Qualitative and Quantitative Analysis of DNA Fragmentation Using Digital Imaging

Apoptosis is an important and common pathway of cellular death. Differentiation from cellular necrosis and quantitation of apoptosis within the milieu of necrosis are analytical challenges. We describe the use of the RIT120 digital imaging software package for quantitative and qualitative analysis of apoptotic DNA ladders induced by a variety of agents, such as serum, tumor necrosis factor-α, transforming growth factor-β1, and nitric oxide. Autoradiographs of DNA ladders are densitometrically scanned to yield a set of curves with peaks corresponding to specific DNA fragments, thereby allowing quantitative subtraction of concurrent DNA degradation from necrotic death. Integration of the areas specifically under the peaks yields a quantitative measure of apoptosis. We provide a useful, rapid, and objective means to quantitate apoptosis, using relatively inexpensive hardware and software.     

Concanavalin A-stimulated Ca2+ uptake in rat splenocytes

Summary Commercially available concanavalin A binds Ca²⁺ with high apparent affinity. In order to dissociate concanavalin A stimulated Ca²⁺ uptake (defined as an increased association of ⁴⁵Ca²⁺ with cells) in rat splenocytes and Ca²⁺ binding to cell-bound concanavalin A, conditions were developed to remove more than 75% of the bound concanavalin A. Under these conditions concanavalin A treated cells showed a considerable increase in ⁴⁵Ca²⁺ uptake over control. The concanavalin A stimulated uptake of ⁴⁵Ca²⁺ occurred within minutes, and required concentrations of concanavalin A which promoted [³H]thymidine uptake into these cells. Succinyl concanavalin A was less potent in promoting Ca²⁺ uptake than concanavalin A. Sodium periodate inhibited Ca²⁺ uptake at concentrations which promoted ³H-thymidine incorporation into splenocytes.It is concluded that con canavalin A promotes Ca²⁺ uptake which is not due to binding of ⁴⁵Ca²⁺ to concanavalin A. Although the concanavalin A-promoted Ca²⁺ uptake occurs at lectin concentrations that cause lymphocyte proliferation as measured by ³H-thymidine incorporation, the role of Ca²⁺ in this event remains unclear.     

Translocation of 14C-assimilates in roses

The upper shoot on decapitated rose branches ( Rosa hybrids cv. Marimba) grows faster than lower shoots on the same branch. Transport of radioactive assimilates to the upper shoot is higher than to the lower ones. Darkening of the uppermost shoot resulted in the reduction of growth and ^I4C-assimilate accumulation in the darkened shoot as well as the promotion of growth and ¹⁴C transport to the lower 2 shoots, thereby rendering dominance to the second shoot. Benzyladenine treatment to the uppermost shoot reversed the effect of darkening and restored the apical control of this shoot.     

Divalent cations and transmitter release at low concentration of tetrodotoxin

Transmitter release from frog motor terminals was studied in the presence of very low concentrations of tetrodotoxin (TTX, 4.10(-10)--6.10(-9) g/ml). TTX reversibly reduced the amplitude of the end-plate potential (epp), while leaving the amplitude distribution to follow Poisson's law. The effects of a number of divalent cations were studied in the presence of TTX. It was found that after the addition of TTX there was an increase in the constant of dissociation of calcium and strontium from a hypothetical membrane "release site," while the dissociation constants of magnesium and manganese remained unaltered. It is concluded that the release site is probably intracellular and that a reduced presynaptic spike amplitude, as well as magnesium and manganese ions, decrease the access of calcium and strontium to the site.     

Mapping of sites on the surface membrane of mammalian cells

Summary Cells transformed by Simian Virus 40 have sites on the surface membrane for Concanavalin A (Con A) and a copolymer of ornithine, leucine (POL). The cells can be rapidly agglutinated by Con A, more slowly aggregated by POL, and they can be killed by both compounds. Treatment with Con A or POL has been used to select resistant cell variants from the transformed cells. Variants selected for resistance to Con A were also resistant to POL, but variants selected for resistance to POL were not resistant to Con A. The POL-selected variants showed less aggregation by POL but no decrease in agglutinability by Con A, whereas Con A-selected variants showed a decrease both in POL aggregation and Con A agglutination. The selection for both sites by Con A and only for POL sites by POL, can be explained in that the sites for POL are part of the sites for Con A and/or are included in clusters of Con A sites.Paper I in this series is Inbar, Ben-Bassat and Sachs (1971a).     

