The timing of synthesis of proteins required for mitotic spindle and phragmoplast in partially synchronized root meristems of Vicia faba L.

PubMed

Olszewska, M J; Marciniak, K; Kuran, H

1990-10-01

After cycloheximide treatment (1 h, 2.5 micrograms/ml) protein synthesis was decreased by 70% and was partially restored after 7 h of postincubation (still 20% decrease). In partially synchronized root meristems of Vicia faba L. treated with cycloheximide at middle G2, a strong decrease of the mitotic index was observed. Exposure to the drug at late G2 did not modify the mitotic index; the changes in the phase indices suggested that the course of mitosis was blocked at prophase-metaphase/anaphase-telophase transitions. The use of indirect immunocytochemical staining of tubulin (second antibody labeled with peroxidase) made it possible to show a decreased number of cells with preprophase bands in cycloheximide-treated meristems and the mitotic spindles and phragmoplasts containing a reduced number of shortened bands of microtubules. As a result of these structural and functional disturbances, binucleate cells and polyploid nuclei were observed.

Cycloheximide and 4-OH-TEMPO suppress chloramphenicol-induced apoptosis in RL-34 cells via the suppression of the formation of megamitochondria.

PubMed

Karbowski, M; Kurono, C; Wozniak, M; Ostrowski, M; Teranishi, M; Soji, T; Wakabayashi, T

1999-02-04

Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6, 6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2, 6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.

Effects of inhibitors on early protein, RNA, and lipid synthesis in germinating vesicular-arbuscular mycorrhizal fungal spores of Glomus caledonium.

PubMed

Beilby, J P

1983-05-01

The incorporation of isotopically labelled precursors, [1-14C]leucine, [2-14C]uracil and [1-14C]acetate, by germinating spores of the vesicular-arbuscular mycorrhizal fungus Glomus caledonium was investigated after prior incubation with several metabolic inhibitors. Inhibition of isotope incorporation was greatest with ethidium bromide and cycloheximide and least with chloramphenicol and actinomycin D. The incorporation of [1-14C]leucine into protein was reduced by 92% within 60 min of incubation in either ethidium bromide at 5 micrograms/mL or cycloheximide at 100 micrograms/mL. For these studies time zero was coincident with dry quiescent spores being first wet. Lipid synthesis during germination of the spores was detected at 120 min by following the incorporation of [1-14C]acetate. At 51 h, incorporation was greater in the triacylglycerides and diacylglycerides than in the free sterols, phospholipids, or free fatty acid. Spores exposed to cycloheximide for 60 min after imbibition showed very little phospholipid synthesis and no free fatty acid synthesis. The synthesis of RNA after spore imbibition is also discussed.

Regulation of Sulfate Assimilation in Plants

PubMed Central

Brunold, Christian; Schmidt, Ahlert

1978-01-01

When 0.5 mm cysteine is added to cultures of Lemna minor L. growing with sulfate as the sole sulfur source, there is a rapid 80% loss of extractable adenosine 5′-phosphosulfate sulfotransferase. This loss is accompanied by an inhibition of sulfate uptake; however, lack of sulfate is not responsible for the decreasing adenosine 5′-phosphosulfate sulfotransferase activity. Cultivation with cysteine causes an increase in the cyst(e)ine pool of L. minor. This fact taken together with the observed inactivation of adenosine 5′-phosphosulfate sulfotransferase in crude extracts by cysteine suggests that the cysteine pool is involved in the in vivo regulation of the enzyme. The activity of adenosine 5′-phosphosulfate sulfotransferase is restored within 24 hours after transfer to a culture medium without cysteine. This restoration is partially blocked by 6-methyl purine and actinomycin D and completely by cycloheximide. Cycloheximide added to cultures of L. minor L. causes a loss of extractable APSTase comparable to the one obtained with cysteine. This loss may be in part due to cysteine, since cycloheximide causes a pronounced increase in the cysteine pool of L. minor. PMID:16660289

Energy metabolism in isolated chick (Gallus domesticus) gastrocnemius and tilapia (Tilapia mossambica) epaxial muscle at various temperatures in vitro.

PubMed

Suzuki, E Y; Early, R J; Patterson, P H

1994-09-01

Muscle respiration experiments on inhibitor dosage (experiment 1), muscle preparation (tendons removed vs. unstretched vs. stretched muscles; chick muscle only; experiment 2) and media temperature (26.5, 32, 37, 42 degrees C; experiment 3) were conducted on chick (Gallus domesticus) gastrocnemius and tilapia (Tilapia mossambica) epaxial muscle in vitro. Experiment 1: The dosage of cycloheximide and ouabain required for maximum inhibition of protein synthesis and Na+,K+ ATPase, respectively, in chick and tilapia muscle was approximately 6 x 10(-5) M. Experiment 2: Removing the tendons of chick muscle decreased (% inhibition, P = 0.05) cycloheximide-sensitive respiration compared to stretched and unstretched muscles (tendons intact). However, muscle preparation had little influence on ouabain-sensitive respiration. Experiment 3: Cycloheximide-sensitive respiration tended to increase (microliter O2/mg DNA.hr, P = 0.054) with media temperature in tilapia muscle. Chick muscle was less responsive in this respect. Ouabain-sensitive respiration increased at lower temperature in chick muscle (% inhibition, cubic relationship, P = 0.001) and at higher temperature in tilapia muscle (% inhibition, quadratic relationship, P = 0.0002).

Mechanism of fever induction in rabbits.

PubMed Central

Siegert, R; Philipp-Dormston, W K; Radsak, K; Menzel, H

1976-01-01

Three exogenous pyrogens (Escherichia coli lipopolysaccharide, synthetic double-stranded ribonucleic acid. Newcastle disease virus) were compared with respect to their mechanisms of fever induction in rabbits. All inducers stimulated the production of an endogenous pyrogen demonstrated in the blood as well as prostaglandins of the E group, and of cyclic adenosine 3',5'-monophosphate in the cerebrospinal fluid. The concentrations of these compounds were elevated approximately twofold as compared to the controls. Independently of the mode of induction, the fever reaction could be prevented by pretreatment with 5 mg of cycloheximide per kg, although the three fever mediators were induced as in febrile animals. Consequently, at least one additional fever mediator that is sensitive to a 30 to 50% inhibition of protein synthesis by cycloheximide has to be postulated. The comparable reactions of the rabbits after administration of different pyrogens argues for a similar fever mechanism. In contrast to fever induction there was no stimulation of endogenous pyrogen, prostaglandins of the E group, and cyclic adenosine 3',5'-monophosphate in hyperthermia as a consequence of exposure of the animals to exogenous overheating. Furthermore, hyperthermia could not be prevented by cycloheximide. PMID:185148

The translational inhibitor cycloheximide represses growth factor depletion-induced apoptosis in an alb-SV40T transgenic rat liver cell line.

PubMed

Bulera, S J; Sattler, C A; Pitot, H C

1996-06-01

A transgenic rat line carrying the alb-SV40A transgene has been described by this laboratory. Several cell lines have been established from the livers of two of these rats. One of these cell lines, L37, exhibits a large nuclear/cytoplasmic ratio and a well-differentiated cytoplasm containing numerous organelles. When L37 cells are placed into culture medium lacking necessary growth factors, cellular proliferation continues for 48 hours after medium change. Subsequent to the initial 48 hours, cells begin to shrink and lose contact with adjacent cells, eventually sloughing off the culture plate surface, with most cell deaths occurring between 48 and 96 hours after medium change. Microscopic examination of sloughing cells indicates they possess highly convoluted and blebbed plasma membranes, a morphological characteristic of apoptosis. Ultrastructural studies demonstrate the ubiquitous presence of apoptotic bodies. When DNA isolated from growth factor-depleted cells is resolved on agarose gels, DNA fragmentation ladders are observed at times of maximum apoptotic change. Quantitative analysis of L37 cells between 48 and 96 hours after the removal of the culture medium shows that 59% +/- 2% of the cells undergo apoptosis. When cycloheximide, puromycin, or actinomycin D is added to the L37 cultures, only cycloheximide is able to repress apoptosis, indicating that the mechanism of apoptosis in the L37 liver-derived cell line requires a cycloheximide-sensitive translational event. The extremely high rate of apoptosis, together with the maintenance of hepatocellular characteristics, indicates the usefulness of this cell line as a model in which to study the mechanisms of hepatocellular apoptosis.

Inhibition of protein synthesis but not β-adrenergic receptors blocks reconsolidation of a cocaine-associated cue memory

PubMed Central

Dunbar, Amber B.

2016-01-01

Previously consolidated memories have the potential to enter a state of lability upon memory recall, during which time the memory can be altered before undergoing an additional consolidation-like process and being stored again as a long-term memory. Blocking reconsolidation of aberrant memories has been proposed as a potential treatment for psychiatric disorders including addiction. Here we investigated of the effect of systemically administering the protein synthesis inhibitor cycloheximide or the β-adrenergic antagonist propranolol on reconsolidation. Rats were trained to self-administer cocaine, during which each lever press resulted in the presentation of a cue paired with an intravenous infusion of cocaine. After undergoing lever press extinction to reduce operant responding, the cue memory was reactivated and rats were administered systemic injections of propranolol, cycloheximide, or vehicle. Post-reactivation cycloheximide, but not propranolol, resulted in a reactivation-dependent decrease in cue-induced reinstatement, indicative of reconsolidation blockade by protein synthesis inhibition. The present data indicate that systemically targeting protein synthesis as opposed to the β-adrenergic system may more effectively attenuate the reconsolidation of a drug-related memory and decrease drug-seeking behavior. PMID:27421890

Inhibition of protein synthesis but not β-adrenergic receptors blocks reconsolidation of a cocaine-associated cue memory.

PubMed

Dunbar, Amber B; Taylor, Jane R

2016-08-01

Previously consolidated memories have the potential to enter a state of lability upon memory recall, during which time the memory can be altered before undergoing an additional consolidation-like process and being stored again as a long-term memory. Blocking reconsolidation of aberrant memories has been proposed as a potential treatment for psychiatric disorders including addiction. Here we investigated of the effect of systemically administering the protein synthesis inhibitor cycloheximide or the β-adrenergic antagonist propranolol on reconsolidation. Rats were trained to self-administer cocaine, during which each lever press resulted in the presentation of a cue paired with an intravenous infusion of cocaine. After undergoing lever press extinction to reduce operant responding, the cue memory was reactivated and rats were administered systemic injections of propranolol, cycloheximide, or vehicle. Post-reactivation cycloheximide, but not propranolol, resulted in a reactivation-dependent decrease in cue-induced reinstatement, indicative of reconsolidation blockade by protein synthesis inhibition. The present data indicate that systemically targeting protein synthesis as opposed to the β-adrenergic system may more effectively attenuate the reconsolidation of a drug-related memory and decrease drug-seeking behavior. © 2016 Dunbar and Taylor; Published by Cold Spring Harbor Laboratory Press.

Human Mycoses.

DTIC Science & Technology

1977-01-01

of Action of Miconazole ; Immunity in Dermatomycoses; Biochemical Studies in Microsporum canis Spore Germination and Mechanism of Cycloheximide Resistance; and Biosynthetic Potentialities of Candida Species.

Protein Breakdown and Formation of Protease in Attached and Detached Cotyledons of Phaseolus vulgaris L.

PubMed

Yomo, H; Srinivasan, K

1973-12-01

In contrast to earlier reported results of similar experiments in peas, in which almost no increase in protease activity occurred in incubated detached cotyledons, we report here an increase in protease activity in both attached and detached bean cotyledons. Detached bean cotyledons showed continually increasing protease activity up to the 12th day, while that in attached cotyledons declined after 6 days. The free amino acid level in detached cotyledons reached a maximum at the 11th day; protease formation leveled off after 50% of the original seed protein was digested. These data suggest that high free amino acid levels may inhibit protease formation.The activity of partially purified protease in aqueous extracts was enhanced by 10 mm 2-mercaptoethanol or cysteine, indicating a sulfhydryl requirement for activation. Protease formation in detached cotyledons was inhibited 30% by 10 mug/ml cycloheximide and 50% by 100 mum abscisic acid. In contrast, alpha-amylase formation was inhibited 90% by 10 mug/ml cycloheximide and 95% by 20 mum abscisic acid. The cycloheximide data suggest that only a part of the protease, but all of the alpha-amylase, is synthesized de novo; the similar pattern of inhibition by abscisic acid emphasizes the concept that protease may exist in two forms.

Protein Breakdown and Formation of Protease in Attached and Detached Cotyledons of Phaseolus vulgaris L. 12

PubMed Central

Yomo, Harugoro; Srinivasan, Komala

1973-01-01

In contrast to earlier reported results of similar experiments in peas, in which almost no increase in protease activity occurred in incubated detached cotyledons, we report here an increase in protease activity in both attached and detached bean cotyledons. Detached bean cotyledons showed continually increasing protease activity up to the 12th day, while that in attached cotyledons declined after 6 days. The free amino acid level in detached cotyledons reached a maximum at the 11th day; protease formation leveled off after 50% of the original seed protein was digested. These data suggest that high free amino acid levels may inhibit protease formation. The activity of partially purified protease in aqueous extracts was enhanced by 10 mm 2-mercaptoethanol or cysteine, indicating a sulfhydryl requirement for activation. Protease formation in detached cotyledons was inhibited 30% by 10 μg/ml cycloheximide and 50% by 100 μm abscisic acid. In contrast, α-amylase formation was inhibited 90% by 10 μg/ml cycloheximide and 95% by 20 μm abscisic acid. The cycloheximide data suggest that only a part of the protease, but all of the α-amylase, is synthesized de novo; the similar pattern of inhibition by abscisic acid emphasizes the concept that protease may exist in two forms. PMID:16658628

Quantitative in vivo detection of brain cell death after hypoxia ischemia using the lipid peak at 1.3 ppm of proton magnetic resonance spectroscopy in neonatal rats.

PubMed

Ahn, So Yoon; Yoo, Hye Soo; Lee, Jang Hoon; Sung, Dong Kyung; Jung, Yu Jin; Sung, Se In; Lim, Keun Ho; Chang, Yun Sil; Lee, Jung Hee; Kim, Ki Soo; Park, Won Soon

2013-07-01

This study was performed to determine the accuracy of proton magnetic spectroscopy ((1)H-MRS) lipid peak as a noninvasive tool for quantitative in vivo detection of brain cell death. Seven day-old Sprague Dawley rats were subjected to 8% oxygen following a unilateral carotid artery ligation. For treatment, cycloheximide was given immediately after hypoxic ischemia (HI). Lipid peak was measured using (1)H-MRS at 24 hr after HI, and then brains were harvested for fluorocytometric analyses with annexin V/propidium iodide (PI) and fluorescent probe JC-1, and for adenosine-5'-triphosphate (ATP) and lactate. Increased lipid peak at 1.3 ppm measured with (1)H-MRS, apoptotic and necrotic cells, and loss of mitochondrial membrane potential (ΔΨ) at 24 hr after HI were significantly improved with cycloheximide treatment. Significantly reduced brain ATP and increased lactate levels observed at 24 hr after HI showed a tendency to improve without statistical significance with cycloheximide treatment. Lipid peak at 1.3 ppm showed significant positive correlation with both apoptotic and necrotic cells and loss of ΔΨ, and negative correlation with normal live cells. Lipid peak at 1.3 ppm measured by (1)H-MRS might be a sensitive and reliable diagnostic tool for quantitative in vivo detection of brain cell death after HI.

Cell stress and translational inhibitors transiently increase the abundance of mammalian SINE transcripts.

PubMed Central

Liu, W M; Chu, W M; Choudary, P V; Schmid, C W

1995-01-01

The abundance of Alu RNA is transiently increased by heat shock in human cell lines. This effect is specific to Alu repeats among Pol III transcribed genes, since the abundance of 7SL, 7SK, 5S and U6 RNAs is essentially unaffected by heat shock. The rapid induction of Alu expression precedes the heat shock induction of mRNAs for the ubiquitin and HSP 70 heat shock genes. Heat shock mimetics also transiently induce Alu expression indicating that increased Alu expression is a general cell-stress response. Cycloheximide treatment rapidly and transiently increases the abundance of Alu RNA. Again, compared with other genes transcribed by Pol III, this increase is specific to Alu. However, as distinguished from the cell stress response, cycloheximide does not induce expression of HSP 70 and ubiquitin mRNAs. Puromycin also increases Alu expression, suggesting that this response is generally caused by translational inhibition. The response of mammalian SINEs to cell stress and translational inhibition is not limited to SINEs which are Alu homologues. Heat shock and cycloheximide each transiently induce Pol III directed expression of B1 and B2 RNAs in mouse cells and C-element RNA in rabbit cells. Together, these three species exemplify the known SINE composition of placental mammals, suggesting that mammalian SINEs are similarly regulated and may serve a common function. Images PMID:7784180

Negative regulation of early polyomavirus expression in mouse embryonal carcinoma cells.

PubMed Central

Cremisi, C; Babinet, C

1986-01-01

Embryonal carcinoma cells are resistant to infection by polyomavirus (Py). We showed that this block was partially removed by inhibiting protein synthesis temporarily. The block was also partially removed when Py was coinfected with simian virus 40. Cycloheximide treatment of cells infected with Py mutants able to grow on PCC4 embryonal carcinoma cells led to 3- to 10-fold increases in the production of T-antigen-positive cells. At 31 degrees C, Py T-antigen expression was enhanced when the cells were treated with cycloheximide. We suggest that a negative labile regulatory protein(s) is synthesized in PCC4 cells, preventing the initiation of early Py transcription by binding to the noncoding sequence, especially the enhancer element B and perhaps also element A, and that the Py mutants retained a binding site(s). PMID:3016339

Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification.

PubMed Central

Gottlieb, R A; Nordberg, J; Skowronski, E; Babior, B M

1996-01-01

We have previously shown that in neutrophils deprived of granulocyte colony-stimulating factor, apoptosis is preceded by acidification and that the protection against apoptosis conferred on neutrophils by granulocyte colony-stimulating factor is dependent upon delay of this acidification. To test the hypothesis that acidification could be a general feature of apoptosis, we examined intracellular pH changes in another cell line. Jurkat cells, a T-lymphoblastoid line, were induced to undergo apoptosis with anti-Fas IgM, cycloheximide, or exposure to short-wavelength UV light. We found that acidification occurred in response to treatment with these agents and that acidification preceded DNA fragmentation. Jurkat cells were also found to possess an acid endonuclease that is active below pH 6.8, compatible with a possible role for this enzyme in chromatin digestion during apoptosis. Incubation of the cells with the bases imidazole or chloroquine during treatment with anti-Fas antibody or cycloheximide or after UV exposure decreased apoptosis as assessed by nuclear morphology and DNA content. The alkalinizing effect of imidazole and chloroquine was shown by the demonstration that the percentage of cells with an intracellular pH below 6.8 after treatment with anti-Fas antibody, cycloheximide, or UV was diminished in the presence of base as compared with similarly treated cells incubated in the absence of base. We conclude that acidification is an early event in programmed cell death and may be essential for genome destruction. Images Fig. 5 PMID:8570610

Inhibition of chitin biosynthesis in cultured imaginal discs: Effects of alpha-amanitin, actinomycin-D, cycloheximide, and puromycin.

PubMed

Oberlander, Herbert; Ferkovich, Stephen; Leach, Eddie; Van Essen, Frank

1980-02-01

Wing imaginal discs isolated from last instar larvae of the Indian meal moth,Plodia interpunctella, produced chitin when incubated in vitro with ≧2×10 -7 M 20-hydroxyecdysone. Chitin biosynthesis was initiated 8 h after the conclusion of a 24-h treatment with hormone. Simulataneous incubation of wing discs with 20-hydroxyecdysone and either inhibitors of RNA synthesis (alpha-amanitin, actinomycin-D) or inhibitors of protein systhesis (cycloheximide, puromycin) prevented chitin biosynthesis. We conclude from our results that RNA and protein synthesis must continue undiminished during the hormone-contact period, and that synthesis of protein, but not of new RNA is required during the posthormone culture period. Our findings are consistent with the hypothesis that ecdysteroids stimulate insect metamorphosis by promoting the synthesis of new RNA and protein during a hormone-dependent phase followed by hormone-independent protein synthesis.

