Surface modification of acid-functionalized mesoporous gamma-alumina for non-fission 99Mo/99mTc generator.

Mesoporous gamma-alumina (MGA) was synthesized for neutron-activated 99Mo adsorbent. Acid functionalization of the MGA was carried out to enhance the Mo adsorption capacity and the 99Mo breakthrough profile. The acid-treated MGA has a more positive particle charge, rougher surface, smaller particle and pore size, larger surface area, and wider pore distance. The acid-treated MGA has a Mo adsorption capacity of 82.8 ± 6.3 mg Mo/g and resulted in 99mTc eluate with the 99Mo breakthrough at the acceptable level.

Caspase-1 cleavage of transcription factor GATA4 and regulation of cardiac cell fate.

Caspase-1 or interleukin-1ß (IL-1ß) converting enzyme is a pro-inflammatory member of the caspase family. An IL-1ß-independent role for caspase-1 in cardiomyocyte cell death and heart failure has emerged but the mechanisms underlying these effects are incompletely understood. Here, we report that transcription factor GATA4, a key regulator of cardiomyocyte survival and adaptive stress response is an in vivo and in vitro substrate for caspase-1. Caspase-1 mediated cleavage of GATA4 generates a truncated protein that retains the ability to bind DNA but lacks transcriptional activation domains and acts as a dominant negative regulator of GATA4. We show that caspase-1 is rapidly activated in cardiomyocyte nuclei treated with the cell death inducing drug Doxorubicin. We also find that inhibition of caspase-1 alone is as effective as complete caspase inhibition at rescuing GATA4 degradation and myocyte cell death. Caspase-1 inhibition of GATA4 transcriptional activity is rescued by HSP70, which binds directly to GATA4 and masks the caspase recognition motif. The data identify a caspase-1 nuclear substrate and suggest a direct role for caspase-1 in transcriptional regulation. This mechanism may underlie the inflammation-independent action of caspase-1 in other organs.

Purification of human recombinant GATA-1 from bacteria: implication for protein-protein interaction studies.

GATA-1 is a key regulator of terminal erythroid differentiation in mammals and birds. The structural and biochemical studies of human GATA-1 (hGATA-1) are limited by the difficulty of its purification in a sufficient amount. Here we describe the procedure for obtaining pure bacterial recombinant hGATA-1 in an active functional state. We demonstrate that this protein may be successfully used for preparing an affinity column, producing GATA-1-specific rabbit polyclonal antibodies, and studying DNA-protein and protein-protein interactions.

Serum response factor-GATA ternary complex required for nuclear signaling by a G-protein-coupled receptor.

Endothelins are a family of biologically active peptides that are critical for development and function of neural crest-derived and cardiovascular cells. These effects are mediated by two G-protein-coupled receptors and involve transcriptional regulation of growth-responsive and/or tissue-specific genes. We have used the cardiac ANF promoter, which represents the best-studied tissue-specific endothelin target, to elucidate the nuclear pathways responsible for the transcriptional effects of endothelins. We found that cardiac-specific response to endothelin 1 (ET-1) requires the combined action of the serum response factor (SRF) and the tissue-restricted GATA proteins which bind over their adjacent sites, within a 30-bp ET-1 response element. We show that SRF and GATA proteins form a novel ternary complex reminiscent of the well-characterized SRF-ternary complex factor interaction required for transcriptional induction of c-fos in response to growth factors. In transient cotransfections, GATA factors and SRF synergistically activate atrial natriuretic factor and other ET-1-inducible promoters that contain both GATA and SRF binding sites. Thus, GATA factors may represent a new class of tissue-specific SRF accessory factors that account for muscle- and other cell-specific SRF actions.

Natural pregnancy in hypothyroid woman complicated by spontaneous ovarian hyperstimulation syndrome.

A unique case of life-threatening spontaneous ovarian hyperstimulation syndrome, resulting from severe untreated hypothyroidism, was observed in a woman who conceived spontaneously and gave birth to a normal viable infant.

Anthracyclines as tumor cell differentiating agents: effects on the regulation of erythroid gene expression.

