Addition Of Various Inhibitors Research Articles

TCR signaling and cellular metabolism regulate the capacity of murine epidermal γδ T cells to rapidly produce IL-13 but not IFN-γ.

Resident epidermal T cells of murine skin, called dendritic epidermal T cells (DETCs), express an invariant γδ TCR that recognizes an unidentified self-ligand expressed on epidermal keratinocytes. Although their fetal thymic precursors are preprogrammed to produce IFN-γ, DETCs in the adult epidermis rapidly produce IL-13 but not IFN-γ early after activation. Here, we show that preprogrammed IFN-γ-producing DETC precursors differentiate into rapid IL-13 producers in the perinatal epidermis. The addition of various inhibitors of signaling pathways downstream of TCR to the in vitro differentiation model of neonatal DETCs revealed that TCR signaling through the p38 MAPK pathway is essential for the functional differentiation of neonatal DETCs. Constitutive TCR signaling at steady state was also shown to be needed for the maintenance of the rapid IL-13-producing capacity of adult DETCs because in vivo treatment with the p38 MAPK inhibitor decreased adult DETCs with the rapid IL-13-producing capacity. Adult DETCs under steady-state conditions had lower glycolytic capacity than proliferating neonatal DETCs. TCR stimulation of adult DETCs induced high glycolytic capacity and IFN-γ production during the late phase of activation. Inhibition of glycolysis decreased IFN-γ but not IL-13 production by adult DETCs during the late phase of activation. These results demonstrate that TCR signaling promotes the differentiation of IL-13-producing DETCs in the perinatal epidermis and is needed for maintaining the rapid IL-13-producing capacity of adult DETCs. The low glycolytic capacity of adult DETCs at steady state also regulates the rapid IL-13 response and delayed IFN-γ production after activation.

Possible ways to minimize nitrosation reactions during post-combustion CO2 capture process

The formation and any emission of nitrosamines in a post-combustion CO2 capture process are of great interest over the past few years even though its concentration is quite low. In the present research, bench scale nitrosation experiments were conducted using morpholine, a representative of secondary amines, with NO2 gas and flue gas. Laboratory experiments with 5M morpholine with 100ppm NO2 gas resulted in 88μg/g nitrosomorpholine (NSMO) in 6h and 450μg/g in 24h. However, coal derived flue gas gave only about 7μg/g NSMO in 24h. To prove the inhibitory effect from SO2 in the flue gas, experiments were conducted with diluted NO2 gas blended with SO2 gas in the feed stream. Formation of NSMO was inhibited by about 53.3% when equal concentrations of SO2 and NO2 existed compared with the same concentration of NO2 gas diluted with N2. The inhibitory effect of SO2 gas was found to be linear with its concentration. Apart from that, the addition of various inhibitors to the amine solution was also evaluated and isoascorbic acid was found the best with 86% inhibition activity under the test conditions. Isoascorbic acid was further examined both under simulated CO2 scrubber and stripper conditions indicating good stability and inhibition performance.

Robustness of Salmonella loop-mediated isothermal amplification assays for food applications

Loop-mediated isothermal amplification (LAMP) assays have been developed recently for Salmonella detection. This study aimed at evaluating the robustness of two Salmonella LAMP assays in comparison with PCR and real-time quantitative PCR for food applications. Performance of the assays was examined under abusive preparation conditions, running temperatures and pH, and with the addition of various inhibitors and food rinses. LAMP achieved robust detection under abusive assay preparation conditions (holding at 22 and 37°C for up to 30 min) and running temperatures (57-68°C). With a hot-start DNA polymerase, PCR obtained comparable results under these temperature ranges. However, PCR performed markedly poorer under abusive pH. LAMP also showed greater tolerance to potential inhibitors than PCR. When food rinses including meat juice, chicken rinse, egg homogenate and produce homogenate were added at 20% of the reaction mix, PCR amplifications were completely inhibited, but LAMP reactions were not. Our results demonstrated that LAMP is a robust alternative to PCR in Salmonella detection for food applications. This study filled important knowledge gaps regarding the robustness of Salmonella LAMP assays. The findings will help bring Salmonella LAMP assays closer to wider applications in food testing.