Cell wall pH and auxin transport velocity

NASA Technical Reports Server (NTRS)

Hasenstein, K. H.; Rayle, D.

1984-01-01

According to the chemiosmotic polar diffusion hypothesis, auxin pulse velocity and basal secretion should increase with decreasing cell wall pH. Experiments were designed to test this prediction. Avena coleoptile sections were preincubated in either fusicoccin (FC), cycloheximide, pH 4.0, or pH 8.0 buffer and subsequently their polar transport capacities were determined. Relative to controls, FC enhanced auxin (IAA) uptake while CHI and pH 8.0 buffer reduced IAA uptake. Nevertheless, FC reduced IAA pulse velocity while cycloheximide increased velocity. Additional experiments showed that delivery of auxin to receivers is enhanced by increased receiver pH. This phenomenon was overcome by a pretreatment of the tissue with IAA. Our data suggest that while acidic wall pH values facilitate cellular IAA uptake, they do not enhance pulse velocity or basal secretion. These findings are inconsistent with the chemiosmotic hypothesis for auxin transport.

Apoptosis induced by microtubule disrupting drugs in cultured human lymphoma cells. Inhibitory effects of phorbol ester and zinc sulphate.

PubMed

Takano, Y; Okudaira, M; Harmon, B V

1993-03-01

The effects of the microtubule disrupting drugs (MDD) vinblastine, vincristine and colchicine on a human lymphoma cell line, BM 13674, were investigated. Twelve hours after administration of vinblastine (10(-3) mg/ml), vincristine (10(-2) mg/ml) or colchicine (10(-2) mg/ml), cell death with the characteristic morphology of apoptosis was observed in 71.6%, 82.2% and 76.9% of the cells respectively. The mode of death was confirmed as apoptotic by the occurrence of internucleosomal DNA cleavage, which was demonstrated by agarose gel electrophoresis. For the purpose of casting light on the mechanism involved, inhibition tests were performed on apoptosis induced by one of these drugs, vinblastine, using a phorbol ester (PDBu), zinc sulphate and cycloheximide. PDBu, an activator of protein kinase C, and zinc sulphate, a putative inhibitor of the endonuclease were thought to be responsible for internucleosomal DNA cleavage; both markedly reduced the induction of apoptosis. The protein synthesis inhibitor cycloheximide, on the other hand, had no inhibitory effect. Moreover, cycloheximide treatment per se enhanced apoptosis. This suggests that new protein synthesis is not required for the execution of vinblastine-induced apoptosis. Such a finding is in accord with recent reports suggesting that the "death program" within many cell types may be primed but unable to proceed due to concomitant production of specific "apoptotic inhibitors". It is suggested that phorbol esters prevent vinblastine-induced apoptosis in the BM 13674 cells by activating one or more of these specific "apoptotic inhibitors", possibly by means of PKC-mediated phosphorylation.

Reconsolidation and update of morphine-associated contextual memory in mice.

PubMed

Escosteguy-Neto, Joao Carlos; Varela, Patricia; Correa-Neto, Nelson Francisco; Coelho, Laura Segismundo; Onaivi, Emmanuel S; Santos-Junior, Jair Guilherme

2016-04-01

Drug addiction can be viewed as a pathological memory that is constantly retrieved and reconsolidated. Since drug abuse takes place in different contexts, it could be considered that reconsolidation plays a role in memory updating. There is consistent evidence supporting the role of reconsolidation in the strength and maintenance of contextual memories induced by drugs of abuse. However, this role is not well established in memory update. The purpose of the current study was to assess the reconsolidation process over memory update. C57BL6 mice were subjected to a morphine-induced, conditioned place preference (CPP) paradigm. Based on CPP results, animals were divided into distinct experimental groups, according to the contextual characteristics of the re-exposure and a second CPP Test. Re-exposure in the original context was important for memory maintenance and re-exposure under discrete contextual changes resulted in memory updating, although original memory was maintained. Interestingly, cycloheximide, an inhibitor of protein synthesis, had different outcomes in our protocol. When the re-exposure was done under discrete contextual changes, cycloheximide treatment just after re-exposure blocked memory updating, without changes in memory maintenance. When re-exposure was done under the original context, only two subsequent cycloheximide injections (3 and 6h) disrupted later CPP expression. Considering the temporal window of protein synthesis in consolidation and reconsolidation, these findings suggest that re-exposure, according to the contextual characteristics in our protocol, could trigger both phenomena. Furthermore, when new information is present on retrieval, reconsolidation plays a pivotal role in memory updating. Copyright © 2016 Elsevier Inc. All rights reserved.

Holding immature equine oocytes in the absence of meiotic inhibitors: effect on germinal vesicle chromatin and blastocyst development after intracytoplasmic sperm injection.

PubMed

Choi, Y H; Love, L B; Varner, D D; Hinrichs, K

2006-09-01

Holding immature oocytes before the onset of maturation simplifies oocyte transport and aids in scheduling later manipulations. We report here a method for holding equine oocytes in the absence of meiotic inhibitors. In Experiment 1, immature oocytes with expanded cumuli were cultured at 38.2 degrees C in medium containing cycloheximide, or were held at room-temperature in M199 with Hanks' salts, for 16-18 h before maturation. Control oocytes were matured immediately after recovery. Oocytes were fertilized by intracytoplasmic sperm injection and cultured for 4d. Embryo development was not different among treatments. In Experiment 2, oocytes were treated as in Experiment 1, but embryos were cultured for 7.5d. Blastocyst development was significantly lower in the cycloheximide-treated group than in controls (7% versus 30%) with the room-temperature group intermediate (16%). In Experiment 3, oocytes were cultured at 38.2 degrees C in medium containing roscovitine, or were held at room temperature in sealed glass vials in a mixture of 40% M199 with Earle's salts, 40% M199 with Hanks' salts, and 20% FBS (EH treatment) for 16-18 h, before maturation, sperm injection, and embryo culture for 7.5d. Blastocyst development of oocytes in the EH treatment was significantly higher than that for roscovitine-treated oocytes (34% versus 12%), but not significantly different from that for controls (25%). Oocytes in the EH treatment did not mature during holding (70% germinal vesicle stage after 18 h holding). Whereas culture with cycloheximide or roscovitine of equine oocytes with expanded cumuli reduced subsequent blastocyst formation, these oocytes could be held in a modified M199 at room temperature overnight without adverse affecting meiotic or developmental competence.

Association of electrophoretic karyotype of Candida stellatoidea with virulence for mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon-Chung, K.J.; Wickes, B.L.; Merz, W.G.

1988-07-01

Seven isolates of Candida stellatoidea were studied for their electrophoretic karyotype, virulence for mice, sensitivity to UV radiation, growth rate in vitro, reaction on cycloheximide-indicator medium, and proteinase activity. The isolates exhibited one of two distinct electrophoretic karyotypes as determined by orthogonal field alternating gel electrophoresis (OFAGE). Four isolates, including the type culture of C. stellatoidea, belonged to electrophoretic karyotype type I by OFAGE, showing eight to nine bands of which at least two bands were less than 1,000 kilobases in size as estimated by comparison with the DNA bands of Saccharomyces cerevisiae. These isolates failed to produce fatal infectionmore » in mice within 20 days when 5 X 10(5) cells were injected intravenously. The yeasts were cleared from the kidneys of two of three mice tested by day 30. Type I showed proteinase activity on bovine serum albumin agar at pH 3.8 and produced a negative reaction on cycloheximide-bromcresol green medium within 48 h. The three grouped in type II by OFAGE showed banding patterns similar to those of a well-characterized isolate of Candida albicans. The isolates of type II had an electrophoretic karyotype of six to seven bands approximately 1,200 kilobases or greater in size. All three type II isolates were highly virulent for mice, producing fatality curves similar to those of a previously studied C. albicans isolate. From 80 to 90% of the mice injected with 5 X 10(5) cells intravenously died within 20 days. The type II isolates produced a positive reaction on cycloheximide-bromcresol green agar and showed no proteinase activity on bovine serum albumin agar at the low pH. In addition, the type II isolates grew faster and were significantly more resistant to UV irradiation than the type I isolates.« less

Japan Report, Science and Technology.

DTIC Science & Technology

1987-04-10

than 0.1 yg/m£. However, several Candida genus yeasts, as well as C. maltosa, possess a cycloheximide resistance and in the case of C. maltosa...Suppose acquisition of the technique is the objective, whether acquisition of such stereotyped techniques is meaningful or not is questionable

The α-Arrestin Bul1p Mediates Lactate Transporter Endocytosis in Response to Alkalinization and Distinct Physiological Signals.

PubMed

Talaia, Gabriel; Gournas, Christos; Saliba, Elie; Barata-Antunes, Cláudia; Casal, Margarida; André, Bruno; Diallinas, George; Paiva, Sandra

2017-11-24

Eukaryotic α-arrestins connect environmental or stress signaling pathways to the endocytosis of plasma membrane transporters or receptors. The Saccharomyces cerevisiae lactate transporter Jen1p has been used as a model cargo for elucidating the mechanisms underlying endocytic turnover in response to carbon sources. Here, we discover a novel pathway of Jen1p endocytosis mediated by the α-arrestin Bul1p in response to the presence of cycloheximide or rapamycin, or prolonged growth in lactate. While cycloheximide or rapamycin modify cells pleiotropically, the major effect of prolonged growth in lactate was shown to be external pH alkalinization. Importantly, employment of specific inactive Jen1p versions showed that Bul1p-dependent endocytosis requires lactate transport, according to the signal imposed. Our results support a model where conformational changes of Jen1p, associated with substrate/H + symport, are critical for the efficiency of Bul1p-dependent Jen1p turnover. Copyright © 2017 Elsevier Ltd. All rights reserved.

Protein synthesis and the recovery of both survival and cytoplasmic "petite" mutation in ultraviolet-treated yeast cells. I. Nuclear-directed protein synthesis.

PubMed

Heude, M; Chanet, R; Moustacchi, E

1975-04-01

The contribution of nuclear-directed protein synthesis in the repair of lethal and mitochondrial genetic damage after UV-irradiation of exponential and stationary phage haploid yeast cells was examined. This was carried out using cycloheximide (CH), a specific inhibitor of nuclear protein synthesis. It appears that nuclear protein synthesis is required for the increase in survival seen after the liquid holding of cells at both stages, as well as for the "petite" recovery seen after the liquid holding of exponential phase cells. The characteristic negative liquid holding effect observed for the UV induction of "petites" in stationary phase cells (increase of the frequency of "petites" during storage) remained following all the treatments which inhibited nuclear protein synthesis. However, the application of photoreactivating light following dark holding with cycloheximide indicates that some steps of the repair of both nuclear and mitochondrial damage are performed in the absence of a synthesis of proteins.

Antibiotics Do Not Control Blister Rust in Eastern White Pine Seedlings

Treesearch

William R. Phelps; Ray Weber

1968-01-01

To prevent blister rust infections in Eastern white pine seedlings, the antibiotics, cycloheximide (acti-dione) and Phytoactin, were tested in root dips, root slurries, and foliar drenches before planting and after planting the trees. None of the methods and materials tested was effective.

Exploring the Ubiquitin-Proteasome Protein Degradation Pathway in Yeast

ERIC Educational Resources Information Center

Will, Tamara J.; McWatters, Melissa K.; McQuade, Kristi L.

2006-01-01

This article describes an undergraduate biochemistry laboratory investigating the ubiquitin-proteasome pathway in yeast. In this exercise, the enzyme beta-galactosidase (beta-gal) is expressed in yeast under the control of a stress response promoter. Following exposure to heat stress to induce beta-gal expression, cycloheximide is added to halt…

The energetic cost of protein synthesis in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss).

PubMed

Pannevis, M C; Houlihan, D F

1992-01-01

To establish the energetic cost of protein synthesis, isolated trout hepatocytes were used to measure protein synthesis and respiration simultaneously at a variety of temperatures. The presence of bovine serum albumin was essential for the viability of isolated hepatocytes during isolation, but, in order to measure protein synthesis rates, oxygen consumption rates and RNA-to-protein ratios, BSA had to be washed from the cells. Isolated hepatocytes were found to be capable of protein synthesis and oxygen consumption at constant rates over a wide range of oxygen tension. Cycloheximide was used to inhibit protein synthesis. Isolated hepatocytes used on average 79.7 +/- 9.5% of their total oxygen consumption on cycloheximide-sensitive protein synthesis and 2.8 +/- 2.8% on maintaining ouabain-sensitive Na+/K(+)-ATPase activity. The energetic cost of protein synthesis in terms of moles of adenosine triphosphate per gram of protein synthesis decreased with increasing rates of protein synthesis at higher temperatures. It is suggested that the energetic cost consists of a fixed (independent of synthesis rate) and a variable component (dependent on synthesis rate).

Effects of cycloheximide on extinction in an appetitively motivated operant conditioning task depend on re-exposure duration.

PubMed

Mierzejewski, Pawel; Olczak, Mieszko; Rogowski, Artur; Kostowski, Wojciech; Samochowiec, Jerzy; Filip, Malgorzata; Przegalinski, Edmund; Bienkowski, Przemyslaw

2008-08-29

Little is known about the role of new protein synthesis in extinction of operant responding for natural and chemical reinforcers. In the present study, the authors investigated whether the effects of a protein synthesis inhibitor, cycloheximide (CHX) on extinction of operant responding for sweet reward depended on the duration of re-exposure sessions. In addition, the authors investigated whether the effects of CHX on extinction could generalize to relapse of saccharin seeking induced by discrete cues. CHX injected after short re-exposure sessions (5min) accelerated extinction of non-reinforced responding. In contrast, the drug injected after long re-exposure sessions (30min) partially inhibited extinction. Reinstatement of saccharin seeking induced by the saccharin-paired discrete cues was not altered by the previous treatment with CHX. Concluding, the results of the present study indicate that: (i) the protein synthesis inhibitor, CHX can alter extinction of operant responding for sweet reward in rats; (ii) the effects of CHX on extinction critically depend on the duration of re-exposure/extinction sessions and do not generalize to relapse of saccharin seeking induced by discrete cues.

Spectral analysis and quantum chemical studies of chair and twist-boat conformers of cycloheximide in gas and solution phases

NASA Astrophysics Data System (ADS)

Tokatli, A.; Ucun, F.; Sütçü, K.; Osmanoğlu, Y. E.; Osmanoğlu, Ş.

2018-02-01

In this study the conformational behavior of cycloheximide in the gas and solution (CHCl3) phases has theoretically been investigated by spectroscopic and quantum chemical properties using density functional theory (wB97X-D) method with 6-31++G(d,p) basis set, for the first time. The calculated IR results reveal that in the ground state the molecule exits as a mixture of the chair and twist-boat conformers in the gas phase, while the calculated NMR results reveal that it only exits as the chair conformer in the solution phase. In order to obtain the contributions coming from intramolecular interactions to the stability of the conformers in the gas and solution phases, the quantum theory of atoms in molecules (QTAIM), noncovalent interactions (NCI) method, and natural bond orbital analysis (NBO) have been employed. The QTAIM and NCI methods indicated that by intramolecular interactions with bond critical point (BCP) the twist-boat conformer is more stabilized than the chair conformer, while by steric interactions it is more destabilized. Considering that these interactions balance each other, the stabilities of the conformers are understood to be dictated by the van der Waals interactions. The NBO analyses show that the hyperconjugative and steric effects play an important role in the stabilization in the gas and solution phases. Furthermore, to get a better understanding of the chemical behavior of this important antibiotic drug we have evaluated and, commented the global and local reactivity descriptors of the both conformers. Finally, the EPR analysis of γ-irradiated cycloheximide has been done. The comparison of the experimental and calculated data have showed the inducement of a radical structure of (CH2)2ĊCH2 in the molecule. The experimental EPR spectrum has also confirmed that the molecule simultaneously exists in the chair and twist-boat conformers in the solid phase.

Problem-Based Test: The Effect of Fibroblast Growth Factor on Gene Expression

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2011-01-01

This paper shows the results of an experiment in which the effects of fibroblast growth factor (FGF), actinomycin D (Act D; an inhibitor of transcription), and cycloheximide (CHX; an inhibitor of translation) were studied on the expression of two genes: a gene called "Fnk" and the gene coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).…

Protein Synthesis Inhibition Blocks Consolidation of an Acrobatic Motor Skill

ERIC Educational Resources Information Center

Kaelin-Lang, Alain; Dichgans, Johannes; Schulz, Jorg B.; Luft, Andreas R.; Buitrago, Manuel M.

2004-01-01

To investigate whether motor skill learning depends on de novo protein synthesis, adult rats were trained in an acrobatic locomotor task (accelerating rotarod) for 7 d. Animals were systemically injected with cycloheximide (CHX, 0.5 mg/kg, i.p.) 1 h before sessions 1 and 2 or sessions 2 and 3. Control rats received vehicle injections before…

Chemical control of blister rust on sugar pine...two fungicides show promise in California tests

Treesearch

Clarence R. Quick

1967-01-01

Among several fungicides tested, Phytoactin L-340 and Dowicide 1 (o-phenylphenol) showed the most promise for systemic chemical control of blister rust on sugar pine in northern California. The trials included 22 tests on 224 sugar pines: five with Acti-dione BR (cycloheximide), one each with five common chemical fungicides,and 12 with phytoactin. Apparent...

Development of New Genetic Manipulation Tools for Metabolic Engineering of Diatoms

DTIC Science & Technology

2008-08-28

protein L41 has been shown in a variety of yeasts to be involved in resistance to anisomycin and cycloheximide 7𔄂. A conserved mutation from proline...atipitis Yarrowia lipolytica Candida troplcalis S. cerevisiae A. nidulans Oryza sativa Homo sapiens P. tricornutum T. pseudonana...Pichia stipitis Yarrowia lipolytica Candida troplcalis S. cerevisiae A. nidulans Oryza sativa Homo sapiens P

Light-stimulated Production of a Chloroplast-localized System for Protein Synthesis in Euglena gracilis1

PubMed Central

Reger, Bonnie J.; Smillie, R. M.; Fuller, R. C.

1972-01-01

Chloroplasts and proplastids isolated respectively from autotrophic and dark-adapted cells of Euglena gracilis strain Z incorporated 14C-l-leucine into protein. In each case the incorporation was inhibited by chloramphenicol (50% inhibition at about 5 μg/ml for chloroplasts and 30 μg/ml for proplastids), but not appreciably by cycloheximide at concentrations up to 200 μg/ml. Chloroplasts from autotrophic cells incorporated leucine into protein at rates of about 10 pg leucine per mg RNA in one minute, but isolated proplastids were only 5 to 10% as active. When dark-adapted cells were illuminated there was little increase in the activity of the chloroplast fraction during the first 12 hr. Between 12 and 24 hr, when there was a rapid increase in the rate of synthesis of chlorophyll, the capacity of the chloroplast fraction for protein synthesis increased markedly. Suppression of the formation of a chloroplast-localized system for protein synthesis by treating the cells with chloramphenicol and the lack of such an effect with cycloheximide suggests that certain of the proteins which form part of a functional chloroplast system for protein synthesis are themselves synthesized within the chloroplasts. PMID:16658126

Polymerase III transcription factor B activity is reduced in extracts of growth-restricted cells.