Tumor cells, and particularly leukemic cells, can be considered as maturation-arrested cells which have escaped some normal control and continue to proliferate. This maturation arrest can be reversed by differentiation agents such as antitumor drugs currently used in conventional cytotoxic chemotherapy. In this respect, anthracyclines have been shown to trigger the differentiation of leukemic and solid tumor cells, but the molecular mechanisms by which such drugs lead to the differentiating phenotype are still poorly understood. Using human leukemic multipotent K562 cells, we have demonstrated that subtoxic concentrations of aclacinomycin (ACLA) and doxorubicin (DOX) preferentially stimulate the hemoglobinic pathway (globins and heme synthesis) and the expression of mRNAs of globins and of porphobilinogen deaminase (PBGD). However, our results indicate that both drugs exert this differentiating effect along distinct regulatory pathways. Indeed, only ACLA and not DOX induces the expression of erythropoietin receptor (EpoR) mRNAs and of membrane EpoR, as well as an overexpression of the erythroid transcription factors GATA-1 and NF-E2 known to play a central role in erythroid gene regulation. Similarly, using transfection assays, ACLA but not DOX activates the regulatory regions (promoters and enhancers) of GATA-1, EpoR, PBGD, epsilon- and gamma-globin genes. Finally, results of run-on assays indicate that ACLA induces an enhancement of the transcription rate of these erythroid genes whereas DOX preferentially increases stability of GATA-1, NF-E2 and PBGD mRNAs. In conclusion, ACLA mainly acts at the transcriptional level via specific activation of erythroid regulatory regions whereas DOX rather acts at the posttranscriptional level by increasing the half-lives of erythroid mRNAs.

Activation of erythroid-specific promoters during anthracycline-induced differentiation of K562 cells.

Anthracycline antitumor drugs such as aclacinomycin (ACM) and doxorubicin (DOX) used in subtoxic concentrations induce erythroid differentiation of the erythroleukemic cell line K562. To elucidate the possible role of erythroid genes of the erythropoietin receptor (EpoR) and the transcription factor GATA-1 in this effect, the regulatory regions of the above genes and human epsilon- and gamma-globin and porphobilinogen deaminase (PBGD) genes were fused to the firefly luciferase gene. The resulting reporter constructs were tested in a transfection assay of the erythroleukemic cell line K562 stimulated to differentiate by treatment with the anthracycline drugs ACM and DOX or hemin (HEM). The results showed activation of the tested promoters after cell treatment with ACM, but not with DOX or HEM. In contrast to the mouse EpoR gene promoter, the activity of the human EpoR gene promoter (-659/-60) in the reporter construct was not modified by addition of the first intron sequence. In ACM-treated K562 cells, EpoR gene promoter activity completely correlated with EpoR and GATA-1 mRNA levels and the degree of erythroid maturation. In addition, ACM strongly activated the erythroid gene promoters that contain GATA binding sites. Nevertheless, less activation was also observed for the GATA-1 gene promoter (-312/-31) lacking any known GATA binding sites. Insertion of the GATA-1 gene enhancer with two canonic GATA binding sites, stimulated the ACM activation effect for EpoR and GATA-1 promoter-containing constructs. Mutation of the enhancer GATA binding sites abolished this effect. All the regulatory regions tested (except gamma-globin promoter) were completely inactive in nonerythroid COS7 cells. These data indicate that (1) two structurally different anthracycline analogues, DOX and ACM, differ in their differentiation mechanisms, and (2) ACM switches on the erythroid program of K562 cells, at least in part because of interaction with a factor(s) that recognizes the GATA binding sites in the promoter region of erythroid genes leading to their activation.

Evidence for distinct regulation processes in the aclacinomycin- and doxorubicin-mediated differentiation of human erythroleukemic cells.

Human erythroleukemic K 562 cells were induced to were induced to differentiate along the erythroid lineage by anthracycline antitumor drugs, such as aclacinomycin (ACLA) and doxorubicin (DOX). Subsequent stimulation of heme and globin synthesis led to a differential quantitative expression of hemoglobins. Gower 1 (epsilon2, zeta2) was the major type for ACLA and X (epsilon2, gamma2) for DOX. Although ACLA and DOX increased both the expression of gamma-globin and porphobilinogen deaminase mRNAs, striking differences were observed in the expression of erythropoietin receptor mRNAs and in erythroid transcription factors GATA-1 and NF-E2, known to play a key role in erythroid gene regulation. Indeed, ACLA induces an increase either in the binding capacity of GATA-1 and NF-E2 or in the accumulation of erythropoietin receptor, GATA-1 and NF-E2 transcripts. In contrast, their expression with DOX was not significantly modified compared to uninduced cells, except for a slight decrease in NF-E2 expression on day 3. In conclusion, these data show that: 1. increased expression of erythroid transcription factors and erythroid genes are associated only with ACLA treatment, and 2. although cytotoxicity of both ACLA and DOX is certainly dependent on DNA intercalation, regulation of differentiation processes by these two drugs involves distinct mechanisms.