RhoA Regulates Calcium-Independent Periodic Contractions of the Cell Cortex

When microtubules are depolymerized in spreading cells, they experience morphological oscillations characterized by a period of about a minute, indicating that normal interactions between the microfilament and microtubule systems have been significantly altered. This experimental system provides a test bed for the development of both fine- and coarse-grained models of complex motile processes, but such models need to be adequately informed by experiment. Using criteria based on Fourier transform analysis, we detect spontaneous oscillations in spreading cells. However, their amplitude and tendency to operate at a single frequency are greatly enhanced by microtubule depolymerization. Knockdown of RhoA and addition of various inhibitors of the downstream effector of RhoA, Rho kinase, block oscillatory behavior. Inhibiting calcium fluxes from endoplasmic reticulum stores and from the extracellular medium does not significantly affect the ability of cells to oscillate, indicating that calcium plays a subordinate regulatory role compared to Rho. We characterized the dynamic structure of the oscillating cell by light, fluorescence, and electron microscopy, showing how oscillating cells are dynamically polarized in terms of their overall morphology, f-actin and phosphorylated myosin light chain distribution, and nuclear position and shape. Not only will these studies guide future experiments, they will also provide a framework for the development of refined mathematical models of the oscillatory process.

Impact of 2-Bromo-5,6-Dichloro-1-β- d -Ribofuranosyl Benzimidazole Riboside and Inhibitors of DNA, RNA, and Protein Synthesis on Human Cytomegalovirus Genome Maturation

Herpesvirus genome maturation is a complex process in which concatemeric DNA molecules are translocated into capsids and cleaved at specific sequences to produce encapsidated-unit genomes. Bacteriophage studies further suggest that important ancillary processes, such as RNA transcription and DNA synthesis, concerned with repeat duplication, recombination, branch resolution, or damage repair may also be involved with the genome maturation process. To gain insight into the biochemical activities needed for herpesvirus genome maturation, 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB) was used to allow the accumulation of human cytomegalovirus concatemeric DNA while the formation of new genomes was being blocked. Genome formation was restored upon BDCRB removal, and addition of various inhibitors during this time window permitted evaluation of their effects on genome maturation. Inhibitors of protein synthesis, RNA transcription, and the viral DNA polymerase only modestly reduced genome formation, demonstrating that these activities are not required for genome maturation. In contrast, drugs that inhibit both viral and host DNA polymerases potently blocked genome formation. Radioisotope incorporation in the presence of a viral DNA polymerase inhibitor further suggested that significant host-mediated DNA synthesis occurs throughout the viral genome. These results indicate a role for host DNA polymerases in genome maturation and are consistent with a need for terminal repeat duplication, debranching, or damage repair concomitant with DNA packaging or cleavage. Similarities to previously reported effects of BDCRB on guinea pig cytomegalovirus were also noted; however, BDCRB induced low-level formation of a supergenomic species called monomer+ DNA that is unique to human cytomegalovirus. Analysis of monomer+ DNA suggested a model for its formation in which BDCRB permits limited packaging of concatemeric DNA but induces skipping of cleavage sites.

Amyloplast Formation in Cultured-Tobacco Cells II: Effects of Transcription/Translation Inhibitors on Accumulation of Starch.

Differentiation of leucoplast-like plastids to amyloplasts occurred when BY-2 cultured tobacco (Nicotiana tabacum L.) cells were grown for 2 days in auxin-depleted culture medium containing cytokinin (benzyladenine, 1 mg/l). Development of the amyloplasts was inhibited by addition of various inhibitors, such as actinomycin D, cycloheximide, and chloramphenicol, indicating that amyloplast formation in BY-2 cells requires de novo synthesis of RNA and proteins in nucleo-cytoplasm and in organelles (i.e., plastids and mitochondria). Actinomycin D and cycloheximide inhibited accumulation of starch effectively and promptly irrespective of the timing of addition, while the inhibitory effect of chloramphenicol decreased as the time until addition grew longer. This indicates that continuous expression of cell-nuclear genes is necessary for amyloplast formation in BY-2 cells, whereas organelle gene expression becomes less necessary in the late phase of amyloplast development.

