Biomedical subjects

O Georgiev

Publications and source records attributed to O Georgiev.

At least 19 recordsLinked to original sources

PubMed2001

Nucleo-cytoplasmic trafficking of metal-regulatory transcription factor 1 is regulated by diverse stress signals.

The metal-regulatory transcription factor 1 (MTF-1) is a key regulator of heavy metal-induced transcription of metallothionein I and II and other genes in mammals and other metazoans. Transcriptional activation of genes by MTF-1 is mediated through binding to metal-responsive elements of consensus TGCRCNC in the target gene promoters. In an attempt to further clarify the mechanisms by which certain external signals activate MTF-1 and in turn modulate gene transcription, we show here that human MTF-1 has a dual nuclear and cytoplasmic localization in response to diverse stress stimuli. MTF-1 contains a consensus nuclear localization signal located just N-terminal to the first zinc finger that contributes to but is not essential for nuclear import. MTF-1 also harbors a leucine-rich, nuclear export signal. Under resting conditions, the nuclear export signal is required for cytoplasmic localization of MTF-1 as indicated by mutational analysis and transfer to the heterologous green fluorescent protein. Export from the nucleus was inhibited by leptomycin B, suggesting the involvement of the nuclear export protein CRM1. Our results further show that in addition to the heavy metals zinc and cadmium, heat shock, hydrogen peroxide, low extracellular pH (pH 6.0), inhibition of protein synthesis by cycloheximide, and serum induce nuclear accumulation of MTF-1. However, heavy metals alone (and not the other stress conditions) induce a significant transcriptional response via metal-responsive element promoter sequences, implying that nuclear import of MTF-1 is necessary but not sufficient for transcriptional activation. Possible roles for nuclear import under non-metal stress conditions are discussed.

Cadmium↗

PubMed2001

Target gene search for the metal-responsive transcription factor MTF-1.

Activation of genes by heavy metals, notably zinc, cadmium and copper, depends on MTF-1, a unique zinc finger transcription factor conserved from insects to human. Knockout of MTF-1 in the mouse results in embryonic lethality due to liver decay, while knockout of its best characterized target genes, the stress-inducible metallothionein genes I and II, is viable, suggesting additional target genes of MTF-1. Here we report on a multi-pronged search for potential target genes of MTF-1, including microarray screening, SABRE selective amplification, a computer search for MREs (DNA-binding sites of MTF-1) and transfection of reporter genes driven by candidate gene promoters. Some new candidate target genes emerged, including those encoding alpha-fetoprotein, the liver-enriched transcription factor C/EBPalpha and tear lipocalin/von Ebner's gland protein, all of which have a role in toxicity/the cell stress response. In contrast, expression of other cell stress-associated genes, such as those for superoxide dismutases, thioredoxin and heat shock proteins, do not appear to be affected by loss of MTF-1. Our experiments have also exposed some problems with target gene searches. First, finding the optimal time window for detecting MTF-1 target genes in a lethal phenotype of rapid liver decay proved problematical: 12.5-day-old mouse embryos (stage E12.5) yielded hardly any differentially expressed genes, whereas at stage 13.0 reduced expression of secretory liver proteins probably reflected the onset of liver decay, i.e. a secondary effect. Likewise, up-regulation of some proliferation-associated genes may also just reflect responses to the concomitant loss of hepatocytes. Another sobering finding concerns gamma-glutamylcysteine synthetase(hc) (gamma-GCS(hc)), which controls synthesis of the antioxidant glutathione and which was previously suggested to be a target gene contributing to the lethal phenotype in MTF-1 knockout mice. gamma-GCS(hc) mRNA is reduced at the onset of liver decay but MTF-1 null mutant embryos manage to maintain a very high glutathione level until shortly before that stage, perhaps in an attempt to compensate for low expression of metallothioneins, which also have a role as antioxidants.

Animals↗

PubMed2001

The Drosophila homolog of mammalian zinc finger factor MTF-1 activates transcription in response to heavy metals.

