Incorporation Of 3H-uridine Research Articles

Abstract 1622: Ecubectedin is a novel transcriptional inhibitor that displays potent antitumor effects in vitro and in vivo

Abstract Background - Ecubectedin (PM14) is a novel transcriptional inhibitor related to ecteinascidins family. In this work, we present its antiproliferative activity and mechanism of action. The in vitro antitumor activity obtained in cellular models has been also demonstrated in in vivo models. Methods and results - The antineoplastic activity of ecubectedin was evaluated in a panel of cell lines representative of different solid human cancers, by MTT assays. Ecubectedin exhibits potent antitumor in vitro activity with mean GI50 values in the very low nanomolar range. Electrophoretic Mobility Shift assays demonstrate that ecubectedin binds to DNA and experiments of 3H-uridine incorporation show that the drug efficiently inhibits mRNA synthesis (85% reduction after 90 min). Moreover, ecubectedin specifically inhibits transactivated transcription, as evaluated in a reporter system of the transcriptional activity mediated by NF-κB after activation with TNFα. In the presence of ecubectedin, transactivation was almost completely abolished. The mechanism of transcriptional inhibition involves the stalling and irreversible proteasomal degradation of elongating RNA Pol II. As a final event, ecubectedin induces double strand breaks in the DNA, inducing cell cycle arrest in the S-phase that can be followed through flow cytometry and triggers apoptotic death of the tumor cell as seen through Annexin V flow cytometry assays. The in vivo antitumor activity of ecubectedin was characterized in xenografted tumors of human origin in athymic nu/nu mice, namely breast (MDA-MB-231), soft tissue sarcoma (HT-1080), SCLC (H526 and H82) and prostate (22Rv1). Ecubectedin was intravenously administered (at 1.25 mg/kg) on days 0, 7 and 14. Compared to placebo, ecubectedin treatment of mice bearing xenografts resulted in a statistically significant reduction of volume in MDA-MB-231 (p≤0.0005 on days 7-35), HT-1080 (p≤0.0004 on days 2-14), H82 (p≤0.0001 on days 5-14), H526 (p≤0.0001 from day 4 to the end of the experimental period), and 22Rv1 (p≤0.0001 on days 3-14) tumors. In mice bearing MDA-MB-231, H526 and 22Rv1 tumors, complete tumor remissions were seen in 1/10 (lasting 103 days), 9/10 (lasting 220 days) and 7/10 (lasting 13 days) ecubectedin-treated animals, respectively. Conclusions - Ecubectedin is a novel transcriptional inhibitor that displays potent antiproliferative activity in different human solid cancer models in vitro and in vivo. The drug is currently in clinical development in Phase II trials. Citation Format: Gema Santamaría Nuñez, Marta Martínez Diez, María José Guillén, Eva Maria Garrido-Martin, Pablo Avilés, Carmen Cuevas. Ecubectedin is a novel transcriptional inhibitor that displays potent antitumor effects in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1622.

Day and night regulation of the HO-1/HSP32 synthesis in the harmful dinoflagellate Prorocentrum minimum: response to salinity stress

Potentially toxic dinoflagellate species Prorocentrum minimum (Pavillard) Schiller, 1933 colonized the brackish-water Baltic Sea in the early 1980s, where thereafter it has been forming regular harmful blooms. Heat shock proteins (HSP) play an important role in their ability to adapt to environmental stress. In this study, we evaluated how the synthesis of heme oxygenase-1 (HO-1/HSP32), an important part of cellular machinery for cell protection, changes in P. minimum under the influence of salinity stress during the day-night cycle. We identified sequences of the HO-1 homologs in the unannotated P. minimum transcriptomes, represented in the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) database. Phylogenetic analysis showed that this protein clustered in a distinct clade and demonstrated an evolutionary relationship with the HO-1 homologs of other taxonomic groups. To identify P. minimum cells that express the HO-1 protein, we used fluorescent flow cytometry. In control conditions (17‰), the number of cells expressing this protein was 2.2 times higher during the day than at night, with the same average fluorescence intensity (1.7 units). During the daytime, the critical salinity conditions of 8‰ did not lead to significant changes in the number of cells expressing HO-1, while in the night culture this salinity shift caused a 2.7-fold increase in the number of cells expressing HO-1, and led to an increase in the intensity of fluorescence up to 2.0 units. The overall DNA and RNA synthesis rates were estimated by the incorporation of 3H-thymidine and 3H-uridine, respectively. Transcription and replication levels were significantly higher under critical salinity conditions (8‰) compared to the control (17‰) and typical sea salinity conditions (35‰). The greatest increase in the DNA synthesis rates (up to 2.7 times) was observed during the daytime in cells exposed to salinity stress at 8‰. In contrast, the greatest increase in the RNA synthesis rates (2.7 times) was registered in cells exposed to salinity of 8‰ at night. Thus, the replication activity of cells in response to salinity stress increased significantly during the day, while transcription levels reached their maximum values at night. These results indicate that a significant increase in the synthesis of both nucleic acids and the HO-1 stress protein can serve as a biomarker of the impact of environmental stress factors, such as salinity. This allowed concluding that the studied parameters demonstrate a high potential for future modeling of harmful algae blooms, particularly those formed by the invasive potentially toxic species under horohaline conditions of the Baltic Sea.