The fate of osmo-accumulated proline in leaf discs of Rape ( Brassica napus L.) incubated in a medium of low osmolarity

When rape leaf discs were submitted in vitro to an upshock osmotic stress they accumulated proline. We used leaf discs treated for 16–20 h in the light with either sucrose 800 mM (−2.3 MPa) or PEG 6000 (400 g/kg H 2O) (−1.69 MPa) to study their capacity to mobilize proline once transferred to media of higher osmotic potential. It was found that proline metabolism took place with no lag provided the external pressure was increased stepwise by 0.3 MPa. The mean rate of proline metabolization, which was lower than the rate of accumulation during the upshock, was dependent on the level of proline available at the beginning of the transfer and related to the external osmotic potential. In the recovery medium, following a hypersomotic stress with sucrose, a significant quantity of proline leakage occurred, indicating organic solute efflux during osmotic adjustment of leaf discs experiencing hypo-osmotic stress. These released solutes were taken up by the leaf disc and mobilized during the later stages of recovery. Fluxes of carbohydrates and proline are much less prominent in PEG-treated discs. The kinetics performed with sucrose and PEG-treated leaf discs showed that a significant fraction of the osmo-accumulated proline was not available for mobilization during recovery. It is suggested that, at the cellular level, this proline could be stored in the vacuole. This contrasts with the main fraction, which presumably accumulated in the cytosol/plastids. Such compartmentation seems to be related to the upshock osmotic stress response, because turgid leaf discs loaded with exogenous l-proline exhibited a high rate of proline mobilization when transferred to the reference medium not supplemented with proline. As demonstrated by the changes, firstly, in the level of individual free amino acids during the recovery of the leaf discs, and secondly induced by added citrate and glutamate on the rate of proline withdrawal, proline metabolization is partly reliant on conversion to, and the subsequent metabolism of glutamate. However changes in the activities at the level of transcription and protein synthesis are also involved, as shown by the addition of various inhibitors of protein synthesis or proline analogs.

A study of the effects of various inhibitors in C/C composites

The oxidation behaviour of carbon/carbon composites, with the addition of various inhibitors, has been studied using thermodynamic analysis and microscopy. The effects of ZrB2, B, SiB4, tetraethylorthosilicate (TEOS), tributylphosphate (TBP) and additions of treatment with H3PO4 were compared using L8 orthogonal array table and analysed by the ANOVA-TM program.

Glucocorticoid-Dependent Induction of the mRNA Coding for Argininosuccinate Lyase in Cultured Fetal Rat Hepatocytes

Dexamethasone increased both argininosuccinate lyase (ASL) activity and specific mRNA level in cultured fetal hepatocytes. Addition of various inhibitors of RNA synthesis showed that the increase in ASL mRNA may be related to an enhancement of ASL gene transcription, but not to a specific messenger stabilization. An apparent half-life of about 12 h for ASL mRNA was found in both untreated and dexamethasone-treated hepatocytes. About 30 h were necessary to observe the maximal effect of dexamethasone, and, in addition, both puromycin and cycloheximide (two inhibitors of protein synthesis) blocked the inducing effect of the steroid. These results suggested the involvement of intermediary protein(s) in the mechanism of induction of ASL mRNA by glucocorticoids.

