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)

Hypothalamic GnRH is a decapeptide that plays a pivotal role in mammalian reproduction by stimulating the synthesis and secretion of gonadotropins via binding to the GnRH receptor on the pituitary gonadotropins. It is hypothesized that sex steroids may regulate GnRH I (a classical form of GnRH), GnRH II (a second form of GnRH), and GnRH I receptor (GnRHRI) at the transcriptional level in target tissues. Thus, in the present study a role for progesterone (P4) in the regulation of GnRH I, GnRH II, and GnRHRI was investigated using a human neuronal medulloblastoma cell line (TE671) as an in vitro model. The cells were transfected with human GnRHRI promoter-luciferase constructs, and promoter activities were analyzed after P4 treatment by luciferase and beta-galactosidase assay. The mRNA levels of GnRH I and GnRH II were analyzed by RT-PCR. Treatment of TE671 cells with P4 resulted in a decrease in GnRHRI promoter activity compared with the control level in a dose- and time-dependent manner. Cotreatment of these cells with RU486, an antagonist of P4, reversed P4-induced inhibition of GnRHRI promoter activity, suggesting that the P4 effect is mediated by P4 receptor (PR). In the cells transfected with a full-length of PR A- or PR B-expressing vector, overexpression of PR A increased the sensitivity toward P4 in an inhibition of GnRHRI promoter, whereas PR B increased transcriptional activity of GnRHRI promoter in the presence of P4. However, PR B itself did not act as a transcriptional activator of GnRHRI promoter. Because TE671 cells have been recently demonstrated to express and synthesize two forms of GnRHs, we also investigated the regulation of GnRH mRNAs by P4. In the present study, P4 increased GnRH I mRNA levels in a time- and dose-dependent manner. This stimulatory effect of P4 in the regulation of GnRH I mRNAs was significantly attenuated by RU486, whereas no significant difference in the expression level of GnRH II was observed with P4 or RU496. Interestingly, although the expression level of PR B was low compared with that of PR A, P4 action on the GnRH I gene was mediated by PR B. In conclusion, these results indicate that P4 is a potent regulator of GnRHRI at the transcriptional level as well as GnRH I mRNA. This distinct effect of P4 on the GnRH system may be derived from different pathways through PR A or PR B.
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PMID:Differential role of progesterone receptor isoforms in the transcriptional regulation of human gonadotropin-releasing hormone I (GnRH I) receptor, GnRH I, and GnRH II. 1556 29

The two gvpA promoters P(cA) and P(pA) of Halobacterium salinarum, and the P(mcA) promoter of Haloferax mediterranei were investigated with respect to growth-phase-dependent expression and regulation in Haloferax volcanii transformants using the bgaH reading frame encoding BgaH, an enzyme with beta-galactosidase activity, as reporter. For comparison, the P(fdx) promoter of the ferredoxin gene of Hbt. salinarum and the P(bgaH) promoter of Haloferax lucentense (formerly Haloferax alicantei) were analysed. P(fdx), driving the expression of a house-keeping gene, was highly active during the exponential growth phase, whereas P(bgaH) and the three gvpA promoters yielded the largest activities during the stationary growth phase. Compared to P(fdx), the basal promoter activities of P(pA) and P(mcA) were rather low, and larger activities were only detected in the presence of the endogenous transcriptional activator protein GvpE. The P(cA) promoter does not yield a detectable basal promoter activity and is only active in the presence of the homologous cGvpE. To investigate whether the P(cA)-TATA box and the BRE element were the reason for the lack of the basal P(cA) activity, these elements and also sequences further upstream were substituted with the respective sequences of the stronger P(pA) promoter and investigated in Hfx. volcanii transformants. All these promoter chimera did not yield a detectable basal promoter activity. However, whenever the P(pA)-BRE element was substituted for the P(cA)-BRE, an enhanced cGvpE-mediated activation was observed. The promoter chimeras harbouring P(pA)-BRE plus 5 (or more) bp further upstream also gained activation by the heterologous pGvpE and mcGvpE proteins. The sequence required for the GvpE-mediated activation was determined by a 4 bp scanning mutagenesis with the 45 bp region upstream of P(mcA)-BRE. None of these alterations influenced the basal promoter activity, but the sequence TGAAACGG-n4-TGAACCAA was important for the GvpE-mediated activation of P(mcA).
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PMID:In vivo analyses of constitutive and regulated promoters in halophilic archaea. 1563 22