Dur3 and nrt2 genes in the bloom-forming dinoflagellate Prorocentrum minimum: Transcriptional responses to available nitrogen sources.

The increasing inflow of nitrogen (N) substrates into marine nearshore ecosystems induces proliferation of harmful algal blooms (HABs) of dinoflagellates, such as potentially toxic invasive species Prorocentrum minimum. In this study, we estimated the influence of NO3-, NH4+ and urea on transcription levels and urea transporter dur3 and nitrate transporter nrt2 genes expression in these dinoflagellates. We identified dur3 and nrt2 genes sequences in unannotated transcriptomes of P.minimum and other dinoflagellates presented in MMETSP database. Phylogenetic analysis showed that these genes of dinoflagellates clustered to the distinct clade demonstrating evolutionary relationship with the other known dur3 and nrt2 genes of microalgae. The evaluation of expression levels of dur3 and nrt2 genes by RT-qPCR revealed their sensitivity to input of the studied N sources. Dur3 expression levels were downregulated after the supplementation of additional N sources and were 1.7-2.6-fold lower than in the nitrate-grown culture. Nrt2 expression levels decreased 1.9-fold in the presence of NH4+. We estimated total RNA and DNA synthesis rates by the analysis of incorporation of 3H-thymidine and 3H-uridine in batch and continuous cultures. Addition of N compounds did not affect the DNA synthesis rates. Transcription levels increased up to 12.5-fold after the N supplementation in urea-limited treatments. Investigation of various nitrogen sources as biomarkers of dinoflagellate proliferation due to their differentiated impact on expression of dur3 and nrt2 genes and transcription rates in P.minimum cells allowed concluding about high potential of the studied parameters for future modeling of HABs under global N pollution.

Influence of hydroxyurea on nucleic acids content and 3H-uridine incorporation in callus and tumorous tobacco tissues cultured in vitro

In callus and tumor tissues of <i>Nicotiana tabacum</i> cultured for 39 days in media supplemented with various concentrations of hydroxyurea (1.3 x 10<sup>-4</sup> M - 1.3 x 10<sup>-3</sup> M) a decrease of DNA content (ca. 24 per cent in callus tissue and ca. 23 per cent in tumour tissue) and a decrease of RNA content (over 10 per cent and ca. 9 per cent in callus and tumour tissue, respectively) was observed. The autoradiographic method showed that a long-lasting action of this com-pound inhibits RNA synthesis. A stronger inhibitory influence of hydroxyurea upon incorporation of 3H-uridine from the incubation medium was revealed.

Further observations on the activity of Y chromosome heterochromatin in Rumex thyrsiflorus

Observation of premeiotic nuclei of plants containing different numbers (from one to five) of Y chromosomes reveal that is such plants prior to meiosis all Y chromosomes are in fuzzy state, while in nuclei of tapetal cells or nuclei from anther stalk the number of big chromocentres is directly correlated with the number of Y chromosomes in given plants. This finding provides a further indication of genetic activity of Y chromosomes in this particular stage of the life cycle (premeiosis). Another indication of genetical activity of Y chromosomes was obtained from analysis of the rate of RNA synthesis in premeiosis. It was found that PMC's in the last premeioitc interphase exhibit a high rate of 3H-uridine incorporation. Thus in PMC's the fuzzy appearance of Y chromosomes coincides with intensive RNA synthesis.