Metallothioneins (MTs) are short, cysteine-rich proteins for heavy metal homeostasis and detoxification; they bind a variety of heavy metals and also act as radical scavengers. Transcription of mammalian MT genes is activated by heavy metal load via the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein whose elimination in mice leads to embryonic lethality due to liver decay. Here we characterize the Drosophila homolog of vertebrate MTF-1 (dMTF-1), a 791-amino-acid protein which is most similar to its mammalian counterpart in the DNA-binding zinc finger region. Like mammalian MTF-1, dMTF-1 binds to conserved metal-responsive promoter elements (MREs) and requires zinc for DNA binding, yet some aspects of heavy metal regulation have also been subject to divergent evolution between Drosophila and mammals. dMTF-1, unlike mammalian MTF-1, is resistant to low pH (6 to 6.5). Furthermore, mammalian MT genes are activated best by zinc and cadmium, whereas in Drosophila cells, cadmium and copper are more potent inducers than zinc. The latter species difference is most likely due to aspects of heavy metal metabolism other than MTF-1, since in transfected mammalian cells, dMTF-1 responds to zinc like mammalian MTF-1. Heavy metal induction of both Drosophila MTs is abolished by double-stranded RNA interference: small amounts of cotransfected double-stranded RNA of dMTF-1 but not of unrelated control RNA inhibit the response to both the endogenous dMTF-1 and transfected dMTF-1. These data underline an important role for dMTF-1 in MT gene regulation and thus heavy metal homeostasis.

Amino Acid Sequence↗

PubMed2001

[Longitudinal study of parameters of respiratory function tests and blood gas analysis in patients with progressive systemic scleroderma].

The longitudinal study encloses 53 patients with PSS followed up for a period of 6 years. The most frequent changes in the parameters of pulmonary function are: diffusing capacity impairment--reduction in DICO (86.8% of the patients), restrictive lung disease pattern (50.9% of the patients) and arterial hypoxemia (43.4% of the patients). The duration of PSS and the follow up period do not cause significant changes in the pulmonary function parameters and the arterial blood-gas analysis in the group of patients studied.

Blood Gas Analysis↗

PubMed2000

Characterization of the mouse gene for the heavy metal-responsive transcription factor MTF-1.

MTF-1 is a zinc finger transcription factor that mediates the cellular response to heavy metal stress; its targeted disruption in the mouse leads to liver decay and embryonic lethality at day E14. Recently, we have sequenced the entire MTF-1 gene in the compact genome of the pufferfish Fugu rubripes. Here we have defined the promoter sequences of human and mouse MTF-1 and the genomic structure of the mouse MTF-1 locus. The transcription unit of MTF-1 spans 42 kb (compared to 8.5 kb in Fugu) and is located downstream of the gene for a phosphatase (INPP5P) in mouse, human, and fish. In all of these species, the MTF promoter region has the features of a CpG island. In both mouse and human, the 5' untranslated region harbors conserved short reading frames of unknown function. RNA mapping experiments revealed that in these two species, MTF-1 mRNA is transcribed from a cluster of multiple initiation sites from a TATA-less promoter without metal-responsive elements. Transcription from endogenous and transfected MTF-1 promoters was not affected by heavy metal load or other stressors, in support of the notion that MTF-1 activity is regulated at the posttranscriptional level. Tissue Northern blots normalized for poly A+ RNA indicate that MTF-1 is expressed at similar levels in all tissues, except in the testes, that contain more than 10-fold higher mRNA levels.

5' Untranslated Regions↗

PubMed2000

[99mTcDTPA-aerosol clearance in early diagnosis of pulmonary changes in patients with active systemic lupus erythematosus].

Nineteen patients with active SLE and a control group of 20 healthy persons were investigated. We have made to all of them 99mTcDTPA-aerosol scintigraphy and the following pulmonary functional tests--Dc CO, Dm, Vc, HRCT. Accelerated clearance of 99mTcDTPA was found in all patients with active SLE. The half-elimination time T1/2 was shortened and varied between 22.6 +/- 4.7 min for the right one. It wasn't established correlative dependence between 99mTcDTPA and the pulmonary functional tests. In 36.8% of the patients HRCT was normal, while 99mTcDTPA clearance was accelerated. This fact shows, that 99mTcDTPA-aerozsol scintigraphy is more sensitive method for early diagnosis of pulmonary changes, than the pulmonary functional tests and HRCT.

