In vitro radiolabeling of galactosyl and N-acetylgalactosaminyl moieties of glycoproteins with carbon-14

Summary The primary alcohol group on the carbon 6 of terminal galactosyl and N-acetylgalactosaminyl moieties of glycoproteins can be oxidized to an aldehyde by treatment with galactose oxidase. By reacting these aldehyde groups with¹⁴C-labeled sodium cyanide,¹⁴C-labeled cyanohydrin derivatives were obtained. Similarly, reduction of these aldehyde groups with tritiated sodium borohydride following standard procedures, yields³H-labeled glycoproteins.¹⁴C- and³H-labeled derivatives of asialofetuin and asialo ovine submaxillary mucin with high specific radioactivities were prepared using these procedures. Mixtures containing microgram amounts of¹⁴C- and³H-labeled glycoproteins were subjected to column chromatography and gradient ultracentrifugation and the position of the individual glycoproteins was determined by simultaneous counting for¹⁴C and³H. These experiments demonstrate the usefulness of this approach for comparative analytical studies using biological specimens available in minute quantities.     

In vitro effects of methionine-enkephalin,somatostatin and insulin on cultured gonadal cells of the snailHelix aspersa

Isolated snail gonadal cells were cultured in the presence of synthetic neuropeptides in order to determine the subsequent effect of these substances on gonadal synthetic activities. Gonadal cells were incubated for 24 h in concentrations of methionine-enkephalin, somatostatin and insulin ranging from 10^–4 M to 10^–9 M, in medium 199 supplemented with 6% Ultroser G. Synthesis of DNA and protein by the cultured cells were simultaneously estimated by measuring incorporation of³H thymidine and³⁵S methionine. The rate of labelled precursor incorporation was measured using the liquid scintillation technique. All substances tested exerted a dose-dependent effect. The synthetic activity of the cultured cells was highest when the concentration of the peptides added to the medium approximated the physiological levels. Methionine-enkephalin, somatostatin and insulin at 2×10^–8 M significantly increased³H thymidine incorporation, by 62%, 69% and 69% respectively, and protein synthesis by 42%, 57% and 57%, respectively. In the case of juvenile gonadal cultured cells, a similar increase in³H and³⁵S incorporation was registered for a 10^–7 M peptide concentration. Both lower and higher peptide concentrations inhibited³H thymidine and³⁵S methionine incorporation. Pharmacological studies suggest the existence of methionine-enkephalin and somatostatin-like receptors on snail gonadal cells. These results indicate that our gonadal cell culture model provides a useful tool for the study of the neuroendocrinological control of the activity of snail gonadal cells.     

Comparison of the biological effects of anemia inducing and polycythemia inducing Friend virus complex

Summary The comparison of the biological effects of FV^P and FV^A showed that leukemogenesis appears to be delayed in FV^A infected mice as compared to FV^P infected animals after injection of comparable quantities of virus as measured in spleen focus forming units. In addition, no CFU-EI, characteristic for FV^P induced leukemia, were found in leukemic spleen or bone marrow of FV^A infected mice. Since it was possible to distinguish both viruses by their different host ranges, which are helper virus determined, it is suggested that the observed differences, especially the lack of CFU-EI in FV^A infected mice, might be due to differences in the helper virus component of the FV complex.Supported by the Deutsche Forschungsgemeinschaft (SFB 112 Zellsystemphysiologie).     

A reappraisal of the hormonal regulation of larval fat body histolysis in femaleDrosophila melanogaster

The histolysis of larval fat body cells in adult femaleDrosophila melanogaster was examined in wild type and mutant animals. The fat body cells of wild type (Canton-S),apterous ^56f homozygotes,apterous ^78jts homozygotes and heterozygotes,apterous ⁴/+, ecdysoneless¹ homozygotes and heterozygotes all underwent histolysis normally during the 72 h following adult eclosion. Only in the case ofap ⁴/ap⁴ adults did the cells fail to histolyze normally. The fat body cells of both diapausing and non-diapausing wild type females underwent histolysis at the same rate. Attempts to demonstrate histolysis in vitro were unsuccessful, even in the presence of juvenile hormones (JHs), larval ring glands, or adult ovaries. In all strains other than theap ⁴ homozygotes, a significant proportion of larval fat body cells were dead at any time while theap ⁴/ap⁴ animals, almost all cells remained viable. It is postulated that fat body cell lysis following eclosion is not a JH-mediated event, but is elicited by an as yet unidentified factor(s), possibly originating in the ovary.     

