Francisco J. Moreno

Silver staining of the nucleolar organizer regions (NORs) on Lowicryl and cryo-ultrathin sections.

Nucleolar organizer region (NOR) silver staining was applied to sections of fixed material. A positive reaction on cryo-ultrathin sections was found as well as on semithin and ultrathin Lowicryl sections. Repeatable staining that was easy to control was obtained by a one-step procedure after aldehyde-Carnoy fixation. Fixation of the material by formaldehyde and glutaraldehyde alone in cacodylate buffer also maintained reaction selectivity when ammonium chloride was used after fixation. Enzymatic digestion by pronase, RNase A, DNase I, or micrococcal nuclease was applied to ultrathin Lowicryl sections. Pronase digestion removed the silver-stained proteins, whereas digestion by the nucleases did not. A routine procedure is proposed for easy NOR silver staining of sections that preserves a good tissue ultrastructure and is also compatible with cytochemical and immunological investigations.

Chronic gastric ulcer healing in rats subjected to selective and non-selective cyclooxygenase-2 inhibitors.

UNLABELLEDnThe influence of different nonsteroidal anti-inflammatory drugs (NSAIDs) and of a proton pump inhibitor on the healing parameters of a chronic gastric ulcer was evaluated. Wistar rats were used after the induction of a chronic acetic acid ulcer. The animals were treated orally for 8 and 15 days, twice daily, with the conventional NSAID, piroxicam (0.35 mg/kg), the non-narcotic analgesic, metamizol (33 mg/kg), the selective cyclooxygenase-2 inhibitor, celecoxib (1.8 mg/kg) and the proton pump inhibitor, omeprazole (0.35 mg/kg). Macroscopic ulcer index, myeloperoxidase activity and prostaglandin E(2) content (both biochemical parameters were evaluated in ulcerated and in intact tissue) as well as histological and immunohistochemical evaluations were carried out at 8 and 15 days. Omeprazole accelerated ulcer healing at 8 and 15 days (P<0.05), while celecoxib delayed healing significantly at 15 days (P<0.01). At 8 days, the prostaglandin E2 content decreased with all NSAIDs at the ulcer site as well as in intact tissue. The same happened at 15 days except for celecoxib, which only diminished prostaglandins in intact mucosa. Immunohistochemistry showed differences in the location of cyclooxygenase-2 and -1. The highest cyclooxygenase-2 expression was found with piroxicam and the lowest expression was with celecoxib.nnnCONCLUSIONSnDown-regulation of cyclooxygenase-2 expression as well as a possible involvement of the chemical structure of celecoxib, a 1,5-dirarylpirazole with a sulphonamide moiety, may account for the delay in ulcer healing.

Characterization and immunolocalization of a nucleolar antigen with anti-NOR serum in HeLa cells.

We have used a serum from a patient with rheumatoid arthritis and found it to immunoblot with a 92- to 88-kDa protein doublet with an isoelectric point of around 7.5 after mono- and two-dimensional electrophoresis in whole HeLa cells. By means of immunofluorescence and immunoelectron microscopy we have found it to specifically react with the nucleolar fibrillar component. After quantitative analysis under the electron microscope, we have demonstrated a similar labeling both in the fibrillar centers and the dense fibrillar component, using two different gold-coupled markers. When transcription was inhibited under physiological conditions (mitosis) or after AMD treatment the antigen remained, as shown by immunoblotting and immunolabeling with anti-NOR serum. These biochemical characteristics, which coincide with those of the ribosomal transcription human upstream binding factor, together with the immunolocalization with anti-NOR serum, allow us to discuss the possible role of these antigens in rDNA transcription.

Ag-NOR proteins and rDNA transcriptional activity in plant cells.

