Biomedical subjects
L Benítez-Bribiesca
Publications and source records attributed to L Benítez-Bribiesca.
Differential staining of DNA strand breaks in dried comet assay slides.
The comet assay involves embedding cells in agarose on microscope slides. After lysis and electrophoresis, staining is usually performed with a fluorescent DNA-binding dye and observation is carried out on fresh wet slides through an epifluorescence microscope. We present here a simple alternative for preservation of the agarose comet slides and a fluorescent staining that allows fine differential analysis of DNA strand breaks under confocal microscopy. Lymphocytes were processed according to previous published methods. Slides were quickly dehydrated in a hot oven at 50C for 20 min. Once the agarose layer was dried and reduced to a thin film, slides were treated with RNase. Image analysis showed higher tail length, total area, and tail moment. Using confocal microscopic optical sectioning, a thickness of approximately 180 microm for wet slides and 12 microm for dehydrated gels was calculated. Acridine orange, used for DNA differential staining, allowed quantitation of metachromasia and orthochromasia with confocal scanning microscopy. Differences between alkaline and neutral comet assay with AO were clear-cut and, in principle, a metachromatic index can be calculated. (J Histochem Cytochem 49:921-922, 2001)
The role of mast cell tryptase in neoangiogenesis of premalignant and malignant lesions of the uterine cervix.
Recently, mast cell tryptase has been identified as another potent proangiogenic factor in tumors, along with fibroblast and vascular endothelial growth factors. Its role has been studied in a number of cancers, including carcinoma of the uterine cervix, with discordant results. Our aim was to study the expression of tryptase and bFGF in mast cells (MCs) during development of neoangiogenesis in premalignant and malignant lesions of the cervix. Biopsy specimens from 21 patients without cancer and from 63 patients with dysplasias and squamous cell carcinomas were used. They were stained with Alcian blue-safranin O (ABSO) and immunostained with specific antibodies against factor VIII, CD105, tryptase, and bFGF. Tryptase-positive mast cells increased with tumor progression and were close to newly formed blood vessels. Vascularization showed a linear increase from dysplasia to invasive cancer. We suggest that MC tryptase may upregulate neoangiogenesis in carcinogenesis of the uterine cervix.
Morphologic, biochemical and molecular mitochondrial changes during reperfusion phase following brief renal ischemia.
Ischemia/reperfusion of organs and cells induces apoptosis through a complicated series of changes in mitochondria, mainly the generation of oxygen free radicals, permeability transitions, calcium translocations, and release of apoptogenic factors such as cytochrome c and Bcl-2 family members. The liberation of these factors occurs very early after reoxygenation and it has been assumed that it takes place without any structural alteration of the mitochondrial membranes. The aim of this study was to detect ultrastructural changes of mitochondria in the initial stages of reperfusion at the time when Bcl-2 and succinic dehydrogenase, located in the outer and inner membranes, respectively, were released. Ischemia/reperfusion was produced in adult rats by clamping one renal artery for 60 min and reoxygenating for 60, 120, 180, and 240 min. A model of chemical hypoxia with intra-arterial 50 mM sodium azide served as comparison, allowing free blood flow for 30, 60, 120 and 180 min. Light and electron microscopy, immunostaining for Bcl-2, and enzyme histochemistry for succinic dehydrogenase were performed. Our results showed mitochondrial swelling, rupture of inner and outer membranes, and leakage of mitochondrial matrix into the cytoplasm in ischemia after 120 min of reperfusion. Bcl-2 immunoreactivity and focal lowering of SDH reactivity were also noted and became more pronounced at the same time that the mitochondrial ultrastructure demonstrated more evident changes including rupture of the inner and outer membranes. Our studies seem to indicate that in early ischemia-reperfusion and in chemical hypoxia-induced apoptosis, the earliest ultrastructural changes take place in mitochondria and that swelling and rupture of mitochondrial membranes occur in parallel with the loss of Bcl-2 and SDH activity.
Oxidative damage, bleomycin, and gamma radiation induce different types of DNA strand breaks in normal lymphocytes and thymocytes. A comet assay study.