PubMed Central

Tower, J; Sollner-Webb, B

1988-01-01

Extracts of cells that are down-regulated for transcription by RNA polymerase I and RNA polymerase III exhibit a reduced in vitro transcriptional capacity. We have recently demonstrated that the down-regulation of polymerase I transcription in extracts of cycloheximide-treated and stationary-phase cells results from a lack of an activated subform of RNA polymerase I which is essential for rDNA transcription. To examine whether polymerase III transcriptional down-regulation occurs by a similar mechanism, the polymerase III transcription factors were isolated and added singly and in pairs to control cell extracts and to extracts of cells that had reduced polymerase III transcriptional activity due to cycloheximide treatment or growth into stationary phase. These down-regulations result from a specific reduction in TFIIIB; TFIIIC and polymerase III activities remain relatively constant. Thus, although transcription by both polymerase III and polymerase I is substantially decreased in extracts of growth-arrested cells, this regulation is brought about by reduction of different kinds of activities: a component of the polymerase III stable transcription complex in the former case and the activated subform of RNA polymerase I in the latter. Images PMID:3352599

Synthesis of protein in intestinal cells exposed to cholera toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peterson, J.W.; Berg, W.D. Jr.; Coppenhaver, D.H.

1987-11-01

The mechanism by which cyclic adenosine monophosphate (AMP), formed by intestinal epithelial cells in response to cholera toxin, ultimately results in alterations in water and electrolyte transport is poorly understood. Several studies have indicated that inhibitors of transcription or translation block much of the transport of ions and water in the intestine and edema formation in tissue elicited by cholera toxin. Data presented in this study confirmed the inhibitory effects of cycloheximide on cholera toxin-induced fluid accumulation in the rabbit intestinal loop model. Neither cycloheximide nor actinomycin D altered the amount of cyclic AMP that accumulated in intestinal cells andmore » Chinese hamster ovary cells exposed to cholera toxin. An increase in (/sup 3/H) leucine incorporation was readily demonstrable in intestinal epithelial cells from rabbits challenged with Vibrio cholerae. Similarly, intestinal epithelial cells incubated with cholera toxin for 4 hr synthesized substantially more protein than controls as determined by relative incorporation of (/sup 35/S) methionine. Most of the new protein synthesized in response to cholera toxin was membrane associated and of high molecular weight. The possible significance of the toxin-induced protein relative to cholera pathogenesis was discussed.« less

Degradation of Phenolic Compounds and Ring Cleavage of Catechol by Phanerochaete chrysosporium

PubMed Central

Leatham, Gary F.; Crawford, R. L.; Kirk, T. Kent

1983-01-01

POL-88, a mutant of the white-rot fungus Phanerochaete chrysosporium, was selected for diminished phenol-oxidizing enzyme activity. A wide variety of phenolic compounds were degraded by ligninolytic cultures of this mutant. With several o-diphenolic substrates, degradation intermediates were produced that had UV spectra consistent with muconic acids. Extensive spectrophotometric and polarographic assays failed to detect classical ring-cleaving dioxygenases in cell homogenates or in extracts from ligninolytic cultures. Even so, a sensitive carrier-trapping assay showed that intact cultures degraded [U-14C]catechol to [14C]muconic acid, establishing the presence of a system capable of 1,2-intradiol fission. Significant accumulation of [14C]muconic acid into carrier occurred only when evolution of 14CO2 from [14C]catechol was inhibited by treating cultures with excess nutrient nitrogen (e.g., l-glutamic acid) or with cycloheximide. l-Glutamic acid is known from past work to repress the ligninolytic system in P. chrysosporium and to mimic the effect of cycloheximide. The results here indicate, therefore, that the enzyme system responsible for degrading ring-cleavage products to CO2 turns over faster than does the system responsible for ring cleavage. PMID:16346340

Cycloheximide- and puromycin-induced heat resistance: different effects on cytoplasmic and nuclear luciferases

PubMed Central

Michels, Annemieke A; Kanon, Bart; Konings, Antonius W.T; Bensaude, Olivier; Kampinga, Harm H

2000-01-01

Inhibition of translation can result in cytoprotection against heat shock. The mechanism of this protection has remained elusive so far. Here, the thermoprotective effects of the translation inhibitor cycloheximide (CHX) and puromycin were investigated, using as reporter firefly luciferase localized either in the nucleus or in the cytoplasm. A short preincubation of O23 cells with either translation inhibitor was found to attenuate the heat inactivation of a luciferase directed into the cytoplasm, whereas the heat sensitivity of a nuclear-targeted luciferase remained unaffected. After a long-term CHX pretreatment, both luciferases were more heat resistant. Both the cytoplasmic and the nuclear luciferase are protected against heat-induced inactivation in thermotolerant cells and in cells overexpressing heat shock protein (Hsp)70. CHX incubations further attenuated cytoplasmic luciferase inactivation in thermotolerant and in Hsp70 overexpressing cells, even when Hsp70-mediated protection was saturated. It is concluded that protection by translation inhibition is unlikely due to an increase in the pool of free Hsps normally engaged in translation and released from the nascent polypeptide chains on the ribosomes. Rather, a decrease in nascent chains and thermolabile polypeptides may account for the heat resistance promoted by inhibitors of translation. PMID:11005376

Mutagenesis of Saccharomyces cerevisiae by sodium azide activated in barley.

PubMed

Velemínský, J; Silhánková, L; Smiovská, V; Gichner, T

1979-07-01

Concentrated dialysate of the extract prepared from barley seeds treated with sodium azide increased up to 100--200 times the frequency of forward mutations to cycloheximide resistance in the excision-deficient UV-sensitive heploid strain rad2-5 of Saccharomyces cerevisiae, when applied to growing cells in complete medium at pH 4.2. Only a slight increase of mutation frequency (less than 4 times) was found in the haploid RAD+ strain treated in the same way as well as in haploid RAD+ and rad2-5 strains treated directly by sodium azide. In contrast with the barley-activated sodium azide, UV irradiation was more effective in the induction of cycloheximide resistance in the RAD+ strain than in the RAD2-5 mutant. The dialysate from azide-treated barley seeds, applied at both pH 4.2 and pH 9, also significantly increased the frequency of locus-specific suppressor mutations to isoleucine independence and -- to a lesser extent -- reversions and/or gene conversions in the trp5 locus in growing cells of the diploid strain D7. The dialysate was also mutagenic in resting cells of strains D7 and rad2-5 but with lower effectiveness.

UV-B light contributes directly to the synthesis of chiloglottone floral volatiles

PubMed Central

Amarasinghe, Ranamalie; Poldy, Jacqueline; Matsuba, Yuki; Barrow, Russell A.; Hemmi, Jan M.; Pichersky, Eran; Peakall, Rod

2015-01-01

Background and Aims Australian sexually deceptive Chiloglottis orchids attract their specific male wasp pollinators by means of 2,5-dialkylcyclohexane-1,3-diones or ‘chiloglottones’, representing a newly discovered class of volatiles with unique structures. This study investigated the hypothesis that UV-B light at low intensities is directly required for chiloglottone biosynthesis in Chiloglottis trapeziformis. Methods Chiloglottone production occurs only in specific tissue (the callus) of the labellum. Cut buds and flowers, and whole plants with buds and flowers, sourced from the field, were kept in a growth chamber and interactions between growth stage of the flowers and duration and intensity of UV-B exposure on chiloglottone production were studied. The effects of the protein synthesis inhibitor cycloheximide were also examined. Key Results Chiloglottone was not present in buds, but was detected in buds that were manually opened and then exposed to sunlight, or artificial UV-B light for ≥5 min. Spectrophotometry revealed that the sepals and petals blocked UV-B light from reaching the labellum inside the bud. Rates of chiloglottone production increased with developmental stage, increasing exposure time and increasing UV-B irradiance intensity. Cycloheximide did not inhibit the initial production of chiloglottone within 5 min of UV-B exposure. However, inhibition of chiloglottone production by cycloheximide occurred over 2 h of UV-B exposure, indicating a requirement for de novo protein synthesis to sustain chiloglottone production under UV-B. Conclusions The sepals and petals of Chiloglottis orchids strongly block UV-B wavelengths of light, preventing chiloglottone production inside the bud. While initiation of chiloglottone biosynthesis requires only UV-B light, sustained chiloglottone biosynthesis requires both UV-B and de novo protein biosynthesis. The internal amounts of chiloglottone in a flower reflect the interplay between developmental stage, duration and intensity of UV-B exposure, de novo protein synthesis, and feedback loops linked to the starting amount of chiloglottone. It is concluded that UV-B light contributes directly to chiloglottone biosynthesis. These findings suggest an entirely new and unexpected biochemical reaction that might also occur in taxa other than these orchids. PMID:25649114

Protein synthesis in the basolateral amygdala complex is required for consolidation of a first-order fear memory, but not for consolidation of a higher-order fear memory.

PubMed

Leidl, Dana M; Lay, Belinda P P; Chakouch, Cassandra; Westbrook, R Frederick; Holmes, Nathan M

2018-04-12

The present series of experiments pursued our recent findings that consolidation of a second-order fear memory requires neuronal activity, but not de novo protein synthesis, in the basolateral amygdala complex (BLA). It used a modified second-order conditioning protocol in which rats were exposed to S1-shock pairings in stage 1 and pairings of the serial S2-S1 compound and shock in stage 2. Experiment 1 showed that responding (freezing) to S2 in this protocol is conditional on its compounding with S1 in stage 2 (Experiment 1), and therefore, the result of associative formation. The remaining experiments then showed that the protein synthesis requirement for consolidation of new learning about S2 varied with the training afforded S1. When S1 was trained in stage 1 and present in stage 2, consolidation of the new S2 fear memory was unaffected by pre- or post-stage 2 infusions of the protein synthesis inhibitor, cycloheximide, into the BLA (Experiments 2 and 5). This result was observed independently of the number of S1-shock pairings in stage 1 (even a single pairing produced the result), and alongside demonstrations that cycloheximide infusions disrupt consolidation of a first-order fear memory (Experiments 2 and 5). However, when S1 was not conditioned in stage 1 (Experiment 3) or was omitted from conditioning in stage 2 (Experiment 4), consolidation of the new S2 fear memory was disrupted by post-stage 2 cycloheximide infusions into the BLA. These results were taken to imply that the consolidation of a higher-order fear memory exploits molecular events associated with consolidation of a reactivated first-order fear memory; hence it occurs independently of de novo protein synthesis in the BLA. Alternatively, the nature of the association formed in higher-order conditioning may be such as to not require de novo protein synthesis for its consolidation. Copyright © 2018 Elsevier Inc. All rights reserved.

Chemoprevention of Prostate Cancer by Phenethyl Isothiocyanate

DTIC Science & Technology

2007-03-01

AD cells were exposed to PEITC, protein synthesis inhibitor cycloheximide (CHX), or proteasome inhibitor MG-132 alone, or to the combination of...The results indicated that down-regulation of Sp1 may require protein synthesis for a proteasome activity, thereby enhancing Sp1 protein degradation...carcinogen N- methyl-N- nitrosourea (MNU) for the formation of prostate tumors. The experimental group of rats was provided with PEITC, to evaluate the

Sensor & Model Enabled Water Quality & Security Assessment System for Situational Awareness of Water Distribution Networks

DTIC Science & Technology

2010-06-01

Scenario – 12 gallons of readily available toxic substance – pump ($150 rental) – wrench to open a fire hydrant ($10)  One (1) terrorist, or...6 Gallons Water General Comments Aflatoxin 7.6 Potent Carcinogen Aldicarb 1.1 Cycloheximide 2.1 LSD 0.2 Highly Toxic , Psychoactive Mercuric Chloride...Chlorfenvinphos, Formetanate Hydrochloride, Acrolein, Chloropicrin, Sodium chloroacetate, Thyoglycolate medium, Crotoxyphos, Glyphosate , Jimsonweed, Methanol

Effect of Antimicrobial Compounds on Balamuthia mandrillaris Encystment and Human Brain Microvascular Endothelial Cell Cytopathogenicity▿

PubMed Central

Siddiqui, Ruqaiyyah; Matin, Abdul; Warhurst, David; Stins, Monique; Khan, Naveed Ahmed

2007-01-01

Cycloheximide, ketoconazole, or preexposure of organisms to cytochalasin D prevented Balamuthia mandrillaris-associated cytopathogenicity in human brain microvascular endothelial cells, which constitute the blood-brain barrier. In an assay for inhibition of cyst production, these three agents prevented the production of cysts, suggesting that the biosynthesis of proteins and ergosterol and the polymerization of actin are important in cytopathogenicity and encystment. PMID:17875991

Development of cytotoxicity-sensitive human cells using overexpression of long non-coding RNAs.

PubMed

Tani, Hidenori; Torimura, Masaki

2015-05-01

Biosensors using live cells are analytical devices that have the advantage of being highly sensitive for their targets. Although attention has primarily focused on reporter gene assays using functional promoters, cell viability assays are still efficient. We focus on long non-coding RNAs (lncRNAs) that are involved in the molecular mechanisms associated with responses to cellular stresses as a new biological material. Here we have developed human live cells transfected with lncRNAs that can be used as an intelligent sensor of cytotoxicity for a broad range of environmental stresses. We identified three lncRNAs (GAS5, IDI2-AS1, and SNHG15) that responded to cycloheximide in HEK293 cells. Overexpression of these lncRNAs sensitized human cells to cell death in response to various stresses (cycloheximide, ultraviolet irradiation, mercury II chloride, or hydrogen peroxide). In particular, dual lncRNA (GAS5 plus IDI2-AS1, or GAS5 plus SNHG15) overexpression sensitized cells to cell death by more cellular stresses. We propose a method for highly sensitive biosensors using overexpression of lncRNAs that can potentially measure the cytotoxicity signals of various environmental stresses. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Texture Analysis of Poly-Adenylated mRNA Staining Following Global Brain Ischemia and Reperfusion

PubMed Central

Szymanski, Jeffrey J.; Jamison, Jill T.; DeGracia, Donald J.

2011-01-01

Texture analysis provides a means to quantify complex changes in microscope images. We previously showed that cytoplasmic poly-adenylated mRNAs form mRNA granules in post-ischemic neurons and that these granules correlated with protein synthesis inhibition and hence cell death. Here we utilized the texture analysis software MaZda to quantify mRNA granules in photomicrographs of the pyramidal cell layer of rat hippocampal region CA3 around 1 hour of reperfusion after 10 min of normothermic global cerebral ischemia. At 1 hour reperfusion, we observed variations in the texture of mRNA granules amongst samples that were readily quantified by texture analysis. Individual sample variation was consistent with the interpretation that animal-to-animal variations in mRNA granules reflected the time-course of mRNA granule formation. We also used texture analysis to quantify the effect of cycloheximide, given either before or after brain ischemia, on mRNA granules. If administered before ischemia, cycloheximide inhibited mRNA granule formation, but if administered after ischemia did not prevent mRNA granulation, indicating mRNA granule formation is dependent on dissociation of polysomes. We conclude that texture analysis is an effective means for quantifying the complex morphological changes induced in neurons by brain ischemia and reperfusion. PMID:21477879

Onychomycosis Caused by Chaetomium globosum

PubMed Central

Kim, Dong Min; Lee, Myung Hoon; Ha, Gyoung Yim; Kim, Heesoo; Choi, Jong Soo

2013-01-01

Onychomycosis is usually caused by dermatophytes, but some nondermatophytic molds and yeasts are also associated with invasion of nails. The genus Chaetomium is a dematiaceous nondermatophytic mold found in soil and plant debris as a saprophytic fungus. We report the first Korean case of onychomycosis caused by Chaetomium globosum in a 35-year-old male. The patient showed brownish-yellow discoloration and subungual hyperkeratosis on the right toenails (1st and 5th) and left toenails (1st and 4th). Direct microscopic examination of scraping on the potassium hydroxide preparation revealed septate hyphae and repeated cultures on Sabouraud's dextrose agar (SDA) without cycloheximide slants showed the same fast-growing colonies, which were initially velvety white then turned to dark gray to brown. However, there was no growth of colony on SDA with cycloheximide slants. Brown-colored septated hyphae, perithecia and ascospores were shown in the slide culture. The DNA sequence of internal transcribed spacer region of the clinical sample was a 100% match to that of C. globosum strain ATCC 6205 (GenBank accession number EF524036.1). We confirmed C. globosum by KOH mount, colony, and light microscopic morphology and DNA sequence analysis. The patient was treated with 250 mg oral terbinafine daily and topical amorolfine 5% nail lacquer for 3 months. PMID:23717019

Regulation of activity in vitro and in vivo of three phospholipases B from Saccharomyces cerevisiae

PubMed Central

2004-01-01

The genome of the yeast, Saccharomyces cerevisiae, contains three highly similar genes coding for phospholipases B/lysophospholipases. These enzymes behave differently with respect to substrate preferences in vitro and relative contributions to phospholipid catabolism in vivo [Merkel, Fido, Mayr, Prüger, Raab, Zandonella, Kohlwein and Paltauf (1999) J. Biol. Chem. 274, 28121–28127]. It is shown in the present study that, in vitro, pH markedly affects the substrate preference of Plb1p and Plb2p, but not of Plb3p. At the pH optimum of 2.5–3.5, the order of substrate preference of Plb1p and Plb2p is PtdSer (phosphatidylserine)>PtdIns>PtdCho (phosphatidylcholine>PtdEtn (phosphatidylethanolamine). At pH values of 5 and above, the substrate preferences change to PtdCho=PtdEtn for Plb1p and PtdSer=PtdEtn for Plb2p. Accordingly, with cultured cells the ratio of PtdIns/PtdCho breakdown, as reflected in the ratio of GroPIns (glycerophosphoinositol)/GroPCho (glycerophosphocholine) released into the culture medium, is inversely related to the pH of the growth medium. This effect is ascribed to the pH response of Plb1p, because Plb2p does not contribute to the degradation of PtdIns and PtdCho in vivo. Bivalent and tervalent cations activate phospholipases B at pH 5.5, but are inhibitory at pH 2.5. Al3+ at a concentration of 20 mM increases Plb1p activity in vitro by 8-fold and leads to a 9-fold increase in GroPCho release by whole cells. In vivo, cycloheximide strongly inhibits the breakdown of PtdIns, and to a lesser extent PtdCho. However, Al3+-stimulated GroPCho release is almost completely inhibited by cycloheximide. Deletion of PLB3 leads to increased sensitivity to toxic Al3+. Addition of SDS or melittin to cultured cells leads to a significant increase in phospholipid degradation, which is insensitive to inhibition by cycloheximide. Deletion mutants defective in the PLB1 gene are significantly more resistant to SDS than are wild-type cells. PMID:15588231

40 CFR Appendix B to Part 355 - The List of Extremely Hazardous Substances and Their Threshold Planning Quantities

Code of Federal Regulations, 2010 CFR

2010-07-01

... Trifluoride Compound With Methyl Ether (1:1) 1,000 1,000 359-06-8 Fluoroacetyl Chloride b 10 10 371-62-0... Phenylhydrazine Hydrochloride 1,000 1,000/10,000 60-34-4 Methyl Hydrazine 10 500 60-41-3 Strychnine sulfate 10 100... 10 10/10,000 65-30-5 Nicotine sulfate 100 100/10,000 66-81-9 Cycloheximide 100 100/10,000 67-66-3...

40 CFR Appendix B to Part 355 - The List of Extremely Hazardous Substances and Their Threshold Planning Quantities

Code of Federal Regulations, 2011 CFR

2011-07-01

... Trifluoride Compound With Methyl Ether (1:1) 1,000 1,000 359-06-8 Fluoroacetyl Chloride b 10 10 371-62-0... Phenylhydrazine Hydrochloride 1,000 1,000/10,000 60-34-4 Methyl Hydrazine 10 500 60-41-3 Strychnine sulfate 10 100... 10 10/10,000 65-30-5 Nicotine sulfate 100 100/10,000 66-81-9 Cycloheximide 100 100/10,000 67-66-3...

Endophytic Streptomyces in the traditional medicinal plant Arnica montana L.: secondary metabolites and biological activity.

