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.

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 1 h 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 24 h 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.

Morphological and functional abnormalities in the ileum of rats with spontaneous hypertension: studies on SGLT1 protein.

BACKGROUND: Na+-dependent D-glucose and D-galactose transport was studied in the ileal brush-border membrane vesicles from both spontaneously hypertensive rats (SHR) and their normotensive genetic control Wistar-Kyoto (WKY) rats. Initial rates and accumulation ratios of the transport of both monosaccharides were significantly lower in the hypertensive rats compared to the WKY rats. METHODS: In order to determine whether such modifications are related to morphological abnormalities, ileal epithelium of SHR and WKY rats was examined by light and electron microscopy. In addition, immunohistochemical and immunocytochemical localization of Na+-glucose cotransporter (SGLT1) was performed. RESULTS: Light microscopy studies showed hypertrophy in the ileal villi of hypertensive rats, with an increase in the villus width when compared to those from normotensive rats. Immunohistochemical studies of SGLT1 showed the protein localized in the apical membrane of the absorptive epithelial cells, along the entire villus. No changes between SHR and WKY rats were noted in the intensity and distribution of the SGLT1 protein along the villus-crypt axis. Electron microscopy studies showed a patchy loss of microvilli in the ileal enterocytes of SHR, compared to those from WKY rats. Immunocytochemical studies of SGLT1 were carried out by the immunogold method. Colloidal gold particles were localized at the ileal microvilli of normotensive rats. No significant presence of SGLT1 was found in the smooth apical surface of ileum from hypertensive rats, although most adjacent microvilli were marked. CONCLUSION: Morphological changes were accompanied by modifications in the sugar transport and in the immunolocalization of SGLT1 in the ileal epithelium of SHR.

Ultrastructural and functional changes in the jejunal epithelium of spontaneously hypertensive rats.

Ultrastructural studies on the epithelium, sugar transport and immunocytochemistry of Na+-glucose cotransporter (SGLT1) were carried out in the jejunum of Spontaneously Hypertensive Rats (SHR) and their normotensive genetic control, Wistar-Kyoto (WKY) rats. Electron microscopy studies showed a regular brush-border membrane in the jejunal enterocytes of WKY rats, with colloidal gold particles, representing SGLTI, localized at the microvilli of the absorptive epithelial cells. However, a patchy loss of microvilli was detected in the jejunal sections from SHR, with no presence of colloidal gold particles, indicating the absence of the SGLT1 protein. Most adjacent microvilli were normal in size like those found in WKY rats, and SGLT1 labeling was observed. All these changes were accompanied by a reduction in Na+-dependent D-glucose and D-galactose uptakes in the jejunal BBMVs isolated from SHR, when compared to WKY rats. We conclude that ultrastructural changes were paralleled by modifications in the sugar transport and in the localization of SGLT1 in the jejunal epithelium of SHR.

Lack of hotspot targets: a constraint for IS30 transposition in Salmonella.

IS30 is an insertion element common in E. coli strains but rare or absent in Salmonella. Transfer of the IS30-flanked transposon Tn2700 to Salmonella typhimurium was assayed using standard delivery procedures of bacterial genetics (conjugation and transduction). Tn2700 'hops' were rare and required transposase overproduction, suggesting the existence of host constraints for IS30 activity. Sequencing of three Tn2700 insertions in the genome of S. typhimurium revealed that the transposon had been inserted into sites with a low homology to the IS30 consensus target, suggesting that inefficient Tn2700 transposition to the Salmonella genome might be caused by a lack of hotspot targets. This view was confirmed by the introduction of an IS30 'hot target sequence', whose sole presence permitted Tn2700 transposition without transposase overproduction. Detection of IS30-induced DNA rearrangements in S. typhimurium provided further evidence that the element undergoes similar activities in E. coli and S. typhimurium. Thus, hotspot absence may be the main (if not the only) limitation for IS30 activity in the latter species. If these observations faithfully reproduce the scenario of natural populations, establishment of IS30 in the Salmonella genome may have been prevented by a lack of DNA sequences closely related to the unusually long (24 bp) IS30 consensus target.

Synthesis of FinP RNA by plasmids F and pSLT is regulated by DNA adenine methylation.