Most anticancer treatments such as chemo- and radiotherapy induce DNA damage and apoptosis in normal cells. The aim of this study was to assess the induction of single and double DNA strand breaks (ssb and dsb, respectively) and apoptosis in normal human lymphocytes and rat thymocytes subjected to the action of H2O2, bleomycin and ionizing radiation. Normal human peripheral thymocytes and young rat thymocytes were subjected to the following treatments: a) H2O2; b) bleomycin, and c) gamma-radiation, all with various doses. DNA strand breaks were studied with the alkaline and neutral comet assay for detection of ssb and dsb. Apoptosis was quantified morphologically and with DNA agarose gel electrophoresis. After H2O2 treatment, a dose-dependent increase of ssb was observed. Bleomycin treatment produced a moderate increase of ssb at lower concentrations and a striking increase of dsb at higher concentrations that coincided with the presence of apoptosis and DNA ladders. Gamma radiation initially induced the formation of ssb, and after three hours an increase of dsb in a dose-dependent manner. Apoptosis and DNA laddering appeared only 3 hours post-irradiation. The biomonitoring of DNA damage inflicted by antineoplastic agents can be easily performed with the comet assay and could be useful to monitor and modulate chemo- and radiotherapeutic regimes in cancer patients.
Dendritic spine pathology in infants with severe protein-calorie malnutrition.
BACKGROUND: Experimental undernutrition in animals, during the critical brain development period, produces retardation of brain growth as well as a number of different morphologic and functional abnormalities in neurons, mainly in the dendritic synaptic apparatus. These alterations are the cause of the poor neurointegrative development that occurs in experimental malnutrition. Severe malnutrition during early postnatal life in humans is known to produce similar neurointegrative disorders as well as mental retardation, but there are very few studies describing the morphology of the dendritic apparatus in infants suffering from this condition. OBJECTIVE: To study the dendritic spine density and morphology in dendrites from cortical neurons in infants dying from severe malnutrition. METHODOLOGY: Brain sections from the somestesic, motor, and occipital cortical areas of 13 infants who died of severe malnutrition and 7 eutrophic infants who died of other causes were studied by means of the rapid Golgi method. Apical dendritic spines from neurons of the fifth cortical layer were studied and counted in all sections. RESULTS: Apical dendrites were significantly shorter in malnourished infants than in the control group (581.54 +/- 54.32 microm in severe malnutrition vs 846.3 microm in normal infants). The number of dendritic spines per dendrite was also significantly diminished (185.3 +/- 36.1 in malnourished vs 374.3 +/- 41.6 in eutrophic infants). There were marked morphologic abnormalities in the dendritic spines of infants dying of severe malnutrition that were classified as dysplastic. CONCLUSIONS: Short apical dendrites, fewer spines, and dendritic spine abnormalities occur in severe infant malnutrition. These anatomic anomalies might be related to the neuropsychological deficits that occur in these children.
Cancer research in Mexico. An overview.
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Proteinase activity in invasive cancer of the breast. Correlation with tumor progression.
Hydrolysis of extracellular matrix is a necessary step for malignant cells to invade, and metastasize. Three groups of proteinases, mainly serine, thiol and metalloproteinases, have been found to be secreted by cancer cells and responsible for the proteolytic cascade triggered during invasion. Previous studies from our group and others have shown that the thiol proteinase cathepsin B1 is a constant indicator of tumor invasion in carcinoma of the cervix, although others point to plasminogen activators and collagenases. So far, there are no systematic studies to correlate cathepsin B and plasminogen activator activity with advancing malignant disease and thus estimate its capability as a marker of progression. The purpose of this study was to determine the activity of cathepsin B like proteinase and plasminogen activators in invasive carcinoma of the breast at various clinical stages and with different estrogen receptor status. One hundred patients with carcinoma of the breast at different clinical stages were studied. Cathepsin B and plasminogen activators activity was assessed in tumor cytosols using different synthetic oligopeptides as substrates following the method of Smith. Estrogen receptor concentration was determined with monoclonal antibodies. A statistical analysis and correlation with different clinical stages was performed. Cathepsin B-like activity had a consistent and progressive elevation in direct correlation with clinical stage (stage I, 1.97 SE +/- 0.46; stage II, 6.67 SE +/- 1.12; stage III, 28.19 SE +/- 3.48; nmol/mg/30 min), while plasminogen activators, although constantly elevated, had no correlation with tumor progression. No relation could be found with estrogen receptor status. It is concluded that cathepsin B, but not plasminogen activator, is a good indicator of tumor progression in invasive carcinoma of the breast.