Adult↗

PubMed1999

Mutagenesis and selection of PDZ domains that bind new protein targets.

PDZ domains are a recently characterized protein-recognition module. In most cases, PDZ domains bind to the C-terminal end of target proteins and are thought thereby to link these target proteins into functional signaling networks. We report the isolation of artificial PDZ domains selected via a mutagenesis screen in vivo, each recognizing a different C-terminal peptide. We demonstrate that the PDZ domains isolated can bind selectively to their target peptides in vitro and in vivo. Two of the target peptides chosen are the C-terminal ends of two cellular transmembrane proteins with which no known PDZ domains have been reported to interact. By targeting these artificial PDZ domains to the nucleus, interacting target peptides were efficiently transported to the same subcellular localization. One of the isolated PDZ domains was tested and shown to be efficiently directed to the plasma membrane when cotransfected with the full-length transmembrane protein in mammalian cells. Thus, artificial PDZ domains can be engineered and used to target intracellular proteins to different subcellular compartments.

Amino Acid Sequence↗

PubMed1999

Characterization of the transcription factor MTF-1 from the Japanese pufferfish (Fugu rubripes) reveals evolutionary conservation of heavy metal stress response.

The pufferfish Fugu rubripes was recently introduced as a new model organism for genomic studies, since it contains a full set of vertebrate genes but only 13% as much DNA as a mammal. Fugu genes tend to be smaller and densely spaced due to shortening of introns and intergenic spacers. We isolated the Fugu gene for the metal-responsive transcription factor MTF-1 (MTF1), a mediator of heavy metal regulation and oxidative stress response previously characterized in mammals. In addition, most of the cDNA sequence was also determined. The 780 amino acid MTF-1 protein of Fugu is very similar to that of mouse and human, with 90% amino acid identity in the DNA binding zinc finger domain and 57% overall identity. Expression of the pufferfish cDNA in mammalian cells shows that Fugu MTF-1 has the same DNA binding specificity as its mammalian counterpart and also induces transcription in response to zinc and cadmium. The protein-coding part of the Fugu MTF-1 gene spans 6.4 kb and consists of 11 exons. Upstream region and first exon constitute a CpG island. The distance between stop codon and polyadenylation motifs is >2 kb, suggesting a very long 3' untranslated mRNA region, followed by another CpG island which may represent the promoter of the next gene downstream. Part of the MTF-1 genomic structure was also determined in the mouse, and some striking similarities were found: for example, the upstream adjacent gene in both species is INPP5P, encoding a phosphatase. The mouse MTF-1 promoter is also embedded in a CpG island, which however shares no sequence similarity to the one of Fugu. The Fugu CpG island is shorter than the one of the mouse and has no elevated [G+C] content; these and other data indicate that CpG islands of fish may represent a primordial stage of CpG island evolution.

Amino Acid Sequence↗

PubMed1999

The heavy metal-responsive transcription factor-1 (MTF-1) is not required for neural differentiation.

The zinc finger transcription factor MTF-1 is essential for proper response to heavy metal load and other stress conditions in vertebrates, and also contributes to the maintenance of the cellular redox state. Target genes include metallothioneins (MT-I and MT-II) and gamma-glutamylcysteine synthetase (gamma-GCS), an enzyme involved in glutathione biosynthesis. Although MTF-1 is expressed ubiquitously, the primary defect in null mutant mice is hepatocyte necrosis, which results in embryonic lethality around day E14 and prevents the analysis of delayed effects on other organs. To assess the impact of MTF-1 deficiency on the function of the mature central nervous system, we employed the neural grafting strategy. Neuroectodermal brain tissue obtained from transgenic mouse embryos at gestational day 12.5 was transplanted into the caudoputamen of adult wild-type mice. 33 days later, grafts derived from MTF-1 deficient mice consisted of fully differentiated neuroectodermal tissue and showed no differences to heterozygous control grafts. This indicates that MTF-1 is dispensable for the development and differentiation of the nervous system. Such transplants devoid of MTF-1 may provide a useful tool for the further investigation of the effect of cell stress, including oxidative stress.

Animals↗

PubMed1998

Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1.

