Characterization of BioID tagging systems in budding yeast and exploring the interactome of the Ccr4-Not complex.

The modified E. coli biotin ligase BirA* was the first developed for proximity labeling of proteins (BioID). However, it has low activity at temperatures below 37°C, which reduces its effectiveness in organisms growing at lower temperatures, such as budding yeast. Multiple derivatives of the enzymes have been engineered, but a comparison of these variations of biotin ligases has not been reported in Saccharomyces cerevisiae. Here, we designed a suite of vectors to compare the activities of biotin ligase enzymes in yeast. We found that the newer TurboID versions were the most effective at labeling proteins, but they displayed low constitutive activity from biotin contained in the culture medium. We describe a simple strategy to express free BioID enzymes in cells that can be used as an appropriate control in BioID studies to account for the promiscuous labeling of proteins caused by random interactions between bait-BioID enzymes in cells. We also describe chemically-induced BioID systems exploiting the rapamycin-stabilized FRB-FKBP interaction. Finally, we used the TurboID version of the enzyme to explore the interactome of different subunits of the Ccr4-Not gene regulatory complex. We find that Ccr4-Not predominantly labeled cytoplasmic mRNA regulators, consistent with its function in mRNA decay and translation quality control in this cell compartment.

Cytotoxic potency of trialkyltins to C6 glioma cells in vitro: impact of exposure conditions.

Because of a possible role of astrocytes in trialkyltin-induced neurotoxicity in vivo various studies have been performed using cultures of astrocytes or glioma cells in vitro. With respect to cytotoxic potencies of trialkyltins these studies gave rather divergent results. Therefore the aim of the present study was to clarify whether variations of experimental conditions could be responsible for the differences of the cytotoxic activities of trimethyltin (TMT), triethyltin (TET) and tributyltin (TBT). Experiments were performed with rat C6 glioma cells. Toxicity was determined by measuring the reduction of the cell protein content. Cultures of proliferating and growth-arrested cells did not differ in their sensitivity. Exposure duration (1-72 h) had a strong but differing influence on the cytotoxic potency of the trialkyltins. After short exposure times the potencies differed largely (TMT < TET < TBT), whereas they became more and more similar with increasing exposure duration. The potency-time relationships for TMT and TET could be described by the equation: EC50 = k x t(-n), while for TBT an incipient value (EC50, infinity) had to be included: EC50 = EC50, infinity + k x t(-n). Addition of serum albumin to the culture medium decreased the cytotoxic potency of the trialkyltins. However, the impact of protein binding on their bioavailability was relatively low. The cytotoxic potency of the alkyltins was not dependent on the concentration of C6 cells. Taken together, neither differences in exposure conditions nor in the proliferative status of the cells are sufficient to account for the discrepancies in published results for trialkyltin cytotoxicity to astrocytes. Instead they may--at least partially--be explained by differing sensitivities of the endpoints used. Furthermore, C6 glioma cells respond considerably more sensitively to trialkytins than primary astrocytes, which questions their applicability as models for astrocyte toxicity.

Factors influencing nominal effective concentrations of chemical compounds in vitro: medium protein concentration.

Quantitative data used to characterise biological activities of chemicals in vitro (e. g. EC50 values) are generally based on nominal concentrations and thus depend on factors influencing the availability of a compound. In this study, the impact of protein binding on the availability of chemicals in vitro is theoretically investigated and experimentally examined using a bovine sperm cell assay to measure the cytotoxic potency of selected compounds at different medium protein concentrations. In agreement with theoretical considerations, linear correlations between EC50 values and medium albumin concentrations were determined with 2,4-dichlorophenol, pentachlorophenol, p,p'-DDT and mercuric chloride. Ratios of EC50 values measured in the presence and absence of 4% (w/v) albumin varied between 500 (hexachlorophene), 258 (pentachlorophenol) and almost 1 (potassium cyanide, dextropropoxyphene). Calculated molar ratios of substance bound to albumin ranged from 0.05 (arsenic trioxide) and 0.1 (potassium cyanide) to 2.5 and 4.7 moles/mole for malathion and xylene, respectively. The fractions bound at 4% albumin varied between 11 and 15% for dextropropoxyphene and potassium cyanide, respectively, and more than 99% for hexachlorophene, pentachlorophenol and mercuric chloride. The results clearly demonstrate that the differing impact of protein binding on the bioavailability of chemicals considerably influences their nominal and relative potencies in the presence of albumin.

