Notch-mediated induction of N-cadherin and α9-integrin confers higher invasive phenotype on rhabdomyosarcoma cells.

BACKGROUND: Rhabdomyosarcoma (RMS) is the commonest type of soft-tissue sarcoma in children. Patients with metastatic RMS continue to have very poor prognosis. Recently, several works have demonstrated a connection between Notch pathway activation and the regulation of cell motility and invasiveness. However, the molecular mechanisms of this possible relationship remain unclear. METHODS: The Notch pathway was manipulated pharmacologically and genetically. The mRNA changes were analysed by quantitative PCR and protein variations by western blot and immunofluorescence. Finally, the capabilities of RMS cells to adhere, heal a wound and invade were assessed in the presence of neuronal cadherin (N-cadherin)- and α9-integrin-blocking antibodies. RESULTS: Cells treated with γ-secretase inhibitor showed lower adhesion capability and downregulation of N-cadherin and α9-integrin. Genetic manipulation of the Notch pathway led to concomitant variations in N-cadherin and α9-integrin. Treatment with anti-N-cadherin-blocking antibody rendered marked inhibition of cell adhesion and motility, while anti-α9-integrin-blocking antibody exerted a remarkable effect on cell adhesion and invasiveness. CONCLUSION: Neuronal cadherin and α9-integrin are postulated as leading actors in the association between the Notch pathway and promotion of cell adhesion, motility and invasion, pointing to these proteins and the Notch pathway itself as interesting putative targets for new molecular therapies against metastases in RMS.

A novel dimeric oxovanadium (IV) species identified in Saccharomyces cerevisiae cells.

Saccharomyces cerevisiae cells stored oxovanadium (IV) ions in a dimeric form. In the late stationary phase Saccharomyces cerevisiae cells grown in rich medium containing concentrations of oxovanadium (V), orthovanadate from 12 to 18 mM, causing growth stasis, a dimeric oxovanadium (IV) species was identified by EPR spectroscopy. The EPR spectrum exhibited at 110 K the low-field forbidden deltaMs = +/-2 transition at g around 4 and the half-field deltaMs = +/-1 15-lines feature at g around 2 out of the presence of a triplet state by the coupling of the oxovanadium (IV) ions in a dimeric form. Hyperfine splitting of 75.2 x 10(-4) cm(-1) and an interionic distance of about 4.4 angstroms was calculated. The dimeric species was localized in the cellular cytoplasmic space.

The prenatal detection of the fragile X chromosome: review of recent experience.

The fragile X chromosome has been identified in specimens from 17 male and 10 female fetuses in 11 laboratories throughout the world, obtained from at least 79 fetuses at increased risk for the fra(X) syndrome. Of these, 19 were confirmed, 6 were pending, 1 was negative and 1 could not be confirmed. Twenty-five of the 79 cases were studied in our laboratory (Institute for Basic Research [IBR]) and resulted in fra(X) demonstration in specimens from 3 male and 5 female fetuses. All 3 males and 2 of the 5 females have been confirmed. When amniocytes from the two confirmed female fetuses were exposed to FUdR after culturing in Chang medium, fra(X) frequencies were virtually negative indicating that Chang medium should not be used in fragile X studies at least when FUdR is used to induce fragility. Finally, amniocytes from a fra(X) male fetus studied in 3 different laboratories exhibited strikingly different frequencies. To date, we have experienced no false-positives or negatives, but the latter case was controversial. It is recommended that laboratories undertaking fra(X) prenatal detection use a combination of at least two different proven induction systems as well as complementary DNA marker studies to prevent false negative diagnosis.

Enrichment with a polyunsaturated fatty acid enhances the survival of Saccharomyces cerevisiae in the presence of tributyltin.