We have shown previously that the heavy metal-responsive transcriptional activator MTF-1 regulates the basal and heavy metal-induced expression of metallothioneins. To investigate the physiological function of MTF-1, we generated null mutant mice by targeted gene disruption. Embryos lacking MTF-1 die in utero at approximately day 14 of gestation. They show impaired development of hepatocytes and, at later stages, liver decay and generalized edema. MTF-1(-/-) embryos fail to transcribe metallothionein I and II genes, and also show diminished transcripts of the gene which encodes the heavy-chain subunit of the gamma-glutamylcysteine synthetase, a key enzyme for glutathione biosynthesis. Metallothionein and glutathione are involved in heavy metal homeostasis and detoxification processes, such as scavenging reactive oxygen intermediates. Accordingly, primary mouse embryo fibroblasts lacking MTF-1 show increased susceptibility to the cytotoxic effects of cadmium or hydrogen peroxide. Thus, MTF-1 may help to control metal homeostasis and probably cellular redox state, especially during liver development. We also note that the MTF-1 null mutant phenotype bears some similarity to those of two other regulators of cellular stress response, namely c-Jun and NF-kappaB (p65/RelA).

Animals↗

PubMed1998

An embryonic demethylation mechanism involving binding of transcription factors to replicating DNA.

In vertebrates, transcriptionally active promoters are undermethylated. Since the transcription factor Sp1, and more recently NF-kappaB, have been implicated in the demethylation process, we examined the effect of transcription factors on demethylation by injecting in vitro methylated plasmid DNA into Xenopus fertilized eggs. We found that various transactivation domains, including a strong acidic activation domain from the viral protein VP16, can enhance demethylation of a promoter region when fused to a DNA binding domain which recognizes the promoter. Furthermore, demethylation occurs only after the midblastula transition, when the general transcription machinery of the host embryo becomes available. Nevertheless, transcription factor binding need not be followed by actual transcription, since demethylation is not blocked by alpha-amanitin treatment. Finally, replication of the target DNA is a prerequisite for efficient demethylation since only plasmids that carry the bovine papilloma virus sequences which support plasmid replication after the midblastula transition are demethylated. No demethylation is detectable in the oocyte system where DNA is not replicated. These results suggest that, in the Xenopus embryo, promoters for which transcription factors are available are demethylated by a replication-dependent, possibly passive mechanism.

Amanitins↗

PubMed1998

Homologous recombination and DNA-end joining reactions in zygotes and early embryos of zebrafish (Danio rerio) and Drosophila melanogaster.

A linear DNA with partial sequence redundancy can be recircularized in cells by either nonhomologous end joining (NEJ) or by homologous recombination (HR). We have studied the relative contributions of these processes in zygotes or early embryos of species that serve as model organisms for developmental genetics. Thus, we have microinjected a linearized plasmid substrate into zygotes of zebrafish (Danio rerio) or into the posterior end of Drosophila melanogaster early embryos before pole cell formation. Similar to the situation observed previously in Xenopus zygotes/early embryos, we detected a large preponderance of DNA-end joining over homologous recombination. A comparison of end-joined junctions revealed that from the three species tested, zebrafish introduced the least number of sequence distortions upon DNA-end joining, while Drosophila produced the largest deletions (average 14 bp) with occasional nucleotide patch insertions, reminiscent of the N nucleotides at V(D)J junctions in mammalian immune receptor genes. Double-strand gap repair by homologous sequences ('homologous recombination') involving a bimolecular reaction was readily detectable in both zebrafish and Drosophila. This involved specifically designed recombination substrates consisting of a mutagenized linear plasmid and DNA fragments carrying the wild-type sequence. Our results show that the basic machinery for homologous recombination is present at early developmental stages of these two genetic model organisms. However, it seems that for any experimental exploitation, such as targeted gene disruption, one would have to inhibit or bypass the overwhelming DNA-end joining activity.

Animals↗

PubMed1997

An epitope tagged mammalian/prokaryotic expression vector with positive selection of cloned inserts.