Most normal somatic cells enter a state called replicative senescence after a certain number of divisions, characterized by irreversible growth arrest. Moreover, they express a pronounced inflammatory phenotype that could contribute to the aging process and the development of age-related pathologies. Among the molecules involved in the inflammatory response that are overexpressed in senescent cells and aged tissues is intercellular adhesion molecule-1 (ICAM-1). Furthermore, ICAM-1 is overexpressed in atherosclerosis, an age-related, chronic inflammatory disease. We have recently reported that the transcriptional activator p53 can trigger ICAM-1 expression in an nuclear factor-kappa B (NF-kappaB)-independent manner (Gorgoulis et al, EMBO J. 2003; 22: 1567-1578). As p53 exhibits an increased transcriptional activity in senescent cells, we investigated whether p53 activation is responsible for the senescence-associated ICAM-1 overexpression. To this end, we used two model systems of cellular senescence: (a) human fibroblasts and (b) conditionally immortalized human vascular smooth muscle cells. Here, we present evidence from both cell systems to support a p53-mediated ICAM-1 overexpression in senescent cells that is independent of NF-kappaB. We also demonstrate in atherosclerotic lesions the presence of cells coexpressing activated p53, ICAM-1, and stained with the senescence-associated beta-galactosidase, a biomarker of replicative senescence. Collectively, our data suggest a direct functional link between p53 and ICAM-1 in senescence and age-related disorders.
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PMID:p53-dependent ICAM-1 overexpression in senescent human cells identified in atherosclerotic lesions. 1571 69

p94/calpain 3 is a skeletal muscle-specific member of the Ca(2+)-regulated cytosolic cysteine protease family, the calpains. Defective p94 protease activity originating from gene mutations causes a muscular dystrophy called calpainopathy, indicating the indispensability of p94 for muscle survival. Because of the existence of the p94-specific regions IS1 and IS2, p94 undergoes very rapid and exhaustive autolysis. To elucidate the physiological relevance of this unique activity, the autolytic profiles of p94 and the effect of the p94 binding protein, connectin/titin, on this process were investigated. In vitro analysis of p94 autolysis showed that autolysis in IS1 proceeds without immediate disassembly into fragments and that the newly identified cryptic autolytic site in IS2 is critical for disassembling autolyzed fragments. As a genetic system to assay p94 autolysis semiquantitatively, p94 was expressed in yeast as a hybrid protein between the DNA binding and activation domains of the yeast transcriptional activator Gal4. Transcriptional activation by the Gal4-p94:WT hybrid protein is precluded by p94 autolysis. Complete or partial loss of autolytic activity by C129S active site mutation, limb girdle muscular dystrophy type 2A pathogenic missense mutations, or PCR-based random mutagenesis could be detected by semiquantitative restoration of Gal4-dependent beta-galactosidase gene expression. Using this system, the N2A connectin fragment that binds to p94 was shown to suppress p94 autolytic disassembly. The proximity of the IS2 autolytic and connectin-binding sites in p94 suggested that N2A connectin suppresses IS2 autolysis. These data indicate the importance of p94-connectin interaction in the control of p94 functions by regulating autolytic decay of p94.
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PMID:Suppressed disassembly of autolyzing p94/CAPN3 by N2A connectin/titin in a genetic reporter system. 1662 76