PubMed

Wardecki, Tina; Brötz, Elke; De Ford, Christian; von Loewenich, Friederike D; Rebets, Yuriy; Tokovenko, Bogdan; Luzhetskyy, Andriy; Merfort, Irmgard

2015-08-01

Arnica montana L. is a medical plant of the Asteraceae family and grows preferably on nutrient poor soils in mountainous environments. Such surroundings are known to make plants dependent on symbiosis with other organisms. Up to now only arbuscular mycorrhizal fungi were found to act as endophytic symbiosis partners for A. montana. Here we identified five Streptomyces strains, microorganisms also known to occur as endophytes in plants and to produce a huge variety of active secondary metabolites, as inhabitants of A. montana. The secondary metabolite spectrum of these strains does not contain sesquiterpene lactones, but consists of the glutarimide antibiotics cycloheximide and actiphenol as well as the diketopiperazines cyclo-prolyl-valyl, cyclo-prolyl-isoleucyl, cyclo-prolyl-leucyl and cyclo-prolyl-phenylalanyl. Notably, genome analysis of one strain was performed and indicated a huge genome size with a high number of natural products gene clusters among which genes for cycloheximide production were detected. Only weak activity against the Gram-positive bacterium Staphylococcus aureus was revealed, but the extracts showed a marked cytotoxic activity as well as an antifungal activity against Candida parapsilosis and Fusarium verticillioides. Altogether, our results provide evidence that A. montana and its endophytic Streptomyces benefit from each other by completing their protection against competitors and pathogens and by exchanging plant growth promoting signals with nutrients.

Androgen-sensitive spermine-binding protein of rat ventral prostate. Purification of the protein and characterization of the hormonal effect.

PubMed Central

Mezzetti, G; Loor, R; Liao, S

1979-01-01

The rat ventral prostate contains a cytosol protein that can non-covalently bind spermine much more tightly than spermidine or other natural diamines. The protein has been purified to homogeneity, as judged by electrophoresis in urea- and sodium dodecyl sulphate-containing polyacrylamide gels. The protein, with or without spermine bound to it, sediments at 3 S in a sucrose gradient with or without 0.4 M-KCl. The molecular weight of the protein is about 30 000. Each molecule of the binding protein can bind one molecule of spermine. In the prostate of rats injected with cycloheximide, the protein appears to have a half-life of about 3.5 h. The spermine-binding activity of an acidic fraction obtained by DEAE-cellulose chromatography of the prostate cytosol proteins is reduced by about 40--60% within 20--40 h after castration. This effect is reversed very rapidly within 15--30 min by intraperitoneal injection of 5 alpha-dihydrotestosterone. The hormonal effect is androgen-specific and is not mimicked by dexamethasone or oestradiol-17 beta. The androgen effect was reduced significantly when rats were injected with cycloheximide or actinomycin D, suggesting that the acidic protein may be one of the earliest proteins induced by androgen in the rat ventral prostate. Images Fig. 1. PMID:534539

Apoptosis-inducing and apoptosis-preventing functions of poliovirus.

PubMed Central

Tolskaya, E A; Romanova, L I; Kolesnikova, M S; Ivannikova, T A; Smirnova, E A; Raikhlin, N T; Agol, V I

1995-01-01

Data showing that an apoptotic reaction (the exit into the cytoplasm and nucleolytic internucleosomal degradation of chromosomal DNA, compaction and fragmentation of chromatin, cellular shrinkage, and cytoplasmic blebbing) developed in a subline of HeLa-S3 cells upon nonpermissive poliovirus infection with either a guanidine-sensitive poliovirus in the presence of guanidine, a guanidine-dependent mutant in the absence of guanidine, or certain temperature-sensitive mutants at a restrictive temperature are presented. Essentially, no apoptotic reaction occurred upon permissive infection of these cells. Both permissive and nonpermissive infections resulted in the inhibition of host protein synthesis. Actinomycin D or cycloheximide also elicited a rapid apoptotic reaction in uninfected cells. However, preinfection or coinfection with poliovirus prevented the apoptotic response to the addition of actinomycin D, and preinfection blocked cycloheximide-induced apoptosis as well. These data fit a model in which the cells used are prepared to develop apoptosis, with their viability due to the presence of certain short-lived mRNA and protein species. Poliovirus infection turns on two oppositely directed sets of reactions. On the one hand, the balance is driven toward apoptosis, probably via the shutoff of host macromolecular synthesis. On the other hand, viral protein exhibits antiapoptotic activity, thereby preventing premature cell death. To our knowledge, this is the first description of an antiapoptotic function for an RNA virus. PMID:7529330

Detection of adenovirus type 2-induced early polypeptides using cycloheximide pretreatment to enhance viral protein synthesis.

PubMed Central

Harter, M L; Shanmugam, G; Wold, W S; Green, M

1976-01-01

(35S) methionine-labeled polypeptides synthesized by adenovirus type 2-infected cells have been analyzed by polyacrylamide gradient gel electrophoresis and autoradiography. Cycloheximide (CH) was added to infected cultures to accumulate early viral mRNA relative to host cell mRNA. This allowed viral proteins to be synthesized in increased amounts relative to host proteins after removal of CH and pulse-labeling with (35S)methionine. During the labeling period arabinosyl cytosine was added to prevent the synthesis of late viral proteins. This procedure facilitated the detection of six early viral-induced polypeptides, designated EP1 through EP6 (early protein), with apparent molecular weights of 75,000 (75K), 42K, 21K, 18K, 15K, and 11K. Supportive data were obtained by coelectrophoresis of (35S)- and (3H)methionine-labeled polypeptides from infected and uninfected cells, respectively. Three of these early polypeptides have not been previously reported. CH pretreatment enhanced the rates of synthesis of EP4 and EP6 20- to 30-fold and enhanced that of the others approximately twofold. The maximal rates of synthesis of the virus-induced proteins varied, in a different manner, with time postinfection and CH pretreatment. Since CH pretreatment appears to increase the levels of early viral proteins, it may be a useful procedure to assist their isolation and functional characterization. Images PMID:950686

Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment.

PubMed

Lim, Pek Siew; Hardy, Kristine; Peng, Kaiman; Shannon, Frances M

2016-03-01

T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA) and calcium ionomycin (I) as stimuli. We were also interested to examine the importance of new protein synthesis in regulating the expression of genes involved in T cell activation. Thus we have pre-treated mouse EL4 T cells with cycloheximide, a protein synthesis inhibitor, and left the cells unstimulated or stimulated with PMA/I for 4 h. We performed microarray expression profiling of these cells to correlate the gene expression with chromatin state of T cells upon T cell activation [1]. Here, we detail further information and analysis of the microarray data, which shows that T cell activation leads to differential expression of genes and inducible genes can be further classified as primary and secondary response genes based on their protein synthesis dependency. The data is available in the Gene Expression Omnibus under accession number GSE13278.

Detoxification function of the Arabidopsis sulphotransferase AtSOT12 by sulphonation of xenobiotics.

PubMed

Chen, Jinhua; Gao, Liqiong; Baek, Dongwon; Liu, Chunlin; Ruan, Ying; Shi, Huazhong

2015-08-01

Cytosolic sulphotransferases have been implicated in inactivation of endogenous steroid hormones and detoxification of xenobiotics in human and animals. Yet, the function of plant sulphotransferases in xenobiotic sulphonation and detoxification has not been reported. In this study, we show that the Arabidopsis sulphotransferase AtSOT12 could sulphonate the bacterial-produced toxin cycloheximide. Loss-of-function mutant sot12 exhibited hypersensitive phenotype to cycloheximide, and expression of AtSOT12 protein in yeast cells conferred resistance to this toxic compound. AtSOT12 exhibited broad specificity and could sulphonate a variety of xenobiotics including phenolic and polycyclic compounds. Enzyme kinetics analysis indicated that AtSOT12 has different selectivity for simple phenolics with different side chains, and the position of the side chain in the simple phenolic compounds affects substrate binding affinity and catalytic efficiency. We proposed that the broad specificity and induced production of AtSOT12 may have rendered this enzyme to not only modify endogenous molecules such as salicylic acid as we previously reported, but also sulphonate pathogen-produced toxic small molecules to protect them from infection. Sulphonation of small molecules in plants may constitute a rapid way to inactivate or change the physiochemical properties of biologically active molecules that could have profound effects on plant growth, development and defence. © 2015 John Wiley & Sons Ltd.

Regulation of glycerolipid metabolism in Tetrahymena species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jones, C.R.

1988-01-01

A simplified extraction system was developed to study the in vivo incorporation of 1-{sup 14}C-acetate into the glycerolipids of Tetrahymena. Cells incubated in the presence of 1-{sup 14}C-sodium acetate were spotted onto pre-acidified TLC plate and extracted by developing the plates in ethanol, and thereafter developing with 90:10:1 (v:v:v) hexane-ether-acetic acid. The synthesis of glycerolipids in Tetrahymena thermophila and Tetrahymena furgasoni, was studied using the above assay system. The level of triacylglycerol was elevated at stationary phase, whereas phospholipid levels were depressed. A shift from phospholipid synthesis to triacylglycerol synthesis occurred when growth was inhibited in Tetrahymena thermophila. The signalmore » for this shift in Tetrahymena thermophila is apparently a decrease in protein synthesis due either to culture age, the presence of cycloheximide, or the removal of amino acid from the synthetic medium. In exponentially growing Tetrahymena thermophila cycloheximide stimulated the incorporation of 1-{sup 14}C-acetate into triacylglycerol and decreased the incorporation into phospholipid, while stopping the incorporation of 1-{sup 14}C-serine into TCA precipitable material from the cells. Similar alterations in glycerolipid synthesis were obtained when amino acids were deleted from the medium. An in vitro assay system was developed to measure the incorporation of 1-{sup 14}C-palmitic acid into triacylglycerol and phospholipid using conditions similar to those of the in vivo studies.« less

Investigating CH4 production in an oxic plant-soil system -a new approach combining isotopic labelling (13C) and inhibitors

NASA Astrophysics Data System (ADS)

Lenhart, Katharina; Keppler, Frank

2017-04-01

Typically, aerated soil are net sinks of atmospheric methane (CH4), being highest in native ecosystems (pristine forests > managed forests > grasslands > crop fields). However, this does not exclude a simultaneous endogenic CH4 production in the plant-soil system, which cannot be detected simply via CH4 flux measurements. Methanogenic archaea producing CH4 under anoxic conditions were thought to be the only biotic source of CH4 in the soil. However, until recently a non-archaeal pathway of CH4 formation is known where CH4 is produced under oxic conditions in plants (Keppler et al. 2006) and fungi (Lenhart et al. 2012). Additionally, abiotic formation of CH4 from soil organic matter was reported (Jugold et al. 2012) and may be ubiquitous in terrestrial ecosystems. The major goal of this project was to determine soil endogenic CH4 sources and to estimate their contribution to the endogenic CH4 production. Especially the effect of plants and fungi on soil CH4 production was investigated. Therefore, a series of experiments was carried out on field fresh soil collected in a grassland and a forest ecosystem under controlled laboratory conditions. By combining selective inhibitors and 13C labelling, CH4 production rates of several CH4 sources were quantified. The major difficulty was to detect the comparatively small flux of CH4 production against the background of the high CH4 consumption rates due to methanotrophic bacteria. Therefore, we supplemented bare soil and soil with vegetation with selective inhibitors and 13C labelled substrates in a closed chamber system. In a first step, CH4 production was determined by the inhibition of CH4 oxidizing bacteria with Difluoromethane (DFM, 2ml l-1). In the following, a 13C labelled substrate (either CO2, Acetate, or Methionine -S-CH3 labelled) was added in combination with a specific inhibitor -either for archaeal methanogenesis (Bromoethanesulfonate), bacteria (Streptomycin), or fungi (Captan, Cycloheximide). Gas samples were taken during the incubation for CH4 and CO2 concentration measurements and isotope ratio mass spectrometry (CH4, CO2). Grassland and forest soils showed differences in CO2 and CH4 production rates. Based on the 13C-CH4 signature we found that all substrates were metabolized to CH4, but to a different degree. Inhibitors reduced CH4 production and conversion of certain substrates to a different degree. Using the example of acetate and cycloheximide, in both soils acetate increased respiration, whereas cycloheximide reduced respiration by 56 and 62 %, respectively. For CH4 production, however, no effect was visible for the grassland soil, but in the forest soil CH4 production increased by 69 %. Cycloheximide inhibited the substrate-induced CH4 production by 63 %, indicating that fungi were responsible for this pathway. Moreover, the finding that fungi use the methyl group of acetate to produce CH4 was also verified with a sterile culture. References Lenhart, K. et al. Evidence for methane production by saprotrophic fungi. Nat Commun 3, 1046, (2012). Keppler, F., et al. Methane emissions from terrestrial plants under aerobic conditions. Nature 439, 187-191 (2006). Jugold, A. et al. Non-microbial methane formation in oxic soils. Biogeosciences 9, 5291-5301, (2012).

A pyrogen derived from human white cells which is active in mice.

PubMed Central

Gordon, A. H.; Parker, I. D.

1980-01-01

An endogenous pyrogen smaller in molecular size than that previously obtained from human white cells has been found in supernatants of these cells after uptake of zymosan and incubation for 18 h. The new pyrogen after separation from other pyrogens which are produced at the same time has been found to produce fever in mice but not in rabbits. Because it is not formed if cycloheximide is present and is inactivated by leucine aminopeptidase, it is believed to be a peptide. PMID:7448120

Shift from posttranscriptional to predominant transcriptional control of the expression of the GM-CSF gene during activation of human Jurkat cells.

PubMed

Razanajaona, D; Maroc, C; Lopez, M; Mannoni, P; Gabert, J

1992-05-01

The expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is differentially regulated in various cell types. We investigated the mechanisms controlling its expression in 12-O-tetradecanoylphorbol-13-acetate plus phytohemagglutinin-stimulated Jurkat cells, a human T-cell line. In unstimulated cells, GM-CSF mRNA was undetectable by Northern blot. Upon activation, it was detected from 3 h onward, with a progressive increase in the levels of the transcript up to 24 h of stimulation. Whereas cycloheximide treatment at the time of stimulation blocked mRNA induction, its addition at later times resulted in a marked increase in transcript levels. Run-on analysis showed that transcription of the GM-CSF gene was low to undetectable in unstimulated cells; stimulation led to transcriptional activation, which was weak at 6 h but had increased 16-fold at 24 h. In addition, the mRNA half-life decreased during activation, from 2.5 h at 6 h down to 45 min at 24 h. Cycloheximide treatment increased GM-CSF mRNA half-life (3- and 4-fold, respectively). Our results show: (a) both transcriptional and posttranscriptional signals regulate GM-CSF mRNA levels in activated Jurkat cells, (b) de novo protein synthesis is required for mRNA induction, whereas destabilizing labile proteins control the transcript stability, and (c) a shift from a posttranscriptional to a predominant transcriptional control of GM-CSF gene expression occurs during activation.

Fasting-induced apoptosis in rat liver is blocked by cycloheximide.

PubMed

Tessitore, L; Tomasi, C; Greco, M

1999-08-01

The effect of cycloheximide (CH) on the fasting-induced changes of rat liver cell and protein turnover has been investigated. Late starvation phase (3-4-day-fasting period) was characterised by a decrease in liver weight and protein and DNA content. The loss of DNA was not related to liver cell necrosis but due not only to depression of cell proliferation as shown by the drop in the labelling index but also induction of apoptosis. This type of apoptosis was documented by the increase in the apoptotic index (cells labelled by TUNEL) and transglutaminase activity as well as by DNA fragmentation. The liver cells of fasted rats appeared smaller as shown by the higher cell density and DNA/protein ratio than in controls. Females were more resistant to fasting-induced apoptosis than males. A single dose of CH, a drug primary known as inhibitor of protein synthesis, induced or enhanced apoptosis in fed and 2-days fasted male rats, respectively, without any sign of cell necrosis. On the contrary, the administration of repeated doses of CH blocked apoptosis induced by fasting. CH "froze" protein and DNA content as well as apoptotic process at the level of 2 days-fasted rats. While fasting-induced liver protein loss resulted from a marked reduction in protein synthesis with a slight decrease in degradation, repeated treatment with CH virtually blocked protein loss by abolishing protein catabolism. These data suggest a direct relationship between the catabolic side of protein turnover and the apoptotic process.

Single neurons in the nucleus of the solitary tract respond selectively to bitter taste stimuli.

PubMed

Geran, Laura C; Travers, Susan P

2006-11-01

Molecular data suggest that receptors for all bitter ligands are coexpressed in the same taste receptor cells (TRCs), whereas physiological results indicate that individual TRCs respond to only a subset of bitter stimuli. It is also unclear to what extent bitter-responsive neurons are stimulated by nonbitter stimuli. To explore these issues, single neuron responses were recorded from the rat nucleus of the solitary tract (NST) during whole mouth stimulation with a variety of bitter compounds: 10 microM cycloheximide, 7 mM propylthiouracil, 10 mM denatonium benzoate, and 3 mM quinine hydrochloride at intensities matched for behavioral effectiveness. Stimuli representing the remaining putative taste qualities were also tested. Particular emphasis was given to activating taste receptors in the foliate papillae innervated by the quinine-sensitive glossopharyngeal nerve. This method revealed a novel population of bitter-best (B-best) cells with foliate receptive fields and significant selectivity for bitter tastants. Across all neurons, multidimensional scaling depicted bitter stimuli as loosely clustered yet clearly distinct from nonbitter tastants. When neurons with posterior receptive fields were analyzed alone, bitter stimuli formed a tighter cluster. Nevertheless, responses to bitter stimuli were variable across B-best neurons, with cycloheximide the most, and quinine the least frequent optimal stimulus. These results indicate heterogeneity for the processing of ionic and nonionic bitter tastants, which is dependent on receptive field. Further, they suggest that neurons selective for bitter substances could contribute to taste coding.

Agrochemicals indirectly increase survival of E. coli O157:H7 and indicator bacteria by reducing ecosystem services.

PubMed

Staley, Zachery R; Rohr, Jason R; Senkbeil, Jacob K; Harwood, Valerie J

Storm water and agricultural runoff frequently contain agrochemicals, fecal indicator bacteria (FIB), and zoonotic pathogens. Entry of such contaminants into aquatic ecosystems may affect ecology and human health. This study tested the hypothesis that the herbicide atrazine and the fungicide chlorothalonil indirectly affect the survival of FIB (Escherichia coli and Enterococcus faecalis) and a pathogen (E. coli O157:H7) by altering densities of protozoan predators or by altering competition from autochthonous bacteria. Streptomycin-resistant E. coli, En. faecalis, and E. coli O157:H7 were added to microcosms composed of Florida river water containing natural protozoan and bacterial populations. FIB, pathogen, and protozoan densities were monitored over six days. Known metabolic inhibitors, cycloheximide and streptomycin, were used to inhibit autochthonous protozoa or bacteria, respectively. The inhibitors made it possible to isolate the effects of predation or competition on survival of allochthonous bacteria, and each treatment increased the survival of FIB and pathogens. Chlorothalonil's effect was similar to that of cycloheximide, significantly reducing protozoan densities and elevating densities of FIB and pathogens relative to the control. Atrazine treatment did not affect protozoan densities, but, through an effect on competition, resulted in significantly greater densities of En. faecalis and E. coli O157:H7. Hence, by reducing predaceous protozoa and bacterial competitors that facilitate purifying water bodies of FIBs and human pathogens, chlorothalonil and atrazine indirectly diminished an ecosystem service of fresh water.

Pharmacological modulation of lipopolysaccharide-induced pleural eosinophilia in the rat; a role for a newly generated protein.

