[Hypopotassemia caused by cloxacillin: a new case].
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Biomedical subjects
S Plaza
Publications and source records attributed to S Plaza.
Back-mutation of the V-Ets to the C-Ets carboxy-terminal amino acids in the P135gag-myb-ets results in chicken neuroretina cells transformation and loss of basic fibroblast growth factor responsiveness.
The v-Myb, v-Ets containing E26 retrovirus (called in this work E26ABC) induces the proliferation of chicken neuroretina (CNR) cells in minimal medium, strongly stimulated by basic Fibroblast Growth Factor (bFGF) which confers on them the ability to form colonies in soft agar. V-Ets differs from its cellular counterpart c-Ets-1 by two point mutations and by the replacement of the 13 last C-terminal amino acids by 16 unrelated residues as a consequence of DNA segment inversion in the viral sequence. It has been documented that this different C-terminal sequence influences DNA binding activity and specificity. Replacement in E26ABC virus of the sequence encoding the 16 v-Ets last C-terminal amino acids by the sequence encoding the 13 c-Ets-1 derived C-terminus (virus E26ABO), results in the production of a P135gag-myb-ets with modified biological properties on CNR cells. E26ABO infected CNR cells proliferate in minimal medium more efficiently than E26ABC, are unresponsive to bFGF and able to grow in soft agar. In contrast, CNR cells infected by viruses encoding Myb and Ets proteins either in the E26ABO or in the E26ABC configuration are bFGF responsive. Since Myb alone is sufficient to induce bFGF responsiveness on CNR cells, these results suggest that the c-Ets-1 C-terminus interferes with the Myb activity of the E26ABO P135gag-myb-ets protein in CNR cells.
Animals↗
Nonstructural proteins NS2 of minute virus of mice associate in vivo with 14-3-3 protein family members.
The nonstructural NS2 proteins of the prototype strain of minute virus of mice (MVMp) were previously shown to be involved in parvoviral DNA amplification as well as in efficient virus production in a host cell-specific manner (L. K. Naeger, N. Salomé, and D. J. Pintel, J. Virol. 67:1034-1043, 1993). NS2 polypeptides were also reported to participate in the cytotoxic activity of parvoviruses (C. Legrand, J. Rommelaere, and P. Caillet-Fauquet, Virology 195:149-155, 1993), for which transformed cells are preferential targets. To identify cellular partners of NS2 proteins, coimmunoprecipitation experiments were performed with various antibodies directed against the parvoviral products. Two cellular proteins with molecular masses of 30 and 32 kDa were found to associate in vivo with the NS2 polypeptides. From amino acid sequence homology, these NS2 partners were assigned to the 14-3-3 family of cellular proteins, showing at least partial identity with the epsilon and beta or zeta 14-3-3 isoforms. In agreement with this assignment, NS2-30/32-kDa protein immune complexes displayed an activating function for exoenzyme S in vitro, a hallmark of 14-3-3 polypeptides. Interactions with 14-3-3 proteins did not appear sufficient for NS2 functions, since they were not disrupted by NS2 C-terminal modifications that impaired virus replication. Binding of NS2 to 14-3-3 proteins was detected in various cells of mouse, rat, hamster, monkey, and human origin, irrespective of NS2 dispensability and host cell transformation or permissiveness. The ubiquitous 14-3-3 proteins were recently reported to associate with several other cellular or viral polypeptides involved in signal transduction and/or cell cycle regulation pathways (A. Aitken, Trends Biochem. Sci. 20:95-97, 1995). The NS2 products may connect with one of these pathways through their interaction with specific 14-3-3 polypeptides.
14-3-3 Proteins↗
Kaposi's sarcoma with an intense parasitization by Leishmania.
Visceral leishmaniasis has been reported as a complication in patients infected with human immunodeficiency virus (HIV) who live in areas where leishmaniasis is endemic or in patients who have traveled to these areas. Kaposi's sarcoma has been found frequently in patients with acquired immunodeficiency syndrome (AIDS). We report a HIV-infected patient having visceral leishmaniasis associated with Kaposi's sarcoma in which a biopsy specimen obtained from a pigmented cutaneous lesion revealed the coexistence of a Kaposi's sarcoma pattern with Leishmania parasitic colonization.
AIDS-Related Opportunistic Infections↗
C-Myb acts as transcriptional activator of the quail PAX6 (PAX-QNR) promoter through two different mechanisms.