Active Attachment of Azospirillum brasilense Cd to Quartz Sand and to a Light-textured Soil by Protein Bridging

Inoculation and incubation of Azospirillurn brasilense Cd in pure quartz sand resulted in their attachment to sand particles by fibrillar material. Addition of low concentrations of nutrients, such as fructose or malate together with NH4C1 or root extract, enhanced bacterial multiplication and attachment. This attachment was relatively weak, grid depended on the presence of living bacterial cells and on growth conditions. Sequential washing after bacterial attachment decreased the number of bacterial cells in the sand. The killing of A. brasilense Cd, either before or after attachment, resulted in elimination of most of the applied cells from the sand. By comparison, light-textured soil adsorbed both dead and live bacteria. Addition of protease or EDTA to attached bacteria in sand significantly reduced the number of bacteria. The addition of various inhibitors or exposure to high temperatures yielded a reduced attachment of bacteria, proportionate to their relative inhibitory effect on growth of A. brasilense Cd. Agitating A. brasilense Cd cells immediately after addition to sand reduced bacterial attachment, but increased bacterial multiplication proportionate to the increase in agitation. Attachment of A. brasilense Cd under microaerophilic conditions was lower than under aerobic conditions, and depended on the amount, quality and composition of the available nutrients. The richer the mixture, the greater the attachment and multiplication of bacteria. Similar trends, but at a lower magnitude, were observed in light-textured soil.

Intracellular transport of 3,5,3'-triiodo-L-thyronine in rat skeletal myoblasts.

Since in skeletal muscle circulating L-T3 is the only source for the hormone bound to nuclei, we investigated the intracellular and intranuclear transport of L-T3 in L6E9 rat skeletal muscle cells. The characteristics of this process were assessed by analyzing the nuclear bound L-T3 as a marker of the internalized hormone and by determining the initial rate of L-T3 uptake. [125I]L-T3 cellular uptake at 37 C reached a plateau at 2 h when the nuclear uptake, after an initial lag phase, was still increasing and represented 4.7% of the cellular uptake. Incubation at 4 C caused [125I]L-T3 cellular uptake to decrease by 77% and nuclear uptake to be abolished. A similar effect on [125I]L-T3 nuclear uptake was obtained after myoblasts were incubated at 37 C with a 1000-fold excess of unlabeled L-T3. The addition of various inhibitors of ATP production, cytoskeleton integrity, endocytosis, and Na+, K+-ATPase that did not interfere with [125I]L-T3 binding to the cell surface or to isolated nuclei caused a dose-dependent reduction of both extranuclear and nuclear uptake, ranging from 34-85%. Scatchard analysis revealed the presence on the myoblast surface of L-T3 high affinity (Ka = 1.6 X 10(9) M-1) and low affinity (Ka = 5.4 X 10(6) M-1) binding sites; other iodothyronines exhibited lower affinity for both sites. Kinetic analysis of L-T3 transport after 60-sec incubation at 23 C defined a process with a Km of 17 +/- 5.6 nM and a maximum velocity of 83 +/- 35 pmol/mg DNA. These results indicate the existence in rat myoblasts of a temperature-dependent, energy-requiring, saturable, and stereospecific L-T3 uptake mechanism, probably mediated through an endocytotic pathway, located on the myoblast plasma membrane, that may regulate L-T3 action in skeletal muscle.

Some properties of a papain–agarose reactor provided with a pH monitor

AbstractA small reactor of immobilized papain was used to gain some knowledge about the effect of immobilization upon the reactivity of the enzyme towards one substrate and various types of inhibitors. A buffer solution containing benzoyl–arginine ethyl ester as substrate was run through a small column of papain immobilized by attachment to agarose beads. The pH of the effluent was measured continuously and provided the data used to calculate the substrate conversion during passage through the reactor. The operation of the system was checked by determining the substrate conversion as a function of flow rate. It proved to operate as theory demanded. The rate and extent of inhibition were measured after addition of various inhibitors to the buffer–substrate solution. The following quantities of immobilized papain were found to be equal within ±20% to those of the free enzyme in solution: the overall activity, the Km of benzoyl–arginine ethyl ester, the Ki of the competitive inhibitor benzoylamino‐acetonitrile, the rate of inactivation by chloroacetic acid and by chloroacetamide, the rate of activation by cysteine of the mixed disulfide of papain and cysteine, and the rate of spontaneous reactivation of the KCNO–papain adduct. The inactivation by KCNO proved to be strongly pH dependent. This may explain why the rate of the latter reaction is only 66% of the rate with free enzyme. It is concluded that the rates and equilibrium constants measured in the present reactor system are within ±20% of the values of the dissolved enzyme, provided that the reactions are not strongly pH dependent. Calculation showed there was no diffusion limitation.