PubMed

Bozza, P T; Castro-Faria-Neto, H C; Martins, M A; Larangeira, A P; Perales, J E; e Silva, P M; Cordeiro, R S

1993-06-01

Intrathoracic injection of endotoxin lipopolysaccharide, LPS into rats induced a dose-dependent increase in the number of eosinophils recovered from the pleural cavity. The pleural eosinophil accumulation peaked within 24-48 h, and returned to basal levels within 120 h. This phenomenon was accompanied by mononuclear cell infiltration, and preceded by massive neutrophil accumulation. Pretreatment with indomethacin, BW 755C (a dual cyclo/lipoxygenase inhibitor), BW A4C (a specific lipoxygenase inhibitor) or the platelet activating factor (PAF) antagonists WEB 2086 and PCA 4248 failed to inhibit the endotoxin-induced pleural eosinophilia, whilst dexamethasone (5-10 micrograms/cavity) or cycloheximide (14-28 micrograms/cavity) abolished this phenomenon. Transfer of the cell-free pleural washing from LPS-treated donor rats to normal recipient rats led to a two-fold increase in the eosinophil counts. Treatment of donors, but not recipients, with cycloheximide or dexamethasone inhibited the eosinophil accumulation induced by the pleural washings, indicating that the generation of the eosinophilotactic activity, but not its effects, depends on protein synthesis. This eosinophilotactic activity was maintained after lyophilization and heating (100 degrees C for 30 min), but was destroyed by trypsin. This substance has a molecular weight ranging between 10 and 50 kDa. The available data suggest that the late eosinophil accumulation induced by LPS is independent of arachidonic acid metabolites and PAF, and probably depends on a newly generated heat-stable soluble protein.

Endosymbiosis in trypanosomatid protozoa: the bacterium division is controlled during the host cell cycle

PubMed Central

Catta-Preta, Carolina M. C.; Brum, Felipe L.; da Silva, Camila C.; Zuma, Aline A.; Elias, Maria C.; de Souza, Wanderley; Schenkman, Sergio; Motta, Maria Cristina M.

2015-01-01

Mutualism is defined as a beneficial relationship for the associated partners and usually assumes that the symbiont number is controlled. Some trypanosomatid protozoa co-evolve with a bacterial symbiont that divides in coordination with the host in a way that results in its equal distribution between daughter cells. The mechanism that controls this synchrony is largely unknown, and its comprehension might provide clues to understand how eukaryotic cells evolved when acquiring symbionts that later became organelles. Here, we approached this question by studying the effects of inhibitors that affect the host exclusively in two symbiont-bearing trypanosomatids, Strigomonas culicis and Angomonas deanei. We found that inhibiting host protein synthesis using cycloheximide or host DNA replication using aphidicolin did not affect the duplication of bacterial DNA. Although the bacteria had autonomy to duplicate their DNA when host protein synthesis was blocked by cycloheximide, they could not complete cytokinesis. Aphidicolin promoted the inhibition of the trypanosomatid cell cycle in the G1/S phase, leading to symbiont filamentation in S. culicis but not in A. deanei. Treatment with camptothecin blocked the host protozoa cell cycle in the G2 phase and induced the formation of filamentous symbionts in both species. Oryzalin, which affects host microtubule polymerization, blocked trypanosomatid mitosis and abrogated symbiont division. Our results indicate that host factors produced during the cell division cycle are essential for symbiont segregation and may control the bacterial cell number. PMID:26082757

[Cladophilaphora bantiana brain abscess treated with voriconazole in an immunocompetent patient].

PubMed

Atalay, Mustafa Altay; Koç, Ayşe Nedret; Koyuncu, Sümeyra; Ulu Kiliç, Ayşegül; Kurtsoy, Ali; Alp Meşe, Emine

2014-07-01

Phaeohyphomycosis is a term used to define infections caused by darkly pigmented fungi with septate hyphae which contain melanin in their cell walls. Although fungi rarely cause central nervous system (CNS) infections, the incidence of CNS infections caused by melanin-containing fungi has been increasing in the recent years. Cladophialophora bantiana is the most frequently isolated species from cerebral phaeohyphomycosis. It mostly affects adult men in the second and third decade of life and about half of the cases occurs in immunocompetent patients. In this report, the isolation of C.bantiana from brain tissue of an immunocompetent patient who was operated with the initial diagnosis of a brain abscess, was presented. A 27 year-old male patient presenting without any chronic disease was admitted to the emergency department of our hospital with the complaints of persistent headache and diplopia. Magnetic resonance imaging (MRI) showed a space-occupying lesion in the right parietal lobe and left frontal lobe. Brain abscess was diagnosed in the patient who was referred to the neurosurgery department. Treatment was initiated with ceftriaxone and metronidazole. The abscess material sent for direct microscopic examination in the mycology laboratory was stained with Gram and Giemsa and cultured in the Sabouraud dextrose agar medium (SDA) with and without antibiotics (cycloheximide and chloramphenicol). Then, it was incubated at 37°C and 25°C. Direct examination and staining revealed a septate hyphae. The patient who received liposomal amphotericin B was referred to the infectious diseases department. Surface colors of all media including SDA with cycloheximide were olive-gray to black and contained velvety colonies. Lemon-like very long and integrated chains of conidium with poor branching in cornmeal Tween 80 agar, as well as growth at 42°C in passages, positive urease test result and cycloheximide resistance suggested C.bantiana. The isolate was confirmed as C. bantiana based on its DNA sequence analysis. Minimum inhibitor concentration (MIC) values for amphotericin B, voriconazole, caspofungin, and posaconazole were 2 µg/ml, 0.03 µg/ml, 0.03 µg/ml and 0.03 µg/ml, respectively. Liposomal amphotericin B was replaced with voriconazole due to the antifungal susceptibility profile. The patient who was symptom-free was discharged at 24 days after hospitalization with oral voriconazole treatment. In conclusion, cerebral phaeohyphomycosis should be considered in immunocompetent individuals. Given the fact that early diagnosis saves lives, such specimens should promptly be sent for mycological analysis.

Phytochelatin synthesis and glutathione levels in response to heavy metals in tomato cells.

PubMed

Scheller, H V; Huang, B; Hatch, E; Goldsbrough, P B

1987-12-01

Cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, produce phytochelatins (poly[gamma-glutamylcysteinyl]glycines) when exposed to cadmium. The synthesis of these peptides is accompanied by a decline in cellular levels of glutathione. Buthionine sulfoximine, an inhibitor of glutathione synthesis, inhibits the sustained production of phytochelatins. However, phytochelatin synthesis can occur in the presence of buthionine sulfoximine provided that sufficient glutathione is available. These results indicate that glutathione is a substrate for phytochelatin synthesis. The protein synthesis inhibitor cycloheximide does not affect the initial production of phytochelatin.

Light-Induced Nuclear Synthesis of Spinach Chloroplast Fructose-1,6-bisphosphatase 1

PubMed Central

Chueca, Ana; Lázaro, Juan José; Gorgé, Julio López

1984-01-01

Etiolated spinach (Spinacia oleracea L. var Winter Giant) seedlings show a residual photosynthetic fructose-1,6-bisphosphatase activity, which sharply rises under illumination. This increase in activity is due to a light-induced de novo synthesis, as it has been demonstrated by enzyme labeling experiments with 2H2O and [35S]methionine. The rise of bisphosphatase activity under illumination is strongly inhibited by cycloheximide, but not by the 70S ribosome inhibitor lincocin, which shows the nuclear origin of this chloroplastic enzyme. Images Fig. 3 PMID:16663662

Prevalence of Pityrosporum orbiculare on normal skin of Iraqi children.

PubMed

Sharquie, K A; Al-Kubaisy, W A; Al-Rubaey, M C

2005-05-01

To determine the age incidence and prevalence of Pityrosporum orbiculare on the normal skin of healthy Iraqi children, we carried out a survey of clinically normal skin of 110 healthy children in Baghdad during April 1998-October 1998. We isolated P. orbiculare on a peptone--glucose--yeast extract medium containing chloramphenicol and cycloheximide, and overlaid with olive oil. The organism was present on the trunk in 77.5% of children 10-14 years, 27.5% of children 5-9 years, and 6.6% of children < 5 years.

Neither Aurora B activity nor histone H3 phosphorylation is essential for chromosome condensation during meiotic maturation of porcine oocytes.

PubMed

Jelínková, Lucie; Kubelka, Michal

2006-05-01

Aurora kinase B (AURKB) is a chromosomal passenger protein that is essential for a number of processes during mitosis. Its activity is regulated by association with two other passenger proteins, INCENP and Survivin, and by phosphorylation on Thr 232. In this study, we examine expression and phosphorylation on Thr-232 of AURKB during meiotic maturation of pig oocytes in correlation with histone H3 phosphorylation and chromosome condensation. We show that histone H3 phosphorylation on Ser-10, but not on Ser-28, correlates with progressive chromosome condensation during oocyte maturation; Ser-10 phosphorylation starts around the time of the breakdown of the nuclear envelope, with the maximal activity in metaphase I, whereas Ser-28 phosphorylation does not significantly change in maturing oocytes. Treatment of oocytes with 50 microM butyrolactone I (BL-I), an inhibitor of cyclin-dependent kinases, or cycloheximide (10 microg/ml), inhibitor of proteosynthesis, results in a block of oocytes in the germinal vesicle stage, when nuclear membrane remains intact; however, condensed chromosome fibers or highly condensed chromosome bivalents can be seen in the nucleoplasm of BL-I- or cycloheximide-treated oocytes, respectively. In these treated oocytes, no or only very weak AURKB activity and phosphorylation of histone H3 on Ser-10 can be detected after 27 h of treatment, whereas phosphorylation on Ser-28 is not influenced. These results suggest that AURKB activity and Ser-10 phosphorylation of histone H3 are not required for chromosome condensation in pig oocytes, but might be required for further processing of chromosomes during meiosis.

Defining the Timing of Action of Antimalarial Drugs against Plasmodium falciparum

PubMed Central

Langer, Christine; Goodman, Christopher D.; McFadden, Geoffrey I.

2013-01-01

Most current antimalarials for treatment of clinical Plasmodium falciparum malaria fall into two broad drug families and target the food vacuole of the trophozoite stage. No antimalarials have been shown to target the brief extracellular merozoite form of blood-stage malaria. We studied a panel of 12 drugs, 10 of which have been used extensively clinically, for their invasion, schizont rupture, and growth-inhibitory activity using high-throughput flow cytometry and new approaches for the study of merozoite invasion and early intraerythrocytic development. Not surprisingly, given reported mechanisms of action, none of the drugs inhibited merozoite invasion in vitro. Pretreatment of erythrocytes with drugs suggested that halofantrine, lumefantrine, piperaquine, amodiaquine, and mefloquine diffuse into and remain within the erythrocyte and inhibit downstream growth of parasites. Studying the inhibitory activity of the drugs on intraerythrocytic development, schizont rupture, and reinvasion enabled several different inhibitory phenotypes to be defined. All drugs inhibited parasite replication when added at ring stages, but only artesunate, artemisinin, cycloheximide, and trichostatin A appeared to have substantial activity against ring stages, whereas the other drugs acted later during intraerythrocytic development. When drugs were added to late schizonts, only artemisinin, cycloheximide, and trichostatin A were able to inhibit rupture and subsequent replication. Flow cytometry proved valuable for in vitro assays of antimalarial activity, with the free merozoite population acting as a clear marker for parasite growth inhibition. These studies have important implications for further understanding the mechanisms of action of antimalarials, studying and evaluating drug resistance, and developing new antimalarials. PMID:23318799

Low-level laser therapy prevents endothelial cells from TNF-α/cycloheximide-induced apoptosis.

PubMed

Chu, Yu-Hsiu; Chen, Shu-Ya; Hsieh, Yueh-Ling; Teng, Yi-Hsien; Cheng, Yu-Jung

2018-02-01

Low-level laser therapy (LLLT), widely used in physiotherapy, has been known to enhance wound healing and stimulate cell proliferation, including fibroblast and endothelial cells. Applying LLLT can increase cell proliferation in many kinds of cells including fibroblasts and endothelial cells. However, the protective mechanisms of LLLT on endothelial apoptosis remain unclear. We hypothesized LLLT can protect endothelial cells from inflammation-induced apoptosis. Human endothelial cell line, EA.hy926 cells, and TNF-α/cycloheximide (TNF/CHX) were used to explore the protective effects of LLLT (660 nm) on inflammation-induced endothelial apoptosis. Cell viability, apoptosis, caspase-3/7/8/9 activity, MAPKs signaling, NF-κB activity, and inducible/endothelial nitric oxide synthase (iNOS/eNOS) expression were measured. Our results showed that LLLT increased EA.hy926 cell proliferation, attenuated the TNF/CHX-induced apoptosis, and reduced the TNF/CHX-mediated caspase-3/7/8/9 activation. In addition, LLLT increased ERK MAPK phosphorylation and suppressed the TNF/CHX-increased p38 MAPK, JNK, IKK phosphorylation, NF-κB translocation, and iNOS expression. The caspases-3 cleavage and cell death were not increased in cells treating with ERK inhibitor U0126, which implicated that ERK is not to be responsible for the protective effects of LLLT. After treating with p38 mitogen-activated protein kinase (MAPK) activator, the protection of LLLT in cell apoptosis was no longer existed, showing that LLLT protected the endothelial cells by suppressing p38 MAPK signaling. Our results provide a new insight into the possible molecular mechanisms in which LLLT protects against inflammatory-induced endothelial dysfunction.

Use of inhibitors for coastal bacteria and phytoplankton: Application to nitrogen uptake measurement

NASA Astrophysics Data System (ADS)

Trottet, Aurore; Fouilland, Eric; Leboulanger, Christophe; Lanouguère, Elodie; Bouvy, Marc

2011-06-01

For several decades, prokaryotic and eukaryotic inhibitors have been used to exclude bacteria from microalgal cultures and for investigating prey-predator relationships. Recently there has been considerable interest in using specific inhibitors for studying the interactions between bacteria and phytoplankton, by selective repression of either organism's activity. The effectiveness of chemical inhibitors must be tested before applying them to natural communities to partition metabolic activities between functional groups. Six different antibiotics selected from the most commonly reported in the literature were tested, at concentrations varying from 12.5 to 100 mg L -1, for their effect on bacterial growth and functional diversity of natural communities from Mediterranean coastal waters. Penicillin and streptomycin each at a final concentration of 100 mg L -1 significantly reduced bacterial growth within 2 h. There was a greater impact on bacterial functional diversity when both antibiotics were mixed together. This mixture did not have any significant effect on the growth of selected cultured phytoplankton strains, whereas the eukaryote inhibitor cycloheximide at 100 mg L -1 reduced growth within 2 h of incubation. The penicillin-streptomycin mixture and cycloheximide alone successfully partitioned NH 4+ and NO 3- uptake between bacteria and phytoplankton bi-weekly sampled in a coastal lagoon in Autumn, where bacterial contribution to total NH 4+ and NO 3- uptake averaged 46 and 41%, respectively. The use of specific inhibitors may be a valuable method for studying interactions, such as competition and mutualism, or lack of interaction between the different components of microbial communities and could be used to study their relative importance in biogeochemical fluxes.

Oxygen requirements for formation and activity of the squalene expoxidase in Saccharomyces cerevisiae

NASA Technical Reports Server (NTRS)

Jahnke, L.; Klein, H. P.

1983-01-01

The effect of oxygen on squalene epoxidase activity in Saccharomyces cerevisiae was investigated. In cells grown in standing cultures, the epoxidase was localized mainly in the 'mitochondrial' fraction. Upon aeration, enzyme activity increased and the newly formed enzyme was associated with the 'microsomal' fraction. At 0.03 percent (vol/vol) oxygen, epoxidase levels doubled, whereas the ergosterol level was only slightly increased. Cycloheximide inhibited the increase in epoxidase under these conditions. An apparent K sub m for oxygen of 0.38 percent (vol/vol) was determined from a crude particulate preparation for the epoxidase.

Effect of Gibberellic Acid on the Plasticity and Elasticity of Avena Stem Segments 1

PubMed Central

Adams, Paul A.; Montague, Michael J.; Tepfer, Mark; Rayle, David L.; Ikuma, Hiroshi; Kaufman, Peter B.

1975-01-01

Extensibility characteristics of Avena stem segments treated with gibberellic acid (GA) were investigated in living internodes using a microgrowth method and in partially extracted cell walls subjected to Instron extensometer analysis. Both techniques showed that treatment with GA greatly increases internodal plasticity, but has virtually no effect on internodal elasticity. The increase in plasticity occurred 1 to 2 hours after the initiation of hormone treatment, which is similar to the time of onset of GA-enhanced growth and cell wall synthesis. Cycloheximide was shown to inhibit the effect of GA on plasticity. PMID:16659388

Osimertinib (AZD9291) decreases programmed death ligand-1 in EGFR-mutated non-small cell lung cancer cells.

PubMed

Jiang, Xiao-Ming; Xu, Yu-Lian; Huang, Mu-Yang; Zhang, Le-Le; Su, Min-Xia; Chen, Xiuping; Lu, Jin-Jian

2017-11-01

Osimertinib (AZD9291) is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that has been approved for the treatment of EGFR-mutated non-small cell lung cancer (NSCLC). In NSCLC patients, an EGFR mutation is likely to be correlated with high levels of expression of programmed death ligand-1 (PD-L1). Here, we showed that osimertinib decreased PD-L1 expression in human EGFR mutant NSCLC cells in vitro. Osimertinib (125 nmol/L) markedly suppressed PD-L1 mRNA expression in both NCI-H1975 and HCC827 cells. Pretreatment with the N-linked glycosylation inhibitor tunicamycin, osimertinib clearly decreased the production of new PD-L1 protein probably due to a reduction in mRNA. After blocking transcription and translation processes with actinomycin D and cycloheximide, respectively, osimertinib continued to reduce the expression of PD-L1, demonstrating that osimertinib might degrade PD-L1 at the post-translational level, which was confirmed by a cycloheximide chase assay, revealing that osimertinib (125 nmol/L) decreased the half-life of PD-L1 from approximately 17.8 h and 13.8 h to 8.6 h and 4.6 h, respectively, in NCI-H1975 and HCC827 cells. Pretreatment with the proteasome inhibitors (MG-132 or bortezomib) blocked the osimertinib-induced degradation of PD-L1, but an inhibitor of autophagy (chloroquine) did not. In addition, inhibition of GSK3β by LiCl prevented osimertinib-induced PD-L1 degradation. The results demonstrate that osimertinib reduces PD-L1 mRNA expression and induces its protein degradation, suggesting that osimertinib may reactivate the immune activity of T cells in the tumor microenvironment in EGFR-mutated NSCLC patients.

Retinoic acid stimulates interstitial collagenase messenger ribonucleic acid in osteosarcoma cells

NASA Technical Reports Server (NTRS)

Connolly, T. J.; Clohisy, J. C.; Shilt, J. S.; Bergman, K. D.; Partridge, N. C.; Quinn, C. O.

1994-01-01

The rat osteoblastic osteosarcoma cell line UMR 106-01 secretes interstitial collagenase in response to retinoic acid (RA). The present study demonstrates by Northern blot analysis that RA causes an increase in collagenase messenger RNA (mRNA) at 6 h, which is maximal at 24 h (20.5 times basal) and declines toward basal level by 72 h. This stimulation is dose dependent, with a maximal response at 5 x 10(-7) M RA. Nuclear run-on assays show a greater than 20-fold increase in the rate of collagenase mRNA transcription between 12-24 h after RA treatment. Cycloheximide blocks RA stimulation of collagenase mRNA, demonstrating the need for de novo protein synthesis. RA not only causes an increase in collagenase secretion, but is known to decrease collagen synthesis in UMR 106-01 cells. In this study, the increase in collagenase mRNA is accompanied by a concomitant decrease in the level of alpha 1(I) procollagen mRNA, which is maximal at 24 h (70% decrease), with a return to near-control levels by 72 h. Nuclear run-on assays demonstrated that the decrease in alpha 1 (I) procollagen expression does not have a statistically significant transcriptional component. RA did not statistically decrease the stability of alpha 1 (I) procollagen mRNA (calculated t1/2 = 8.06 +/- 0.30 and 9.01 +/- 0.62 h in the presence and absence of RA, respectively). However, transcription and stability together probably contribute to the major decrease in stable alpha 1 (I) procollagen mRNA observed. Cycloheximide treatment inhibits basal level alpha 1 (I) procollagen mRNA accumulation, demonstrating the need for on-going protein synthesis to maintain basal expression of this gene.