To understand the regulation of the Pax-6 gene, which plays an important role in eye development, we have characterized the promoter region of the quail Pax-6(Pax-QNR) gene. In addition to TATA and CAAT boxes, sequence analysis revealed several putative cis-regulatory elements among which three myb-responsive elements (MRE). C-myb encodes a nuclear, DNA-binding phosphoprotein that functions as transcriptional regulator. Co-transfection in quail embryo cells of the Pax-QNR/pax-6 promoter with a vector expressing the 75 kDa c-myb protein resulted in an increase in Pax-QNR promoter activity. By footprinting experiments we identified multiple binding sites for the myb protein within the promoter region. Protein containing the myb DNA-binding domain fused to the VP16-transactivation domain was fully efficient in Pax-QNR promoter transactivation, demonstrating that myb can transactivate through a direct binding on DNA. However, a myb truncated protein devoid of DNA-binding domain was also able to transactivate the Pax-QNR promoter. These results show that this promoter can be transactivated by the myb protein directly as well as indirectly. Finally we show by in situ hybridization that c-myb is strongly expressed in the developing neuroretina, simultaneously with Pax-QNR. These observations suggest that the c-myb protein may be a regulator of Pax-QNR/pax-6.
Animals↗
NF-Y controls transcription of the minute virus of mice P4 promoter through interaction with an unusual binding site.
Electrophoretic mobility shift assays performed with nuclear extracts from human fibroblasts revealed the formation of two major protein complexes with an oligonucleotide (nucleotides 78 to 107) from the palindromic region located upstream from the minute virus of mice (MVM) P4 promoter. It was shown that this oligonucleotide bound USF at the enhancer E box CACATG. The second complex contained the transcription factor NF-Y, whose association was surprising because its target sequence lacks the canonical CCAAT motif present in all mammalian NF-Y binding sites identified so far. The MVM NF-Y recognition element instead contains the CCAAC sequence. USF and NF-Y had distinct but overlapping sequence requirements for binding, suggesting that their associations with MVM DNA were mutually exclusive. Because of the palindromic nature of MVM DNA terminal sequences, NF-Y associated with the three nucleotide configurations corresponding to the hairpin structure and to the external and internal arms of the extended duplex replication form, respectively. However, owing to the imperfection of the palindrome, the binding of USF was restricted to the internal arm. Point mutations that suppressed the in vitro binding of NF-Y to the internal palindromic arm reduced the activity of the resident P4 promoter, while those preventing complex formation with USF did not, as determined by transient expression assays using the luciferase reporter gene. The data led to the identification of a novel P4 upstream regulatory region capable of interacting with two transcription factors, from which one (NF-Y) appeared to upmodulate the activity of the promoter.
Base Sequence↗
Isolation of a fully infectious variant of parvovirus H-1 supplanting the standard strain in human cells.
A variant H-1 virus, designated H-1 dr virus, was isolated from stock of the standard H-1 virus strain propagated in the newborn human kidney cell line NB-E. Molecular cloning and sequence analysis revealed an in-frame deletion at map positions 39 to 41. This deletion affects the open reading frames encoding the nonstructural proteins NS-1 and NS-2 and the untranslated leader sequence of the R3 transcripts encoding the capsid proteins. In addition, H-1 dr virus harbors a 58-nucleotide duplication inboard from the right-hand terminal palindrome. Internal deletions and terminal reiterations are hallmarks of H-1 virus type I variants that typically are defective interfering particles. Indeed, H-1 dr virus was found to progressively supplant the standard strain in serially coinfected NB-E cell cultures. However, H-1 dr virus differed from previously described type I variants in its full infectivity, as was apparent from its ability to give yields of replication and progeny virus production that were similar to those of the standard virus strain in NB-E cells. Hence, the interference of H-1 dr virus in the propagation of standard H-1 virus in coinfected cells was not accompanied by a drop in the titer of infectious virus. Moreover, H-1 dr virus proved to induce the same pathogenic effects in newborn hamsters as the standard virus strain did.
Base Sequence↗
Identification and characterization of a neuroretina-specific enhancer element in the quail Pax-6 (Pax-QNR) gene.