The role of zeatin and gibberellic acid in breaking of the abscisic acid-induced dormancy in Triticale caryopses

The investigations were conducted on the germinating embryos and the whole caryopses of <em>Triticale</em>. During preimbibition and 24 hours germination caryopses were treated with abscisic acid (ABA), which produced 63% inhibition of embryo growth. Gibberellin-A<sub>3</sub> (GA<sub>3</sub>) reversed the ABA effect in 18%, while zeatin in 22%. The clear synergic reaction was observed (36%) when both stimulators acted together. There was no significant effect of ABA, ABA and GA<sub>3</sub>, as well as ABA and zeatin on the synthesis of polyribosomal RNA in the initial period of germination of excised embryos. However, during 24 hours germination of whole caryopses ABA caused a twofold decrease in <sup>3</sup>H-uridine incorporation into the total fraction of embryonic ribosomes. While the incorporation of <sup>14</sup>C-aminoacid mixture into ribosomal proteins was even three-fold lower. Effect of GA<sub>3</sub> and zeatin on breaking of the ABA-induced "dormancy" was studied. It was confirmed that the higher polyribosome contribution to the sum total of ribosomes the more intensive synthesis of ribosomal proteins. No higher <sup>3</sup>H-uridine incorporation into polyribosomal fraction was observed. From the results it may be inferred that in the initial period of germination of <em>Triticale</em> caryopses regulation of protein biosynthesis occurs rather at the translation than transcription level.

Ribonucleic acids and ribosomal proteins synthesis during germination of unripe and aged wheat caryopses

Caryopses collected at milk, wax and full ripeness as well as aged ones stored at air humidity of 30, 60 and 80 per cent were subjected to germination. The more unripe were the caryopses the lower was their germination capacity and dry mass increment of the sprouts, the same was true for fully ripe but aged caryopses. After 48 h of germination in the presence of radioactive precursors the embryos were removed from the caryopses and used for isolation of the total ribosomal fraction and RNA. Distinct synthesis of three basic total RNA fractions and of the polyribosomal fraction was observed in all samples and in embryos from grain which had completely lost its germination capacity. High radioactivity was recorded in preparations of total RNA and polyribosomal ones from 48-h sprouts growing from caryopses collected at milk ripeness and in those of aged caryopses stored under air humidity of 30 and 60 per cent. According to the authors, the depressed amount of latent forms of RNA (preformed mRNA, tRNA and ribosomes) is the cause of intensive <sup>3</sup>H-uridine incorporation into RNA in embryos from unripe and aged caryopses. For germination of grains with a reduced amount of latent forms the biosynthesis mechanisms must be based to a much higher extent on newly synthesised molecules of ribonucleic acids.

Ribonucleic acids in the embryos of germinating wheat grains of different ripeness

During 48 h germination of wheat grains of different ripeness the amount of RNA in the germinating embryos doubles, while the rate of synthesis is to a large extent correlated with the level of ribonucleic acids accumulated during the development and ripening of the grain. The highest RNA content was noted in the germs developing from grain harvested at the stage of full ripeness, the lowest - in germs from grain at milk ripeness. Intensity of <sup>3</sup>H-uridine incorporation into the RNA fraction of the germs during 24-h germination also depends on ripeness. Significantly lower RNA synthesis was noted in germinating embryos from wheat grain harvested during milk ripeness as compared to wax or full ripeness.

Studies on the mechanism which prevents germination of unripe Triticale caryopses

<em>Triticale</em> (MT-3) caryopses were collected at three developmental stages (milk, milk-wax, and full ripeness i.e. 25, 39, 53 days after flowering) and germinated for 72 hours. The highest polyribosome contribution to the sum total of ribosomes, as well as, the highest <sup>3</sup>H-uridine incorporation into the total ribosomal fraction of embryos (seedlings) were found at full ripeness, lower - at milk ripeness, and the lowest at milk-wax ripeness. Treatment of caryopses with gibberellin-A<sub>3</sub> (GA<sub>3</sub>) and benzyloadenine (BA) caused an increase in the percentage of embryonic polyribosomes in caryopses which were collected at milk and full ripeness. Whereas the significant increase in <sup>3</sup>H-uridine incorporation intothe total ribosomal fractions of embryos (seedlings) was observed only during the germination of the least ripe caryopses. This was characteristic of samples with caryopses at milk ripeness treated with BA, or BA and GA<sub>3</sub> together. The studies proved that the mechanism which prevents the germination of unripe Triticale cyryopses and the formation of polyribosomes which were germination-induced, originated at the final stage of grain development, before its full ripeness.