Wachstum von Calciumoxalatkristallen in vitro: Einfache Methode zur Messung der Hemmwirkung menschlichen Urins

A technically simple and reproducible method is described for the determination of the growth of crystals of calcium oxalate monohydrate in vitro. In a system made metastable with respect to calcium and oxalate ion concentrations, the time course of crystal growth was measured by the decrease of free calcium ions, following the addition of a fixed quantity of calcium oxalate seed crystals. Inhibition of crystal growth caused by the addition of various inhibitors or urine was determined, and the possibility of supersaturation of crystal-forming ions was excluded by using low concentrations. The known inhibitors, ethane 1-hydroxy-1,1-bisphosphonate, heparin and chondroitin sulphate C were tested, and ethane 1-hydroxy-1,1-bisphosphonate showed the strongest inhibition of crystal growth. The inhibitory action of human urine was treated by addition of urine (30 ml/l) from resting and strongly hydrated probands.

Effect of inorganic iodide on thyroglobulin hydrolysis in cultured thyroid glands.

The process of thyroglobulin hydrolysis in mouse thyroid glands labelled in vitro was studied from 2-24 h after they had been maintained in tissue culture. The culture medium was supplemented with mononitrotyrosine to prevent deiodination of iodotyrosines. Hydrolysis of labelled thyroglobulin, under these conditions, led to the release of labelled iodotyrosines and iodothyronines. The rate of formation of these compounds was measured as an index of thyroglobulin hydrolysis (TH). TH was markedly stimulated by TSH. NaI inhibited TSH stimulation of TH at a concentration of 10(-5)M or greater. NaI, at similar concentrations,also markedly diminished or abolished the incorporation of 131I into thyroidal proteins from radioiodide-supplemented media. The addition of various inhibitors of iodination effectively blocked the effect of iodide on TH. In experiments where radioimmunossay was used to measure medium hormone concentrations, the release of unlabelled thyroxine (T4) and triiodothyronine (T3) induced by TSH was found to be significantly decreased in the presence of 10(-4)M NaI. These studies demonstrate that iodide inhibits the hydrolysis of thyroglobulin at or near concentrations which also inhibit iodination of thyroidal proteins. The present data suggest that formation of an iodinated compound is necessary for the effect of iodide. In addition, these studies demonstrate the utility of this in vitro system for the investigation of thyroid physiology.

Studies on the phosphorylated intermediates of A K +-stimulated ATPase from rabbit gastric mucosa

A density gradient-purified microsomal membrane preparation from rabbit fundic gastric mucosa was used for a detailed study of the K +-stimulated ATPase and associated intermediate reactions. Membranes incubated with γ-[ 32P]ATP show the rapid incorporation of 32P into phosphoprotein. Phosphoprotein levels were markedly reduced (1) when ATP hydrolysis went to completion or (2) upon addition of unlabeled ATP, thus suggesting the participation of a rapid turnover phosphorylated intermediate in the gastric microsomal ATPase. Addition of K +, Rb + or Tl + greatly reduced the level of the intermediate while stimulating ATPase activity; the observed affinities of these cations were similar for the effects on both ATPase and intermediate levels, with Tl + > K + > Rb +. Neither ATPase nor intermediate were stimulated by Na +, and ouabain was without effect on the reactions, thus differentiating this system from the (Na + + K +)-ATPase. Addition of various inhibitors showed differential effects on the partial reactions of the gastric ATPase system. N-ethylmaleimide and Zn 2+ showed characteristics of completely abolishing the K +-stimulated component of ATPase as well as the effects of K + in reducing the level of intermediate, thus suggesting that these agents exert their inhibitory effect on a phosphoprotein phosphatase partial reaction. F − abolished the K +-stimulated ATPase, but its more complex effects on the intermediate suggested an additional reaction step within the domain of the phosphorylated intermediate. Results are consistent with a model system for the gastric microsomal ATPase involving a Mg 2+-dependent protein kinase, a phosphorylated intermediate(s), and a K +-stimulated phosphoprotein phosphatase.