Toxicity of organotin compounds in primary cultures of rat cortical astrocytes.

The neurotoxic organotin compounds trimethyl (TMT) and triethyltin (TET) are known to induce astrogliosis in vivo, which is indicated by an increased synthesis of glial fibrillary acidic protein (GFAP) in astrocytes. In contrast, tributyltin (TBT) does not induce astrogliosis. The aim of this study was to investigate whether trialkyltin derivatives can induce an increased GFAP synthesis in astrocyte cultures in the absence of neurons and whether differences between the action of TMT, TET, and TBT can be detected. Primary cultures of rat cortical astrocytes from 2-day-old rats were grown in 96-well plates until confluency and then exposed to various concentrations of TMT, TET, and TBT for 40 h. Effects on basal cell functions were measured by colorimetric determination of cell protein contents and by assessment of viability by means of the MTT assay. An indirect sandwich ELISA for 96-well plates was used for quantitative measurements of the GFAP content of the cells. All three compounds induced a concentration-dependent cytotoxicity indicated by parallel decreases of protein contents and MTT reduction. Half-maximum cytotoxic concentrations were 3 micromol/L (TBT), 30 micromol/L (TET), and 800 micromol/L (TMT). Cellular GFAP contents were reduced in parallel to cytotoxic action but no increase in GFAP expression at subcytotoxic concentrations could be observed. Thus, the astrocytes were not able to respond to TMT or TET exposure by an increased synthesis of GFAP in the absence of neuronal signals.

Factors influencing nominal effective concentrations of chemical compounds in vitro: cell concentration.

In vitro potency data (e.g. EC(50) values), used to characterise the biological activity of chemicals, are generally based on nominal effective concentrations and thus depend on any factor influencing the availability of a compound. In this study the significance of cell binding for the availability of chemicals in vitro is (i) theoretically investigated by means of a simple equilibrium distribution model and (ii) experimentally examined using a bull sperm assay to measure the cytotoxic potency of selected compounds at different cell concentrations. Compounds were selected either to cover a wide range of hydrophobicity (log K(ow)=2.52-5.69) or to represent modes of cell binding other than partitioning into cellular lipids. With the exception of xylene, the EC(50) values increased with increasing cell concentration. The ratios of EC(50) values determined at about 120 x 10(6) and 15 x 10(6) cells/ml were: pentachlorophenol. 1.2, 1-nitronaphthalene: 1.9, thioridazine: 2.7, dieldrin: 4.1, hexachlorophene: 4.1, digitonin: 5.1, methylmercury chloride: 7.9, antimycin A: 10.1 and p,p'-dichlorodiphenyl dichloroethylene (p,p'-DDE): >19.1. The influence of partitioning into cell lipids was rather well predicted by the equilibrium distribution model, except for p,p'-DDE. The results show that cell binding can significantly affect the availability of compounds in vitro and thus toxic potencies and toxic equivalency factors.

Synthesis, in vitro binding, and tissue distribution of radioiodinated 2-[125I]N-(N-benzylpiperidin-4-yl)-2-iodo benzamide, 2-[125I]BP: a potential sigma receptor marker for human prostate tumors.