DNA adenine methylase mutants of Salmonella typhimurium contain reduced amounts of FinP, an antisense RNA encoded by the virulence plasmid pSLT. Lowered FinP levels are detected in both Dam- FinO+ and Dam- FinO- backgrounds, suggesting that Dam methylation regulates FinP production rather than FinP half-life. Reduced amounts of F-encoded FinP RNA are likewise found in Dam- mutants of Escherichia coli. A consequence of FinP RNA scarcity in the absence of DNA adenine methylation is that Dam- mutants of both S. typhimurium and E. coli show elevated levels of F plasmid transfer. Inhibition of F fertility by the S. typhimurium virulence plasmid is also impaired in a Dam- background.

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 anti-nucleolin 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.

DNA adenine methylase mutants of Salmonella typhimurium and a novel dam-regulated locus.

Mutants of Salmonella typhimurium lacking DNA adenine methylase were isolated; they include insertion and deletion alleles. The dam locus maps at 75 min between cysG and aroB, similar to the Escherichia coli dam gene. Dam- mutants of S. typhimurium resemble those of E coli in the following phenotypes: (1) increased spontaneous mutations, (2) moderate SOS induction, (3) enhancement of duplication segregation, (4) inviability of dam recA and dam recB mutants, and (5) suppression of the inviability of the dam recA and dam recB combinations by mutations that eliminate mismatch repair. However, differences between S. typhimurium and E. coli dam mutants are also found: (1) S. typhimurium dam mutants do not show increased UV sensitivity, suggesting that methyl-directed mismatch repair does not participate in the repair of UV-induced DNA damage in Salmonella. (2) S. typhimurium dam recJ mutants are viable, suggesting that the Salmonella RecJ function does not participate in the repair of DNA strand breaks formed in the absence of Dam methylation. We also describe a genetic screen for detecting novel genes regulated by Dam methylation and a locus repressed by Dam methylation in the S. typhimurium virulence (or "cryptic") plasmid.

New methods in Salmonella genetics.

This review summarizes several recent developments in Salmonella genetics; some of the procedures described can be easily adapted to Escherichia coli and have also potential applications in non-enteric bacteria. The novel methods outlined include genetic mapping procedures, ancillary tools for cloning, a strategy for analyzing DNA-protein interactions in vivo, a method for plasmid curing and a procedure for the detection of bacterial virulence genes.

Anemia megalobástica Asociada a Diarrea crónica.Estudio prospectivo y multicéntrico en Lima / Megaloblástic anemia associated to chronic diarrhea. Prospective and multicentric study in Lima

A partir de 1986 hemos observado un incremento de Anemia Megaloblástica (AM) asociada a diarrea crónica, en 60 por ciento no se encontró ninguna relación causal. En los últimos 3 años hemos utilizado un protocolo multicéntrico prospectivo en Lima (Perú), se incluyeron adultos con AM confirmada por aspirado de médula ósea, excluyendo: ancianos, gestantes, alcohólicos, portadores de neoplasias, etc. Los pacientes fueron 45 con promedio de edad de 37.5 años. Se encontraron dosajes disminuídos de B12 + ácido fólico: 64 por ciento, B12: 20 por ciento, y ácido fólico:16 por ciento. Las biopsias gástricas demostraron atrofia: 33 por ciento (fondo), 7,6 por ciento (cuerpo) y 12 por ciento(antro). El pH gástrico menor o igual a 4.5 en 50 por ciento. El cultivo microbiológico del jugo duodenal fué positivo en 35.2 por ciento 96/17), la mayoría coliformes gram negativos. Presentaron diversas alteraciones estructurales 5/8 (62.5 por ciento) biopsias duodenales, 5/6 (83 por ciento) biopsias yeyunales y 4/4 (100 por ciento) biopsias ileales. Los estudios parasitológicos excluyeron diphillobothrium pacificum. Estos hallazgos nos llevan a sugerir que un significativo número de pacientes con AM y diarrea crónica (con o sin síndrome espruiforme) en Lima, son consecuencia del sobrecrecimiento bacteriano intestinal, lo que los configuraría como casos de Esprue Tropical

[Megaloblastic anemia associated with chronic diarrhea. A prospective and multicenter study in Lima]. / Anemia megaloblástica asociada a diarrea crónica. Estudio prospectivo y multicéntrico en Lima.