(+/-)-3-[4-(2-dimethylamino-1-methylethoxy)-phenyl]-1H-pyrazolo[3,4- B]pyridine-1-acetic acid (Y-25510) stimulates production of IL-1 beta and IL-6 at the level of messenger RNA expression in cultured human monocytes.

PubMed

Kusuhara, H; Komatsu, H; Hisadome, M; Ikeda, Y

1996-12-01

(+/-)-3-[4-(2-Dimethylamino-1-methylethoxy)phenyl]-1H-pyrazolo[3, 4-b]pyridine-1-acetic acid (Y-25510) stimulated the mRNA expression for interleukin-1 beta (IL-1 beta), and enhanced the expression induced by lipopolysaccharide (LPS) in cultured human peripheral blood mononuclear cells (PBMC) and THP-1 cells, a cell-line derived from human monocytic leukemia. Y-25510 also stimulated the mRNA expression for IL-6 in both types of the cells, however, the stimulation required the presence of LPS. In THP-1 cells, the stimulation of IL-1 beta mRNA expression by Y-25510 was suppressed by cycloheximide, an inhibitor of protein synthesis. This phenomenon indicates that the stimulation requires de norv protein synthesis. In contrast, the stimulation of mRNA expression for IL-6 by Y-25510 was not suppressed by cycloheximide but suppressed by N alpha-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of nuclear transcription factor-kappa B (NF-kappa B) activation, in the presence of LPS, suggesting that the stimulation requires NF-kappa activation. These results demonstrate that Y-25510 stimulates the mRNA expression for IL-1 beta and IL-6 by different mechanisms. Dexamethasone suppressed the LPS-induced expression of mRNA for IL-1 beta and IL-6 in THP-1 cells, whereas the drug never suppressed the mRNA expression for these cytokines in the presence of Y-25510. The result indicates that Y-25510 stimulates the mRNA expression for IL-1 beta and IL-6 by different mechanisms from those of LPS.

Generation and Characterization of Human Heme Oxygenase-1 Transgenic Pigs

PubMed Central

Yang, Jaeseok; Cho, Bumrae; Hwang, Jong-Ik; Park, Sol Ji; Hurh, Sunghoon; Kim, Hwajung; Lee, Eun Mi; Ro, Han; Kang, Jung Taek; Kim, Su Jin; Won, Jae-Kyung; O'Connell, Philip J.; Kim, Hyunil; Surh, Charles D.; Lee, Byeong-Chun; Ahn, Curie

2012-01-01

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation. PMID:23071605

Evaluation of a 99mTc-labeled AnnexinA5 variant for non-invasive SPECT imaging of cell death in liver, spleen and prostate.

PubMed

Greupink, Rick; Sio, Charles F; Ederveen, Antwan; Orsel, Joke

2009-12-01

We investigate radio-labeling and pharmacokinetics of a new AnnexinA5 variant (HYNIC-cys-AnxA5) and then assess its utility for the non-invasive detection of cell death in liver, spleen and prostate. AnnexinA5 binds to phosphatidylserine expressed on the surface of apoptotic and necrotic cells. Contrary to other AnnexinA5 variants, the new cys-AnxA5 allows for site-specific conjugation of a hydrazinonicotinamide-maleimide moiety and subsequent radio-labeling with (99m)Tc at a position not involved in the AnxA5-phosphatidylserine interaction. Distribution of (99m)Tc-HYNIC-cys-AnxA5 was studied in rats, both invasively and via SPECT/CT. Cycloheximide was used to induce cell death in liver and spleen, whereas apoptosis in the prostate was induced by castration. HYNIC-cys-AnxA5 was efficiently and reproducibly labeled with (99m)Tc. Blood clearance of radioactivity after iv-injection was adequately described by a two-compartment model, the renal cortex representing the main site of accumulation. Cycloheximide treatment resulted in increased accumulation of intravenous-injected (99m)Tc-HYNIC-cys-AnxA5 in liver and spleen over controls, which correlated well with TUNEL staining for cell death in corresponding tissue sections. However, the increase in TUNEL-positive prostate epithelial cells observed following castration was not paralleled by greater (99m)Tc-HYNIC-cys-AnxA5 accumulation. (99m)Tc-HYNIC-cys-AnxA5 appears a suitable tracer for assessment of cell death in liver and spleen, but not prostate.

Effects of the novel compound aniracetam (Ro 13-5057) upon impaired learning and memory in rodents.

PubMed

Cumin, R; Bandle, E F; Gamzu, E; Haefely, W E

1982-01-01

The effect of aniracetam (Ro 13-5057, 1-anisoyl-2-pyrrolidinone) was studied on various forms of experimentally impaired cognitive functions (learning and memory) in rodents and produced the following effects: (1) almost complete prevention of the incapacity to learn a discrete escape response in rats exposed to sublethal hypercapnia immediately before the acquisition session; (2) partial (rats) or complete (mice) prevention of the scopolamine-induced short-term amnesia for a passive avoidance task; (3) complete protection against amnesia for a passive avoidance task in rats submitted to electroconvulsive shock immediately after avoidance acquisition; (4) prevention of the long-term retention- or retrieval-deficit for a passive avoidance task induced in rats and mice by chloramphenicol or cycloheximide administered immediately after acquisition; (5) reversal, when administered as late as 1 h before the retention test, of the deficit in retention or retrieval of a passive avoidance task induced by cycloheximide injected 2 days previously; (6) prevention of the deficit in the retrieval of an active avoidance task induced in mice by subconvulsant electroshock or hypercapnia applied immediately before retrieval testing (24 h after acquisition). These improvements or normalizations of impaired cognitive functions were seen at oral aniracetam doses of 10-100 mg/kg. Generally, the dose-response curves were bell-shaped. The mechanisms underlying the activity of aniracetam and its 'therapeutic window' are unknown. Piracetam, another pyrrolidinone derivative was used for comparison. It was active only in six of nine tests and had about one-tenth the potency of aniracetam. The results indicate that aniracetam improves cognitive functions which are impaired by different procedure and in different phases of the learning and memory process.

Biodegradation of zearalenone by Saccharomyces cerevisiae: Possible involvement of ZEN responsive proteins of the yeast.

PubMed

Zhang, Hongyin; Dong, Manjia; Yang, Qiya; Apaliya, Maurice Tibiru; Li, Jun; Zhang, Xiaoyun

2016-06-30

The mycotoxin zearalenone, also known as F-2 mycotoxin or RAL is a potent estrogenic metabolite produced by some Gibberella and Fusarium species. It is a common contaminant of cereal crops, livestock and poultry products. However, detoxification of zearalenone (ZEN) remains a challenge. Recently, biological approach for ZEN detoxification is being explored. In this study, we investigated the biodegradation of ZEN by using Saccharomyces cerevisiae and the possible mechanisms involved. The findings revealed that, after 48h of incubation of S. cerevisiae in combination with ZEN, the ZEN was completely degraded by S. cerevisiae. On the contrary, heat-killed cells and cell-free culture filtrates of S. cerevisiae could not degrade ZEN. Furthermore, addition of cycloheximide to S. cerevisiae combined with ZEN at time 0h prevented ZEN degradation, while addition of cycloheximide at 12h significantly slowed down degradation. The results also indicated cellular proteomics of S. cerevisiae. Several differential proteins were identified, most of which were related to basic metabolism. The findings revealed that, after 48h of incubating ZEN together with S. cerevisiae, ZEN was completely degraded by S. cerevisiae. The mechanisms involved in the degradation of ZEN by S. cerevisiae may be the production of associated intracellular and extracellular enzymes, which have the ability to degrade ZEN. In addition, there were some functional proteins produced by S. cerevisiae, indicating that the basic metabolism of S. cerevisiae was improved when ZEN was added. This novel discovery by the authors, will greatly contribute to the field of biodegradation of mycotoxin by antagonists. The authors also believed this innovation will open the grounds for further research and improvement of S. cerevisiae in the field of biodegradation. Copyright © 2016 Elsevier B.V. All rights reserved.

Heparin-benzyl alcohol enhancement of biofilms formation and antifungal susceptibility of vaginal Candida species isolated from pregnant and nonpregnant Saudi women

PubMed Central

Al-akeel, Raid A; El-kersh, Talat A; Al-Sheikh, Yazeed A; Al-Ahmadey, Ziab Zakey

2013-01-01

Biofilm formation by Candida species is a major contribute to their pathogenic potential.The aim of this study was to determine in vitro effects of EDTA, cycloheximide, and heparin-benzyl alcohol preservative on C. albicans (126) and non-albicans (31)vaginal yeast isolates biofilm formations and their susceptibility against three antifungal Etest strips. Results of the crystal violet-assay, indicated that biofilms formation were most commonly observed [100%] for C. kefyr, C. utilis, C. famata, and Rhodotorula mucilaginosa, followed by C. glabrata [70%], C. tropicalis [50%], C. albicans [29%], Saccharomyces cerevisiae [0.0%]. EDTA (0.3mg/ml) significantly inhibited biofilm formation in both C. albicans and non-albicans isolates (P=0.0001) presumably due to chelation of necessary metal cations for the process-completion. In contrast, heparin (-benzyl alcohol preservative) stimulated biofilm formation in all tested isolates, but not at significant level (P=0.567). Conversely, cycloheximide significantly (P=0.0001) inhibited biofilm formation in all C. albicans strains(126) and its effect was even 3 fold more pronounced than EDTA inhibition, probably due to its attenuation of proteins (enzymes) and/or complex molecules necessary for biofilm formation. Results also showed that all nonalbicans yeasts isolates were susceptible to 5-flucytosine (MIC50, 0.016 µg/ml; MIC90, 0.064 µg/ml), but 14% of C. albicans isolates were resistant (MIC50, 0.064 µg/ml; MIC90 >32 µg/ml). The MIC50 value of amphotricin B for all C. albicans and non-albicans isolates was at a narrow range of 0.023 µg /ml, and the MIC90 values were 0.047 µg/ml and 0.064 µg/ml respectively, thereby confirming its efficacy as a first line empiric- treatment of Candida spp infections. PMID:23750080

Generation and characterization of human heme oxygenase-1 transgenic pigs.

PubMed

Yeom, Hye-Jung; Koo, Ok Jae; Yang, Jaeseok; Cho, Bumrae; Hwang, Jong-Ik; Park, Sol Ji; Hurh, Sunghoon; Kim, Hwajung; Lee, Eun Mi; Ro, Han; Kang, Jung Taek; Kim, Su Jin; Won, Jae-Kyung; O'Connell, Philip J; Kim, Hyunil; Surh, Charles D; Lee, Byeong-Chun; Ahn, Curie

2012-01-01

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.

Use of Antibiotics for Maintenance of Axenic Cultures of Amphidinium carterae for the Analysis of Translation

PubMed Central

Liu, Chieh-Lun; Place, Allen R.; Jagus, Rosemary

2017-01-01

Most dinoflagellates in culture are bacterized, complicating the quantification of protein synthesis, as well as the analysis of its regulation. In bacterized cultures of Amphidinium carterae Hulbert, up to 80% of protein synthetic activity appears to be predominantly bacterial based on responses to inhibitors of protein synthesis. To circumvent this, axenic cultures of A. carterae were obtained and shown to respond to inhibitors of protein synthesis in a manner characteristic of eukaryotes. However, these responses changed with time in culture correlating with the reappearance of bacteria. Here we show that culture with kanamycin (50 μg/mL), carbenicillin (100 μg/mL), and streptomycin sulfate (50 μg/mL) (KCS), but not 100 units/mL of penicillin and streptomycin (PS), prevents the reappearance of bacteria and allows A. carterae protein synthesis to be quantified without the contribution of an associated bacterial community. We demonstrate that A. carterae can grow in the absence of a bacterial community. Furthermore, maintenance in KCS does not inhibit the growth of A. carterae cultures but slightly extends the growth phase and allows accumulation to somewhat higher saturation densities. We also show that cultures of A. carterae maintained in KCS respond to the eukaryotic protein synthesis inhibitors cycloheximide, emetine, and harringtonine. Establishment of these culture conditions will facilitate our ability to use polysome fractionation and ribosome profiling to study mRNA recruitment. Furthermore, this study shows that a simple and fast appraisal of the presence of a bacterial community in A. carterae cultures can be made by comparing responses to cycloheximide and chloramphenicol rather than depending on lengthier culture-based assessments. PMID:28763019

Reversal of cycloheximide-induced memory disruption by AIT-082 (Neotrofin) is modulated by, but not dependent on, adrenal hormones.

PubMed

Yan, Rongzi; Nguyen, Quang; Gonzaga, James; Johnson, Mai; Ritzmann, Ronald F; Taylor, Eve M

2003-04-01

AIT-082 (Neotrofin), a hypoxanthine derivative, has been shown to improve memory in both animals and humans. In animals, adrenal hormones modulate the efficacy of many memory-enhancing compounds, including piracetam and tacrine (Cognex). To investigate the role of adrenal hormones in the memory-enhancing action of AIT-082. Plasma levels of adrenal hormones (corticosterone and aldosterone) in mice were significantly reduced by surgical or chemical (aminoglutethimide) adrenalectomy or significantly elevated by oral administration of corticosterone. The effects of these hormone level manipulations on the memory-enhancing activity of AIT-082 and piracetam were evaluated using a cycloheximide-induced amnesia/passive avoidance model. As previously reported by others, the memory enhancing action of piracetam was abolished by adrenalectomy. In contrast, the memory enhancement by 60 mg/kg AIT-082 (IP) was unaffected. However, a sub-threshold dose of AIT-082 (0.1 mg/kg, IP) that did not improve memory in control animals did improve memory in adrenalectomized animals. These data suggested that, similar to piracetam and tacrine, the memory enhancing action of AIT-082 might be inhibited by high levels of adrenal hormones. As expected, corticosterone (30 and 100 mg/kg) inhibited the action of piracetam, however no dose up to 100 mg/kg corticosterone inhibited the activity of AIT-082. These data suggest that while AIT-082 function is not dependent on adrenal hormones, it is modulated by them. That memory enhancement by AIT-082 was not inhibited by high plasma corticosterone levels may have positive implications for its clinical utility, given that many Alzheimer's disease patients have elevated plasma cortisol levels.

Cycloheximide and actinomycin D delay death and affect bcl-2, bax, and Ice gene expression in astrocytes under in vitro ischemia.

PubMed

Yu, Albert Cheung Hoi; Yung, Hon Wa; Hui, Michael Hung Kit; Lau, Lok Ting; Chen, Xiao Qian; Collins, Richard A

2003-10-15

An in vitro ischemia model was established and the effect of the metabolic inhibitors cycloheximide (CHX) and actinomycin D (ActD) on apoptosis in astrocytes under ischemia studied. CHX decreased by 75% the number of cells dying after 6 hr of ischemia compared with control cultures. TdT-mediated dUTP nick end labelling (TUNEL) staining of comparable cultures was reduced by 40%. ActD decreased cell death by 60% compared with controls. The number of TUNEL-positive cells was reduced by 38%. The nuclear shrinkage in TUNEL-positive astrocytes in control cultures did not occur in ActD-treated astrocytes, indicating that nuclear shrinkage and DNA fragmentation during apoptosis are two unrelated processes. Expression of bcl-2 (alpha and beta), bax, and Ice in astrocytes under similar ischemic conditions, as measured by quantitative reverse transcription-polymerase chain reaction, indicated that ischemia down-regulated bcl-2 (alpha and beta) and bax. Ice was initially down-regulated from 0 to 4 hr, before returning to control levels after 8 hr of ischemia. ActD decreased the expression of these genes. CHX reduced the expression of bcl-2 (alpha and beta) but increased bax and Ice expression. It is hypothesized that the balance of proapoptotic (Bad, Bax) and antiapoptotic (Bcl-2, Bcl-Xl) proteins determines apoptosis. The data suggest that the ratio of Bcl-2/Bad in astrocytes following ActD and CHX treatment does not decrease as much in untreated cells during ischemia. Our data indicate that it is the ratio of Bcl-2 family members that plays a critical role in determining ischemia-induced apoptosis. It is also important to note that ischemia-induced apoptosis involves the regulation of RNA and protein synthesis. Copyright 2003 Wiley-Liss, Inc.

Thyroid hormone stimulates progesterone release from human luteal cells by generating a proteinaceous factor.

PubMed

Datta, M; Roy, P; Banerjee, J; Bhattacharya, S

1998-09-01

Blood samples collected from 29 women (aged between 19 and 35 years) during the luteal phase of the menstrual cycle (between days 18 and 23 of the cycle) showed that deficiency in thyroid hormone level is related to a decrease in progesterone (P4) secretion. To observe the effect of thyroid hormone on human ovarian luteal cells, 3,5,3'-triiodothyronine (T3; 125 ng/ml) was added to luteal cells in vitro. T3 significantly stimulated progesterone release (P < 0.01) from luteal cells and this could be blocked by cycloheximide, indicating a protein mediator for the T3 effect. The T3 stimulatory effect was inhibited by anti-T3 antibody suggesting specificity of T3 action. Addition of T3 caused a more than threefold increase in cellular protein synthesis which was inhibited by cycloheximide. Preparation of partially purified thyroid hormone-induced factor (TIF) (from peak II of Sephadex G 100 chromatography of T3-incubated cells), and its addition to luteal cell incubations caused a significant increase in P4 release (P < 0.05). Incubation with trypsin or treatment with heat destroyed the stimulatory effect of TIF on P4 release, indicating the proteinaceous nature of TIF. Purified thyroid hormone-induced protein. (TIP) from rat granulosa cells and fish ovarian follicles greatly stimulated P4 release from human luteal cells. These results suggest that T3 stimulation of P4 release from human luteal cells is not direct, but is mediated through a putative protein factor, which appears to be a protein conserved through evolution as far as its biological activity is concerned.

Evidence for an Inactivating System of Nitrate Reductase in Hordeum vulgare L. during Darkness That Requires Protein Synthesis 1

PubMed Central

Travis, R. L.; Jordan, W. R.; Huffaker, R. C.

1969-01-01

The disappearance of nitrate reductase activity in leaves of Hordeum vulgare L. during darkness was inhibited by cycloheximide, actinomycin D, and low temperature. Thus, protein synthesis was probably required for the disappearance of nitrate reductase in the dark. Since chloramphenicol did not affect the rate of loss of activity, the degradation or inactivation apparently required protein synthesis by the cytoplasmic ribosomal system. Consistent with this observation, nitrate reductase is also reportedly located in the cytoplasm. Thus, the amount of nitrate reductase activity present in leaves of barley may be controlled by a balance between activating and inactivating systems. PMID:16657182

Model for Stress-induced Protein Degradation in Lemna minor1

PubMed Central

Cooke, Robert J.; Roberts, Keith; Davies, David D.

1980-01-01

Transfer of Lemna minor fronds to adverse or stress conditions produces a large increase in the rate of protein degradation. Cycloheximide partially inhibits stress-induced protein degradation and also partially inhibits the protein degradation which occurs in the absence of stress. The increased protein degradation does not appear to be due to an increase in activity of soluble proteolytic enzymes. Biochemical evidence indicates that stress, perhaps acting via hormones, affects the permeability of certain membranes, particularly the tonoplast. A general model for stress-induced protein degradation is presented in which changes in membrane properties allow vacuolar proteolytic enzymes increased access to cytoplasmic proteins. PMID:16661588

Granule swelling and cleavage of mitogen-activated protein kinases in human neutrophils undergoing apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kato, Takayuki, E-mail: tkato@med.osaka-cu.ac.jp; Ikemoto, Masaru; Hato, Fumihiko

2009-04-10

Extracellular signal-regulated kinase and p38 have been shown to be cleaved in human neutrophils undergoing apoptosis induced by tumor necrosis factor-{alpha} and cycloheximide. However, the cleavage products of these molecules were undetected when apoptotic neutrophils were pretreated with phenylmethylsulfonyl fluoride or disrupted by nitrogen cavitation before preparation of cell lysates. The electron microscopy revealed that granules in apoptotic neutrophils were significantly swollen than those in control cells. These findings suggest that granule membrane may become destabilized during neutrophil apoptosis, leading to rapid proteolysis of these molecules by granule-derived serine proteases during preparation of cell lysates with the conventional lysis buffer.