The toxicity of inorganic metal species towards Saccharomyces cerevisiae has been shown to be markedly dependent on cellular fatty acid composition. In this investigation, the influence of fatty acid supplementation on the toxicity of the lipophilic organometal, tributyltin was investigated. Growth of S. cerevisiae was increasingly inhibited when the tributyltin concentration was increased from 0 to 10 microM. However, the inhibitory effect was partly alleviated by supplementation of the medium with 1 mM linoleate (18:2), a treatment that leads to large-scale incorporation of this polyunsaturated fatty acid (to > 60% of total fatty acids) in yeast membrane lipids. Cells that were previously enriched with 18:2 also showed reduced loss of vitality compared to cells grown in the absence of a fatty acid supplement, when exposed to tributyltin. For example, addition of tributyltin to a concentration of 0.1 microM was associated with an approximate 10% reduction in the H+ efflux activity of 18:2-enriched cells, but a 70% reduction in that of fatty acid-unsupplemented cells. Despite the increased tributyltin resistance of 18:2-enriched S. cerevisiae, the level of cell-associated tributyltin was found to be approximately two-fold higher in these organisms than in fatty acid-unsupplemented cells. These results demonstrate an increased resistance of 18:2-enriched membranes to the direct toxic action(s) of tributyltin. This is in contrast to the previously reported effect of 18:2 enrichment on sensitivity of S. cerevisiae to inorganic metal cations.

Vanadium affects vacuolation and phosphate metabolism in Hansenula polymorpha.

The yeast Hansenula polymorpha is able to grow on vanadate concentrations that are toxic to other organisms. Transmission electron microscopy analysis showed that H. polymorpha cells growing on a vanadate-containing medium undergo a significant increase in cell vacuolation and a thickening of the cell wall; the presence of small cytoplasmic vesicles and an increase in cristae at the level of the plasma membrane were also observed. These ultrastructural modifications were accompanied by a change in the intracellular polyphosphate level, as shown by in vivo 31P-NMR. The involvement of these observed changes in vanadium detoxification is discussed.

Relationship between Helicobacter pylori infection and gastric follicular hyperplasia. A retrospective study.

BACKGROUND: The occurrence of lymphoid follicles in the gastric mucosa was recently considered to be acquired in respose to infection by Helicobacter pylori. METHODS: In order to investigate whether there is any correlation between follicular hyperplasia and Helicobacter pylori colonization, a morphological study was performed in antral biopsy specimens from 267 patients with H. pylori-associated gastritis, and from 312 subjects with histological evidence of chronic gastritis, but without H. pylori colonization. RESULTS: Follicular hyperplasia was observed in 33 (12.3%) specimens with H. pylori-associated gastritis, and in 56 (9%) samples from non-infected group (p=ns). In both H. pylori-positive and H. pylori-negative specimens lymphoid follicular hyperplasia showed a significant correlation with the degree of the gastritis. In these two groups the prevalence of follicular hyperplasia was also significantly higher in the elderly. CONCLUSIONS: On the basis of these results, we were unable to find any significant correlation between H. pylori infection and occurrence of lymphoid follicular hyperplasia. The appearance of organized follicular structures in the gastric mucosa seems to be acquired in response to multiple events, and not exclusively in consequence of the colonization by Helicobacter pylori.

[The role of Campylobacter pylori in gastroduodenal pathology]. / Contributo alle indagini sul ruolo del Campylobacter pylori nella patologia gastroduodenale.

Biopsy specimens from the gastric antral mucosa of 194 patients with upper gastrointestinal symptoms were studied with the Warthin-Starry silver stain to demonstrate the presence of Campylobacter Pylori (CP). Of these patients, 44 had peptic ulcer visible on endoscopic investigation. Spiral bacteria were detected in 70 out of 75 specimens with histologically active gastritis (93%), in 34 of 54 specimens with inactive form of gastritis (63%) and in 26 of 65 specimens without morphological changes (40%). Of 44 patients found to have gastric or duodenal ulcers, 33 also had bacteria. No significant differences between patients with endoscopically visible peptic ulcer and those without ulcer were seen. Our results indicate a close relationship between the active form of chronic gastritis and Campylobacter; however they do not show a definite association with peptic ulcer.

Linfedema asociado al cáncer de mama: factores de riesgo, diagnóstico y tratamiento quirúrgico / Breast cancer related lymphedema: risk factors, diagnosis and surgical treatment