The preclinical evaluation of a sigma receptor-specific radiopharmaceutical that binds to human prostate tumor cells with a high affinity is described. We have synthesized and radioiodinated 2-[125I]-N-(N-benzylpiperidin-4-yl)-2-iodobenzamide (2-[125I]BP) that possesses high affinity for both sigma-1 and sigma-2 receptor subtypes that are expressed on a variety of tumor cells. 2-IBP was synthesized, purified and characterized by routine spectroscopic and analytical methods. Radioiodination was accomplished using an oxidative iododestannylation reaction in the presence of chloramine T in high yields (76%-93%) with a very high-specific activity (1700-1900 Ci/mmol). The in vitro competition binding studies of 2-[125I]BP with various sigma receptor ligands in LnCAP human prostate tumor cells showed a dose-dependent saturable binding. The inhibition constants (Ki, nM) for binding of 2-[125I]BP to human prostate tumor cells for 4-IBP, haloperidol and 2-IBP were 4.09, 6.34 and 1.6 nM, respectively. The clearance of 2-[125I]BP, in Sprague-Dawley rats, was rapid from the blood pool, other normal tissues and the total body. Tissue distribution studies in nude mice bearing human prostate tumor (DU-145) also showed a fast clearance from normal organs. The tumor had the highest percentage of injected dose per gram (%ID/g) of all tissues at 4 h as well as 24 h (2.0 +/- 0.05 and 0.147 +/- 0.038 ID/g, respectively) postinjection. The in vivo receptor binding specificity was demonstrated using haloperidol (a known high-affinity sigma receptor ligand). A significant decrease (> 50%, p = 0.001) was observed in tumor concentration when haloperidol was used as a blocking agent. The high affinity of 2-[125I]BP for sgma receptor-binding sites, its fast in vivo clearance from normal organs and its high uptake and retention in tumor implies that 2-[123I]BP or 2-[131I]BP may be a promising tracer for noninvasive imaging of human prostate tumors.

Influence of protein binding and lipophilicity on the distribution of chemical compounds in in vitro systems.

Concentration-effect relationships determined in vitro depend not only on the activity of the chemical compounds or the sensitivity of the targets but also on the distribution of the compounds in the in vitro systems. In cell cultures, protein binding and lipid/water partition can be major determinants of distribution. Both are dependent on substance-specific factors, such as affinity to protein binding sites and lipophilicity, and on system-specific factors, such as protein concentration and lipid/water relationships. With this in mind, an algorithm has been developed that relates the total concentration of a compound to the free concentration in the aqueous phase. This algorithm can be used to describe theoretically the quantitative influence of the various factors relevant for distribution on concentration-response relationships. Conditions can be defined where protein binding and lipid/water partition are quantitatively important and have to be considered when comparing EC(50) values obtained in different in vitro systems. Provided that substance- and system-related parameters are known, the algorithm can be used in comparative cell toxicology to estimate equivalent effective concentrations for differently composed systems.

Synthesis, in vitro validation and in vivo pharmacokinetics of [125I]N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl) ethylamine: a high-affinity ligand for imaging sigma receptor positive tumors.

N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl)ethylamine, IPEMP, and the corresponding bromo derivative, BrPEMP, have been synthesized and characterized. Both BrPEMP and IPEMP were evaluated for sigma-1 and sigma-2 subtype receptor affinities and found to possess very high affinities for both receptor subtypes. The precursor for radioiodination n-tributylstannylphenylethylpiperidinylethylamine was prepared from its bromo derivative by palladium-catalyzed stannylation reaction. Radioiodinated 4-[125I]PEMP was readily prepared in high yields and high specific activity by oxidative iododestannylation reaction using chloramine-T as oxidizing agent. Sites labeled by 4-[125I]PEMP in guinea pig brain membranes showed high affinity for BD1008, haloperidol, and (+)-pentazocine (Ki = 5.06 +/- 0.40, 32.6 +/- 2.75, and 48.1 +/- 8.60 nM, respectively), which is consistent with sigma receptor pharmacology. Competition binding studies of 4-[125I]PEMP in melanoma (A375) and MCF-7 breast cancer cells showed a high affinity, dose-dependent inhibition of binding with known sigma ligand N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl) ethylamine, BD1008 (Ki = 5, 11 nM, respectively), supporting the labeling of sigma sites in these cells. Haloperidol, however showed a weaker (Ki = 100-200 nM) affinity for the sites labeled by 4-[125I]PEMP in these cells. Biodistribution studies of 4-[125I]PEMP in rats showed a fast clearance of this radiopharmaceutical from blood, liver, lung, and other organs. A co-injection of 4-IPEMP with 4-[125I]PEMP resulted in 37%, 69%, and 35% decrease in activity in liver, kidney, and brain (organs possessing sigma receptors), respectively at 1-h postinjection. These results suggest that 4-[125I]PEMP is a promising radiopharmaceutical for pursuing further studies in animal models with tumors.