The role of nickel in urea assimilation by algae.

PubMed

Rees, T A; Bekheet, I A

1982-12-01

Nickel is required for urease synthesis by Phaeodactylum tricornutum and Tetraselmis subcordiformis and for growth on urea by Phaeodactylum. There is no requirement for nickel for urea amidolyase synthesis by Chlorella fusca var. vacuolata. Neither copper nor palladium can substitute for nickel but cobalt partially restored urease activity in Phaeodactylum. The addition of nickel to nickel-deficient cultures of Phaeodactylum or Tetraselmis resulted in a rapid increase of urease activity to 7-30 times the normal level; this increase was not inhibited by cycloheximide. It is concluded that nickel-deficient cells over-produce a non-functional urease protein and that either nickel or the functional urease enzyme participates in the regulation of the production of urease protein.

Nitrate Utilization by the Diatom Skeletonema costatum

PubMed Central

Serra, Juan L.; Llama, Maria J.; Cadenas, Eduardo

1978-01-01

Nitrate uptake has been studied in nitrogen-deficient cells of the marine diatom Skeletonema costatum. When these cells are incubated in the presence of nitrate, this ion is quickly taken up from the medium, and nitrite is excreted by the cells. Nitrite is excreted following classical saturation kinetics, its rate being independent of nitrate concentration in the incubation medium for nitrate concentration values higher than 3 micromolar. Nitrate uptake shows mixed-transfer kinetics, which can be attributed to the simultaneous contributions of mediated and diffusion transfer. Cycloheximide and p-hydroxymercuribenzoate inhibit the carrier-mediated contribution to nitrate uptake, without affecting the diffusion component. When cells are preincubated with nitrate, the net nitrogen uptake is increased. PMID:16660652

The effect of DNA replication on mutation of the Saccharomyces cerevisiae CDC8 gene.

PubMed

Zaborowska, D; Zuk, J

1990-04-01

Incubation in YPD medium under permissive conditions when DNA replication is going on, strongly stimulates the induction of cdc+ colonies of UV-irradiated cells of yeast strains HB23 (cdc8-1/cdc8-3), HB26 (cdc8-3/cdc8-3) and HB7 (cdc8-1/cdc8-1). Inhibition of DNA replication by hydroxyurea, araCMP, cycloheximide or caffeine or else by incubation in phosphate buffer pH 7.0, abolishes this stimulation. Thus the replication of DNA is strongly correlated with the high induction of cdc+ colonies by UV irradiation. It is postulated that these UV-induced cdc+ colonies arise as the result infidelity in DNA replication.

Gene expression as a sensitive endpoint to evaluate cell differentiation and maturation of the developing central nervous system in primary cultures of rat cerebellar granule cells (CGCs) exposed to pesticides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hogberg, Helena T.; Department of Physiology, Wenner-Gren Institute, Stockholm University; Kinsner-Ovaskainen, Agnieszka

The major advantage of primary neuronal cultures for developmental neurotoxicity (DNT) testing is their ability to replicate the crucial stages of neurodevelopment. In our studies using primary culture of cerebellar granule cells (CGCs) we have evaluated whether the gene expression relevant to the most critical developmental processes such as neuronal differentiation (NF-68 and NF-200) and functional maturation (NMDA and GABA{sub A} receptors), proliferation and differentiation of astrocytes (GFAP and S100{beta}) as well as the presence of neural precursor cells (nestin and Sox10) could be used as an endpoint for in vitro DNT. The expression of these genes was assessed aftermore » exposure to various pesticides (paraquat parathion, dichlorvos, pentachlorophenol and cycloheximide) that could induce developmental neurotoxicity through different mechanisms. All studied pesticides significantly modified the expression of selected genes, related to the different stages of neuronal and/or glial cell development and maturation. The most significant changes were observed after exposure to paraquat and parathion (i.e. down-regulation of mRNA expression of NF-68 and NF-200, NMDA and GABA{sub A} receptors). Similarly, dichlorvos affected mainly neurons (decreased mRNA expression of NF-68 and GABA{sub A} receptors) whereas cycloheximide had an effect on neurons and astrocytes, as significant decreases in the mRNA expression of both neurofilaments (NF-68 and NF-200) and the astrocyte marker (S100{beta}) were observed. Our results suggest that toxicity induced by pesticides that target multiple pathways of neurodevelopment can be identified by studying expression of genes that are involved in different stages of cell development and maturation, and that gene expression could be used as a sensitive endpoint for initial screening to identify the compounds with the potential to cause developmental neurotoxicity.« less

Oocyte transport: Developmental competence of bovine oocytes arrested at germinal vesicle stage by cycloheximide under air.

PubMed

Hashimoto, Shu; Kimura, Kouji; Iwata, Hisataka; Takakura, Ryo

2003-02-01

The effects of the medium (TCM 199 or SOFaa) and temperature (20 or 39 C) during meiotic arrest by cycloheximide (CHX) under air on the developmental competence of bovine oocytes after in vitro maturation (IVM) and fertilization (IVF) were investigated. Oocytes were maintained in meiotic arrest by 10 microg/ml CHX in a 50-microl droplet of 25-mM HEPES-buffered TCM 199 (H199) at 39 C or synthetic oviduct fluid (HSOFaa) at 20 or 39 C in air for 24 h. After release from the arrest, the oocytes was matured and fertilized in vitro and their developmental competence was examined. The developmental rate of oocytes arrested in HSOFaa at 20 C to the blastocyst stage was similar to that of non-arrested oocytes but was significantly higher (P<0.05) than that of oocytes arrested at 39 C in H199 or in HSOFaa. In consideration of oocyte transport conditions, we also investigated the meiotic arrest of oocytes maintained in a 0.25-ml straw by CHX individually with 10 microl HSOFaa or as a group (40-50 oocytes) with 170-200 microl HSOFaa at 20 C in air for 24 h. After release from meiotic arrest, the developmental competence of these oocytes was assessed similarly. The developmental rate of oocytes treated with CHX individually was similar to that of those treated with CHX in 50-microl droplet of HSOFaa at 20 C. However, the developmental rate of oocytes treated with CHX as a group was lower than that of oocytes treated with CHX in a 50-microl droplet. Five blastocysts developed from oocytes maintained in meiotic arrest in a plastic straw were transferred to five recipient heifers. Consequently, three recipients became pregnant and 2 calves were delivered. The results of the present study indicate that bovine oocytes treated with CHX in HSOFaa at 20 C under air retain the same developmental competence as non-arrested oocytes.

Mechanism for the decrease in the FIP1L1-PDGFRalpha protein level in EoL-1 cells by histone deacetylase inhibitors.

PubMed

Ishihara, Kenji; Kaneko, Motoko; Kitamura, Hajime; Takahashi, Aki; Hong, Jang Ja; Seyama, Toshio; Iida, Koji; Wada, Hiroshi; Hirasawa, Noriyasu; Ohuchi, Kazuo

2008-01-01

Acetylation and deacetylation of proteins occur in cells in response to various stimuli, and are reversibly catalyzed by histone acetyltransferase and histone deacetylase (HDAC), respectively. EoL-1 cells have an FIP1L1-PDGFRA fusion gene that causes transformation of eosinophilic precursor cells into leukemia cells. The HDAC inhibitors apicidin and n-butyrate suppress the proliferation of EoL-1 cells and induce differentiation into eosinophils by a decrease in the protein level of FIP1L1-PDGFRalpha without affecting the mRNA level for FIP1L1-PDGFRA. In this study, we analyzed the mechanism by which the protein level of FIP1L1-PDGFRalpha is decreased by apicidin and n-butyrate. EoL-1 cells were incubated in the presence of the HDAC inhibitors apicidin, trichostatin A or n-butyrate. The protein levels of FIP1L1-PDGFRalpha and phosphorylated eIF-2alpha were determined by Western blotting. Actinomycin D and cycloheximide were used to block RNA synthesis and protein synthesis, respectively, in the chasing experiment of the amount of FIP1L1-PDGFRalpha protein. When apicidin- and n-butyrate-treated EoL-1 cells were incubated in the presence of actinomycin D, the decrease in the protein level of FIP1L1-PDGFRalpha was significantly enhanced when compared with controls. In contrast, the protein levels were not changed by cycloheximide among these groups. Apicidin and n-butyrate induced the continuous phosphorylation of eIF-2alpha for up to 8 days. The decrease in the level of FIP1L1-PDGFRalpha protein by continuous inhibition of HDAC may be due to the decrease in the translation rate of FIP1L1-PDGFRA. Copyright 2008 S. Karger AG, Basel.

Cycloheximide: No Ordinary Bitter Stimulus

PubMed Central

Hettinger, Thomas P.; Formaker, Bradley K.; Frank, Marion E.

2007-01-01

Cycloheximide (CyX), a toxic antibiotic with a unique chemical structure generated by the actinomycete, Streptomyces griseus, has emerged as a primary focus of studies on mammalian bitter taste. Rats and mice avoid it at concentrations well below the thresholds for most bitter stimuli and T2R G-protein-coupled receptors specific for CyX with appropriate sensitivity are identified for those species. Like mouse and rat, golden hamsters, Mesocricetus auratus, also detected and rejected micromolar levels of CyX, although 1 mM CyX failed to activate the hamster chorda tympani nerve. Hamsters showed an initial tolerance for 500 μM CyX, but after that, avoidance of CyX dramatically increased, plasticity not reported for rat or mouse. As the hamster lineage branches well before division of the mouse-rat lineage in evolutionary time, differences between hamster and mouse-rat reactions to CyX are not surprising. Furthermore, unlike hamster LiCl-induced learned aversions, the induced CyX aversion neither specifically nor robustly generalized to other non-ionic bitter stimuli; and unlike adverse reactions to other chemosensory stimuli, aversions to CyX were not mollified by adding a sweetener. Thus, CyX is unlike other bitter stimuli. The gene for the high-affinity CyX receptor is a member of a cluster of 5 orthologous T2R genes that are likely rodent specific; this “CyX clade” is found in the mouse, rat and probably hamster, but not in the human or rabbit genome. The rodent CyX-T2R interaction may be one of multiple lineage-specific stimulus-receptor interactions reflecting a response to a particular environmental toxin. The combination of T2R multiplicity, species divergence and gene duplication results in diverse ligands for multiple species-specific T2R receptors, which confounds definition of ‘bitter’ stimuli across species. PMID:17400304

Effects of (-)-S-2,8-dimethyl-3-methylene-1-oxa-8-azaspiro[4,5]decane L-tartrate monohydrate (YM796), a novel muscarinic agonist, on disturbance of passive avoidance learning behavior in drug-treated and senescence-accelerated mice.

PubMed

Suzuki, M; Yamaguchi, T; Ozawa, Y; Ohyama, M; Yamamoto, M

1995-11-01

Effects of YM796 (-)-S-2,8-dimethyl-3-methylene-1-oxa-8-azaspiro[4,5]decane L-tartrate monohydrate; a novel muscarinic agonist, were observed on disturbance of passive avoidance learning behavior in drug- (protein synthesis inhibitor and anticholinergic drugs) treated and senescence-accelerated mice in comparison with those of a muscarinic agonist (AF102B) and acetylcholinesterase inhibitors (E2020 (1-benzyl-4-[(5,6-dimethoxy-1-indanone-2-yl) methyl] piperidene hydrochloride), NIK247 [9-amino-2,3,5,6,7,8-hexahydro-1H-cyclopenta(b)-quinoline monohydrate hydrochloride], THA (9-amino-1,2,3,4-tetrahydroacridine) and physostigmine). All tested drugs administered before training significantly prolonged the shortened latency of step-through induced by the protein synthesis inhibitor cycloheximide (150 mg/kg s.c.). This shortened latency was also significantly prolonged when YM796 was administered immediately after training, but not when administered before the test trial. The ameliorating effect of YM796 on the impairment in learning behavior by cycloheximide was significantly suppressed by pirenzepine (0.1 micrograms/mouse i.c.v.). When administered before training, all test drugs prolonged the shortened latency of step-through induced by treatment with the anticholinergic drugs [scopolamine (1 mg/kg s.c.) and hemicholinium-3 (0.3 microgram/mouse i.c.v.)], suggesting that they ameliorated the impairment of learning behavior. This shortened latency in scopolamine-treated mice was also significantly prolonged by YM796, AF102B, E2020, NIK247 and physostigmine when administered immediately after training, but not when administered before the test trial. The pharmacological actions of YM796 administered immediately after training and before the test trial in hemicholinium-3-treated mice were similar to those in scopolamine-treated mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotropin-dependent oocyte maturational competence requires activation of the protein kinase A pathway and synthesis of RNA and protein in ovarian follicles of Nibe, Nibea mitsukurii (Teleostei, Sciaenidae)

USGS Publications Warehouse

Yoshizaki, G.; Shusa, M.; Takeuchi, T.; Patino, R.

2002-01-01

Luteinizing hormone- (LH)-dependent ovarian follicle maturation has been recently described in two stages for teleost fishes. The oocyte's ability to respond to the steroidal maturation-inducing hormone (MIH), also known as oocyte maturational competence (OMC), is acquired during the first stage; whereas the MIH-dependent resumption of meiosis occurs during the second stage. However, studies directly addressing OMC have been performed with a limited number of species and therefore the general relevance of the two-stage model and its mechanisms remain uncertain. In this study, we examined the hormonal regulation of OMC and its basic transduction mechanisms in ovarian follicles of the sciaenid teleost, Nibe (Nibea mitsukurii). Exposure to MIH [17,20??-dihydroxy-4-pregnen-3-one or 17,20??,21-trihydroxy-4-pregnen-3-one] stimulated germinal vesicle breakdown (index of meiotic resumption) in full-grown follicles primed with human chorionic gonadotropin (HCG, an LH-like gonadotropin) but not in those pre-cultured in plain incubation medium. The induction of OMC by HCG was mimicked by protein kinase A (PKA) activators (forskolin and dibutyryl cyclic AMP), and blocked by specific inhibitors of PKA (H89 and H8) as well as inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Forskolin-induced OMC was also inhibited by actinomycin D and cycloheximide. A strong activator of protein kinase C, PMA, inhibited HCG-dependent OMC. In conclusion, OMC in Nibe ovarian follicles is gonadotropin-dependent and requires activation of the PKA pathway followed by gene transcription and translation events. These observations are consistent with the two-stage model of ovarian follicle maturation proposed for other teleosts, and suggest that Nibe can be used as new model species for mechanistic studies of ovarian follicle differentiation and maturation in fishes.

Replication of adeno-associated virus in cells irradiated with UV light at 254 nm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yakobson, B.; Hrynko, T.A.; Peak, M.J.

1989-03-01

Irradiation of simian virus 40 (ori mutant)-transformed Chinese hamster embryo cells (OD4 line) with UV light induced a cellular capacity which supported a full cycle of helper-independent adeno-associated virus replication. Monochromatic UV light at 254 nm was about 1,000-fold more effective than UV light at 313 nm, indicating that cellular nucleic acid is the primary chromophore in the UV-induced process leading to permissiveness for adeno-associated virus replication. The UV irradiation and the infection could be separated for up to 12 h without substantial loss of permissiveness. During this time interval, the induction process was partly sensitive to cycloheximide, suggesting amore » requirement for de novo protein synthesis.« less

Schizosaccharomyces pombe Polysome Profile Analysis and RNA Purification.

PubMed

Wolf, Dieter A; Bähler, Jürg; Wise, Jo Ann

2017-04-03

Polysome profile analysis is widely used by investigators studying the mechanism and regulation of translation. The method described here uses high-velocity centrifugation of whole cell extracts on linear sucrose gradients to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes. Cycloheximide is included in the lysis buffer to "freeze" polysomes by blocking translation. After centrifugation, the gradient is fractionated and RNA (and/or protein) is prepared from each fraction for subsequent analysis of individual species using northern or western blots. The entire RNA population in each fraction can be analyzed by hybridization to microarrays or by high-throughput RNA sequencing, and the proteins present can be identified by mass spectrometry analysis. © 2017 Cold Spring Harbor Laboratory Press.

REFORMATION OF NUCLEOLI AFTER ETHIONE-INDUCED FRAGMENTATION IN THE ABSENCE OF SIGNIFICANT PROTEIN SYNTHESIS

PubMed Central

Shinozuka, Hisashi; Farber, Emmanuel

1969-01-01

The rat liver nucleolus, after fragmentation induced by ethionine treatment, has been found to undergo complete reformation by adenine in the presence of a dose of cycloheximide sufficient to cause inhibition of protein synthesis by 90–95%. In contrast, actinomycin D given along with adenine was followed by the appearance of a small compact mass containing only the fibrillar component with no evident granules. This structure resembled pseudonucleoli seen in the anucleolate mutant of Xenopus laevis or in certain early stages of amphibian oocytes. Actinomycin D administered 2 hr after adenine induced a segregation of the fibrillar and granular components of nucleoli similar to that induced in the normal nucleolus. The implications of these findings in relation to nucleolar organization are briefly discussed. PMID:5775789

TIF-IA, the factor mediating growth-dependent control of ribosomal RNA synthesis, is the mammalian homolog of yeast Rrn3p.

PubMed

Bodem, J; Dobreva, G; Hoffmann-Rohrer, U; Iben, S; Zentgraf, H; Delius, H; Vingron, M; Grummt, I

2000-08-01

Cells carefully modulate the rate of rRNA transcription in order to prevent an overinvestment in ribosome synthesis under less favorable nutritional conditions. In mammals, growth-dependent regulation of RNA polymerase I (Pol I) transcription is mediated by TIF-IA, an essential initiation factor that is active in extracts from growing but not starved or cycloheximide-treated mammalian cells. Here we report the molecular cloning and functional characterization of recombinant TIF-IA, which turns out to be the mammalian homolog of the yeast factor Rrn3p. We demonstrate that TIF-IA interacts with Pol I in the absence of template DNA, augments Pol I transcription in vivo and rescues transcription in extracts from growth-arrested cells in vitro.

TIF-IA, the factor mediating growth-dependent control of ribosomal RNA synthesis, is the mammalian homolog of yeast Rrn3p

PubMed Central

Bodem, Jochen; Dobreva, Gergana; Hoffmann-Rohrer, Urs; Iben, Sebastian; Zentgraf, Hanswalter; Delius, Hajo; Vingron, Martin; Grummt, Ingrid

2000-01-01

Cells carefully modulate the rate of rRNA transcription in order to prevent an overinvestment in ribosome synthesis under less favorable nutritional conditions. In mammals, growth-dependent regulation of RNA polymerase I (Pol I) transcription is mediated by TIF-IA, an essential initiation factor that is active in extracts from growing but not starved or cycloheximide-treated mammalian cells. Here we report the molecular cloning and functional characterization of recombinant TIF-IA, which turns out to be the mammalian homolog of the yeast factor Rrn3p. We demonstrate that TIF-IA interacts with Pol I in the absence of template DNA, augments Pol I transcription in vivo and rescues transcription in extracts from growth-arrested cells in vitro. PMID:11265758

Aldosterone sensitizes connecting tubule glomerular feedback via the aldosterone receptor GPR30

PubMed Central

Ren, YiLin; D'Ambrosio, Martin A.; Garvin, Jeffrey L.; Leung, Pablo; Kutskill, Kristopher; Wang, Hong; Peterson, Edward L.

