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)

Ternary complexes of plasmid DNA, histone H1 protein and amphipathic polyamines (PAPA) were able to mediate the efficient transfection of 3T3. HeLa and COS cells in culture. Using both the beta-galactosidase and luciferase reporter gene systems, the transfection efficiency of PAPA complexes was comparable to that of DOSPA/PE cationic liposomes, considered to be a highly-efficient transfection reagent. Using three different assays of cellular toxicity (propidium iodide, BCECF-AM and Trypan Blue), the PAPA complexes caused minimal cellular toxicity. These results indicate that PAPA complexes are useful transfection reagents for the study of gene expression and function in cultured cells.
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PMID:Protein/amphipathic polyamine complexes enable highly efficient transfection with minimal toxicity. 923 46

Transient transfection of COS-1 cells with an expression vector for NIPP-1, a nuclear subunit of protein phosphatase-1, did not result in an overexpression of NIPP-1 protein, although the levels of mRNA encoding NIPP-1 increased dramatically. Moreover, high concentrations of NIPP-1 mRNA inhibited the translation in reticulocyte lysates of various unrelated mRNAs. This inhibition of translation was caused by the NIPP-1 messenger and not by the translation product, since mutation of the start codon abolished NIPP-1 protein production, but had no influence on the translational inhibition. Analysis of deletion mutants showed that the inhibition was mediated by a 0.5-kb fragment in the 5'-end of the NIPP-1 mRNA. This region, when inserted in the 5'-untranslated region of the beta-galactosidase messenger, inhibited the translation of beta-galactosidase mRNA in COS-1 cells. A predicted highly stable secondary structure deltaG = -239.5 kJ/mol) is present between residues 300 and 500 of NIPP-1 mRNA. The possible importance of this structure in the translational inhibition is discussed.
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PMID:Inhibition of translation by mRNA encoding NIPP-1, a nuclear inhibitor of protein phosphatase-1. 924 54

Gene transfer with replication-deficient adenovirus is a potentially useful tool to study vascular biology. We have constructed a replication-deficient adenovirus (AdRSVeNOS) that carries cDNA for endothelial nitric oxide synthase (eNOS). Transfection of COS-1 cells with AdRSVeNOS increased nitric oxide synthase activity (measured as production of L-citrulline from L-arginine) that was calcium dependent and inhibited by N omega-nitro-L-arginine methyl ester. To investigate effects of overexpression of eNOS on vascular function, we incubated common carotid arteries from rabbits in organ culture with AdRSVeNOS or AdRSV beta gal encoding beta-galactosidase. Transgene expression and responses to vasoactive agents were examined 1 day after transduction. Histochemical staining of beta-galactosidase and immunohistochemistry for eNOS indicated transgene expression in endothelium and adventitial cells. After precontraction with phenylephrine, vessels treated with AdRSVeNOS demonstrated greater relaxation to acetylcholine than vessels treated with vehicle or AdRSV beta gal. Relaxation to calcium ionophore A-23187 was much greater in vessels treated with AdRSVeNOS than in vessels treated with vehicle or AdRSV beta gal. Augmented relaxation in response to A-23187 was also observed after denudation of endothelium in vessels treated with AdRSVeNOS and was inhibited by N omega-nitro-L-arginine. Thus vasorelaxation in response to stimuli that release nitric oxide is augmented after adenovirus-mediated overexpression of eNOS. Transgene expression in adventitial cells appears to be sufficient to alter vasomotor function.
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PMID:Altered vascular function after adenovirus-mediated overexpression of endothelial nitric oxide synthase. 924 99

In Escherichia coli, the topology of inner membrane proteins can be studied conveniently with the alkaline phosphatase/beta-galactosidase (PhoA/LacZ) gene fusion system. PhoA is enzymatically active only when fused to external domains, LacZ when fused to cytoplasmic domains. In eukaryotic cells, only time consuming methods exist to study the topology of membrane proteins. We have extended in the first systematic study the PhoA/LacZ gene fusion system originally developed for E.coli for use in eukaryotic COS.M6 cells. We have fused PhoA and LacZ to the putative external and cytoplasmic loops of rat aquaporin 2 (AQP2), for which a model with six transmembrane domains was proposed previously. The fusion proteins were expressed in E.coli and COS.M6 cells and immunoblot analyses and enzyme activity assays were performed to localize the protein domains in both cell types. The data obtained in E.coli correlated mostly with the predictions of the six transmembrane domain model. However, two fusions were found to exhibit both high PhoA and high LacZ activity, thereby complicating the construction of a complete AQP2 model. In COS.M6 cells, the PhoA fusions were inactive. In contrast, the LacZ fusions succeeded and showed an activity pattern in complete agreement with the predictions of the six transmembrane domain model. Therefore, LacZ fusions can localize cytoplasmic loops in COS.M6 cells by means of a simple enzymatic assay with high reliability and may be used in future studies to develop topological models of other eukaryotic membrane proteins in their authentic cell systems.
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PMID:Topology of eukaryotic multispanning transmembrane proteins: use of LacZ fusions for the localization of cytoplasmic domains in COS.M6 cells. 927 85

