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)

The primary interaction of tetanus toxin and toxoid with mouse neuroblastoma cells (C 1300, clone NB2A) in tissue culture was studied using direct immunofluorescence. Experiments were done in standard routine cultures and also those influenced by chemical modulators. There is a difference in the characteristic binding response between the growth culture cells (grown in presence of fetal calf serum) and differentiating culture cells (grown in absence of serum). Exposure to the toxin gives no visible effect on the cell division or viability in growth cultures; whereas in differentiating cells the processes are shortened and the adherence to the glass is diminished without involving significant cell death. The toxoid did not bind at all under the same experimental conditions. Since there was no biological effect in growth cultures we have called this binding ineffective, and in the case of the differentiating cells, effective binding. Stimulation of pinocytosis increases the uptake of toxin in both cultures. Presence of some surface bound toxin still remaining on the differentiating cells indicates the possibility of another sort of mechanism for internalization. Pre-treatment of the cells with neuraminidase or beta-galactosidase to alter the membrane gangliosides eliminates binding in growth cultures but not in differentiating cultures. From these results we suggest that even though the toxin may well bind to gangliosides, at least in the differentiating cultures they are not solely responsible for the fixation. The morphologically observed effective binding is probably that not related to gangliosides.
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PMID:Interaction of tetanus toxin and toxoid with cultured neuroblastoma cells. Analysis by immunofluorescence. 32 Apr 89

An enzymatically active probe (beta-galactosidase-anti-beta-galactosidase complex) is used to measure circulating immune complexes (CIC), in a competition assay where probe and CIC are confronted with a 'recognition unit'. The latter is bovine conglutinin in the original description of this method. Here we describe a version utilizing human or bovine C1q. The two techniques are compared for their sensitivity and specificity, on both in vitro formed tetanus toxoid-anti-toxoid complexes and on sera from patients with selected diseases. The results confirm that the two recognition units are sensitive to families of CIC that only partially overlap. The parallel use of conglutinin and C1q yields both quantitative and qualitative information on the nature of CIC in individual sera.
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PMID:An enzymatically active antigen-antibody probe to measure circulating immune complexes. II. E. coli beta-galactosidase in the probe and C1q as the recognition unit. 640 86

Peptides of melanosomal proteins have recently been shown to be recognized in an HLA-restricted mode by specific cytolytic T lymphocytes in melanoma patients. Dendritic antigen-presenting cells (DC) are considered to be the most effective stimulators of T cell responses, and the use of these cells has therefore been proposed to generate therapeutic responses to tumor antigens in cancer patients. We, therefore, generated DC from peripheral blood of normal donors in the presence of granulocyte/macrophage colony-stimulating factor and interleukin-4. Flow cytometric analysis of the cells during a 2-week culture revealed a loss of CD14 and CD34 expression, a concomittent increase of CD1a, CD11a,b and c, CD44, CD45, CD54, HLA-class I and II, and intermediate levels of CD26, CD80 and CD86. Cultured DC stimulated proliferation of allogeneic T cells and induced a marked, up to 20-fold, stimulation of T cell proliferation after pulsing with tetanus toxoid. To achieve independence of already-identified antigenic peptides presented in HLA class I-restricted fashion, which limits the general applicability of such peptides for vaccination of melanoma patients, we tested whether DC are transfectable with eukaryotic expression plasmids. DC transfected with two reporter genes (CAT, beta-galactosidase) using a liposome-based transfection technique, exhibited only low levels of enzymatically active proteins, but were able to degrade rapidly intracellular proteins and to process peptides efficiently. Chloramphenicol acetyltransferase as well as tyrosinase mRNA were detectable after transfection by reverse-transcriptase-polymerase chain reaction, and enzyme activities became measurable. Furthermore, DC transfected with the tyrosinase gene were able to induce specific T cell activation in vitro, indicating appropriate peptide processing and presentation in DC after transfection. These data suggest new approaches to future tumor vaccination strategies.
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PMID:Dendritic cells generated from peripheral blood transfected with human tyrosinase induce specific T cell activation. 748 49

