Biomedical subjects
Publications and source records attributed to C Quintana.
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In a previous paper (Quintana et al, Biol Cel 72, (1991) 167-180) we reported the anti-ovalbumin antibody immunolabeling in the cytoplasm of tubular gland cells from cryofixed, cryosubstituted and acrylic-resin embedded quail oviduct. To confirm these preliminary visual observations, we have carried out a semi-automatic quantitative study of immunogold labeling. The quantitative results confirm the previous data. In addition, we found a significant higher immunolabeling with low temperature Lowicryl K11M embedding procedure as compared with high temperature LR White embedding.
Surgery has an important role in the treatment of Crohn's disease (CD). The objective of this communication is to assess the results of surgical treatment in CD, trying to identify recurrence risk factors. A retrospective review of the charts of 120 patients with CD seen between 1963 and 1990 was made. One third of these patients required surgical treatment. The series was divided in two groups. Group A comprised patients treated from 1963 to 1977. Of these, 30% had ileal localization of the disease and 20 patients were submitted to 20 surgical procedures. The major surgical indication was intestinal obstruction and resection was performed in 85% of patients. Morbidity was 20% and mortality 10%. Group B comprised patients treated from 1978 to 1990. Nineteen patients were submitted to 22 surgical procedures. The disease was localized in the ileon in 50% of these patients. Twenty one surgical procedures were resective with a 9% complication rate and no surgical mortality. Eighty percent of patients in this group has a complete follow up. In 47% the disease recurred 37 months (as a mean) after surgery. No effect of age, sex or localization of the disease on the risk of recurrence was observed. It is concluded that morbidity and mortality of surgical treatment for Crohn's disease has decreased and a prolonged control of symptoms has been achieved. No recurrence risk factors were identified in this study.
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We studied the chromosome periphery in human HeLa and TG cells using cryomethods in electron microscopy. A contrasted layer of peripheral chromosomal material (PCM) was visible in cryo-ultrathin sections of mitotic cells. This PCM was composed of closely packed fibrils associated with granules. The PCM did not cover the entire chromosome surface but was found around most of the chromosomes and even between two chromatids. The organization of the PCM was not affected by colchicine treatment of mitotic cells. In cells prepared by quick-freezing, the PCM appeared to be a fibrous material at the chromosome periphery, and was also associated with granules that resembled inter-chromatin granules in size and shape. At higher magnification, direct contacts between the chromosomes and the fibrils of the PCM were observed. The cryotechniques used are known to preserve the native organization of cells. Therefore, the architecture of the perichromosomal region analysed presumably corresponds to that in vivo during mitosis. These observations show that in HeLa and TG cells, a particular structure present at the chromosome periphery in the form of PCM is persistent and ubiquitous. In addition, we showed by immunolabelling that the PCM is the specific site of accumulation of nucleolar antigens during mitosis. These two results, i.e. the identification of specific morphological structures and the compartmentation of proteins, indicate that this layer is a specific region of mitotic cells.
We evaluated a pre-tinning leukocyte labeling technique using Glucoheptonate-Sn lyophilized kit, in the detection of activity in 15 patients with clinical and laboratory signs of active inflammatory bowel disease (IBD). 25 patients, without gastrointestinal disease were the control group. In 7/8 patients with Crohn's disease and in 7/7 with Ulcerative Colitis the study was positive. The negative study corresponded to a patient who has only involvement of the duodenum, an area of difficult evaluation due to the high liver uptake. 3/25 patients of the control group had activity in the intestine at 4 hour images. In spite of the small group evaluated, we believe that this labeling technique is a promising procedure for evaluation of activity in patients with IBD (Sensitivity 93.3%, Specificity 88.0%).
The principal component analysis, a multivariate statistical analysis of data, has been used to process X-ray microanalytical data from cell nuclei. Sixty-seven measurements from different areas of chromatin, nucleoli of rat follicular cells, and nucleoli of rat oocyte cells in their antral stage have been studied. The variables are the X-ray characteristic signals for P, S, Al, Fe, Cu, and Zn. This method demonstrates four distinct groups, the chromatin area, which is associated with a higher concentration of P; the compact mass of oocyte nucleolus which possesses the highest content in S, Al, and Zn, and two groups of nucleolar areas. The fibrillar component is richer in S, Al, and Zn than the granular component. The high degree of correlation between these three elements proves the chemical affinity of metals for the proteins (S being the signature for proteins). Cryoembedding in Lowicryl resin at even lower temperatures (213 degrees K in K11M) after quick cryofixation and cryosubstitution in the absence of chemical fixatives gives good ultrastructural preservation and the possibility of simultaneously performing X-ray microanalysis and immunocytochemistry.
The contribution of French science to the field of biological X-ray microanalysis is reviewed. The main analytical microscopy centers are listed, and their methods and main results are summarized.