KIAA1363 affects retinyl ester turnover in cultured murine and human hepatic stellate cells

Large quantities of vitamin A are stored as retinyl esters (REs) in specialized liver cells, the hepatic stellate cells (HSCs). To date, the enzymes controlling RE degradation in HSCs are poorly understood. In this study, we identified KIAA1363 (also annotated as arylacetamide deacetylase 1 or neutral cholesterol ester hydrolase 1) as a novel RE hydrolase. We show that KIAA1363 is expressed in the liver, mainly in HSCs, and exhibits RE hydrolase activity at neutral pH. Accordingly, addition of the KIAA1363-specific inhibitor JW480 largely reduced RE hydrolase activity in lysates of cultured murine and human HSCs. Furthermore, cell fractionation experiments and confocal microscopy studies showed that KIAA1363 localizes to the endoplasmic reticulum. We demonstrate that overexpression of KIAA1363 in cells led to lower cellular RE content after a retinol loading period. Conversely, pharmacological inhibition or shRNA-mediated silencing of KIAA1363 expression in cultured murine and human HSCs attenuated RE degradation. Together, our data suggest that KIAA1363 affects vitamin A metabolism of HSCs by hydrolyzing REs at the endoplasmic reticulum, thereby counteracting retinol esterification and RE storage in lipid droplets.     

Cutting edge: MHC class I-Ly49 interaction regulates neuronal function

MHC class I molecules (MHC-I) have been implicated in nervous system development in the mouse. In this study we present evidence for the interaction of MHC-I with the NK cell receptor Ly49 in primary cortical neuronal cultures. We show that MHC-I and Ly49 are expressed on neuronal soma and axon surfaces, with Ly49 also present on dendrites. Anti-MHC-I Abs reduce synapsin-I expression and enhance neurite outgrowth and neuronal death. Conversely, anti-Ly49 mAbs increase synapsin-I expression, reduce neurite outgrowth, and promote neuron viability. Because we show that Ly49 genes are selectively expressed in the adult brain, these findings suggest an unsuspected role for the MHC-I-Ly49 interaction in the development and function of the brain.     

Relationship between Nitrite Reduction and Active Phosphate Uptake in the Phosphate-Accumulating Denitrifier Pseudomonas sp. Strain JR 12

Phosphate uptake by the phosphate-accumulating denitrifier Pseudomonas sp. JR12 was examined with different combinations of electron and carbon donors and electron acceptors. Phosphate uptake in acetate-supplemented cells took place with either oxygen or nitrate but did not take place when nitrite served as the final electron acceptor. Furthermore, nitrite reduction rates by this denitrifier were shown to be significantly reduced in the presence of phosphate. Phosphate uptake assays in the presence of the H⁺-ATPase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD), in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or with osmotic shock-treated cells indicated that phosphate transport over the cytoplasmic membrane of this bacterium was mediated by primary and secondary transport systems. By examining the redox transitions of whole cells at 553 nm we found that phosphate addition caused a significant oxidation of a c-type cytochrome. Based on these findings, we propose that this c-type cytochrome serves as an intermediate in the electron transfer to both nitrite reductase and the site responsible for active phosphate transport. In previous studies with this bacterium we found that the oxidation state of this c-type cytochrome was significantly higher in acetate-supplemented, nitrite-respiring cells (incapable of phosphate uptake) than in phosphate-accumulating cells incubated with different combinations of electron donors and acceptors. Based on the latter finding and results obtained in the present study it is suggested that phosphate uptake in this bacterium is subjected to a redox control of the active phosphate transport site. By means of this mechanism an explanation is provided for the observed absence of phosphate uptake in the presence of nitrite and inhibition of nitrite reduction by phosphate in this organism. The implications of these findings regarding denitrifying, phosphate removal wastewater plants is discussed.     

Raised Interleukin‐6 Levels in Obese Patients

Objective: Obese patients demonstrate a variety of biochemical, metabolic, and pulmonary abnormalities. Inflammatory mediators such as tumor necrosis factor‐α and interleukin‐6 (IL‐6) may have a direct effect on glucose and lipid metabolism. Hypoxemia in itself induces release of IL‐6. The aim of this study was to examine the relationship between IL‐6 levels in healthy volunteers (control group) and three different groups of obese patients: patients without obstructive sleep apnea syndrome (OSAS), patients with OSAS, and patients with obesity hypoventilation syndrome (OHS) (daytime baseline oxygen saturation of <93%). Research Methods and Procedures: We measured serum IL‐6 levels in 25 obese patients (body mass index of >35 kg/m²) and 12 healthy women. Results: The results demonstrate statistically significant differences in serum IL‐6 levels between the control group (1.28 ± 0.85 pg/mL) and obese patients without OSAS (7.69 ± 5.06 pg/mL, p < 0.05) and with OSAS (5.58 ± 0.37 pg/mL, p < 0.0005). In the patients with OHS, IL‐6 concentrations were highest (43.13 ± 24.27 pg/mL). Discussion: We conclude that serum IL‐6 is increased in obese patients. The highest IL‐6 levels were found in the patients with OHS.