Resumen El linfedema asociado al cáncer de mama es una de las complicaciones más subestimadas y debilitantes del tratamiento de esta entidad. Ocurre como resultado de la interrupción del flujo linfático en asociación a otros factores. La incidencia varía dependiendo del tipo tratamiento recibido, existiendo mayor riesgo en los casos en los que se realiza mastectomía total, disección axilar, radioterapia, y en los pacientes en los cuales los ganglios están positivos para cáncer, hubo una mayor cantidad de linfonodos resecados, se utilizaron taxanos o padecen de obesidad. El diagnóstico clínico y a través de técnicas de imágenes es fundamental para evaluar el estado funcional del sistema linfático. Los objetivos principales en el manejo del linfedema son limitar la morbilidad del paciente, mejorar la funcionalidad y la calidad de vida. Existen procedimientos que buscan prevenir el desarrollo del linfedema asociado al cáncer de mama. Una vez establecido, el tratamiento puede ser conservador y quirúrgico. El tratamiento quirúrgico incluye procedimientos fisiológicos (reconstructivos) y resectivos. El éxito depende de una buena selección de los pacientes y la realización de un tratamiento individualizado. A continuación, se presenta una revisión en cuanto a la incidencia, factores de riesgo, estrategias diagnósticas y técnicas quirúrgicas con énfasis en el tratamiento microquirúrgico.

Breast cancer related lymphedema is one of the most underestimated and debilitating complications of the treatment of this entity. Occurs as result of the interruption of the lymphatic flow in association with other factors. The incidence varies depending on the type of treatment received; being a higher risk in cases in which total mastectomy, axillary dissection, radiotherapy are performed; and in patients in whom the lymph nodes are positive for cancer, there was a greater number of harvested lymph nodes, taxanes were used or in obese patients. Clinical diagnosis and imaging techniques are essential to assess the functional status of the lymphatic system. The main objectives in the management of lymphedema are to limit patient morbidity, improve functionality and quality of life. There are procedures that seek to prevent the development of breast cancer related lymphedema. Once established, the treatment can be conservative and surgical. Surgical treatment includes physiological (reconstructive) and excisional procedures. Success depends on a good selection of patients and the performance of an individualized treatment. The following is a review regarding the incidence, risk factors, diagnostic strategies and surgical techniques with emphasis on microsurgical treatment.

Combined use of liquid chromatography triple quadrupole mass spectrometry and liquid chromatography quadrupole time-of-flight mass spectrometry in systematic screening of pesticides and other contaminants in water samples.

As a suitable way for routine screening of pesticides and control of other organic contaminants in water, the combination of liquid chromatography triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS) and liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) has been applied to the analysis of 63 surface and waste water samples after conventional solid-phase extraction (SPE). The extracts were screened for 43 pesticides or degradation products by LC-QqQ-MS/MS achieving limits of detection (LOD) ranged from 0.04 to 2 ng L(-1). Of the 43 selected pesticides, 33 were detected in water samples. The ESI-QTOF MS instrument was run using two simultaneous acquisition functions with low and high collision energy (MS(E) approach) and acquiring the full mass spectra. A home-made database containing more than 1100 organic pollutants was used for substance identification. Around 250 of these compounds were available at the laboratory as reference standards. Five pesticides and 3 of their degradation products, different to those selected in the QqQ method, were detected by QqTOF-MS. Thirteen pharmaceuticals and two drugs of abuse were also identified in the samples. In practice, the sample preparation proved to be suitable for both techniques and for a wide variety of substances with different polarity. Mutual confirmation and evidence of co-occurrence of several other organic contaminants were the main advantages of the combination of both techniques.

Multiresidue analysis of organic pollutants by in-tube solid phase microextraction coupled to ultra-high performance liquid chromatography-electrospray-tandem mass spectrometry.

In this work, in-tube solid phase microextraction (IT-SPME) coupling with ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) multiresidue analytical method has been proposed for the first time for on-line enrichment of 9 analytes included in Water Frame Directive 2000/60/EC (WFD). The device was equipped with a GC TRB-5 capillary column, used as pre-concentration loop, and two conventional six-port injection valves. Water sample and desorption solvent volumes were tested. The optimum conditions were 4mL of processed sample followed by elution with 40µL of methanol. The analytes were detected with a mass spectrometer after being ionized positively using an electrospray ionization (ESI) source. The method presents good linearity over the range assayed, 0.025-2.5µg/L for chlorpyriphos and 0.25-25µg/L for the other tested compounds and LODs between 0.025µg/L and 2.5µg/L. Enrichment factors ranged from 2.5 to 10. Intra and inter-day variation coefficients were <26 and 31.6% respectively. Once validated, the method was applied to several water samples from different sources demonstrating that it achieves the on-line enrichment of the analytes with the advantage of minimum sample manipulation, and the identification and quantification of some organic pollutants in water samples in the range of low parts-per-billion. The method provided similar analytical characteristics as those obtained in the established couple IT-SPME-Capillary Liquid Chromatography (CapLC).