Effect of aclacinomycin-doxorubicin association on differentiation and growth of human erythroleukemic K562 cells.

Anthracycline antitumor drugs such as doxorubicin (DOX) and aclacinomycin (ACM) represent potent candidates for the induction of differentiation of leukemic cells. Human multipotent K562 cells were induced by DOX and ACM to differentiate towards the erythroid lineage. After 3 days of culture, DOX-induced differentiation was dose-related whereas ACM did not require total cell growth arrest to induce its optimum effect, indicating that both drugs act differently on the coupling of growth and differentiation. Simultaneous exposure to ACM and DOX and sequential exposure to ACM (30 min) first, followed immediately by DOX did not improve erythroid differentiation. However, it led to either a synergistic or a subadditive inhibition of cell growth. In contrast, DOX (30 min) first, followed by ACM, produced in a narrow range of concentrations (DOX 1000 nM/ACM 1.85 nM, 3.75 nM or 7.5 nM), a synergistic induction of differentiation. Thus, DOX 1000 nM/ACM 3. 75 nM resulted in 81% of differentiated cells compared to 63% for ACM 15 nM and 43% for DOX 30 nM when these were used alone (at their concentration inducing optimum differentiation). In conclusion, these data emphasize the importance of schedules for the combination of chemotherapeutic drugs acting as differentiation inducers.

Increased expression of GATA-1 and NFE-2 erythroid-specific transcription factors during aclacinomycin-mediated differentiation of human erythroleukemic cells.

Anthracycline antitumor drugs, particularly aclacinomycin (ACM) have been shown to be potent inducers of erythroid differentiation in human leukemic K562 cells. Here we report that such an event is associated with an overexpression of the erythroid-specific transcription factors GATA-1 and NFE-2. Using the electrophoretic mobility shift assay, during differentiation over 3 days of culture, we have observed an increase in the binding either of GATA-1 to the promoter of the gamma-globin gene (region -201 to -156) or NFE-2 to the promotor of the porphobilinogen deaminase gene (region -170 to -142). Both events were paralleled by a recruitment of hemoglobinized cells and a stimulation of heme synthesis. Enhanced binding capacity of GATA-1 was confirmed by an increase in its mRNAs. Moreover, GATA-1 and NFE-2 overexpression has been shown to be specific of the differentiating effect of the drug and not of its growth inhibitory effect. In contrast, no change was observed in the binding of the ubiquitous factors OTF-1 and AP-1, except on day 3, where AP-1 decreased. Although ACM is a DNA-intercalating agent, it did not directly affect transcription factors binding to their cis-sequences as assessed by the preincubation of the oligonucleotides probes with increasing concentrations of ACM. Taken together, these results strongly suggest that ACM could exert their erythroid-differentiating activity by modulating the expression of transcription factors which specifically regulate the transcription of erythroid genes.

[Congenital cervico-vaginal atresia and pelvic endometriosis. Clinical case and review of the literature]. / Angeborene zerviko-vaginale Atresie und Beckenendometriose. Klinischer Fall und Literaturübersicht.

Congenital cervico-vaginal atresia is a rare malformation; the presence of a functioning endometrium combined with this anomaly causes a retrograde menstruation and then a greater likelihood of endometriosis. Up to the present literature relates only thirteen cases in which congenital cervico-vaginal atresia was combined with a functioning endometrium. Primary amenorrhea and cyclic, cramping lower abdominal pain was the common symptoms of all the patients. In this work we report a new case of such pathology in a 23 old woman affected also by harmonic nanism. After the review of the literature the embryologic mechanisms involved in lower Mullerian tract malformations, the surgical treatment and the mechanism of formation of endometriosis in association with an outflow tract obstruction are discussed.