Energy dispersive x-ray microanalysis of the concentration of elements in relation to cell reproduction in normal and in cancer cells.
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Biomedical subjects
Publications and source records attributed to N K Smith.
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We have adapted the cryopreparative methods designed for the radioautography of diffusible substances (Stumpf and Roth, 1966, 1967) to produce freeze-fried sections of soft tissues for electron probe microanalysis. This report concerns the evaluation of these methods for use with X-ray microanalysis with particular reference to: (1) the preservation of cellular morphology, (2) the introduction of structural artefact by ice crystal formation, (3) the preservation of natural elemental distributions under experimental conditions where known quantities of diffusible elements are present, (4) the effects of ice crystal formation upon possible artefactual elemental redistributions, (5) the effect of section thickness on elemental quantitation, and (6) the effect of tissue excision on element translocation. Freeze-dried sections were prepared from mouse pancreas and from 20% (w/v) solutions of bovine serum albumin (BSA) and gelatin containing known amounts of inorganic salts and were analysed in a scanning electron microscope fitted with energy dispersive X-ray detecting equipment. With the preparative methods used, the morphology of pancreatic acinar cells was well preserved. Acinar cell boundaries, nuclear boundaries, chromatin, nucleoli, ergastoplasm and zymogen granules were readily discernible. Ice crystals were present within sections of BSA, but 70% had a cut surface area < 1 micrometer2 and over 90% had a cut surface area of < 2 micrometer2. Characteristic peak-to-continuum ratios of elements in the BSA sections remained constant over an order of magnitude change in magnification. Elemental redistributions were not detected until the magnification was such that the analysed area fell totally within the confines of a single ice crystal. No differences in elemental peak-to-continuum values were obtained between 2 and 4 micrometer sections of the gelatin-salt solution. Tissue excision did not cause element translocations when compared to tissues frozen in situ. We conclude that this method is valid for preparing tissues for microanalysis under our conditions (analysis of nuclear, cytoplasmic, and secretory compartments) and is limiting only when analyses are conducted at very high magnifications.
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Chief cells of the carotid and aortic body chemoreceptors possess numerous cytoplasmic dense-core vesicles which are known to contain primarily dopamine. Following fixation in solutions containing 50 mM CaCl2, a 20--30 nm electron-dense particle (EDP) is often observed eccentrically located in many of the vesicles. Approximately 44% of the carotid body and 16% of the aortic body vesicles contain an EDP. The EDP probably represents the Ca++ binding site critical to the stimulus-secretion coupling events culminating in exocytosis of these vesicles. The presence of Ca++ in the cytoplasmic vesicles was verified by electron probe X-ray microanalysis.
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Elemental concentrations in the cytoplasm and nucleus of t tests post-natal mouse myocytes were measured at 2, 4, 8, 16, 32 days and in adults using electron probe X-ray microanalysis. Using an analysis of variance test, significant age dependent changes were found in intracellular potassium, sulfur, phosphorus, and chlorine concentrations (mg/kg dry weight), while sodium and magnesium concentrations did not show significant changes. The application of t tests following linear regression analyses (age versus concentration for each element) did, however, give a significant slope for cytoplasmic sodium, potassium, sulfur, and phosphorus values. The findings correspond closely with an emission spectroscopy-titrimetric study of whole heart ventricle of the same developmental period (Hazelwood and Nichols, '70).
Electron probe energy dispersive X-ray microanalysis was performed on freeze-dried tissue sections. The dry weight concentration of elements (mmole/kg dry weight) was measured in the cytoplasm of several cell types from adult mice and rats. This comparative investigation showed: (1) That the energy dispersive X-ray spectrum of element concentration from the cytoplasm of a specific cell type allows one to distinguish this specific cell type from other cell types with considerable accuracy. (2) That there is a relationship between the concentration of the various elements and the ultrastructural features of the cytoplasmic regions being analyzed. For example, areas rich in ribosomes are also rich in P, K and Mg. (3) These data support the idea that K is directly involved in the control of protein synthesis. The catalog of element concentrations in the cytoplasm of 13 cell types from both mice and rats should be of value to others who seek to answer various questions about these cell types.