Synthesis and pharmacological characterization of 4-[125I]-N-(N-benzylpiperidin-4-yl)-4-iodobenzamide: a high affinity sigma receptor ligand for potential imaging of breast cancer.

The synthesis of [125I]-N-(N-benzylpiperidin-4-yl)-4-iodobenzamide (4-[125I]BP), a novel radiopharmaceutical that possesses high affinity for both sigma-1 and sigma-2 receptor subtypes, and its binding characteristics to MCF-7 breast cancer cells are described. To obtain high yields (with high specific activity) of radioiodinated ligand, (N-benzylpiperidin-4-yl)-4-tri-butylstannyl benzamide was synthesized. Radiolabeled 4-[125I]BP was prepared from tri-butylstannyl precursor with the use of chloramine-T or hydrogen peroxide as an oxidizing agent in high yields (71-86%). The competition binding studies of 4-[125I]BP in MCF-7 breast tumor cells with haloperidol and DTG (known sigma ligands) showed a dose-dependent displacement and high affinity binding (Ki = 4.6 and 56 nM, respectively), demonstrating that sigma receptors are expressed in MCF-7 breast tumor cells. Scatchard analysis of 4-[125I]BP binding in MCF-7 cells revealed saturable binding, with a Kd = 26 nM and a Bmax = 4000 fmol/mg protein. Furthermore, the Scatchard analysis of [3H]DTG binding in MCF-7 cells gave a Kd of 24.5 nM and a Bmax of 2071 fmol/mg of protein. The biodistribution and clearance of 4-[125I]BP was studied in rats. The radiopharmaceutical cleared quickly from the blood pool but rather slowly from the hepatobiliary system. The in vivo specificity was demonstrated by blocking the receptor binding in the presence of haloperidol. A decrease of 55, 63, 43, and 68% was found at 1 h postinjection in brain, kidney, heart, and lung, respectively. These results demonstrate that a high density of sigma receptors are expressed in MCF-7 cells and that radioiodinated 4-IBP may be useful for imaging breast cancer by targeting sigma sites.

In vitro toxicity screening using cultured rat skeletal muscle cells. II. Agents affecting excitable membranes.

The screening of chemicals for their potential to interfere with excitable cell membranes should be an important element of in vitro testing for acute toxicity. The suitability for this purpose of a test system using primary cultured rat skeletal muscle cells was evaluated. The test protocol involved the determination of the concentration-dependent effects on three endpoints: (1) spontaneous contractility, (2) membrane integrity and (3) energy metabolism. The chemicals investigated were: NaCl, KCl and CaCl(2); cardiac glycosides (ouabain, digoxin); sodium channel toxins (tetrodotoxin, saxitoxin, veratridine, Anemonia sulcata toxin II, Bolocera tuediae toxin II); an acetylcholine agonist (carbachol); a calcium antagonist (D600) and three membrane-directed insecticides (deltamethrin, DDT, lindane). The response pattern of most of these substances-alteration of contractility at concentrations that neither affected the energy metabolism nor were cytolethal-characterized them as acting either on the excitable membrane or on the excitation-contraction coupling and the contractile apparatus. The results indicate that the test system is suited to assess chemical effects resulting in: (i) changes of resting membrane and threshold potentials, (ii) altered sodium channel function, (iii) opening of endplate channels, (iv) blockade of calcium channels, and (v) inhibition of Na(+)/K(+)-ATPase.