Potential labels for identifying embryonic raphe neurons and a clonal, neuronally differentiating, raphe-derived cell line, RN33B, in CNS transplantation studies were tested by first characterizing the labels in vitro. The labels that were tested included 4',6-diamidino-2-phenylindole hydrochloride, 1,1'-dioctadecyl-3,3,3'-tetramethylindocarbocyanine perchlorate, the Escherichia coli lacZ gene, Fast Blue, Fluoro-Gold, fluorescein-conjugated latex microspheres, fluorescein isothiocyanate-conjugated or nonconjugated Phaseolus vulgaris leucoagglutinin, methyl o-(6-amino-3'-imino-3H-xanthen-9-yl) benzoate monohydrochloride, or tetanus toxin C fragment. Subsequently, the optimal in vitro labels for embryonic raphe neurons and for RN33B cells were characterized in vivo after CNS transplantation. In vitro, 1,1'-dioctadecyl-3,3,3'-tetramethylindocarbocyanine perchlorate (DiI) optimally labeled embryonic neurons. The Escherichia coli lacZ gene optimally labeled RN33B cells. Most labels were rapidly diluted in cultures of embryonic astrocytes and proliferating RN33B cells. Some labels were toxic and were often retained in cellular debris. In vivo, DiI was visualized in transplanted, DiI-labeled raphe neurons, but not in astrocytes up to 1 mo posttransplant. DiI-labeled host cells were seen after transplantation of lysed, DiI-labeled cells. beta-Galactosidase was visualized in transplanted, Escherichia coli lacZ gene-labeled RN33B cells after 15 days in vivo. No beta-galactosidase was seen in host cells after transplantation of lysed, lacZ-labeled RN33B cells. The results demonstrate that labels for use in CNS transplantation studies should be optimized for the specific population of donor cells under study, with the initial step being characterization in vitro followed by in vivo analysis. Appropriate controls for false-positive labeling of host cells should always be assessed.
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PMID:In vitro labeling strategies for identifying primary neural tissue and a neuronal cell line after transplantation in the CNS. 814 80

B lymphocytes committed to the production of antibodies binding to antigens on pathogenic bacteria and viruses (natural antibodies) are common components of the normal human B cell repertoire. A major proportion of natural antibodies is capable of binding multiple antigens (polyreactive antibodies). Using B cells from three HIV-1 seronegative healthy subjects, and purified HIV-1 and beta-galactosidase from Escherichia coli as selecting antigen, we generated three natural IgM mAb to HIV-1 and a natural IgM mAb to beta-galactosidase. The three HIV-1-selected antibodies (mAb102, mAb103, and mAb104) were polyreactive. They bound with different affinities (Kd = 10(-6) to 10(-8) M) to the HIV-1 envelope gp160, the p24 core protein, and the p66 reverse transcriptase, but not to the 120 glycosylated env protein. They also bound to beta-galactosidase (Kd approximately 10(-7) M), tetanus toxoid, and various various self antigens. In contrast, the natural mAb selected for binding to beta-galactosidase (mAb207.F1) was monoreactive, in that it bound with a high affinity (Kd < 10(-8) M) to this antigen, but to none of the other antigens tested, including HIV-1. Structural analysis of the VH and VL segments revealed that the natural mAb utilized three segments of the VHIV gene family and one of the VHIII family, in conjunction with VL segments of the V lambda I, V lambda II, V lambda III, or V kappa IV subgroups. In addition, the natural mAb VH and VL segments were in unmutated or virtually unmutated (germline) configuration, including those of the monoreactive mAb207.F1 to beta-galactosidase, and were identical or closely related to those utilized by specific autoantibodies or specific antibodies to viral and/or bacterial pathogens. Thus, the present data show that both polyreactive and monoreactive natural antibodies to foreign antigen can be isolated from the normal human B cell repertoire. They also suggest that the VH and VL segments of not only polyreactive but also monoreactive natural antibodies can be encoded in unmutated or minimally mutated genes, and possibly provide the templates for the specific high affinity antibodies elicited by self or foreign antigens.
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PMID:Structure of the VH and VL segments of polyreactive and monoreactive human natural antibodies to HIV-1 and Escherichia coli beta-galactosidase. 831 22

The nontoxic proteolytic C fragment of tetanus toxin (TTC peptide) has the same ability to bind nerve cells and be retrogradely transported through a synapse as the native toxin. We have investigated its potential use as an in vivo neurotropic carrier. In this work we show that a hybrid protein encoded by the lacZ-TTC gene fusion retains the biological functions of both proteins in vivo-i.e. , retrograde transynaptic transport of the TTC fragment and beta-galactosidase enzymatic activity. After intramuscular injection, enzymatic activity could be detected in motoneurons and connected neurons of the brainstem areas. This strategy could be used to deliver a biological activity to neurons from the periphery to the central nervous system. Such a hybrid protein could also be used to map synaptic connections between neural cells.
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PMID:Construction of hybrid proteins that migrate retrogradely and transynaptically into the central nervous system. 925 94

Perforant path long-term potentiation (LTP) in intact mouse hippocampal dentate gyrus increased the neuron-specific, growth-associated protein GAP-43 mRNA in hilar cells 3 days after tetanus, but surprisingly not in granule cells, the perforant path target. This increase was positively correlated with level of enhancement and restricted to central hilar cells on the side of stimulation. Blockade of LTP by puffing DL-aminophosphonovalerate (APV), an N-methyl-D-aspartate (NMDA) receptor blocker into the molecular layer, eliminated LTP-induced GAP-43 mRNA elevation in hilar cells. To determine whether the mRNA elevation was mediated by transcription, LTP was studied in transgenic mice bearing a GAP-43 promoter-lacZ reporter gene. Promoter activity as indexed by Transgene expression (PATE) increased as indicated by blue staining of the lacZ gene product, beta-galactosidase. Potentiation induced a blue band bilaterally in the inner molecular layer of the dentate gyrus along the entire septotemporal axis. Because mossy cells are the only neurons in the central hilar zone that project to the inner molecular layer bilaterally along the entire septotemporal axis and LTP-induced activation of PATE in this zone was confined to the side of stimulation, we concluded that mossy cells were unilaterally activated, increasing synthesis of beta-galactosidase, which was transported bilaterally. Neither granule cells nor pyramidal cells demonstrated increased PATE or increased GAP-43 mRNA levels. These results and recent evidence indicating the necessity of hilar neurons for LTP point to previously unheralded mossy cells as potentially critical for perforant path LTP and the GAP-43 in these cells as important for LTP persistence lasting days.
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PMID:Long-term potentiation activates the GAP-43 promoter: selective participation of hippocampal mossy cells. 932 69