A dual eukaryotic/prokaryotic expression vector has been developed which combines the features of positive selection for cloned inserts along with the production of an epitope-tagged cDNA insert by transient transfection in mammalian cells as well as high level induced expression in E. coli cells harbouring T7 RNA polymerase. This vector, pZilch, has two MCSs flanking a mutant E. coli phenylalanyl-tRNA synthetase gene, pheS, which when expressed in combination with the phenylalanine analog p-CI-Phe, results in termination of host cell protein synthesis. Cloning of inserts using unique sites in the flanking MCS regions results in loss of the pZilch pheS allele and hence permits growth of colonies harbouring recombinants on p-Cl-Phe plates. Additional features of the vector include an optimal Kozak consensus sequence for high level eukaryotic cell expression and an efficient prokaryotic translation initiation site in frame and downstream from the eukaryotic initiation site. Recombinant proteins can be produced with an N-terminal FLAG epitope which can be removed via a specific protease cleavage site. Flanking T7 and SP6 RNA polymerase promoter sites permit in vitro transcription and translation of cloned inserts. A derivative of the vector has also been constructed enabling nuclear accumulation of the tagged proteins via an SV40 nuclear localisation signal upstream of the 5' MCS.

Amino Acid Sequence↗

PubMed1997

A serine/arginine-rich nuclear matrix cyclophilin interacts with the C-terminal domain of RNA polymerase II.

The largest subunit of RNA polymerase II shows a striking difference in the degree of phosphorylation, depending on its functional state: initiating and elongating polymerases are unphosphorylated and highly phosphorylated respectively. Phosphorylation mostly occurs at the C-terminal domain (CTD), which consists of a repetitive heptapeptide structure. Using the yeast two-hybrid system, we have selected for mammalian proteins that interact with the phosphorylated CTD of mammalian RNA polymerase II. A prominent isolate, designated SRcyp/CASP10, specifically interacts with the CTD not only in vivo but also in vitro . It contains a serine/arginine-rich (SR) domain, similar to that found in the SR protein family of pre-mRNA splicing factors, which is required for interaction with the CTD. Most remarkably, the N-terminal region of SRcyp includes a peptidyl-prolyl cis - trans isomerase domain characteristic of immunophilins/cyclophilins (Cyp), a protein family implicated in protein folding, assembly and transport. SRcyp is a nuclear protein with a characteristic distribution in large irregularly shaped nuclear speckles and co-localizes perfectly with the SR domain-containing splicing factor SC35. Recent independent investigations have provided complementary data, such as an association of the phosphorylated form of RNA polymerase II with the nuclear speckles, impaired splicing in a CTD deletion background and inhibition of in vitro splicing by CTD peptides. Taken together, these data indicate that factors directly or indirectly involved in splicing are associated with the elongating RNA polymerases, from where they might translocate to the nascent transcripts to ensure efficient splicing, concomitant with transcription.

Amino Acid Isomerases↗

PubMed1997

The VP16 paradox: herpes simplex virus VP16 contains a long-range activation domain but within the natural multiprotein complex activates only from promoter-proximal positions.

Removal of core promoter elements like the TATA box converts several regulatory upstream regions of viral and cellular genes into classical enhancers, i.e., cis-regulatory elements capable of activating transcription over long distances in an orientation-independent manner. This is not the case with herpes simplex virus (HSV) immediate-early gene promoters, which are strongly induced by the viral transactivator VP16 (Vmw65, alphaTIF, ICP25) complexed with the cellular factors Oct-1 and HCF. Here we report that the VP16 complex can readily bring about strong activation from a promoter-proximal position but fails to induce transcription from a distal downstream enhancer position. This is in striking contrast to results obtained with GAL fusion proteins: in this context, the C-terminal "general" activation domain of VP16 activates transcription to high levels over long distances. Thus, this paradoxical behavior suggests that the VP16 activation domain is not accessible to the transcription machinery when the VP16-Oct-1-HCF complex is bound in a remote position. Only upon specific interactions in a promoter-proximal position, perhaps with the basal transcription factors, can transcription be strongly induced. In agreement with such a proposed mechanism, VP16 proteins to which a heterologous general activation domain has been added strongly activate transcription from a downstream position. The biological role of this unexpected and sophisticated mechanism is most probably a limitation of the VP16 activity to the associated immediate-early genes, without undesired long-range effects on other viral promoters within the tightly packed HSV genome.

Enhancer Elements, Genetic↗

PubMed1997

A novel SR-related protein specifically interacts with the carboxy-terminal domain (CTD) of RNA polymerase II through a conserved interaction domain.