The use of cultured skeletal muscle cells in testing for acute systemic toxicity.

A test system using primary cultured, spontaneously contracting, rat skeletal muscle cells has been developed to detect chemical interactions with excitable membranes. Taking into account some basic toxicokinetic differences between the in vivo and in vitro conditions, results obtained with the first 30 MEIC reference chemicals were quantitatively compared with in vivo acute toxicity data. The results show that the muscle cell test system is suitable for use in acute toxicity assessment and, for instance, for toxicity classification of chemicals.

An in vitro toxicity testing strategy for the classification and labelling of chemicals according to their potential acute lethal potency.

An in vitro test battery is described which consists of five systems using different types of cells: (1) bovine sperm cells, (2) Balb/c 3T3 cells, (3) primary cultures of rat hepatocytes, (4) primary cultures of rat muscle cells, and (5) co-cultures of microcarrier-attached rate hepatocytes and Balb/c 3T3 cells. This combination of in vitro systems covers various aspects of cellular toxicity and permits determination of the intrinsic activity of chemicals with respect to general cytotoxicity, selective cytotoxicity and interference with selected cell-specific functions (Seibert et al., 1992). During the current phase of evaluation the different test systems are used in parallel, resulting in in vitro toxicity profiles which are the basis (a) for interpretation with respect to toxic potential, and (b) for the selection of appropriate assays for inclusion in a hierarchical approach to testing. Based on the experience with this test battery, a preliminary stepwise approach is proposed for the classification of chemicals according to their acute lethal potency. The principle steps and candidate tests are: (1) determination of cytotoxic activity (cytolethal and cytostatic)-sperm cells, 3T3 cell line; (2) determination of hepatocyte-specific cytotoxicity and of the role of bioactivation for cytotoxic activity-co-cultures of hepatocytes and 3T3 cells; and (3) determination of the potential of chemicals to interfere with electrically excitable membranes-muscle cell cultures.

In vitro toxicity screening using cultured rat skeletal muscle cells. I. Surfactants and mitochondrial poisons.

The suitability of a test system using primary cultures of spontaneously contracting rat skeletal muscle cells for assessing effects of chemicals relevant to their toxicity in vivo was evaluated. The effects of ethanol and dimethyl sulphoxide, selected surfactants (digitonin, sodium dodecyl sulphate and Triton X-100) and selected mitochondrial poisons (antimycin A, 2,4-dinitrophenol and pentachlorophenol) on these cultures were assessed. Three endpoints, (1) spontaneous contractility, (2) gross structural membrane integrity and (3) energy metabolism, were studied by determining the reduction in the number of contracting muscle cells, leakage of cytosolic creatine kinase and changes in glucose consumption, respectively. Cells were also screened microscopically for morphological signs of intoxication. Exposure times of 1 and 24 hr were used. Contractility and glucose consumption frequently proved to be more sensitive parameters than loss of intracellular creatine kinase indicating cell death. Most often prominent morphological alterations became obvious at concentrations causing minimal cytolethality. The combination of different endpoints and exposure times revealed distinct patterns of concentration-effect relationships, giving some indication of the different modes of action of the model substances. Non-cytotoxic functional alterations of the muscle cell membrane could be distinguished from cytotoxic action. The results indicate that the test system is suitable for assessing specific qualitative and quantitative aspects of toxicity at the cellular level.

Effects of toxin isolated from the sea anemone Bolocera tuediae on electrical properties of isolated rat skeletal muscle and cultured myotubes.