The metabolism of aged seeds. The formation of polyribosomes in germinating field bean ( Vicia faba sp. minor) seeds of different ages

Total dehydrogenase activity and formation of polyribosomes in embryonic axes and cotyledons of field bean seeds from different harvest years were studied. <sup>3</sup>H-uridine incorporation experiments showed that seed ageing was accompanied by decreased capability for RNA synthesis and polyribosome formation. The embryonic axes of seeds with reduced vigor contained lower levels of polyribosomes.

Cytometry of DNA replication and RNA synthesis: Historical perspective and recent advances based on “click chemistry”

This review covers progress in the development of cytometric methodologies designed to assess DNA replication and RNA synthesis. The early approaches utilizing autoradiography to detect incorporation of (3) H- or (14) C-labeled thymidine were able to identify the four fundamental phases of the cell cycle G(1) , S, G(2) , and M, and by analysis of the fraction of labeled mitosis (FLM), to precisely define the kinetics of cell progression through these phases. Analysis of (3) H-uridine incorporation and RNA content provided the means to distinguish quiescent G(0) from cycling G(1) cells. Subsequent progress in analysis of DNA replication was based on the use of BrdU as a DNA precursor and its detection by the quenching of the fluorescence intensity of DNA-bound fluorochromes such as Hoechst 33358 or acridine orange as measured by flow cytometry. Several variants of this methodology have been designed and used in studies to detect anticancer drug-induced perturbations of cell cycle kinetics. The next phase of method development, which was particularly useful in studies of the cell cycle in vivo, including clinical applications, relied on immunocytochemical detection of incorporated halogenated DNA or RNA precursors. This approach however was hampered by the need for DNA denaturation, which made it difficult to concurrently detect other cell constituents for multiparametric analysis. The recently introduced "click chemistry" approach has no such limitation and is the method of choice for analysis of DNA replication and RNA synthesis. This method is based on the use of 5-ethynyl-2'deoxyuridine (EdU) as a DNA precursor or 5-ethynyluridine (EU) as an RNA precursor and their detection with fluorochrome-tagged azides utilizing a copper (I) catalyzed [3+2] cycloaddition. Several examples are presented that illustrate incorporation of EdU or EU in cells subjected to DNA damage detected as histone H2AX phosphorylation that have been analyzed by flow or laser scanning cytometry.

Effect of short- and long-lasting chilling on pre-rRNA synthesis and transport in root meristematic cells of three soybean cultivars

<p>Autoradiographic studies of <sup>3</sup>H-uridine incorporation (20-min incubation) and dynamics of radioactive particle translocation from nucleolus into cytoplasm (following 80-min postincubation in non-radioactive medium) in root meristematic cells of soybean have been carried out. The experiment was performed with plants subjected to 4-day acclimation in chilling or subjected to 2-hour cold stress and control plants. Three cultivars of soybean: Mazowia, Polan and Progres (cultivated in Poland) were used in the experiment.</p><p>It has been shown that in control conditions the greatest number of RNA precursor is incorporated into nucleoli after 20-min incubation. Following 80-min postincubation cytoplasm is the most radioactive area of the cell - this mainly testifies to dynamic translocation of radioactive ribosome subunits from nucleolus into cytoplasm.</p><p>In chilling conditions the reduction of <sup>3</sup>H-uridine incorporation into cells occurs, as compared to control conditions. Plants subjected to a 4-day acclimation incorporate the radioactive precursor more intensively than plants subjected to cold stress.</p><p>Following 80-min postincubation - in the case of acclimated plants - the nucleolus is the most radioactive area of the cell, which testifies to accumulation of pre-rRNA in it. After the cold stress cytoplasm is more radioactive than the nucleolus. In all three cultivars the processes of synthesis and transport of pre-rRNA particles are similar, only their intensity is different.</p><p>Morphometric measurements of nucleoli in all cultivars subjected to 4-day chilling have shown that root cell nucleoli are larger than those in control. This phenomenon can be connected with stronger inhibition of rRNA transport than its synthesis.</p>

6-Nitro-2-(3-hydroxypropyl)-1H-benz[de]isoquinoline-1,3-dione, a potent antitumor agent, induces cell cycle arrest and apoptosis