The largest subunit of the RNA polymerase II (pol II) contains at the carboxy-terminus a peculiar repetitive sequence that consists of 52 tandem repeats of the consensus motif Tyr-Ser-Pro-Thr-Ser-Pro-Ser, referred to as the C-terminal domain (CTD). Upon transcriptional initiation/promoter clearance, the CTD becomes extensively phosphorylated and apparently remains so during elongation. While the underphosphorylated CTD plays a role in transcriptional initiation, recent evidence couples the highly phosphorylated CTD to RNA processing, namely polyadenylation and splicing. Using a yeast two-hybrid screen, we have selected for human proteins that interact with the CTD of RNA polymerase II. The CTD-GAL fusion protein used as a bait is highly phosphorylated in yeast and, accordingly, we did not isolate proteins implicated in transcriptional regulation but rather proteins with possible roles in RNA splicing. One major cDNA clone isolated this way encodes SRrp129/CASP11, a protein that contains a conserved CTD-interaction domain at the C-terminus and an internal serine-arginine rich domain (SR domain). Proteins of the SR family have been implicated in RNA splicing, notably in the regulation of alternative splicing. Thus we consider it likely that SRrp129 is an auxiliary splice factor. We also improved our method to quickly map domains involved in protein-protein interaction (Stagljar et al., 1996, BioTechniques 21, 430-432). Instead of using sonication for the production of a random DNA fragment library, we took advantage of the fact that DNAse I in the presence of manganese (II) produces double strand rather than single strand DNA breaks. The DNA fragment library of the SRrp129 clone was then used in the yeast two-hybrid system to identify the 100-amino acid domain that interacts with the CTD of RNA polymerase II.

Alternative Splicing↗

PubMed1997

Silencing of RNA polymerases II and III-dependent transcription by the KRAB protein domain of KOX1, a Krüppel-type zinc finger factor.

The so-called KRAB domain, which is present in about one third of the vertebrate Kruppel-type zinc finger factors, has previously been shown to inhibit transcription in cis when tethered to promoter regions. Here we analyze this effect with fusions of the KRAB domain derived from KOX1/ZNF10 zinc finger protein to the heterologous DNA binding domains of both LexA and GAL4 factors. In transfected human cells, repression of reporter gene transcription is observed not only from proximal promoter positions, but also when KRAB is tethered to DNA at a remote position more than 1.8 kb downstream of the initiation site of transcription. Furthermore, KRAB-mediated silencing over short and long distances is not restricted to RNA polymerase II, since transcription by RNA polymerase III is also repressed. However, transcription by RNA polymerase I and by phage T7 RNA polymerase in mammalian cells are not significantly influenced by the KRAB domain. These latter results may indicate that repression by the KRAB domain, at least under our assay conditions, involves specific inhibition of some component(s) of RNA polymerase II and III transcription, rather than inducing some gross physical alteration of template chromatin structure.

Binding Sites↗

PubMed1997

Lipid-lowering effects of fluvastatin in renal transplant patients. A clinical observation.

A Atherosclerosis-related cardiovascular disease remains an important cause of morbidity and mortality in renal transplant patients. We assessed the efficacy and safety of the newer synthetic HMG-CoA reductase inhibitor, fluvastatin, in 12 renal transplant patients who remained hypercholesterolemic, despite having been on the American Heart Association (AHA) Step I diet for 6 weeks. At 8 weeks, compared to the control phase, fluvastatin therapy, 20 mg/day, reduced the total cholesterol (TC) from 321 +/- 57 [+/-SD] to 301 +/- 123 mg/dl (p = 0.3); low-density lipoprotein cholesterol (LDL-C), from 209 +/- 56 to 176 +/- 81 mg/dl (p = 0.2); and the triglyceride (TG) levels from 343 +/- 119 to 277 +/- 117 mg/dl (p = 0.06); all these changes were statistically insignificant. However, the therapy significantly increased the high-density lipoprotein cholesterol (HDL-C) from 37 +/- 11 to 46 +/- 13 mg/dl (p = 0.006). During this short-term treatment period no adverse biochemical effects were noted with the therapy.

Adult↗