The NAD+ precursors, nicotinic acid and nicotinamide protect cells against apoptosis induced by a multiple stress inducer, deoxycholate.

The bile salt, sodium deoxycholate (NaDOC), is a natural detergent that promotes digestion of fats. At high physiologic levels, NaDOC activates many stress-response pathways and induces apoptosis in various cell types. NaDOC induces DNA damage and activates poly(ADP-ribose) polymerase (PARP), an enzyme that utilizes NAD+ as a substrate to repair DNA. NaDOC also induces oxidative stress, endoplasmic reticulum (ER) stress and contributes to protein malfolding. The NAD+ precursors, nicotinic acid (NA) and nicotinamide (NAM) were found to protect cells against NaDOC-induced apoptosis. NA and NAM also decreased constitutive levels of both activated NF-kappaB and GRP78, two proteins that respond to oxidative stress. However, the mechanism by which NA and NAM protects cells against apoptosis does not involve a reduction in constitutive levels of oxidative stress. NA or NAM treatment increased the protein levels of glyceraldehyde-3-phosphate dehydrogense (GAPDH), a multi-functional enzyme, in the nucleus and cytoplasm, respectively. NAM did not activate the promoter/response elements of 13 stress response genes nor reduce intracellular non-protein thiols, suggesting that it is non-toxic to cells. NAM thus has promise as a dietary supplement to help prevent disorders involving excessive apoptosis.

Inhibitory effect of selenomethionine on the growth of three selected human tumor cell lines.

Selenium supplementation has been shown for many years to work as an anticarcinogenic agent both in epidemiology and in in vitro studies. Selenium supplementation has recently been shown to decrease total cancer incidence. However, the mechanism of action of selenium as an anticarcinogenic agent has yet to be elucidated. Selenomethionine was the predominant form of selenium in the dietary supplement in the study by Clark et al. (Clark, L.C., Combs, G.F., Turnbull, W.B., Slate, E.H., Chalker, D.K., Chow, J., Davis, L.S., Glover, R.A., Graham, G.F., Gross, E.G., Krongrad, A., Lesher, J.L., Park, H.K., Sanders, B.B., Smith, C.L., Taylor, J.R. and The Nutritional Prevention of Cancer Study Group (1996) Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin: a randomized controlled trial. J. Am. Med. Assoc., 276 (24), 1957-1963) and therefore we evaluated the growth inhibitory effects of selenomethionine against human tumor cells. Selenomethionine was tested against each of three human tumor cell lines (MCF-7/S breast carcinoma, DU-145 prostate cancer cells and UACC-375 melanoma) and against normal human diploid fibroblasts. All cell lines demonstrated a dose-dependent manner of growth inhibition by selenomethionine. Selenomethionine inhibited the growth of all of the human tumor cell lines in the micromolar (microM) range (ranging from 45 to 130 microM) while growth inhibition of normal diploid fibroblasts required 1 mM selenomethionine, approximately 1000-fold higher than for the cancer cell lines. In short, normal diploid fibroblasts were less sensitive than the cancer cell lines to the growth inhibitory effects of selenomethionine. Furthermore, we show that selenomethionine administration to these cancer cell lines results in apoptotic cell death and aberrant mitoses. These results demonstrate the differential sensitivity of tumor cells and normal cells to selenomethionine.

Limitations of ZAF correction factors in the determination of calcium/phosphorus ratios: important forensic science considerations relevant to the analysis of bone fragments using scanning electron microscopy and energy-dispersive x-ray microanalysis.

A series of calcium phosphate standards having calcium/phosphorus (Ca/P) molar ratios of 0.50, 1.00, 1.50, and 1.67, respectively, was prepared for bulk specimen analysis using scanning electron microscopy (SEM) and energy-dispersive X-ray microanalysis (EDXA). The standards were mounted on carbon planchettes as either pure crystals or crystals embedded in epoxy resin. Ten different samples of each embedded and non-embedded standard were analyzed in a JEOL 100 CX electron microscope interfaced with a Kevex 8000 EDXA system using a lithium-drifted silicon detector and a multichannel analyzer. The Ca/P ratios were determined by calculating both net peak intensities without matrix corrections and atomic kappa-ratios using the MAGIC V computer program with ZAF correction factors for quantitative analysis. There was such extensive absorption of phosphorus X-rays in standards embedded in an epoxy matrix that the observed Ca/P ratios were statistically compatible with four different standards ranging in theoretical Ca/P ratios from 1.0 to 1.67. Although the non-embedded crystals showed a greater separation in the Ca/P ratios, both methods of preparation produced serious flaws in analysis. Direct application of the discovery of this caveat to the identification of suspected bone fragments for forensic science purposes is discussed.

An ultrastructural cytochemical stain specific for neuroendocrine neoplasms.