BackgroundAnticancer activities of several substituted naphthalimides (1H-benz[de]isoquinoline-1,3-diones) are well documented. Some of them have undergone Phase I-II clinical trials. Presently a series of ten N-(hydroxyalkyl) naphthalimides (compounds 1a-j) were evaluated as antitumor agents.MethodsCompounds 1a-j were initially screened in MOLT-4, HL-60 and U-937 human tumor cell lines and results were compared with established clinical drugs. Cytotoxicities of compounds 1d and 1i were further evaluated in a battery of human tumor cell lines and in normal human peripheral blood mononuclear cells. Cell cycle analysis of compound 1i treated MOLT-4 cells was studied by flow cytometry. Its apoptosis inducing effect was carried out in MOLT-4 and HL-60 cells by flow cytometry using annexin V-FITC/PI double staining method. The activities of caspase-3 and caspase-6 in MOLT-4 cells following incubation with compound 1i were measured at different time intervals. Morphology of the MOLT-4 cells after treatment with 1i was examined under light microscope and transmission electron microscope. 3H-Thymidine and 3H-uridine incorporation in S-180 cells in vitro following treatment with 8 μM concentration of compounds 1d and 1i were studied.Results6-Nitro-2-(3-hydroxypropyl)-1H-benz[de]isoquinoline-1,3-dione (compound 1i), has exhibited maximum activity as it induced significant cytotoxicity in 8 out of 13 cell lines employed. Interestingly it did not show any cytotoxicity against human PBMC (IC50 value 273 μM). Cell cycle analysis of compound 1i treated MOLT-4 cells demonstrated rise in sub-G1 fraction and concomitant accumulation of cells in S and G2/M phases, indicating up-regulation of apoptosis along with mitotic arrest and/or delay in exit of daughter cells from mitotic cycle respectively. Its apoptosis inducing effect was confirmed in flow cytometric study in MOLT-4 and the action was mediated by activation of both caspase 3 and 6. Light and transmission electron microscopic studies corroborated its apoptosis inducing efficacy at a concentration of 10 μM in MOLT-4 cells. Its apoptosis induction was also observed in HL-60 cells to an extent much greater than well known apoptosis inducing agents as camptothecin and cis-platin at 10 μM concentration each. It significantly inhibited DNA and RNA synthesis in S-180.ConclusionsIn essence, compound 1i showed potential as an antitumor agent.

Quantitative EEG and neurochemical aspects of memory and learning.

Different, established or putative animal models of learning were studied by electrophysiological (EEG) and neurochemical methodologies. The training of rats to new behavioral patterns proved to result in the stimulation of total RNA synthesis rate in specific brain structures, as well as in modifications of the EEG organization of hippocampus. This two-fold approach to the assessment of modifications in learning-involved brain structures was extended to the study of mirror focus and kindling, to verify the suitability of these phenomena as experimental models of learning. Reduced (3H) uridine incorporation and proportion of poly (A)-associated RNA were found in mirror focus in comparison with control regions. These variations seem related to brain damage rather than be congruent with learning-related modifications. Further studies are necessary to verify whether the kindling phenomena are to be equated to learning models.

Aluminium(III), chromium(III) and iron(III) complexes with 5-iodouracil and 5-iodouracil-histidine and their antitumour activity

Complexes of the type [Al(HL)(OH)Cl2], [M(HL)(OH)2Cl] and [M′(HL)(L′)(OH)Cl], where HL = 5-iodouracil; HL′ = histidine; M = Cr(III), Fe(III) and M′ = Al(III), Cr(III), Fe(III), were synthesized and characterized. The complexes are polymeric showing high decomposition points and are insoluble in water and common organic solvents. The μeff values, electronic spectral bands and ESR spectra suggest a polymeric 6-coordinate spin-free octahedral stereochemistry for the Cr(III) and Fe(III) complexes. 5-Iodouracil acts as a monodentate ligand coordinating to the metal ion through the O atom of C(4) = O while histidine through the O atom of –COO− and the N atom of –NH2 group. In vivo antitumour effect of 5-iodouracil and its complexes was examined on C3H /He mice against P815 murine mastocytoma. As evident from their T/C values, Cr(III) and Fe(III) complexes display significant and higher antitumour activity compared to the 5-iodouracil ligand. The in vitro results of the complexes on the same cells indicate that Cr(III) and Fe(III) complexes show higher inhibition on 3H-thymidine and 3H-uridine incorporation in DNA and RNA replication, respectively, at a dose of 5 μg/mL.