SRY and SOX9, members of the family of high-mobility group (HMG) domain transcription factors, are both essential for testis formation during human embryonic development. The HMG domain is a DNA-binding and DNA-bending motif comprising about 80 amino acid residues. It has been shown that SRY and SOX9 are nuclear proteins. Using normal or mutant SRY-beta-galactosidase and SOX9-beta-galactosidase fusion proteins in transfection studies involving COS-7 cells, we have identified two nuclear localization signals (NLSs) within the HMG domains of both proteins that can independently direct the fusion proteins into the nucleus. Only mutational inactivation of both NLS motifs resulted in complete exclusion of the fusion proteins from the nucleus. The NLS sequences are located at the N and C termini of the HMG domain and are a bipartite NLS motif and a basic cluster NLS motif, respectively. Both NLS motifs are conserved in the HMG domains of other transcription factors. The implications of the present results are discussed regarding (a) the apparent dual function of certain basic amino acid residues in the HMG domain of SRY in both DNA binding and in nuclear localization and (b) the possible control of SOX9 in early gonadal differentiation at the level of nuclear translocation.
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PMID:Two independent nuclear localization signals are present in the DNA-binding high-mobility group domains of SRY and SOX9. 934 31

We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target beta-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican beta-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes.
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PMID:Glypican and biglycan in the nuclei of neurons and glioma cells: presence of functional nuclear localization signals and dynamic changes in glypican during the cell cycle. 936 4

Defects in peptide processing are associated with several disorders, including central diabetes insipidus (CDI). In the Brattleboro (BB) rat with CDI, the mRNA and protein of arginine vasopressin (AVP) are present in the hypothalamus, but no circulating AVP is detectable, thus suggesting a processing defect. The present study examined AVP secretion in cultured COS cells transfected with various constructs from wild-type and mutated Brattleboro AVP gene precursors. The precursor contains three exons encoding for vasopressin (VP), neurophysin (NP), and glycopeptide (GP). The Brattleboro rat has a deletion of a single base, guanine (G), in the NP coding region that leads to a frameshift, resulting in the loss of normal stop codon. The wild-type pcVP (22.0 +/- 5.2 pg/10[-2] U beta-galactosidase [beta-gal]), but not the mutated BB AVP gene pcBB (1.2 +/- 0.4 pg/10[-2] U beta-gal), was associated with AVP secretion from the COS cells as measured by RIA. The wild-type AVP gene without the GP coding region was associated with AVP release greater (47.4 +/- 13.5 pg/10[-2] U beta-gal, n = 5, P < 0.05, versus pcVP) than the pcVP with intact VP, NP, and GP coding regions. However, the wild-type AVP gene with VP coding region alone was not processed and secreted. Normalizing the pcBB total length with the insertion of a stop codon at the site of the normal stop codon was not associated with AVP secretion (3.0 +/- 1.4 pg/10[-2] U beta-gal). However, insertion of a stop codon so that the pcBB length equaled the length of VP and NP coding regions of the wild type was associated with AVP secretion (13.5 +/- 4.0 pg/10[-2] U beta-gal). When a stop codon was inserted into the wild-type NP coding region at the same site as the G deletion in the pcBB, the AVP secretion was significantly lower (15.1 +/- 5.0 pg/10[-2] U beta-gal) than pcVP with VP + NP but no GP coding regions (47.4 +/- 13.5 pg/10[-2] U beta-gal, n = 5, P < 0.05). In summary, (1) both VP and intact NP, but not GP, coding regions are necessary for AVP processing and secretion; (2) decreasing the length of the NP coding region diminishes but does not abolish AVP processing and secretion; and (3) shortening of the pcBB length with a stop codon at a site comparable to wild-type VP + NP allows AVP secretion, albeit less than with wild-type gene precursor. Thus, the CDI in BB rats is caused by the G deletion in NP coding region. This defect leads to abnormalities that contribute to the abnormal AVP processing. Specifically, the frameshift and absence of a stop codon cause a mutated extended C terminus, which, along with the mutated NP, contribute to the abnormal steps of AVP processing, transport, and secretion in the BB rat. These defects no doubt impair the folding and configuration necessary for normal processing of the AVP gene precursor.
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PMID:Arginine vasopressin secretion with mutants of wild-type and Brattleboro rats AVP gene. 940 88