Since 1986 we have been observing an increased number of patients with megaloblastic anaemia (MA) associated to chronic diarrhea. In 60% of the cases we could not identify any etiologic factor. In the last three years a prospective study in Lima (Peru) has been carried on aimed to investigate this aspect; patients with diseases recognized to be associated to MA were excluded. 45 patients were included age average 37.5 years, all of them have a confirmed diagnosis by bone marrow; 64% with low serum B12 and folic acid, 20% with low serum B12, and 16% with low serum folic acid. Gastric biopsies did not show atrophy in 67%; intragastric pH was lower than 4 in 50% duodenal content culture was positive in 35% (6/17) to aerobic gram negative agents; 62% (5/8) of duodenal biopsies, 83% (5/6) of jejunal biopsies, 4/4 (100%) of ileal biopsies, showed diverse structural changes; 100% did not show Diphyllobothrium pacificum. All these findings make us suggest that a significative number of patients with MA and chronic diarrhea in Lima are related to small bowel bacterial overgrowth. These bacteria can "sequestrate" or consume folates and cobalamines besides the direct damage they can cause to intestinal morphology. Future studies are needed to confirm our proposal and define if these cases belong to a variety of tropical sprue.

[Pneumatic dilatation in patients with esophageal achalasia]. / Dilatación neumatica en pacientes con acalasia de esófago.

From October 1984 to March 1992, 21 patients of Hospital Nacional Guillermo Almenara Irigoyen-IPSS, Lima, Perú, with esophageal achalasia were treated with pneumatic dilatation using a 3.5 cm diameter Rider-Moeller balloon. The mean age was 40.5 years (range: 24-54). Six were men and 15 women. The mean time with dysphagia previous to treatment was 5.3 years. A total of 29 sessions were performed, 1.38 sessions per patient. The follow-up of the first 10 patients was carried for a mean time of 48.3 months (range:6-91). A satisfactory response to treatment was obtained in 8 patients (80%). Two patients (20%) relapsed after 2 and 3 treatment sessions needing surgery. One patient suffered a esophageal perforation recovering after surgical treatment. We conclude that pneumatic dilatation with Rider-Moeller balloon is a safe and not difficult medical procedure for esophagus achalasia.

Characterization and immunolocalization of RNA polymerase I transcription factor UBF with anti-NOR serum in protozoa, higher plant and vertebrate cells.

We have used anti-NOR serum from a patient with rheumatoid arthritis, to study its reactivity on different phylogenetically separated species such as protozoa, higher plants, birds and mammals. The biochemical characteristics of the antigens detected after applying mono- and two-dimensional electrophoresis and electrophoretic transfers confirm that they correspond to the rRNA polymerase I transcription factor UBF. We have demonstrated the different molecular sizes, depending on the cell complexity, but the same neutral isoelectric points in whole cell extracts of the different species. We have also demonstrated an immunolocalization of this transcription factor to the fibrillar component in all the species studied. These results suggest a high conservation of UBF throughout evolution and the possibility of using this anti-NOR serum as a tool for the study of the structure, nucleolar organization and functional roles of the different nucleolar components.

[Dermatomyositis in children: clinical course of 9 cases]. / Dermatomiositis infantil: evolución clínica de nueve casos.

Eight cases of dermatomyositis and one of polymyositis were studies among five male and four female patients. All had criteria definitive of the disease. All nine patients were give prednisone; six of them responded favorably to treatment and after discontinuing still remain asymptomatic. Three male patients have had a worse response to therapy, two of them deceased and the third still remains symptomatic, but very slowly improving after fourteen months with steroids and cyclophosphamide. These three patients have had chronic cutaneous vasculitic ulcers and this is a sign of bad prognosis. Both deceased patients exhibited important visceral complications (digestive and pulmonary). A more aggressive therapy has been suggested in these forms of worse clinical evolutions, including use of immunosuppressors.

[Pathology of the phagocytic mononuclear system. Apropos of 4 cases]. / Patología del sistema mononuclear fagocítico. A propósito de cuatro observaciones.

Four patients, with disorders belonging to mononuclear phagocyte system diseases are described: a case of malignant histiocytosis; one of Weber-Christians disease and two siblings affected by familiar erythrophagocytic lymphohistiocytosis. An attempt is made to update classification of this group of diseases previously known as reticulosis, reticulohistiocytosis, reticuloendotheliosis, etcetera.

[Congenital factor VII deficiency (author's transl)]. / Déficit de factor VII.

A new case of factor VII congenital deficiency is presented in a four year old girl, with a factor VII level of 3.9%. The patient had no history of bleeding and her coagulation disorder was a casual finding. Clinical features of the disease and its therapeutic guidelines are discussed. It seemed appropriate to continue the study in order to evaluate the factor VII capacity of inhibiting an specific antibody, to confirm heterozygosity of partents and to prove that this case is a genetic variant of factor VII deficiency with good prognosis.