Differential control of mesangial cell proliferation by interferon-gamma

Rat mesangial cells were shown to be sensitive to recombinant interferon-gamma (IFN-gamma). IFN-gamma reduced thymidine uptake by these cells and inhibited cell proliferation. Incubation of the cells with 1000 U/ml IFN-gamma decreased thymidine uptake by up to 64% and cell numbers were decreased by 17%. The effects of IFN-gamma were dose and time dependent and were partially reversible by the anti-IFN-gamma monoclonal antibody DB-1. This lymphokine did not reduce incorporation of RNA and protein precursors however. Measurements of 3H-uridine and 3H-leucine incorporation indicated significant increases in RNA and protein synthesis (37% and 45%, respectively) on a per cell basis. The mitogenic effects of IL-1 and platelet-derived growth factor (PDGF) were also susceptible to IFN-gamma-mediated inhibition but the mitogenic response to epidermal growth factor (EGF) was much less sensitive. We conclude that while IFN-gamma may act to modulate the mitogenic signals provided by some factors such as IL-1 and PDGF, the response to EGF appears to be unaffected.

The role of topoisomerases and RNA transcription in the action of the antitumour benzonaphthyridine derivative SN 28049

SN 28049 (N-[2-(dimethylamino)ethyl]-2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b]-1,6-naphthyridine-4-carboxamide) is a DNA intercalating drug that binds selectively to GC-rich DNA and shows curative activity against the Colon 38 adenocarcinoma in mice. We wished to investigate the roles of topoisomerase (topo) I, topo II and RNA transcription in the action of SN 28049. We used clonogenic assays to study the cytotoxicity of SN 28049; RNA interference and enzyme assays to examine the role of topo I in SN 28049 action; 3H uridine incorporation and reporter assays to study its effects on transcription; and RT-PCR to examine its ability to reduce endogenous h-TERT expression. In clonogenic assays, SN 28049 showed a biphasic cytotoxic dose response curve in H460 cells typical of acridine derivatives such as N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) although it was approximately 16-fold more potent. Down-regulation of topo IIalpha in HTETOP cells reduced the cytotoxicity of SN 28049, establishing its action as a topo IIalpha poison. Surprisingly, down-regulation of topo I in H460 cells by RNA interference sensitised them to the actions of SN 28049 and other topo II poisons. SN 28049 also inhibited topo I-mediated relaxation of supercoiled plasmid DNA. SN 28049 was also an inhibitor of transcription in HEK293 cells and was more potent at reducing luciferase expression from a GC-rich SP-1 binding promoter than from a non-GC-rich AP-1 binding promoter. The drug also reduced luciferase reporter gene expression driven by the SP-1-binding survivin promoter as well as reducing endogenous h-TERT expression in HEK293 cells whose promoter also contains SP-1 binding sites. We conclude that SN 28049 has a complex action that may involve poisoning of topo IIalpha, suppression of topo I and inhibition of gene transcription from promoters with SP-1 sites. These actions may contribute to the promising experimental solid tumour anticancer activity of SN 28049.