Using nuclear run-on assays, we showed that the tissue-specific expression of quail Pax-6 (Pax-QNR) P0-initiated mRNAs is due in part to regulation of the gene at the transcriptional level. Regulatory sequences governing neuroretina-specific expression of the P0-initiated mRNAs were investigated. By using reporter-based expression assays, we characterized a region within the Pax-QNR gene, located 7.5 kbp downstream from the P0 promoter, that functions as an enhancer in neuroretina cells but not in nonexpressing P0-initiated mRNA cells (quail embryo cells and quail retinal pigment epithelial cells). This enhancer element functioned in a position- and orientation-independent manner both on the Pax-QNR P0 promoter and the heterologous thymidine kinase promoter. Moreover, this enhancer element exhibited a developmental stage-specific activity during embryonic neuroretina development: in contrast to activity at day E7, the enhancer activity was very weak at day E5. This paralleled the level of expression of P0-initiated mRNAs observed at the same stages. Using footprinting, gel retardation, and Southwestern (DNA-protein) analysis, we demonstrated the existence of four neuroretina-specific nuclear protein-binding sites, involving multiple unknown factors. In addition we showed that the quail enhancer element is structurally and functionally conserved in mice. All of these results strongly suggest that this enhancer element may contribute to the neuroretina-specific transcriptional regulation of the Pax-6 gene in vivo.
Animals↗
Quail Pax-6 (Pax-QNR) mRNAs are expressed from two promoters used differentially during retina development and neuronal differentiation.
During investigations on the regulation of the Pax-6 gene, we characterized a cDNA from quail neuroretina showing a 5' untranslated region distinct from that previously described and initiated from an internal promoter. Using RNase protection and primer extension mapping, we localized this second quail Pax-6 promoter, termed P1. As reported for the already described P0 promoter, P1 was also transactivated in vitro by the p46Pax-QNR protein. RNase protection assays performed with quail neuroretina RNA showed that P1-initiated mRNAs were detected before the P0-initiated mRNAs, remained constant up to embryonic day 8, and decreased slowly thereafter whereas, P0-initiated mRNAs accumulated up to embryonic day 8. In contrast, quail retinal pigmented epithelium expressed only the P1-initiated mRNAs. Transformation of these cells by the v-myc oncogene induced neuronal traits in the culture, which thereafter, in addition to the P1-initiated mRNAs, expressed Pax-QNR from the P0 promoter. These results suggest that expression of the quail Pax-6 gene is under the control of different regulators through alternate promoters, P0 being activated at the onset of neuronal differentiation.
Animals↗
Nuclear localization signals, DNA binding, and transactivation properties of quail Pax-6 (Pax-QNR) isoforms.
We reported previously the characterization of Pax-QNR/Pax-6 products expressed in the avian neuroretina. Five proteins (48, 46, 43, 33, and 32 kDa) were characterized, among which the 33 and 32 kDa proteins are devoid of the paired domain. In contrast to the 48-kDa (containing an alternative paired exon 4a) and 46-kDa proteins exclusively located in the nucleus, the 43- (in which the paired exon 5 is spliced out), 33-, and 32-kDa proteins were also found in the cytoplasmic compartment. We report the identification of two nuclear targeting sequences: the basic LKRKLQR region (amino acids 206-212) located in the NH2 terminus of the homeodomain used by the p43 and 33/32 kDa proteins; and the paired exon 5 sequence. A case of human aniridia, where arginine 208 of LKRKLQR is mutated into a tryptophan, has been reported recently. We introduced this mutation into the Pax-QNR p46, p43, and p33/32 proteins. No effect on the nuclear localization or in transactivation potential of the proteins could be observed. Among the several Pax-QNR isoforms characterized, only p46 exhibited DNA-binding and transactivating properties on the Pax-QNR promoter. Deletions of parts of the protein showed that the Pax-6 transactivation domain is located in the carboxyl terminus of the protein.
Amino Acid Sequence↗
Induced expression of the conditionally cytotoxic herpes simplex virus thymidine kinase gene by means of a parvoviral regulatory circuit.
As a step toward the achievement of targeted expression of toxic genes, we have established a model system using the selective trans-activation of the late promoter P38 of Minute Virus of Mice (MVMp) by the parvoviral nonstructural protein NS-1. The conditionally toxic herpes simplex virus type 1 thymidine kinase (tk) gene (HSV1-tk) was cloned under the control of the P38 promoter and transfected into NIH-3T3 TK- cells. Treatment of the stably transfected cells with acyclovir (ACV) followed by infection with MVMp reduced cell survival by 3.5- to 5-fold compared to the toxic effects of ACV or MVMp alone. These results indicate that it should be possible to combine the genuine cytopathic action of parvoviruses with a specific activation of toxic genes driven by parvoviral promoters, to achieve the targeted destruction of parvovirus-expressing (in particular tumor) cells.