Lack of conversion of C29-phytosterols to cholesterol in the khapra beetle,Trogoderma granarium Everts

Summary Metabolic studies in which³H-sitosterol,³H-stigmasterol, and¹⁴C-desmosterol were administered by feeding and injection to the khapra beetle,Trogoderma granarium Everts, provided strong evidence that this insect is unable to dealkylate phytosterols and convert them to cholesterol.     

The effect of physostigmine on (Na++K+)-ATPase activity in different rat brain regions

Summary The activity of (Na⁺+K⁺)-ATPase and acetylcholine esterase were folloed in rat brain cerebral cortex, caudate, thalamus, hippocampus and medulla after i.v. administration of physostigmine. Both enzymes were found to be inhibited in a dose-dependent manner. The most pronounced inhibition of (Na⁺+K⁺)-ATPase was found in caudate. where the highest activity of acetylcholine esterase is found.These studies were supported by a grant from the Union of Science of Republic Serbia, No. 40404-14.     

Identification of cancer stem cell-like cells from human epithelial ovarian carcinoma cell line

Cancer stem cells (CSCs) play an important role in the development, invasion, and drug resistance of carcinoma, but the exact phenotype and characteristics of ovarian CSCs are still disputable. In this study, we identified cancer stem cell-like cells (CSC-LCs) and investigated their characteristics from the ovarian adenocarcinoma cell line 3AO. Our results showed that CSC-LCs were enriched in sphere-forming test and highly expressed CD44⁺CD24⁻. The spheres and CD24⁻ cells possessed strong tumorigenic ability by transplantation into nonobese diabetic/severe combined immunodeficient mice. CD44⁺CD24⁻ cells expressed stem cell markers and differentiated to CD44⁺CD24⁺ cells by immunofluorescence assay and fluorescence-activated cell-sorting analysis. In vitro experiments verified that CD44⁺CD24⁻ cells were markedly resistant to carboplatin and paclitaxol. In conclusion, our study identifies the CD44⁺CD24⁻ phenotype, self-renewal, high tumorigenicity, differentiation potential, and drug resistance of ovarian CSC-LCs. Our findings may provide the evidence needed to explore a new strategy in the treatment of ovarian cancer.     

Lack of pressor effect of dopamine in the pentobarbital-anesthetized rat

Summary The blood pressure and heart rate responses to intravenous dopamine infusion at 2.5, 5.0 and 10.0 g·min^–1·100 g^–1 were studied in conscious and pentobarbital-anesthetized Sprague — Dawley rats. In the conscious rats, dopamine caused a significant dose-related increase in the mean arterial blood pressure which was abolished in the anesthetized rats. The heart rate increased significantly only at the highest dose infused. The responses to equipressor doses of noradrenaline (40 ng·min^–1·100 g^–1) and phenylephrine (1.0 g·min^–1·100 g^–1) were also suppressed in the anesthetized rats. The results suggest that pentobarbital anesthesia depresses the blood pressure response to dopamine infusion in the rat through a depression of activation of alpha-adrenoceptors.16 June 1986     

Uptake of3H-GABA (γ-aminobutyric acid) and3H-leucine in the pancreatic islets and substantia nigra of the rat

Summary Isolated pancreatic islets and thin slices of substantia nigra (SN) of the rat were incubated in a medium containing³H-GABA or³H-leucine to test the activity of both tissues in the uptake of those substances. Pancreatic islets showed a low uptake of both³H-GABA and³H-leucine, but SN had a high activity in the uptake of³H-GABA, though not for³H-leucine. This suggests that GABA contained at high levels in the pancreatic islets plays some functional role other than in neurotransmission as in the central nervous system (CNS).     