Electrophysiological effects of a polypeptide toxin isolated from the sea anemone Bolocera tuediae (BTTX II) on isolated fast and slow rat skeletal muscle and on cultured rat myotubes are described in this paper. Nanomolar concentrations of BTTX II cause a concentration-dependent depolarization. This effect is prevented by the presence of tetrodotoxin. Action potential duration is prolonged in all preparations investigated, with the fast muscle being the least sensitive. The positive overshoot and the maximum rate of rise of the action potential is decreased by BTTX II. Cultured myotubes which are less susceptible to the sodium channel blocking activity of tetrodotoxin have the highest sensitivity to all actions of BTTX II. It is further shown that the proportion of spontaneously contracting myotubes is diminished and the frequency of contractions is increased by the toxin. The effects of Bolocera tuediae toxin II resemble those observed with Anemonia sulcata toxin II on mammalian skeletal muscle. From the results it is concluded, that BTTX II may interfere with both the activation and inactivation of sodium channels of the muscle membrane.

Cultured myotubes from skeletal muscle of adult rats. Characterization and action of Anemonia sulcata toxin II.

Mononucleated myogenic cells (satellite cells) were isolated from skeletal muscle of adult rats and grown in culture. These cells replicated and, beginning with the 6th day in culture, they fused and differentiated into multinucleated myotubes, which accumulated creatine kinase and developed cross striation and spontaneous contractions. The differentiation of the excitable membrane and the action of sea anemone toxin ATX II were investigated with microelectrode techniques. Mature myotubes reached a stable membrane potential of -47.3 mV (+/- 6.5 mV) with the 11th day in culture. Action potentials could be generated in all myotubes. During maturation they became faster (increasing rate of rise) and shorter in duration. In spontaneously contracting myotubes spontaneous action potentials were recorded, which were often associated with subthreshold oscillations of membrane potential. ATX II reduced the membrane potential and prolonged the action potential duration with the lowest effective concentrations being 1 nmol/l and 0.5 nmol/l, respectively. Furthermore, ATX II induced electrical activity in quiescent myotubes. After fusion the development of the membrane electrical properties of satellite cell derived muscle cells followed essentially the same pattern as in primary cultures of embryonic myotubes. Electrophysiologically and with respect to their sensitivity to ATX II the mature myotubes resemble denervated muscle fibres.

Restitution of the thymus in lethally irradiated mice after transplantation of syngeneic or allogeneic bone marrow.

The early restitution of the thymus of bone marrow chimeras was investigated by the immunoperoxidase technique using monoclonal antibodies against Thy-1 and Lyt-1, Lyt-2, Lyt-3. Within two weeks, normal thymus histology was restored in mice which received untreated syngeneic BM or syngeneic or allogeneic BM pretreated with SAL (specificed antilymphocytic serum). Irradiation depleted the thymic cortex of small Thy-1+, Lyt-1+2+3+ cells but did not affect a medullary population of medium sized weakly stained Thy-1+, strongly stained Lyt-1+ cells. Preceded by the appearance of an increasing number of large Thy-1+, Lyt-1- blasts (days 2 and 4), the thymic cortex was repopulated (beginning on day 6) by smaller Thy-1+ cells which acquired Lyt-1, Lyt-2 and Lyt-3 though, obviously not in a strictly sequential manner. Simultaneously, the medullary radioresistant cells disappeared, nd the medulla was subsequently repopulated (beginning on day 8) by thymocytes of a mature phenotype. Early restitution of the thymus in radiation control mice was similar to the bone marrow chimeras. The results indicate that the histological restitution of the thymus originates substantially from radioresistant precursors of host origin. Graft-versus-host reaction induced by untreated allogeneic bone marrow cells prevented normal thymic restitution. A delayed localized cortical repopulation with small Thy-1+, Lyt-1+2+3+ cells, progressive destruction of thymic architecture and almost no restoration of the medullary immunocompetent thymocytes were noted. T cell differentiation obviously was seriously affected by the injuries to the thymic microenvironment due to alloreactive T cells.