Upregulation of the MDR1 (multidrug resistance 1) gene is involved in the development of resistance to antifungal agents in clinical isolates of the pathogen Candida albicans. To better understand the molecular mechanisms underlying the phenomenon, the cis-acting regulatory elements present in the MDR1 promoter were characterized using a beta-galactosidase reporter system. In an azole-susceptible strain, transcription of this reporter is transiently upregulated in response to either benomyl or H(2)O(2), whereas its expression is constitutively high in an azole-resistant strain (FR2). Two cis-acting regulatory elements within the MDR1 promoter were identified that are necessary and sufficient to confer the same transcriptional responses on a heterologous promoter (CDR2). One, a benomyl response element (BRE), is situated at position -296 to -260 with respect to the ATG start codon. It is required for benomyl-dependent MDR1 upregulation and is also necessary for constitutive high expression of MDR1. A second element, termed H(2)O(2) response element (HRE), is situated at position -561 to -520. The HRE is required for H(2)O(2)-dependent MDR1 upregulation, but dispensable for constitutive high expression. Two potential binding sites (TTAG/CTAA) for the bZip transcription factor Cap1p (Candida AP-1 protein) lie within the HRE. Moreover, inactivation of CAP1 abolished the transient response to H(2)O(2). Cap1p, which has been previously implicated in cellular responses to oxidative stress, may thus play a trans-acting and positive regulatory role in the H(2)O(2)-dependent transcription of MDR1. A minimal BRE (-290 to -273) that is sufficient to detect in vitro sequence-specific binding of protein complexes in crude extracts prepared from C. albicans was also defined. Interestingly, the sequence includes a perfect match to the consensus binding sequence of Mcm1p, raising the possibility that MDR1 may be a direct target of this MADS box transcriptional activator. In conclusion, while the identity of the trans-acting factors that bind to the BRE and HRE remains to be confirmed, the tools developed during this characterization of the cis-acting elements of the MDR1 promoter should now serve to elucidate the nature of the components that modulate its activity.
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PMID:Identification of promoter elements responsible for the regulation of MDR1 from Candida albicans, a major facilitator transporter involved in azole resistance. 1715 23

gamma-Secretase is a multisubunit membrane protein complex consisting of presenilin (PS1), nicastrin (NCT), anterior pharynx-1, and presenilin enhancer 2. To analyze the activity of familial Alzheimer disease mutants and to understand the roles of the subunits, we established a yeast transcriptional activator Gal4p system with artificial gamma-secretase substrates containing amyloid precursor protein or Notch fragments. The gamma-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4p release from the membrane-bound substrates, i.e. growth of yeast on histidine and adenine, or beta-galactosidase assay. We screened and evaluated gamma-secretase mutants using this reconstitution system in yeast, which does not possess endogenous gamma-secretase activity. When we introduced familial Alzheimer mutants of PS1 in this system, their activities were shown to be loss of function. Although the protease activity of wild type PS1 depends on the other three subunits introduced, we obtained 15 new PS1 mutants, which are active in the absence of NCT. They possessed a S438P mutation at the ninth transmembrane domain (TM9) together with one missense mutation distributed through transmembrane and loop regions. These mutations were not related to familial Alzheimer mutations of PS1 as identified so far. The S438P mutant was partially active but required other mutations for full activation. Results of the beta-galactosidase assay suggested that they have wild type protease activities, which were further confirmed by the endoproteolysis of PS1, amyloid beta peptides, and Notch intracellular domain production in mammalian cells. These results suggest that NCT is dispensable for the protease activity of gamma-secretase.
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PMID:Nicastrin is dispensable for gamma-secretase protease activity in the presence of specific presenilin mutations. 1925 53

Aft1 is a transcriptional activator in Saccharomyces cerevisiae that responds to iron availability and regulates the expression of genes in the iron regulon, such as FET3, FTR1 and the ARN family. Using a two-hybrid screen, we found that Aft1 physically interacts with the FOB (ferrioxamine B) transporter Arn3. This interaction modulates the ability of Arn3 to take up FOB. The interaction between Arn3 and Aft1 was confirmed by beta-galactosidase, co-immunoprecipitation and SPR (surface plasmon resonance) assays. Truncated Aft1 had a stronger interaction with Arn3 and caused a higher FOB-uptake activity than full-length Aft1. Interestingly, only full-length Aft1 induced the correct localization of Arn3 in response to FOB. Furthermore, we found Aft1 affected Arn3 ubiquitination. These results suggest that Aft1 interacts with Arn3 and may regulate the ubiquitination of Arn3 in the cytosolic compartment.
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PMID:A novel function of Aft1 in regulating ferrioxamine B uptake: Aft1 modulates Arn3 ubiquitination in Saccharomyces cerevisiae. 1946 13