Cryomethods occupy a privileged position among the procedures used for the preparation of biological samples for the various studies that may be performed in electron microscopy (ultrastructural, immunocytochemical and microanalysis in situ). In general, cryomethods are specific to one, or a maximum of 2 types of application. The (3-Cryo) methods (cryofixation, cryosubstitution without fixatives and cryoembedding in the new Lowicryl resins (K11M or HM23) are a set of methods for correlating new structural information with analytical and biochemical data. However, these 3-Cryomethods are delicate, complicated and expensive. To demonstrate that they can be performed, at least in part, with home-made systems at a reasonable cost, we have carried out a structural and immunocytochemical study on the oviduct of the laying quail. We studied the localization of 2 proteins, one cytoplasmic (ovalbumin) and the other nucleolar (B-36). The results provided by the 3-Cryomethods are compared with those obtained with other immunocytochemical methods, including tissue processed by conventional chemical fixation and high or low temperature embedding, or by 2-Cryomethods (cryofixation and cryosubstitution).
A multivariate statistical analysis (the principal component analysis) has been used to process electron probe microanalytical data from cell nuclei. Fifty-seven measurements from different areas of chromatin and nucleolus in follicular rat cells have been studied. The variables are the X-ray characteristic signals for P, S, Al, Fe, Cu and Zn. This method demonstrates three groups of individuals - the chromatin area which is associated with a stronger concentration of P and two groups of nucleolar areas, one of them being connected with a higher content in S, Al and Zn. This high degree of correlation between these three elements proves the chemical affinity of the metals with the protein, S being the signature for proteins.
Out of 103 consecutive patients with ulcerative cholitis, 37 required surgery. We compared perioperative morbidity and mortality in 18 patients receiving intensive nutritional care with 9 controls. An increase in serum albumin form 2.8 +/- 0.5 to 3.4 +/- 0.4 mg/dl (p less than 0.01) and lymphocytes from 1210 +/- 614 to 1736 +/- 770 (p less than 0.05) was observed in the first group, but not in the second. No deaths, shorter hospital stay and fewer complications were observed in the first group. We conclude that intensive nutritional care should be routinely used in patients with severe ulcerative cholitis requiring surgery.
Crystalline accumulations of ferritin-like particles are present within the cytoplasma and the nucleus in midgut epithelial cells of the homopteran Philaenus spumarius. A structural study at the electron microscope level reveals that these particles have the morphological characteristics of the ferritin molecule: crystals have a face-centered cubic structure with a lattice parameter of 14 +/- 1 nm; negatively stained isolated particles have the appearance of ferritin; on rotary-shadowed particles 3 axes of symmetry are clearly seen; image processing performed on selected molecules demonstrates a 4-fold symmetry. A semiquantitative electron microprobe analysis effected on aggregates of microcrystals in thin sections reveals a high atomic ratio Fe/P. Analyzed by SDS-PAGE, the protein subunit has a molecular weight of 18,600. The amino acid composition of the protein bears the general characteristics of the ferritin molecule in terms of polar and nonpolar residues. But in terms of sequences, this protein displays a strong dissimilarity to rat liver ferritin as demonstrated with a common amino acid index test and with immunoelectrophoresis experiments.
The aim of the present work is to gather new information on the ferritin molecule. Natural crystals of ferritin occurring in the yolk platelets of a mollusc oocyte were studied. Their crystallographic structure was found to be equivalent to one of the structures obtained by artificial crystallization (fcc; a = 15 nm). Individual ferritin particles isolated from horse spleen were studied by microdiffraction techniques, using field emission gun transmission electron microscopy. The iron core crystals display a hexagonal structure; our results confirm the value of the unit cell parameter a (0.51 nm) and, for the first time, we have been able to extract the value of the unit cell parameter c (0.95 nm). Thus, among the three models described in the literature for the crystalline structure of the iron complex, our results corroborate that of Towe and Bradley (J. Colloid. Interf. Sci., 1967, 25, 384-392).
Cell nuclei may contain significant quantities of the metals Fe, Ni, Cu, Zn, since they are present in the nucleo-enzymes and/or nucleic acids. These metals have been detected by X-ray microanalysis in situ in dinoflagellates (Kearns et al). Aluminum was only detected in cell nuclei in cases of natural or provoked intoxication. We observed at the ultrastructural level, in situ, the presence of Al, Fe, Ni, Cu, Zn in nuclei of different types of non-intoxicated animal cells. Moreover, we measured the concentration of these metals in the nucleolus and chromatin and compared it with the concentration of P and S.
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A microcomputer reconstruction technique has been developed in order to permit a larger exploitation of stereomicroscopy. The microcomputer facility consists of a digitizing tablet, a microcomputer, a graphics terminal, a graphics plotter and a printer. The technique has been applied to the study of HVEM stereopairs, performed by recording two images of the same area of a specimen (thick section of araldite-embedded leech ganglion neurons), tilted relative to the beam axis through an angle 0/20 degrees. Coordinates of N conjugate points of interest, expressed in a common reference system were obtained with the help of a digitizing tablet and the misorientation between the two images was determined by a method based on a least square technique. New projections of the object on different planes are provided by the microcomputer facility. Also the microcomputer method permits to obtain new stereopairs drawings, in various orientations and slices from a three-dimensional reconstruction of the object oriented in any direction in space. The method permits to obtain computed anaglyph drawings, printed here, which are stereoviews of the same object.