3T3 Cells↗
Pax-QNR/Pax-6, a paired box- and homeobox-containing gene expressed in neurons, is also expressed in pancreatic endocrine cells.
After differential screening of a cDNA library constructed from quail neuroretina cells infected with the v-myc containing avian retrovirus MC29, we have isolated a cDNA clone Pax-QNR, homologous to the murine Pax6 which is mutated in the autosomal dominant mutation small eye (Sey) of the mouse and aniridia in man. Here we report the characterization of Pax-QNR/Pax-6 expression in the chicken, quail, and mouse pancreas. In situ hybridization performed with E3 chick embryos demonstrated that, in addition to the documented expression of Pax-QNR/Pax-6 in the neural tube, this gene is also expressed in the pancreatic bud. This expression is later restricted to discrete parts of the organ. From bacterially expressed Pax-QNR peptides we obtained rabbit antisera (paired domain, serum 11; domain between paired and homeo, serum 12; homeodomain, serum 13; and carboxyl-terminal part, serum 14) capable of specifically recognizing Pax-QNR/Pax-6 proteins (48, 46 kilodaltons) in cell lines derived from alpha- and beta-pancreatic cells, but not from exocrine derived cell lines. We conclude that Pax-QNR/Pax-6 represents another gene expressed both in the endocrine pancreas and neuro-ectodermic tissues.
Animals↗
Pax-QNR/Pax-6, a paired- and homeobox-containing protein, recognizes Ets binding sites and can alter the transactivating properties of Ets transcription factors.
We have previously isolated a cDNA clone encoding a protein with a paired- and homeodomain from MC29-transformed quail neuroretina cells that we have termed Pax-QNR. Pax-QNR is homologous to the murine Pax-6, which is mutated in the autosomal dominant mutation small eye (Sey) of the mouse and aniridia in man. The 46 kDa Pax-QNR protein binds specifically to the e5 DNA recognition sequence present upstream of the Drosophila even-skipped gene. The Pax-QNR paired and homeobox domains expressed separately in bacteria are both able to recognize this sequence. The core sequence recognized by the paired domain of Pax genes is TTCC (GGAA), and this sequence is also present in the core recognition site bound specifically by Ets family-encoded proteins. Ets proteins are a family of transcription factors sharing a highly conserved 85 amino acid DNA binding domain. In this article we demonstrate that Pax-QNR/Pax-6 expressed in reticulocyte lysate is able to specifically recognize several Ets binding sites. In addition, we found that the transactivation mediated by the p68c-ets-1 pr p55erg through the Polyomavirus enhancer sequence is specifically inhibited by the p46kDaPax-QNR in transient transfection assay.
Animals↗
Identification and analysis of the chicken c-mil promoter: possible involvement of Sp1- and Ets-related proteins.
We have determined the exon organization in the 5' region of the chicken c-mil gene and identified its promoter. A 0.44-kb fragment containing the 5' terminus of the c-mil gene showed strong promoter activity when placed upstream of the bacterial chloramphenicol acetyltransferase gene and transfected into chicken embryo fibroblasts (CEF). By primer extension analysis, multiple transcriptional start sites were detected within the promoter region. Nucleotide sequence analysis revealed that the c-mil promoter had a high G + C content (71.8%) and contained multiple GC box-like sequences, but no TATA or CAAT boxes. Deletion analysis of 5' upstream sequences showed that the minimal region required for maximal promoter activity in CEF resides in the 99 bp located immediately upstream of the major initiation site. This region contains two putative Sp1 binding sites and one PU box/PEA3 motif, defined as a recognition element for members of the Ets gene family. These sequences bound proteins present in nuclear extracts of CEF as well as in vitro synthesized Ets-related proteins, suggesting that the binding of Sp1 or related proteins and of Ets-related proteins within the promoter is important for modulation of the mil gene.
Animals↗
Characterization of quail Pax-6 (Pax-QNR) proteins expressed in the neuroretina.