[Chromosomal translocations in soft tissue sarcomas: from molecular biology to clinical application]. / Translocaciones cromosómicas en los sarcomas de partes blandas: de la biología molecular a la aplicación clínica.

Recent advances in the knowledge of the molecular biology of paediatric sarcomas, especially the characterisation of chromosomal translocations associated specifically with particular types of cancer, have established bases for the introduction of new diagnostic tools. This article reviews the main chromosomal translocations associated with paediatric tumours, and summarises their molecular characteristics regarding their oncogenic capabilities, possible usefulness as a differential diagnostic tools and possible correlation with clinical parameters.

Helicobacter pylori and intestinal metaplasia in antral mucosa. A study on their correlation.

To investigate the possible relation between Helicobacter pylori (HP) colonization and incidence of intestinal metaplasia (IM), antral biopsy specimens from a total of 263 subjects (167 had chronic gastritis, 12 gastric cancer, and 84 no histological changes) were examined. IM was observed in 27.7% of the HP positive specimens and in 18.3% of the HP negative biopsies (p = NS). Histochemical characterization of mucins showed that the percentage of HP colonization was not significantly different within various subtypes of IM. We do not find in this study any evidence to support the existence of a pathogenetic role of HP on the morphogenesis of IM in antral mucosa.

Helicobacter pylori in Barrett's esophagus and in normal or inflamed esophageal mucosa: a retrospective study.

To investigate on the presence and significance of Helicobacter pylori (Hp) in Barrett's mucosa, esophageal biopsy specimens from 34 patients with columnar-lined epithelium were retrospectively examined. The incidence of colonization was compared with that observed in a control group of 76 subjects with normal esophageal mucosa or moderate esophagitis. Three of the 34 Barrett's specimens were Hp-positive and only 1 of these showed evidence of inflamed mucosa (p = NS). We do not find in this study any evidence to support the existence of association between Hp and Barrett's esophagus or esophageal inflammation. The presence of Hp in antral mucosa of all positive cases suggest the possibility that esophageal colonisation might merely be a consequence of reflux from the gastric antrum.

Electron paramagnetic resonance studies and effects of vanadium in Saccharomyces cerevisiae.

Vanadium uptake by whole cells and isolated cell walls of the yeast Saccharomyces cerevisiae was studied. When orthovanadate was added to wild-type S. cerevisiae cells growing in rich medium, growth was inhibited as a function of the VO4(3-) concentration and the growth was completely arrested at a concentration of 20 mM of VO4(3-) in YEPD. Electron paramagnetic resonance (EPR) spectroscopy was used to obtain structural and dynamic information about the cell-associated paramagnetic vanadyl ion. The presence of EPR signals indicated that vanadate was reduced by whole cells to the vanadyl ion. On the contrary, no EPR signals were detected after interaction of vanadate with isolated cell walls. A 'mobile' and an 'immobile' species associated in cells with small chelates and with macromolecular sites, respectively, were identified. The value of rational correlation time tau r indicated the relative motional freedom at the macromolecular site. A strongly 'immobilized' vanadyl species bound to polar sites mainly through coulombic attractions was detected after interaction of VO2+ ions with isolated cell walls.

Structural and biochemical aspects of peach fruit abscission (Prunus persica L. Batsch).

The physiological drop of immature fruits was studied in relation to the activation of the abscission zone located between the fruit and the receptacle. Light- and electron-microscopy observations demonstrated that this zone consisted of two types of parenchymatous cells: in the distal region, closer to the fruit, were groups of small thick-walled cells with few intercellular spaces; in the proximal region, closer to the pedicel, the stillgrouped cells were larger, polyphenolic-rich, and thick-walled but with many wide intercellular spaces. Separation of the fruit occurred by dissolution of the middle lamella of the cells of this zone followed by an increase in the size of the intercellular spaces. Lysis of the middle lamella began at the corners of the cells and spread from there across the entire wall surface. Structural changes were paralleled by an increase in soluble proteins, endo-cellulase and exo-polygalacturonase activity. Isoelectric focusing indicated that both enzymes were present as isoenzymes whose patterns were affected by embryoctomy and 2-chloroethylphosphonic acid treatments.

Effect of Helicobacter pylori colonization on quantitative distribution of antral G-cells in apparently healthy subjects and in duodenal ulcer patients. A morphometric study.