Previous work from our laboratory has indicated that the uranaffin reaction, when run under specific conditions, will stain neurosecretory granules. In this ultrastructural cytochemical study, we analyzed the granule-staining properties of 13 normal, 10 abnormal (non-neoplastic), and 138 neoplastic tissues in an attempt to evaluate the specificity of the uranaffin reaction for diagnostic purposes when compared with routinely processed specimens. For the uranaffin reaction, previously fixed tissue stored in buffer was rinsed with 0.9% NaCl and reacted with a 4% aqueous solution of uranyl acetate (pH 3.9) for 48 hours. After three NaCl rinses, the tissue was dehydrated and processed for electron microscopy. The granules of normal or non-neoplastic neuroendocrine cells that stained positively with the uranaffin reaction included pancreatic islet cells, thyroid C cells, adrenal medullary cells, parathyroid chief cells, and the neuroendocrine cells of the intestine. All 42 neuroendocrine neoplasms studied possessed abundant uranaffin-positive granules and included carcinoids, oat cell carcinomas, islet cell neoplasms, medullary carcinomas of the thyroid, pheochromocytomas, carotid body paragangliomas, a pituitary adenoma, Merkel cell carcinomas, parathyroid adenomas, and a neuroblastoma. All 96 control neoplasms that were not classified as neuroendocrine in nature were negative for neurosecretory granules when studied with the uranaffin reaction and included 13 neoplasms derived from endocrine glands, 57 neoplasms from secretory epithelium, 10 of hematopoietic origin, and 16 miscellaneous neoplasms. It was determined that the uranaffin reaction is a useful ultrastructural cytochemical marker for neuroendocrine granules and helped distinguish these cytoplasmic organelles from ultrastructurally similar granules derived from non-neuroendocrine cells.

A quantitative ultrastructural evaluation of the cell organelle specificity of the uranaffin reaction in normal human platelets.

The purpose of this study was to determine the normal variation in the number of uranaffin-positive organelles in normal human platelets and to study the effect of pH and fixation on the uranaffin reaction. The normal variation in uranaffin organelles was determined by studying platelets from nine normal subjects. At pH 3.9, the mean number of reactive sites/platelet profile was 0.43 +/- 0.10 when platelets were fixed with lower glutaraldehyde concentrations and 0.56 +/- 0.24 with higher glutaraldehyde concentrations. Fixed platelets were reacted at pH 2.8, 3.9, 5.0, and 7.0 with four different uranaffin procedures that varied in the extent of fixation and rinse steps (isotonic saline vs. cacodylate buffer). The number of uranaffin-positive sites in 200 platelet profiles was scored under the electron microscope. There was a progressive increase in the number of reactive sites/platelet profile as the pH increased from 2.8 to 7.0. In general, higher pH favored granule matrix and core staining, whereas low pH favored both the staining of granule membranes and their contents. The uranaffin reaction showed organelle specificity when run under certain experimental conditions. At low pH and using isotonic saline in the rinse steps, only the dense bodies and ribosomes stained. The biochemical content of the dense body responsible for uranaffin reactivity is discussed.

An ultrastructural evaluation of the cell organelle specificity of the uranaffin reaction in two human endocrine neoplasms.

The organelle specificity of the uranaffin reaction was determined by subjecting two human neoplasms (a pheochromocytoma and islet cell carcinoma) to four different experimental conditions. In Uranaffin Procedure (UP) I, fixed tissue was immersed in 0.9% sodium chloride (NaCl) before reacting with 4% uranyl acetate (pH 3.9) for 24 hours. In UP II, the tissue was prepared as in UP I with the exception that the tissue was immersed in uranyl acetate for 48 hours. In UP III, fixed tissue was prepared as in UP I with the exception that tissue was immersed in 0.1M cacodylate buffer (pH 7.2) instead of 0.9% NaCl. In UP IV, fixed tissue was prepared as in UP III with the exception that the tissue was immersed in uranyl acetate for 48 hours instead of 24 hours. When UPs I and II were utilized, only three cell organelles showed electron-dense reactivity: the nucleus, ribosomes, and cytoplasmic neurosecretory-like granules. In the nucleus, the nuclear chromatin, nucleolus, interchromatinic granules and perichromatinic granules were intensely stained. The reaction product in all of the uranaffin-positive organelles had a finely granular appearance. When fixed tissue was immersed in cacodylate buffer instead of isotonic saline, a non-specific reactivity was observed. The reaction product in some areas had a distinct crystalline appearance and filled some areas of the cytosol, the cisternae of the endoplasmic reticulum, the Golgi apparatus, the perinuclear cisternae, the nucleus, nucleolus, mitochondria, neurosecretory-like granules and larger lysosome-like bodies. There was a statistically significant (p less than 0.05) increase in the number of uranaffin-positive granules/mu2 when both endocrine neoplasms were reacted with uranyl acetate for 48 hours instead of 24 hours. The increase in uranaffin-positive granules using UP II did not result in an increase in non-specificity of the staining reaction.