In this work we used Allium cepa root meristem cells in actively growing conditions and under treatment with the protein synthesis inhibitors cycloheximide (CHM) and puromycin. Morphological and quantitative results indicate that these drugs induce dramatic alterations in nucleolar structure reflected by a decrease of nucleolar size, much more evident under treatment with CHM, and by segregation of its main components. Quantitative analysis shows a decrease in NOR-silver staining after treatment with CHM, whereas in cells treated with puromycin NOR-silver staining remains constant. Our results reveal a decrease in the Ag-NOR proteins under conditions of diminished cell activity, suggesting a direct relationship between the quantity of Ag-NOR proteins and transcriptional activity. Using two-dimensional gel electrophoresis and NOR-silver staining in gels, we have characterized some proteins corresponding to molecular weights of 28 and 31 KD and pI of approximately 5.2. After treatment with CHM, reactivity of these proteins against NOR-silver staining is diminished. By means of a morphological study, analysis of NOR-silver staining, and of anti-DNA and RNAse-gold labeling, we have tried an approach to the nucleolar organization in plant cells. Our results suggest that the fibrillar component shows a reticular distribution where fibrillar centers, as described in animal cells, are not distinguished.

DNA STRAND‐BREAKS INDUCED BY THE TOPOISOMERASE I INHIBITOR CAMPTOTHECIN IN UNSTIMULATED HUMAN WHITE BLOOD CELLS

Camptothecin (CPT) and actinomicyn‐induced strand‐breaks, repair and apoptosis in unstimulated human blood cells were studied using the DNA comet assay, and electrophoresis of low molecular weight DNA extracts. On the one hand, incubation of G0 leukocytes for 1h with CPT induced DNA strand‐breaks that were observed using the single cell gel electrophoresis technique. On the other hand, internucleosomal DNA fragments were not observed, suggesting that apoptosis had not occurred. DNA‐strand‐breaks caused by CPT were repaired 24h after treatment; the migration of DNA fragments was assessed by a reduction in the number of comets. These data strongly suggest that the unexpected clastogenic effect of this topoisomerase I inhibitor is not due to the collision of the cleavage complex with the replication fork, since replication does not occur in G0. In our opinion, this effect could be due instead to the topoisomerase I enzyme being able to bind DNA in the absence of replication, probably in a way that is not strictly related to the progression of the cell cycle. Thus, CPT does not provoke apoptosis in quiescent leukocytes.

Nucleolar component behaviour in plant cells under different physiological conditions. A morphological, cytochemical and quantitative study

Different nucleolar components of Allium cepa L. root meristem cells under physiological conditions of proliferation and quiescence were quantitatively analyzed after the application of morphometric and cytochemical techniques.

Inhibition of nucleolar protein nucleolin by electroporation with anti‐nucleolin antibodies results in an increase of the nucleolar size

Electroporation of exponentially growing human larynx epidermoid carcinoma cells (HEp‐2) with a serum against nucleolin, one of the most abundant non‐histone nuclear proteins, has shown, 24 h after electroporation, a significant increase in the size of the nucleolus of these cells compared with normal HEp‐2 cells (non‐electroporated) and electroporated HEp‐2 cells in the absence of antinucleolin serum (P < 0.01). Image analysis evaluation of the different nucleolar components proved a major contribution of the dense fibrillar component to the total nucleolar size in cells electroporated with anti‐nucleolin antibodies, more than that corresponding to the dense fibrillar component in cells from any of the control groups (P < 0.01), indicating that the reported increase in nucleolar size was due to a marked enlargement of the dense fibrillar regions. These results, in agreement with previous biochemical and molecular biology studies, suggest a pivotal role for nucleolin in pre‐rRNA processing and constitute morphological evidence supporting this role. Following nucleolin inhibition, impaired pre‐rRNA processing might result in an accumulation of this molecular species in the dense fibrillar component of the nucleolus, where pre‐rRNA is first present.

The comet assay differentiates efficiently and rapidly between genotoxins and cytotoxins in quiescent cells

Our main aim was to establish the efficiency of the single cell electrophoresis technique for differentiating between drugs that bind DNA and those that do not. The alkaline comet assay was used to test the responses of human leukocytes (quiescent cells) to damage induced by reportedly genotoxic and reportedly cytotoxic agents. Incubation of G0 leukocytes for 1 h with the genotoxic agents camptothecin and actinomycin C provoked DNA migration, observed as comet figures. On the other hand, when cells were treated with the cytotoxic agents cordycepin, fluorodeoxyuridine and puromycin, the leukocyte nuclei were indistinguishable from those of untreated cells. In addition, we have developed a rapid method using non‐proliferating cells that requires neither culture nor lymphocyte isolation. This method promises to be useful as a rapid in vitro screening assay.