A simplified histoscore for the estrogen receptor assay in breast cancer.
Different histoscores combining the number of positive cells and the intensity of staining have been used to evaluate the estrogen receptor immunocytochemical assay (ER-ICA). Our aim was to investigate if the simple estimation of the amount of positive cells could be sufficient for the semiquantitative analysis of ER-ICA. Tissue from 51 women with ductal breast carcinoma was used. Half of each sample was processed with the quantitative assay (ER-EIA) and the other half with ER-ICA. Microscopical analysis was performed by two independent observers and classified on a simple scale from 0 to 4+. With EIA 31 cases (60.78%) were positive and 20 (39.21%) negative. With ER-ICA 29 (56.86%) had immunostaining, whereas 22 (43.13%) did not. 95.83% of the ER-ICA positive cases and 77.7% of ER-ICA negative had a good correlation with EIA values. Statistical analysis showed a high degree of correlation (r = 0.88 p 0.001). Hence, simple semiquantitative estimation in ER-ICA is sufficient to provide useful information for clinical use about ER content in tissue sections.
Neutral proteinase activities in different strains and clones of Entamoeba histolytica. Correlation with virulence.
Although several factors are involved in the invasive behavior of E. histolytica, proteinases seem to play a key role. Different proteinases have been found in virulent trophozoites of this parasite. Cytosols of clones A, 32-1 462-1 and L-6 of E. histolytica exhibiting various degrees of virulence were used to study the activity of trypsin-like, plasminogen activator and cathepsin B neutral proteinases with specific synthetic oligopeptides. Cathepsin-B like activity showed the highest values in highly virulent clone A, which is derived from virulent strain HM1:IMSS. On the contrary, non virulent clones had very low activity. Clone L-6, a non virulent subclone of strain HM1:IMSS, retained some cathepsin B-like activity. Trypsin-like and plasminogen activator assays revealed low activity and no differences between virulent and non-virulent clones were found. It is concluded that the Arg-Arg-thiol proteinase (Cathepsin B-like) is a good virulence marker.
Cathepsin B and antiproteases in patients with carcinoma of the cervix.
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Protease and antiprotease concentrations in serum and vaginal fluid of patients with carcinoma of the cervix.
The capacity of malignant cells to invade and disseminate throughout the tissues is partly due to the fact that these cells synthesize and secrete proteases and plasminogen activators. Its growth can be inhibited by antiproteolytic substances in vitro or by natural inhibitors such as alpha-1-antitrypsin (A1AT) and alpha-2-macroglobulin (A2MG). It is a known fact that in cervical cancer, serum A1AT levels are elevated, but so far it has not been determined if serum proteolytic activity is elevated and if both occur simultaneously in vaginal fluid. 32 cases of invasive carcinoma of the cervix, and 25 non-cancerous women were studied. A1AT, A2MG, cathepsin B, trypsin inhibitory capacity (TIC), plasminogen and proteins in plasma and vaginal fluid were studied. In cancer cases, proteolytic activity of serum and vaginal fluid (cathepsin B) is elevated, when compared to controls (p less than 0.001). A1AT and A2MG levels are clearly elevated in plasma, but not in the vaginal fluid; TIC was elevated in serum in cancer cases (1.31 vs. 1.74 p less than 0.005) but was not altered in vaginal fluid. It was found that in cases of carcinoma in situ cathepsin B, plasminogen, A1At and A2MG and CIT were significantly increased (p less than 0.001, p less than 0.001, p less than 0.001 and p less than 0.01 respectively). In vaginal fluid the only statistically significant figure increased was cathepsin B (p less than 0.001). These studies show an altered protease-antiprotease balance in patients with carcinoma of the cervix uteri.
Immunofluorescent localization of alpha-1-antitrypsin in human polymorphonuclear leukocytes.
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The effect of intermediate respiratory chain inhibitors upon the tetrazolium-reductase activity of human polymorphonuclear leukocytes.
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Diffusion rate of 131I labeled human albumin in the ear of the rabbit.
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[Acquired syndrome of neutrophil dysfunction].
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Cytochemical demonstration of proteolytic activity of human and rat spermatozoa.
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Occurrence of alkaline phosphatase-containing muscle fibers after denervation.
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