LARGE-SCALE CHROMATIN STRUCTURE AND TRANSCRIPTIONAL ACTIVITY DURING FINAL DIFFERENTIATION OF BOVINE OOCYTE

The oocyte genome is subject to several levels of regulation, and functional differentiation of large-scale chromatin structure provides an important epigenetic mechanism for the developmental control of global gene expression. Dynamic changes in chromatin structure and function occur during oocyte growth in several mammalian species. In cow, we recently characterized the morphological transitions in the germinal vesicle (GV) during final stages of oogenesys in which chromatin acquires different configurations. In particular, we identified four discrete stages of GV (from GV0 to GV3), characterized by an increase of chromatin condensation, which was related to the sequential achievement of meiotic and embryonic developmental competencies. However, in bovine species, little is known concerning the unique molecular mechanisms involved in the regulation of large-scale chromatin structure and its impact on global transcription and the expression of specific genes involved in the later aspect of oocyte differentiation as well as in the control of the subsequent embryogenesis. The aim of the present study was to analyze the total transcriptional activity and the expression of a panel of genes which could be essential for the developmental competence in bovine oocytes, collected from early and middle antral follicles and sorted on the basis of their chromatin configuration. Global transcriptional activity was analyzed by H3-Uridine incorporation and subsequent autoradiography on semithin Epon sections, while gene expression was examined by RT-PCR with specific primers designed on the basis of sequence database available. In particular, we investigated on the expression pattern of DNA methyl transferase-1, which is involved in the maintenance of methylation pattern of imprinted genes after fertilization, nucleoplasmin-1, nucleoplasmin-2 and nucleoplasmin-3, which are involved in the organization of the nuclear compartment and zygotic arrest-1 that plays an important role after fertilization during the embryonic genome activation. The results indicate that an increase in chromatin condensation is related to global transcriptional silencing in bovine oocyte. In fact, while oocytes with GV0 configuration exhibited high level of transcription, GV1 and GV2 stages showed a remarkable decrease of transcription and the acquisition of GV3 configuration was associated with global repression of transcriptional activity. The gene expression analysis revealed that all the transcripts were present in the different GV categories, regardless of the chromatin configuration, suggesting the potential action of additional levels of gene regulation. Ongoing semi-quantitative analysis aim at clarifying the regulation of gene expression during the chromatin remodeling process and may provide a better understanding of the mechanisms involved in multiple and hierarchical large-scale chromatin modifications that regulate global transcription before the onset of meiosis. (Work supported by MIUR PRIN 2005Work supported by MIUR PRIN 2005073071-003 and “L'Oreal Italy For Women in Science”) (poster)

ICV injection of orexin A induces synthesis of total RNA and mRNA encoding preorexin in various cerebral regions of the rat

This experiment evaluated the induction of RNA synthesis in neurons of various cerebral areas during hyperthermia induced by an intracerebroventricular injection of orexin A. The firing rates of the sympathetic nerves to interscapular brown adipose tissue, along with interscapular brown adipose tissue and colon temperatures, and heart rate were monitored in urethane-anesthetized male Sprague–Dawley rats before and after an injection of orexin A (1.5 nmol) into the lateral cerebral ventricle. Furthermore, the incorporation of 3H-uridine in total RNA and the expression of mRNA encoding the precursor of orexin A were measured in the cerebral areas at the 4th hour after the injection. The same variables were monitored in control rats with an injection of saline. The results show that orexin A increases the sympathetic firing rate, interscapular brown adipose tissue and colonic temperatures, heart rate, along with: (1) the incorporation of 3H-uridine in total RNA of the hypothalamus, hippocampus, cortex and cerebellum, (2) the expression of mRNA encoding the precursor of orexin A in the hypothalamus, cortex and cerebellum. These findings indicate that orexin A induces polyhedral gene expressions in several cerebral regions. Furthermore, we insist to suggest the name “hyperthermine A” as an additional denomination of “orexin A” by considering the strong influence of this neuropeptide on body temperature.

Influence of neopterin and 7,8-dihydroneopterin on the replication of Coxsackie type B5 and influenza A viruses

Pteridine derivatives neopterin and 7,8-dihydroneopterin are produced by human macrophages and dendritic cells upon stimulation with interferon-gamma (IFN-gamma) and therefore become detectable in increased amounts in humans during cell-mediated (Th1-type) immune response. Compounds produced upon influence of cytokine IFN-gamma often exert antiproliferative and antiviral activity. The aim of this study was to investigate the effect of neopterin and 7,8-dihydroneopterin on the replication of Coxsackie type B5 and influenza A viruses. The changes in the replication of these viruses were evaluated by the degree of cytopathic effect and their ability to form plaques in Coxsackie B5-infected human larynx carcinoma epithelial (Hep-2) cells and in influenza A-infected canine kidney epithelial cells (MDCK). Potential toxicity of neopterin and 7,8-dihydroneopterin was estimated by the incorporation of (3)H-thymidine and (3)H-uridine into Hep-2 and MDCK cells. Whereas 30 nmol/l neopterin delayed the development of the cytopathic effect of Coxsackie B5 virus in Hep-2 cells (P < 0.01), 7,8-dihydroneopterin did not have any essential influence at any of the concentrations tested between 10 nmol/l and 1,000 micromol/l. However, 100-1,500 micromol/l 7,8-dihydroneopterin significantly suppressed the propagation of influenza A virus. Neopterin and 7,8-dihydroneopterin were practically nontoxic for Hep-2 and MDCK cells even at high microM concentration. Results suggest that the increased production of neopterin derivatives by activated macrophages and dendritic cells may represent part of the antiviral armature induced by IFN-gamma. The mechanisms of the inhibitory effects of neopterin and 7,8-dihydroneopterin on virus replication apparently are different.