Many inherited neurological diseases and cancers could potentially benefit from efficient targeted gene delivery to neurons of the central nervous system. The nontoxic fragment C (HC) of tetanus toxin retains the specific nerve cell binding and transport properties of tetanus holotoxin. The HC fragment has previously been used to promote the uptake of attached proteins such as horseradish peroxidase, beta-galactosidase and superoxide dismutase into neuronal cells in vitro and in vivo. We report the use of purified recombinant HC fragment produced in yeast and covalently bound to polylysine [poly(K)] to enable binding of DNA. We demonstrate that when used to transfect cells, this construct results in nonviral gene delivery and marker gene expression in vitro in N18 RE 105 cells (a neuroblastoma x glioma mouse/rat hybrid cell line) and F98 (a glioma cell line). Transfection was dependent on HC and was neuronal cell type specific. HC may prove a useful targeting ligand for future neuronal gene therapy.
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PMID:Non-viral neuronal gene delivery mediated by the HC fragment of tetanus toxin. 1009 62

This study describes the construction and analysis of three in vivo-inducible promoter expression plasmids, containing pnirB, ppagC, and pkatG, for the delivery of foreign antigens in the DeltaaroAD mutant of Salmonella enterica var. Typhimurium (hereafter referred to as S. typhimurium). The reporter genes encoding beta-galactosidase and firefly luciferase were used to assess the comparative levels of promoter activity in S. typhimurium in vitro in response to different induction stimuli and in vivo in immunized mice. It was determined that the ppagC construct directed the expression of more beta-galactosidase and luciferase in S. typhimurium than the pnirB and pkatG constructs, both in vitro and in vivo. The gene encoding the C fragment of tetanus toxin was expressed in the aroAD mutant of S. typhimurium (BRD509) under the control of the three promoters. Mice orally immunized with attenuated S. typhimurium expressing C fragment under control of the pagC promoter [BRD509(pKK/ppagC/C frag)] mounted the highest tetanus toxoid-specific serum antibody response. Levels of luciferase expression in vivo and C-fragment expression in vitro from the pagC promoter appeared to be equivalent to if not lower than the levels of expression detected with the constitutive trc promoter. However, mice immunized with BRD509(pKK/ppagC/C frag) induced significantly higher levels of tetanus toxoid-specific antibody than BRD509(pKK/C frag)-immunized mice, suggesting that the specific location of foreign antigen expression may be important for immunogenicity. Mutagenesis of the ribosome binding sites (RBS) in the three promoter/C fragment expression plasmids was also performed. Despite optimization of the RBS in the three different promoter elements, the expression levels in vivo and overall immunogenicity of C fragment when delivered to mice by attenuated S. typhimurium were not affected. These studies suggest that in vivo-inducible promoters may give rise to enhanced immunogenicity and increase the efficacy of S. typhimurium as a vaccine vector.
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PMID:Use of in vivo-regulated promoters to deliver antigens from attenuated Salmonella enterica var. Typhimurium. 1049 87

We have investigated the ability of the growth phase regulated promoters dps and spv, to drive expression of heterologous antigens in Salmonella vaccine strains. Reporter plasmids were constructed which directed beta-galactosidase expression from dps (pDpslacZ) or spv (pSpvlacZ) and these were introduced independently into the Salmonella typhimurium vaccine strain SL3261 (aroA(-)). beta-galactosidase expression was induced 20-fold and 100-fold when broth cultures of SL3261 (pDpslacZ) or SL3261 (pSpvlacZ) respectively, entered the stationary phase of growth. Within macrophages, beta-galactosidase expression was induced 3.5-fold with SL3261 (pDpslacZ) and 7-fold with SL3261 (pSpvlacZ). The spv and dps promoters were used to drive independent expression of the C fragment domain of tetanus toxin (TetC) from plasmids harboured in S. typhimurium SL3261. Levels of anti-TetC antibodies were significantly higher in the sera of BALB/c mice perorally inoculated with SL3261 (pSpvtetC) or SL3261 (pDpstetC) compared to unvaccinated controls. This suggests that these promoter systems may be used to drive foreign antigen expression in live oral Salmonella vaccines.
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PMID:Use of the stationary phase inducible promoters, spv and dps, to drive heterologous antigen expression in Salmonella vaccine strains. 1061 25


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