Studies on the phosphorylated intermediates of a K +-stimulated ATPase from rabbit gastric mucosa

A density gradient-purified microsomal membrane preparation from rabbit fundic gastric mucosa was used for a detailed study of the K +-stimulated ATPase and associated intermediate reactions. Membranes incubated with γ-[ 32P]ATP show the rapid incorporation of 32P into phosphoprotein. Phosphoprotein levels were markedly reduced (1) when ATP hydrolysis went to completion or (2) upon addition of unlabeled ATP, thus suggesting the participation of a rapid turnover phosphorylated intermediate in the gastric microsomal ATPase. Addition of K +, Rb + or Tl + greatly reduced the level of the intermediate while stimulating ATPase activity; the observed affinities of these cations were similar for the effects on both ATPase and intermediate levels, with Tl + > K + > Rb +. Neither ATPase nor intermediate were stimulated by Na +, and ouabain was without effect on the reactions, thus differentiating this system from the (Na + + K +)-ATPase. Addition of various inhibitors showed differential effects on the partial reactions of the gastric ATPase system. N-ethylmaleimide and Zn 2+ showed characteristics of completely abolishing the K +-stimulated component of ATPase as well as the effects of K + in reducing the level of intermediate, thus suggesting that these agents exert their inhibitory effect on a phosphoprotein phosphatase partial reaction. F − abolished the K +-stimulated ATPase, but its more complex effects on the intermediate suggested an additional reaction step within the domain of the phosphorylated intermediate. Results are consistent with a model system for the gastric microsomal ATPase involving a Mg 2+-dependent protein kinase, a phosphorylated intermediate(s), and a K +-stimulated phosphoprotein phosphatase.

Protein Synthesis in Isolated Thyroid Mitochondria

Amino acid incorporation into sheep thyroid mitochondria was reassessed since the only previous results on the subject showed some contradictions with most data obtained with rat liver mitochondria. The incorporation of leucine or of a mixture of amino acids into thyroid mitochondria was linear up to 90 min. Leucine concentration in the incubation medium was an important rate limiting factor of thyroid mitochondrial protein synthesis. Chloramphenicol, puromycin and ethidium bromide were strong inhibitors, while RNAase, actinomycin D, rifampicin and cycloheximide did not exert any inhibitory effect on amino acid incorporation. Addition of various inhibitors and uncouplers of mitochondrial metabolism further showed that optimal mitochondrial protein synthesis required intact electron transport, and oxidative phophorylation, and exogenous substrate and phosphate, while the endogenous pool of adenine nucleotides seemed to be selfsufficient.(Endocrinology91: 817, 1972)

The effect of long-chain fatty acids on orthophosphate-adenosine 5′-triphosphate exchange activity associated with oxidative phosphorylation

1.Various saturated and unsaturated long-chain fatty acids inhibited [32P]Pi-ATP exchange activity associated with respiratory-chain oxidative phosphorylation. Inhibition by a given concentration of long-chain fatty acid was considerably augmented by conditions which prevented fatty acid oxidation during the course of the reaction. This was accomplished by addition of various inhibitors of respiration (KCN, antimycin A, azide, or amytal) or by conducting the reaction under anaerobic conditions.2.Investigation of the effect of various saturated long-chain fatty acids at 3 · 10−5 M and in the presence of 1 mM KCN revealed that myristic, lauric, capric, palmitic, caprylic and caproic acids, in decreasing order of effectiveness, inhibited [32P]Pi-ATP exchange activity. Investigation of the effect of unsaturated fatty acids under similar conditions showed that oleic, palmitoleic, linoleic, erucic and elaidic acids, in decreasing order of effectiveness, inhibited [32P]Pi-ATP exchange activity.3.The inhibition of [32P]Pi-ATP exchange activity by long-chain fatty acids was not accompanied by significant increase in ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity or evidence indicative of mitochondrial disruption.