Eukaryotic initiation factor 2 (eIF2) B is a guanine nucleotide exchange factor that plays a central role in translation initiation and its control, especially in response to diverse cellular stresses. In addition, inherited mutations in human eIF2B subunits cause a fatal brain disorder commonly called childhood ataxia with central nervous system hypomyelination or leukoencephalopathy with vanishing white matter. In yeast, inhibiting activity of eIF2B up-regulates expression of the transcriptional activator general control nondepressible (GCN) 4. We report here evaluation of high-throughput screening (HTS) using a yeast-based reporter gene assay, in which strains containing either wild-type or a mutant eIF2B were screened in parallel to identify compounds modifying eIF2B-dependent responses. The goals of the HTS were twofold: first, to discover compounds that restore normal function to mutant eIF2B, which may have therapeutic utility for the fatal human disease; and second, to identify compounds that activate a GCN4 response, which might be useful experimental tools. The HTS assay measured cell growth by absorbance, and activation of gene expression via a beta-galactosidase reporter gene fusion. Because mutant eIF2B activates GCN4 in the absence of stress inducers, the mutant strain was screened for reduction in GCN4 activation. HTS revealed apparent mutant-selective inhibitors but did not reliably predict selectivity as these hits affected both wild-type and mutant strains equally on dose-response confirmation. Wild-type strain results from the HTS identified two GCN4 response activators, both of which were confirmed to be wild-type selective in dose-response testing, suggesting that these compounds may activate GCN4 by a mechanism that down-regulates eIF2B activity.
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PMID:Discovery of chemical modulators of a conserved translational control pathway by parallel screening in yeast. 1971 53

The Epstein-Barr virus basic leucine zipper transcriptional activator ZEBRA was shown recently to cross the outer membrane of live cells and to accumulate in the nucleus of lymphocytes. We investigated the potential application of the Epstein-Barr virus trans-activator ZEBRA as a transporter protein to facilitate transduction of cargo proteins. Analysis of different truncated forms of ZEBRA revealed that the minimal domain (MD) required for internalization spans residues 170-220. MD efficiently transported reporter proteins such as enhanced green fluorescent protein (EGFP) and beta-galactosidase in several normal and tumor cell lines. Functionality of internalized cargo proteins was confirmed by beta-galactosidase activity in transduced cells, and no MD-associated cell toxicity was detected. Translocation of MD through the cell membrane required binding to cell surface-associated heparan sulfate proteoglycans as shown by strong inhibition of protein uptake in the presence of heparin. We found that internalization was blocked at 4 degrees C, whereas no ATP was required as shown by an only 25% decreased uptake efficiency in energy-depleted cells. Common endocytotic inhibitors such as nystatin, chlorpromazine, and wortmannin had no significant impact on MD-EGFP uptake. Only methyl-beta-cyclodextrin inhibited MD-EGFP uptake by 40%, implicating the lipid raft-mediated endocytotic pathway. These data suggest that MD-reporter protein transduction occurs mostly via direct translocation through the lipid bilayer and not by endocytosis. This mechanism of MD-mediated internalization is suitable for the efficient delivery of biologically active proteins and renders ZEBRA-MD a promising candidate for therapeutic protein delivery applications.
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PMID:Characterization of the cell-penetrating properties of the Epstein-Barr virus ZEBRA trans-activator. 2038 49

The Salmonella tdc operon encodes enzymes belonging to a metabolic pathway that degrades L-serine and L-threonine. The upregulation of the tdc operon and increased virulence of Salmonella grown under oxygen-limiting conditions prompted us to investigate the role of the tdc operon in the pathogenesis of Salmonella Typhimurium. A Salmonella strain carrying a null mutation in tdcA, which encodes the transcriptional activator of the tdc operon, was impaired in mice infected intraperitoneally with the bacterium. In addition, the Salmonella tdcA mutant showed reduced replication compared with the parental strain in cultured animal cells, although their growth rates were similar in various culture media. To understand the function of TdcA in pathogenesis, we performed two-dimensional gel electrophoresis and found that flagellar and PhoP-regulated proteins were affected by the tdcA mutation. The results of beta-galactosidase assays and FACS analysis showed that, among the four PhoP-dependent genes tested, the expression of ssaG, which is located in Salmonella pathogenicity island 2 (SPI2), was reduced in the tdcA mutant, especially in the intracellular environment of macrophages. Taken together, our data suggest that tdcA plays an important role in the pathogenesis of Salmonella.
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PMID:A mutation in tdcA attenuates the virulence of Salmonella enterica serovar Typhimurium. 2039 61


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