions might govern expression of the transgene in hematopoietic cells growing usually in suspension, but not endocytosis of liposome/DNA particles and plasmid migration ot the cell nucleus.
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PMID:Transgene expression, but not gene delivery, is improved by adhesion-assisted lipofection of hematopoietic cells. 1050 19

HIV-2 GH-1 is a molecular clone derived from an AIDS patient from Ghana. In contrast to the prototypic molecular clone ROD, GH-1 exhibits a narrow range of target cell specificity. By an infectious assay using HeLa-CD4 cells stably transfected with an HIV-1 LTR-beta-galactosidase reporter gene and transiently expressing various cloned chemokine receptors, we have examined the coreceptor usage of GH-1. In contrast to ROD, which uses principally CXCR4, GH-1 was found to use mainly if not exclusively CCR5 but not CXCR4. The distinct coreceptor usage of these two molecular clones allowed us to further map the region of gp120 that is important for the coreceptor specificity. By constructing a series of chimeric viruses between GH-1 and ROD, we have demonstrated that the C-terminal half of the V3 loop region of gp120 determines the differential coreceptor usage between GH-1 and ROD, and only a few amino acid differences in this region appear to be able to shift the specificity between CCR5 and CXCR4. Notably, the shift in the coreceptor usage from CCR5 to CXCR4 is associated with an increase in the net positive charge in the V3 region.
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PMID:Small amino acid changes in the V3 loop of human immunodeficiency virus type 2 determines the coreceptor usage for CXCR4 and CCR5. 1054 50


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