To investigate whether antral colonization by Helicobacter pylori (Hp) modifies gastrin-cell population, the number of G-cells was evaluated in antral biopsy specimens from 22 apparently healthy subjects and from 48 duodenal ulcer patients using a morphometric method. The level of serum immunoreactive gastrin in a sample of fasting serum obtained at the time of biopsy was also measured. In healthy subjects the G-cell count (evaluated according to G/I index) and the serum gastrin levels were not significantly different than those found in duodenal ulcer patients. When the antral colonization by Hp was assessed, we found that, both controls and duodenal ulcer Hp-positive patients had a mean G-cell count and fasting serum gastrin levels not significantly higher than in patients without Hp.

A retrospective study on the incidence of lymphocytic gastritis in patients with Helicobacter pylori infection.

To investigate the relationship between lymphocytic gastritis and H. pylori infection, antral biopsy specimens from 162 patients with histological evidence of H. pylori infection, and from 177 subjects without colonization were retrospectively examined. Among patients with antral colonization, lymphocytic gastritis was identified in 8 cases (4.9%). Lymphocytic gastritis was not detected in specimens without H. pylori infection. Quantitation of the epithelial lymphocytic infiltration was carried out in all specimens and showed that cases with lymphocytic gastritis had a mean of 35.3 lymphocytes per 100 epithelial cells, compared with a mean of 2.6 and 2.4 respectively in chronic gastritis and normal mucosa. Among the 331 patients without lymphocytic gastritis, specimens colonized by H. pylori had a mean of epithelial lymphocyte counts significantly higher than those observed in both chronic gastritis and normal mucosa. These findings show a positive correlation between antral colonization by H. pylori and occurrence of lymphocytic gastritis, and suggest that the bacteria may have an important role in the induction of such particular form of antral inflammation.

Chromosomal abnormalities in amniotic fluid cell cultures: a comparison of apparent pseudomosaicism in Chang and RPMI-1640 media.

The finding of chromosome mosaicism is one of the most difficult problems in fetal chromosome analysis. Whether the finding indicates true mosaicism or pseudomosaicism must be investigated. Studies detailing the incidence of true mosaicism and pseudomosaicism have been reported (Hsu & Perlis 1984, Bui et al. 1984, Worton & Stern 1984) but do not correlate pseudomosaicism with any particular type of culture media. Benn & Hsu (1985) compared cell growth and chromosome abnormalities in amniotic fluid cell cultures grown in Chang medium and RPMI-1640 medium and found no statistically significant difference in the rate of abnormalities in the two media. We have previously shown that Chang medium exhibited more abnormalities which were not verified in second and third cultures (Masia et al. 1986). In the current study we examined 212 cases grown in both Chang and RPMI-1640 media, and compared apparent single and multiple cell pseudomosaic abnormalities to medium type. The number of observed abnormalities was 22, occurring in 19 of the cases studied. Apparent pseudomosaic chromosome anomalies were observed in 18 Chang cultures and in 4 RPMI-1640 cultures. Statistical analysis found significant correlation between medium type and the degree of observed pseudomosaic cells. We conclude that the rate at which pseudomosaic cells are observed is partly a function of medium type, and in our laboratory Chang medium caused apparent pseudomosaicism at a greater level than RPMI-1640 medium.

Low frequencies of apparently fragile X chromosomes in normal control cultures: a possible explanation.

Low frequencies of apparently fragile X [fra(X)] chromosomes have been reported in normal control, short-term, whole blood cultures, and they have been noted in both amniocyte and fetal blood cultures. However, there is currently no universal agreement on the lowest frequency for fra(X)(q27) that is diagnostic for the fragile X syndrome. Here, we present our observations on low levels of apparently fra(X) chromosomes in normal samples. We observed frequencies of 0.5% in short-term whole blood cultures and 0.9% in amniotic fluid cell cultures. In 1982, Steinbach et al. described nonspecific telomeric structural changes (TSC) and suggested that such low frequencies of apparently fra(X) chromosomes in normal material may be occurring by the same mechanism that is responsible for TSC formation. To determine if TSC formation can explain the significant baseline frequencies of fra(X) in normal controls, 10,457 cells were screened from 178 individuals referred for fra(X) analysis. Our findings indicated that TSC are not randomly distributed across chromosomes but tend to occur at specific sites. Based on our observations, we offer the hypothesis that the low frequency of apparent fra(X) in normal individuals may be due to nonrandom TSC distribution.