Ca<Superscript>2+</Superscript> signals,cell membrane disintegration,and activation of TMEM16F during necroptosis

Activated receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like (MLKL) are essential components of the necroptotic pathway. Phosphorylated MLKL (pMLKL) is thought to induce membrane leakage, leading to cell swelling and disintegration of the cell membrane. However, the molecular identity of the necroptotic membrane pore remains unclear, and the role of pMLKL for membrane permeabilization is currently disputed. We observed earlier that the phospholipid scramblase and ion channel TMEM16F/anoctamin 6 cause large membrane currents, cell swelling, and cell death when activated by a strong increase in intracellular Ca²⁺. We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNFα, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT₂₉, 16HBE, H441, and L929. Time-dependent changes in intracellular Ca²⁺, cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca²⁺, which was paralleled by the activation of outwardly rectifying Cl^? currents, which were typical for TMEM16F/ANO6. Ca²⁺ oscillations were due to Ca²⁺ release from endoplasmic reticulum, and were independent of extracellular Ca²⁺. The initial TSZ-induced cell swelling was followed by cell shrinkage. Using typical channel blockers and siRNA-knockdown, the Cl^? currents were shown to be due to the activation of ANO6. However, the knockdown of ANO6 or inhibitors of ANO6 did not inhibit necroptotic cell death. The present data demonstrate the activation of ANO6 during necroptosis, which, however, is not essential for cell death.     

Metabolic studies of Hg-203 onChlamydomonas reinhardi

Summary Sterile cultures ofChlamydomonas reinhardi, WT⁺, were treated with Hg-203 at 25°C to identify probably formed volatile mercury compounds. Experiments were performed with living and dead cells under aerobic or anaerobic conditions, respectively, and the mercury concentration was measured in the system algae/nutrient medium. We found a timerelated decrease of mercury concentration in the cell suspension and the cell-free nutrient medium due to a reduction of Hg⁺⁺ to Hg⁰, probably caused by extracellular enzymes; monomethyl or dimethyl mercury could not be detected.This work was supported by a grant of Österreichisches Bundesministerium für Gesundheit und Umweltschutz.     

Studies of total and protein-bound plasma Mg++ in wild and hatchery-reared coho salmon smolts in freshwater and in seawater

Summary Total plasma Mg⁺⁺ and Ca⁺⁺, Mg⁺⁺ in erythrocytes as well as protein-bound plasma Mg⁺⁺ were investigated in wild and hatchery-reared smolts. The proportion of plasma Mg⁺⁺ which was bound to plasma protein did not change significantly during entry into seawater, even though the in vitro addition of exogenous Mg⁺⁺ to the plasma showed that additional binding was possible.     

Calcium-induced cleavage of DNA topoisomerase I involves the cytoplasmic-nuclear shuttling of calpain 2

Important to the function of calpains is temporal and spatial regulation of their proteolytic activity. Here, we demonstrate that cytoplasm-resident calpain 2 cleaves human nuclear topoisomerase I (hTOP1) via Ca²⁺-activated proteolysis and nucleoplasmic shuttling of proteases. This proteolysis of hTOP1 was induced by either ionomycin-caused Ca²⁺ influx or addition of Ca²⁺ in cellular extracts. Ca²⁺ failed to induce hTOP1 proteolysis in calpain 2-knockdown cells. Moreover, calpain 2 cleaved hTOP1 in vitro. Furthermore, calpain 2 entered the nucleus upon Ca²⁺ influx, and calpastatin interfered with this process. Calpain 2 cleavage sites were mapped at K¹⁵⁸ and K¹⁸³ of hTOP1. Calpain 2-truncated hTOP1 exhibited greater relaxation activity but remained able to interact with nucleolin and to form cleavable complexes. Interestingly, calpain 2 appears to be involved in ionomycin-induced protection from camptothecin-induced cytotoxicity. Thus, our data suggest that nucleocytoplasmic shuttling may serve as a novel type of regulation for calpain 2-mediated nuclear proteolysis.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Summary Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptornegative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [³H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10^–6 M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10^–5 M linoleic acid or 10^–5 M arachidonic acid but not by 10^–6 M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (K_d 0.54 nM±0.11 nM; n=6) than growth in medium supplemented with untreated serum (complete medium) (K_d=1.68 nM±0.48 nM; n=6) (p<0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10^–5 M linoleic acid or 10^–5 M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10^–5 M stearic acid or 10^–5 M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10^–5 M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [³H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [³H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation

Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5^KD cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5^KD cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5^KD compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5^KD cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5^KD cells is due to a role of Cdk5 in other pathways or the altered polymer levels.     

Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors

Snake myotoxins have a great impact on human health worldwide. Most of them adopt a phospholipase A2 fold and occur in two forms which often co-exist in the same venom: the Asp49 toxins hydrolyse phospholipids, whilst Lys49 toxins are enzymatically inactive. To gain insights into their mechanism of action, muscle cells were exposed to Bothrops myotoxins, and cytosolic Ca²⁺ and cytotoxicity were measured. In both myoblasts and myotubes, the myotoxins induced a rapid and transient rise in cytosolic [Ca²⁺], derived from intracellular stores, followed, only in myotubes, by a large Ca²⁺ influx and extensive cell death. Myoblast viability was unaffected. Notably, in myotubes Asp49 and Lys49 myotoxins acted synergistically to increase the plasma membrane Ca²⁺ permeability, inducing cell death. Therefore, these myotoxins may bind to acceptor(s) coupled to intracellular Ca²⁺ mobilization in both myoblasts and myotubes. However, in myotubes only, the toxins alter plasma membrane permeability, leading to death. Received 21 January 2009; received after revision 05 March 2009; accepted 11 March 2009     

Biological effects of trilostane in vitro on oocyte maturation and fertilization in the hamster

Summary The effects of the inhibition of steroidogenesis by trilostane on oocyte maturation were examined by studying spontaneous maturation and fertilization in vitro. 10^–6 M trilostane had no influence on the meiotic process, whether the oocytes were naked or not. At a concentration of 10^–6 M and 10^–7 M trilostane, low normal pronuclear formation and high polyspermy were found during in vitro fertilization. However, no retarded male pronuclear development could be detected in the trilostane-treated group. Thus, steroid producing activity within ova is apparently necessary to prevent multiple sperm penetration, but it has no effect on meiosis or the action of the so-called male pronucleus growth factor (MPGF).     

Effects of Ag+ on frog skin: Interactions with oxytocin,amiloride and ouabain

Summary Different biological effects of Ag⁺ (10^–4 M) were found depending on its presence in the outer or the inner solution bathing the frog skin. A marked increase in the electrical conductance and an interference with the action of oxytocin and amiloride were found only when Ag⁺ was added to the outer solution. Results suggest that Ag⁺ affects several transport processes, in particular the permeability of the Na entry pathways.This work was supported by the Swiss National Science Foundation, grant No. 1.300.73. We thank Mrs A. Cergneux for skilful secretarial assistance.     

Further evidence for the dissociation of digoxin-like immunoreactivity from Na+, K+-ATPase inhibitory activity

Summary The effects of adrenalectomy or nephrectomy, carried out one hour previously, on the levels of endogenous digitalis-like factors were determined in rat plasma. Factors were assayed by digoxin-like immunoreactivity and direct Na⁺, K⁺-ATPase inhibitory activity. Digoxin-like immunoreactivity significantly decreased one hour after bilateral ablation of adrenals, while Na⁺, K⁺-ATPase inhibitory activity remained unaltered. There were no changes in either activity one hour after bilateral nephrectomy. These results suggest that digoxin-like immunoreactivity may be derived from the adrenal gland or under adrenal control and the major substances detected by digoxin-like immunoreactivity and direct Na⁺, K⁺-ATPase inhibitory activity may be different.     

The effects of sodium and amiloride on the motility of the caudal epididymal spermatozoa of the rat

Summary The forward motility of the rat caudal epididymal spermatozoa has been studied in different Na⁺ concentrations. When spermatozoa were suspended in a completely Na⁺-free solution, the forward motility suffered a progressive fall and after 3 h was completely suppressed. This effect was fully reversible on resuspending the spermatozoa in a solution containing Na⁺. Amiloride caused a fall in motility and the effect was similar to that of Na⁺ removal. The inhibition by amiloride of the motility was concentration dependent and the dose response curve showed an IC₅₀-value of about 5×10^–5 M. The role of Na⁺ influx in the maintenance of sperm motility was discussed.This work was supported by the World Health Organization.The technical assistance of Mr C.M. Li and the gift of amiloride from Merck, Sharp and Dohme are gratefully acknowledged.