Unfixed freeze-dried and uncoated tissue sections of the mouse duodenum were suspended across a hole in a carbon planchet and analyzed in a scanning electron microscope fitted with energy-dispersive x-ray analytical equipment. Computer analysis of the x-ray spectra allowed elemental microanalysis of the nucleus, cytoplasm, and late anaphase-early telophase chromatin regions in the cryptal and villus enterocytes. Elemental concentrations (mmol/kg dry wt) were measured for Na, Mg, P, S, Cl, K, and Ca. None of the elements were compartmentalized preferentially in either the nucleus or the cytoplasm of interphase enterocytes of crypts or in postmitotic enterocytes of villi. In contrast, Ca, S, and Cl are detectable in significantly higher concentrations in mitotic chromatin of dividing enterocytes of the crypt as compared to surrounding mitotic cytoplasm, but Na, Mg, and P are in lower concentrations in the mitotic chromatin as compared to mitotic cytoplasm. Interphase enterocytes of crypts have higher concentrations of Mg, P, and K, and lower concentrations of Na than do postmitotic enterocytes of villi.
Capillaria hepatica infection was diagnosed by histologic examination in 21 of 71 (30%) coyotes in Louisiana. All infected coyotes were at least 1 year old, and most (19 of 21) were more than 2 year years old. Capillaria hepatica was not found in any livers from 31 red foxes and 20 gray foxes examined.
Tubal segments obtained from patients at cesarean section and at intervals during the first 5 postpartum days were examined to evaluate puerperal changes in the tubal epithelium. The specimens of tubal epithelium were examined under the scanning and transmission electron microscopes. Ciliated cells were most densely distributed on the fimbria and in the ampulla, and were relatively sparsely distributed in the isthmus. Progressive diminution of numbers of ciliated cells and deciliated of individual cells were noted in specimens obtained during the puerperium. Nonciliated cells were in the resting stage at term pregnancy. Secretory activity returned during the puerperium.
Microsurgical reconstruction of the rabbit oviduct was undertaken utilizing either through-and-through sutures including the mucosa, or sutures penetrating the serosa and myosalpinx but not the mucosa. Patency and pregnancy rates did not seem to vary with the suturing technique. However, scanning electron microscopic observation revealed abnormal mucosal fold patterns at 3 weeks after surgery. Fibrinous exudates over the surface of the intraluminal sutures increased with time until the entire surface was thickly veiled by 9 weeks. The observation also revealed regeneration of epithelial cells along the suture. These cells included many abnormal forms such as giant cells and misshapen cells. The majority of these were nonciliated. In theory, the intraluminal suture could form a nidus for epithelial hyperplasia which might cause future tubal obstruction. However, the patency and pregnancy rates obtained with through-and-through suturing were similar to those obtained when the endosalpinx was excluded, both in this study and in previous studies from these laboratories.
Contributing factors to effective continence service provision include funding, organization, and expert knowledge among the individuals providing care. Expert knowledge can be gained through clinical experience and appropriate ongoing education. It has been widely reported that undergraduate education in this area for nurses, doctors and physiotherapists is limited (Brocklehurst, 1990; Swaffield, 1994; Laycock, 1995). Many nurses providing continence care have accumulated knowledge through experience and trial and error. Little is known about the effectiveness of advanced postgraduate education of 'experts' in continence care. This article outlines a continence education module developed to prepare a specialist group of nurses to provide a high standard of continence care that is both safe and effective in a clinical environment. This module was designed and evaluated specifically as part of the Leicestershire Medical Research Council (MRC) Incontinence Study. Changes in continence knowledge, attitudes to research, and acceptability of the module have been explored. When setting up a new nurse-led continence service, it is of great importance to systematically detail the components of the educational preparation of the nurses providing the service. Open discussion of any problems in the design and implementation of this module may inform future modules in this and other areas.
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The effects of recombinant human interleukin-4 (rhu IL-4) on immunological parameters in patients receiving increasing doses of IL-4 in a Phase I trial were investigated. Peripheral blood mononuclear cells were phenotyped for a variety of lymphocyte markers, but no consistent effects were observed. However, increases in HLA Class II expression on monocytes were detected in four patients. NK and LAK activity were neither induced nor augmented by IL-4 treatment. Slight increases in proliferative responses to mitogens and cytokines were observed in some patients. The latter observations and other clinical studies suggest that a combination of IL-4 with IL-2 may be more effective in Phase II clinical trials.