Sialylation is a biosynthetic process occurring in the trans compartments of the Golgi apparatus. Corresponding evidence is based on localization and biochemical studies of alpha2, 6(N)-sialyltransferase (ST6Gal I) as previously reported. Here we describe generation and characterization of polyclonal antibodies to recombinant rat alpha2,3(N)-sialyltransferase (ST3Gal III) expressed as a soluble enzyme in Sf9 cells or as a beta-galactosidase-human-ST3Gal III fusion-protein from E.coli , respectively. These antibodies were used to localize ST3Gal III by immunofluorescence in various cell lines and rat kidney tissue sections. In transiently transfected COS cells the antibodies directed to soluble sialyltransferase or the sialyltransferase portion of the fusion-protein only recognized the recombinant antigen retained in the endoplasmic reticulum. However, an antibody fraction crossreactive with beta-galactosidase recognized natively expressed ST3Gal III which was found to be colocalized with beta1, 4-galactosyltransferase in the Golgi apparatus of several cultured cell lines. Antibodies affinity purified on the beta-galactosidase-ST3Gal III fusion-protein column derived from both antisera have then been used to localize the enzyme in perfusion-fixed rat kidney sections. We found strong staining of the Golgi apparatus of tubular epithelia and a brush-border-associated staining which colocalized with cytochemical staining of the H+ATPase. This subcellular localization was not observed for ST6Gal I which localized to the Golgi apparatus. These data show colocalization in the Golgi apparatus and different post-Golgi distributions of the two sialyltransferases.
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PMID:Immunocytochemical localization of alpha2,3(N)-sialyltransferase (ST3Gal III) in cell lines and rat kidney tissue sections: evidence for golgi and post-golgi localization. 945 Oct 34

Our previous studies showed immunological and functional similarities, as well as partial sequence homology, between the enzymatically inactive alternatively spliced variant of human beta-galactosidase (S-gal) and the 67-kDa elastin/laminin-binding protein (EBP) from sheep. To define the genetic origin of the EBP further, a full-length human S-gal cDNA clone was constructed and subjected to in vitro transcription/translation. The cDNA was also transfected into COS-1 cells and into the EBP-deficient smooth muscle cells (SMC) from sheep ductus arteriosus (DA). In vitro translation yielded an unglycosylated form of the S-gal protein, which immunoreacted with anti-beta-galactosidase antibodies and bound to elastin and laminin affinity columns. S-gal cDNA transfections into COS-1 and DA SMC increased expression of a 67-kDa protein that immunolocalized intracellularly and to the cell surface and, when extracted from the cells, bound to elastin. The S-gal-transfected cells displayed increased adherence to elastin-covered dishes, consistent with the cell surface distribution of the newly produced S-gal-encoded protein. Transfection of DA SMC additionally corrected their impaired elastic fiber assembly. These results conclusively identify the 67-kDa splice variant of beta-galactosidase as EBP.
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PMID:The 67-kDa enzymatically inactive alternatively spliced variant of beta-galactosidase is identical to the elastin/laminin-binding protein. 949 60

The Borna disease virus (BDV) replicates in the nucleus. The viral p40 protein (N), which is found abundantly in the nucleus in BDV-infected cells, may play an important role in virus replication. To analyze the amino acid residues involved in the nuclear targeting of BDV N, a series of eukaryotic expression plasmids encoding deletion mutants of N was constructed and transfected into COS-7 cells. In indirect immunofluorescence assays with a rabbit anti-BDV N antiserum, wild-type N was located in the nucleus of transfected cells in the absence of other viral constituents. In contrast, mutants lacking the 13 NH2-terminal amino acid residues 1MPPKRRLVDDADA13 in common gave a cytoplasmic localization pattern. Similarly, a mutant with substitution of 4KRR6 by 4NSG6 was retained in the cytoplasm. Furthermore, a nonapeptide, 3PKRRLVDDA11, derived from the NH2-terminal region of N conferred nuclear targeting activity to beta-galactosidase, which normally resides in the cytoplasm. Thus, we have identified the nuclear targeting signal of the BDV N and narrowed it to the NH2-terminal region where 4KRR6 basic amino acid residues are located.
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PMID:Nuclear targeting activity associated with the amino terminal region of the Borna disease virus nucleoprotein. 952 28


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