After differential screening of a cDNA library constructed from quail neuroretina cells (QNR) infected with the v-myc-containing avian retrovirus MC29, we have isolated a cDNA clone, Pax-QNR, homologous to the murine Pax-6, which is mutated in the autosomal dominant mutation small eye of mice and in the disorder aniridia in humans. Here we report the characterization of the Pax-QNR proteins expressed in the avian neuroretina. From bacterially expressed Pax-QNR peptides, we obtained rabbit antisera directed against different domains of the protein: paired domain (serum 11), domain between the paired domain and homeodomain (serum 12), homeodomain (serum 13), and carboxyl-terminal part (serum 14). Sera 12, 13, and 14 were able to specifically recognize five proteins (48, 46, 43, 33, and 32 kDa) in the neuroretina. In contrast to proteins of 48, 46, and 43 kDa, proteins of 33 and 32 kDa were not recognized by the paired antiserum (serum 11). Paired-less and paired-containing proteins exhibited the same half-life (6 h) and were phosphorylated mostly on serine residues. Immunoprecipitations performed with subcellular fractions of neuroretinas showed that the paired-containing proteins were located in the nucleus, whereas the 33- and 32-kDa proteins were found essentially in the cytoplasmic compartment. However, immunofluorescence experiments performed after transient transfections showed that p46 and p33/32 were also located in vivo into the nucleus. Thus, the Pax-QNR/Pax-6 gene can produce proteins with two DNA-binding domains as well as proteins containing only the DNA-binding homeodomain.
Animals↗
Quail Pax-6 (Pax-QNR) encodes a transcription factor able to bind and trans-activate its own promoter.
Proper growth and development of multicellular organisms requires precise regulation of developmental genes. One aspect of this regulation is at the level of transcription from the gene promoters. As an initial approach to understanding the regulation of the Pax-6 gene, which plays an important role in eye development and perhaps in other developmental processes, we characterized a promoter region of the quail Pax-6 (Pax-QNR) gene. Sequence analysis of the 5' flanking region revealed a TATA-like box and a CAAT box as well as several putative cis-regulatory elements. A 1.5-kilobase pair fragment, containing 1386 base pairs of 5' flanking sequence, the first exon, and a portion of the first intron, was able to efficiently promote expression of the bacterial CAT gene in quail neuroretina cells. Cotransfection of the Pax-QNR promoter with a vector expressing the 46 kilodalton Pax-QNR protein resulted in an increase in Pax-QNR promoter activity. By electrophoretic migration shift assay and immunoselection experiments, we showed that the Pax-QNR protein can interact directly with the Pax-QNR promoter. By footprinting experiments, we identified the binding sites for the Pax-QNR protein within the promoter region. These results show that Pax-QNR encodes a transcriptional activator and that it potentially trans-activates its own promoter.
Animals↗
Fos expression induces growth arrest in tumorigenic neuroretina cells.
E26-infected chicken neuroretina (CNR) cells expressing P135gag-myb-ets are non-transformed and growth can be stimulated by basic fibroblast growth factor (bFGF). In contrast, MHE226-infected CNR cells, which express p61/63myc in addition to the P135gag-myb-ets of E26, are transformed, tumorigenic cells and are growth inhibited by bFGF. We looked for differences in both cells types which could help to understand the molecular basis of the bFGF response. We found marked differences in c-fos expression. In MHE226-CNR cells c-fos mRNA was induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA) and bFGF, both inhibitors of MHE226-infected cell growth. In contrast, serum, a strong growth inducer, did not stimulate c-fos expression. In E26-infected cells all of these factors induced cell growth and c-fos expression. MHE226-CNR cells superinfected with v-fosFBJ-expressing retrovirus were inhibited in their growth and unresponsive to bFGF. Introduction of v-fosFBJ in E26 CNR cells transformed them but they remained sensitive to bFGF. These results suggest that fos is involved in the induced growth arrest of MHE226-CNR cells.
Animals↗
New human erg isoforms generated by alternative splicing are transcriptional activators.
The erg gene is a member of the ets gene family. ETS proteins have been shown to bind specifically the (GGA-A/T) motif and to transactivate via this consensus sequence. The human erg products exhibit approximately 70% homology with ETS proteins in their DNA-binding domain. We have isolated three erg cDNAs from a human fetal liver library. Two of them are different from the previously described erg-1 and erg-2 cDNAs (Rao et al., Science, 1987, 237, 635-639), in the middle of their coding sequence and in their 5' part where a novel initiation codon is introduced. These isoforms are generated by alternative RNA splicing from a single gene that leads to the inclusion or exclusion of different exon sequences. The three cDNAs expressed by an in vitro transcription-translation system direct the synthesis of proteins of approximately 38, 49 and 55 kDa. These in vitro erg products were tested for their DNA-binding activity by gel mobility-shift assays with different probes containing the ETS-specific binding site. The results indicated that all these erg isoforms are able to bind the ETS binding site in a specific manner. Our data using transient transfection assays indicate that erg protein isoforms function as transcriptional activators.
3T3 Cells↗