2014-01-01

Increasing Na delivery to epithelial Na channels (ENaC) in the connecting tubule (CNT) dilates the afferent arteriole (Af-Art), a process we call connecting tubule glomerular feedback (CTGF). We hypothesize that aldosterone sensitizes CTGF via a nongenomic mechanism that stimulates CNT ENaC via the aldosterone receptor GPR30. Rabbit Af-Arts and their adherent CNTs were microdissected and simultaneously perfused. Two consecutive CTGF curves were elicited by increasing luminal NaCl in the CNT. During the control period, the concentration of NaCl that elicited a half-maximal response (EC50) was 37.0 ± 2.0 mmol/l; addition of aldosterone 10−8 mol/l to the CNT lumen caused a left-shift (decrease) in EC50 to 19.3 ± 1.3 mmol/l (P = 0.001 vs. control; n = 6). Neither the transcription inhibitor actinomycin D nor the translation inhibitor cycloheximide prevented the effect of aldosterone (control EC50 = 34.7 ± 1.9 mmol/l; aldosterone+actinomycin D EC50 = 22.6 ± 1.6 mmol/l; P < 0.001 and control EC50 = 32.4 ± 4.3 mmol/l; aldosterone+cycloheximide EC50 = 17.4 ± 3.3 mmol/l; P < 0.001). The aldosterone antagonist eplerenone prevented the sensitization of CTGF by aldosterone (control EC50 = 33.2 ± 1.7 mmol/l; aldosterone+eplerenone EC50 = 33.5 ± 1.3 mmol/l; n = 7). The GPR30 receptor blocker G-36 blocked the sensitization of CTGF by aldosterone (aldosterone EC50 = 16.5 ± 1.9 mmol/l; aldosterone+G-36 EC50 = 29.0 ± 2.1 mmol/l; n = 7; P < 0.001). Finally, we found that the sensitization of CTGF by aldosterone was mediated, at least in part, by the sodium/hydrogen exchanger (NHE). We conclude that aldosterone in the CNT lumen sensitizes CTGF via a nongenomic effect involving GPR30 receptors and NHE. Sensitized CTGF induced by aldosterone may contribute to renal damage by increasing Af-Art dilation and glomerular capillary pressure (glomerular barotrauma). PMID:24966088

Recessive constitutive mutant Chinese hamster ovary cells (CHO-K1) with an altered A system for amino acid transport and the mechanism of gene regulation of the A system.

PubMed Central

Moffett, J; Englesberg, E

1984-01-01

Chinese hamster ovary cells (CHO-K1) starved for 24 h for amino acids show a severalfold increase in velocity of proline transport through the A system (Vmax is five times that of unstarved cells). This increase is inhibited by cycloheximide, actinomycin D, N-methyl-alpha-amino isobutyric acid (MeAIB, a non-metabolizable specific A system amino acid analog), and by other amino acids that are generally transported by the A system. However, transport by the A system is not a prerequisite for this repression, and all compounds that have affinity for the A system do not necessarily act as "co-repressors." The addition of proline, MeAIB, or other amino acids, as described above, to derepressed cells results in a rapid decrease in A system activity. As shown with proline and MeAIB, this decrease in activity is in part due to a rapid trans-inhibition and a slow, irreversible inactivation of the A system. Neither process is inhibited by cycloheximide or actinomycin D. Alanine antagonizes the growth of CHO-K1 pro cells by preventing proline transport, and alanine-resistant mutants (alar) have been isolated (Moffett et al., Somatic Cell Genet. 9:189-213, 1983). alar2 and alar4 are partial and full constitutive mutants for the A system and have two and six times the Vmax for proline uptake by the A system, respectively. The A system in alar4 is also immune to the co-repressor-induced inactivation. Both alar2 and alar4 phenotypes are recessive. Alar3 shows an increase in Vmax and Km for proline transport through the A system, and this phenotype is codominant. All three mutants have a pleiotropic effect, producing increases in activity of the ASC and P systems of amino acid transport. This increase is not due to an increase in the Na+ gradient. The ASC and P phenotypes behave similarly to the A system in hybrids. A model has been proposed incorporating these results. PMID:6538929

Thrombin stimulates increased plasminogen activator inhibitor-1 release from liver compared to lung endothelium.

PubMed

Huebner, Benjamin R; Moore, Ernest E; Moore, Hunter B; Gonzalez, Eduardo; Kelher, Marguerite R; Sauaia, Angela; Banerjee, Anirban; Silliman, Christopher C

2018-05-01

Plasminogen activator inhibitor-1 (PAI-1) is a major regulator of the fibrinolytic system, covalently binding to tissue plasminogen activator and blocking its activity. Fibrinolysis shutdown is evident in the majority of severely injured patients in the first 24 h and is thought to be due to PAI-1. The source of this PAI-1 is thought to be predominantly endothelial cells, but there are known organ-specific differences, with higher levels thought to be in the liver. Thrombin generation is also elevated in injured patients and is a potent stimulus for PAI-1 release in human umbilical endothelial cells. We hypothesize that thrombin induces liver endothelial cells to release increased amounts of PAI-1, versus pulmonary endothelium, consisting of both stored PAI-1 and a larger contribution from de novo PAI-1 synthesis. Human liver sinusoidal endothelial cells (LSECs) and human microvascular lung endothelial cells (HMVECs) were stimulated in vitro ± thrombin (1 and 5 IU/mL) for 15-240 min, the supernatants were collected, and PAI-1 was measured by enzyme-linked immunosorbent assays. To elucidate the PAI-1 contribution from storage versus de novo synthesis, cycloheximide (10 μg/mL) was added before thrombin in separate experiments. While both LSECs and HMVECs rapidly stimulated PAI-1 release, LSECs released more PAI-1 than HMVECs in response to high-dose thrombin, whereas low-dose thrombin did not provoke immediate release. LSECs continued to release PAI-1 over the ensuing 240 min, whereas HMVECs did not. Cycloheximide did not inhibit early PAI-1 release from LSECs but did at the later time points (30-240 min). Thrombin elicits increased amounts of PAI-1 release from liver endothelium compared with lung, with a small presynthesized stored contribution and a later, larger increase in PAI-1 release via de novo synthesis. This study suggests that the liver may be an important therapeutic target for inhibition of the hypercoagulable surgical patient and the associated complications that result. Copyright © 2017 Elsevier Inc. All rights reserved.

CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

PubMed

Busi, Florent; Cresteil, Thierry

2005-09-01

The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

Upregulation of contractile endothelin type B receptors by lipid-soluble cigarette smoking particles in rat cerebral arteries via activation of MAPK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sandhu, Hardip, E-mail: sandhu.hardip@gmail.co; Xu, Cang Bao; Edvinsson, Lars

2010-11-15

Cigarette smoke exposure increases the risk of stroke. However, the underlying molecular mechanisms are poorly understood. Endothelin system plays key roles in the pathogenesis of stroke. The present study was designed to examine if lipid-soluble (dimethyl sulfoxide-soluble) cigarette smoke particles (DSP) induces upregulation of contractile endothelin type B (ET{sub B}) receptors in rat cerebral arteries and if activation of mitogen activated protein kinase (MAPK) and nuclear factor-kappaB (NF-{kappa}B) mediate the upregulation of contractile endothelin receptors in the cerebral arteries. Rat middle cerebral arteries were isolated and organ cultured in serum free medium for 24 h in the presence of DSPmore » with or without specific inhibitors: MEK specific (U0126), p38 specific (SB202190), JNK specific (SP600125), NF-{kappa}B specific (BMS-345541) or (IMD-0354), transcription inhibitor (actinomycin D), or translation blocker (cycloheximide). Contractile responses to the ET{sub B} receptor agonist sarafotoxin 6c were investigated by a sensitive myograph. The expression of the ET{sub B} receptors were studied at mRNA and protein levels using quantitative real time PCR and immunohistochemistry, respectively. Results show that organ culture per se induced transcriptional upregulation of contractile ET{sub B} receptors in the cerebral vascular smooth muscle cells. This upregulation was further increased at the translational level by addition of DSP to the organ culture, but this increase was not seen by addition of nicotine or water-soluble cigarette smoke particles to the organ culture. The increased upregulation of contractile ET{sub B} receptors by DSP was abrogated by U0126, SP600125, actinomycin D, and cycloheximide, suggesting that the underlying molecular mechanisms involved in this process include activation of MEK and JNK MAPK-mediated transcription and translation of new contractile ET{sub B} receptors. Thus, the MAPK-mediated upregulation of contractile ET{sub B} receptors in cerebral arteries might be a pharmacological target for the treatment of smoke-associated cerebral vascular disease like stroke.« less

Chlamydia trachomatis can protect host cells against apoptosis in the absence of cellular Inhibitor of Apoptosis Proteins and Mcl-1.

PubMed

Ying, Songmin; Christian, Jan G; Paschen, Stefan A; Häcker, Georg

2008-01-01

Infection with Chlamydia protects mammalian host cells against apoptosis. Hypotheses have been proposed to explain this molecularly, including the up-regulation of host anti-apoptotic proteins such as cellular Inhibitor of Apoptosis Protein (IAP) 2 and the Bcl-2 protein Mcl-1. To test for the importance of these proteins, we used mouse embryonic fibroblasts from gene-targeted mice that were deficient in cIAP1, cIAP2, cIAP1/cIAP2, XIAP, or Mcl-1. Infection with Chlamydia trachomatis protected all cells equally well against apoptosis, which was induced either with tumour necrosis factor/cycloheximide (IAP-knock-out cells) or staurosporine (Mcl-1-knock-out). Therefore, these cellular anti-apoptotic proteins are not essential for apoptosis-protection by C. trachomatis.

Three distinct cell phenotypes of induced-TNF cytotoxicity and their relationship to apoptosis

NASA Technical Reports Server (NTRS)

Woods, K. M.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1993-01-01

We have identified three distinct cell phenotypes with respect to the conditions under which cells became susceptible to TNF-mediated lysis. These conditions include: 1) treatment with the protein synthesis inhibitor, cycloheximide; 2) contact with activated macrophages, and 3) infection with vaccinia virus. Whereas vaccinia virus-infected 3T3 cells became sensitive to soluble TNF, F5b cells required contact with activated macrophages. We showed that the "macrophage-resistant" F5m cells did not become sensitive to TNF or to killing by activated macrophages after infection with vaccinia virus. Therefore, vaccinia infection does not sensitize all cells to TNF. We also determined the pathways of lysis for cells after sensitization. Whereas 3T3, LM929, and F5b cells were killed by the process of necrosis, F5m cells lysis was characterized by the release of low mol wt DNA fragments (apoptosis).

Incorporation of carbohydrate residues into peroxidase isoenzymes in horseradish roots.

PubMed

Lew, J Y; Shannon, L M

1973-11-01

Sliced root tissue of the horseradish plant (Armoracia rusticana), when incubated with mannose-U-(14)C, incorporated radioactivity into peroxidase isoenzymes. Over 90% of the radioactivity in the highly purified peroxidase isoenzymes was present in the neutral sugar residues of the molecule, i.e. fucose, arabinose, xylose, mannose. When the root slices were incubated simultaneously with leucine-4,5-(3)H and mannose-U-(14)C, cycloheximide strongly inhibited leucine incorporation into the peptide portion of peroxidase isoenzymes but had little effect on the incorporation of (14)C into the neutral sugars. These results indicated that synthesis of the peptide portion of peroxidase was completed before the monosaccharide residues were attached to the molecule. This temporal relationship between the synthesis of protein and the attachment of carbohydrate residues in the plant glycoprotein, horseradish peroxidase, appears to be similar to that reported for glycoprotein biosynthesis in many mammalian systems.

Incorporation of Carbohydrate Residues into Peroxidase Isoenzymes in Horseradish Roots

PubMed Central

Lew, Jow Y.; Shannon, Leland M.

1973-01-01

Sliced root tissue of the horseradish plant (Armoracia rusticana), when incubated with mannose-U-14C, incorporated radioactivity into peroxidase isoenzymes. Over 90% of the radioactivity in the highly purified peroxidase isoenzymes was present in the neutral sugar residues of the molecule, i.e. fucose, arabinose, xylose, mannose. When the root slices were incubated simultaneously with leucine-4,5-3H and mannose-U-14C, cycloheximide strongly inhibited leucine incorporation into the peptide portion of peroxidase isoenzymes but had little effect on the incorporation of 14C into the neutral sugars. These results indicated that synthesis of the peptide portion of peroxidase was completed before the monosaccharide residues were attached to the molecule. This temporal relationship between the synthesis of protein and the attachment of carbohydrate residues in the plant glycoprotein, horseradish peroxidase, appears to be similar to that reported for glycoprotein biosynthesis in many mammalian systems. PMID:16658584

Construction of a recombinant wine yeast strain expressing beta-(1,4)-endoglucanase and its use in microvinification processes.

PubMed Central

Pérez-González, J A; González, R; Querol, A; Sendra, J; Ramón, D

1993-01-01

A genetic transformation system for an industrial wine yeast strain is presented here. The system is based on the acquisition of cycloheximide resistance and is a direct adaptation of a previously published procedure for brewing yeasts (L. Del Pozo, D. Abarca, M. G. Claros, and A. Jiménez, Curr. Genet. 19:353-358, 1991). Transformants arose at an optimal frequency of 0.5 transformant per microgram of DNA, are stable in the absence of selective pressure, and produce wine in the same way as the untransformed industrial strain. By using this transformation protocol, a filamentous fungal beta-(1,4)-endoglucanase gene has been expressed in an industrial wine yeast under the control of the yeast actin gene promoter. Endoglucanolytic wine yeast secretes the fungal enzyme to the must, producing a wine with an increased fruity aroma. Images PMID:8215355

Chlorophyll degradation in a Chlamydomonas reinhardtii mutant: an accumulation of pyropheophorbide a by anaerobiosis.

PubMed

Doi, M; Inage, T; Shioi, Y

2001-05-01

Chlorophyll degradation was investigated in cells of a chlorophyll b-less mutant of Chlamydomonas reinhardtii under aerobic and anaerobic conditions. During degradation of chlorophyll under anaerobic conditions, chlorophyll catabolite P535, an open-tetrapyrrole, was not excreted, but pyropheophorbide a was accumulated as the end product with a transient accumulation of chlorophyllide a and pheophorbide a in cells, in contrast to the breakdown under aerobic conditions. It is likely that in the absence of oxygen, degradation of chlorophyll a proceeds to pyropheophorbide a by three consecutive reactions, dephytylation, metal-releasing and demethoxycarbonylation, and then stops due to a limitation of the oxygen that the monooxygenase reaction requires for bilin formation. A novel enzyme catalyzing demethoxycarbonylation of pheophorbide a was partially purified. The enzyme activity increased dependent on the age of cells, and its increase was completely suppressed by cycloheximide. Production of P535 was also dependent on cytoplasmic protein synthesis.

Factors affecting the palmitoyl-coenzyme A desaturase of Saccharomyces cerevisiae

NASA Technical Reports Server (NTRS)

Klein, H. P.; Volkmann, C. M.

1975-01-01

The activity and stability of the palmitoyl-coenzyme A (CoA) desaturase complex of Saccharomyces cerevisiae was influenced by several factors. Cells, grown nonaerobically and then incubated with glucose, either in air or under N2, showed a marked increase in desaturase activity. Cycloheximide, added during such incubations, prevented the increase in activity, suggesting de novo synthesis. The stability of the desaturase from cells grown nonaerobically was affected by subsequent treatment of the cells; enzyme from freshly harvested cells, or from cells that were then shaken under nitrogen, readily lost activity upon washing or during density gradient analysis, whereas aerated cells, in the presence or absence of glucose, yielded stable enzyme preparations. The loss of activity in nonaerobic preparations could be reversed by adding soluble supernatant from these homogenates and could be prevented by growing the cells in the presence of palmitoleic acid and ergosterol, but not with several other lipids tested.

Rat Sertoli cells acquire a beta-adrenergic response during primary culture.

PubMed Central

Kierszenbaum, A L; Spruill, W A; White, M G; Tres, L L; Perkins, J P

1985-01-01

Two-dimensional polyacrylamide gel electrophoresis and the radioligand (-)-[125I]iodopindolol (125I-Pin) have been used to study isoproterenol-dependent protein phosphorylation and beta-adrenergic receptor availability, respectively, in cultured Sertoli cells and freshly isolated seminiferous tubular segments of sexually immature and mature rats. Sertoli cells prepared from sexually immature rats show progressive 125I-Pin binding in primary cultures that correlates with isoproterenol-induced cell shape changes, redistribution of immunoreactive vimentin, and phosphorylation of this intermediate filament protein. The development of 125I-Pin binding to Sertoli cell lysates is blocked by cycloheximide. Seminiferous tubules do not show significant isoproterenol-dependent vimentin phosphorylation nor 125I-Pin binding. However, vimentin phosphorylation can be induced by follicle-stimulating hormone or a cyclic nucleotide analog. This study stresses the need for correlating pharmacological-induced responses observed in Sertoli cell primary cultures with those in the intact seminiferous tubule. Images PMID:2984678

Characterization of the phase dependent pulmonary response following irritant inhalation exposure to nitrogen dioxide gas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Siegel, P.D.

1988-01-01

The present study utilized NO{sub 2} to fingerprint the biochemical reaction of the pulmonary compartment to oxidative damage and to correlate this with histopathology following acute and subacute exposures to NO{sub 2}. Acute exposure to NO{sub 2} produced dose-dependent immediate increases in the nonenzymatic parameters of pulmonary protein content, protease inhibitor activity and lung weight. The enzymatic activities of lactate dehydrogenase (LDH), choline kinase and beta-glucuronidase were elevated by two days following acute exposure. All of the above parameters were elevated following subacute exposure, however, nonenzymatic manifestations were attenuated with respect to enzymatic alterations. Hydroxyurea-induced granulocytopenia attenuated the increases inmore » activities of LDH and beta-glucuronidase following acute, but not subacute exposures. Cycloheximide-induced protein synthesis inhibition decrease the LDH and beta-glucuronidase response to NO{sub 2} without altering the increases in protein content or protease inhibitor activity.« less

Characteristics of injury and recovery of net NO3- transport of barley seedlings from treatments of NaCl

NASA Technical Reports Server (NTRS)

Klobus, G.; Ward, M. R.; Huffaker, R. C.

1988-01-01

The nature of the injury and recovery of nitrate uptake (net uptake) from NaCl stress in young barley (Hordeum vulgare L, var CM 72) seedlings was investigated. Nitrate uptake was inhibited rapidly by NaCl, within 1 minute after exposure to 200 millimolar NaCl. The duration of exposure to saline conditions determined the time of recovery of NO3- uptake from NaCl stress. Recovery was dependent on the presence of NO3- and was inhibited by cycloheximide, 6-methylpurine, and cerulenin, respective inhibitors of protein, RNA, and sterol/fatty acid synthesis. These inhibitors also prevented the induction of the NO3- uptake system in uninduced seedlings. Uninduced seedlings exhibited endogenous NO3- transport activity that appeared to be constitutive. This constitutive activity was also inhibited by NaCl. Recovery of constitutive NO3- uptake did not require the presence of NO3-.

A Study of the Mechanisms by Which Adrenocorticotropic Hormone Maintains Adrenal Steroidogenic Responsiveness*

PubMed Central

Ney, Robert L.; Dexter, Richard N.; Davis, Warren W.; Garren, Leonard D.

1967-01-01

Following hypophysectomy in the rat, there was a progressive decline in the rate of adrenal protein synthesis in vivo during the ensuing 24-48 hr, and an accompanying decrease in the acute corticosterone secretory response to an intravenous injection of ACTH. There was a similar decrease in the in vitro conversion of Δ5-pregnenolone, progesterone, and deoxycorticosterone to corticosterone. These in vivo and in vitro effects of hypophysectomy could be reversed by the administration of depot ACTH for an additional 7 hr period. However, if cycloheximide, an inhibitor of protein synthesis, was administered concomitantly with the depot ACTH, then the restorative actions of ACTH on the steroid biosynthetic pathway were prevented. These experiments suggest that ACTH maintains not only the general structure of the adrenal cortex, but also the level of the steroid biosynthetic mechanism, through its effects on adrenal protein synthesis. PMID:4294571

Comparison of Several Selective Media for Isolation and Differentiation of Coagulase-Positive Strains of Staphylococcus aureus1

PubMed Central

McDivitt, Maxine E.; Topp, Eleanor B.

1964-01-01

Six coagulase-positive strains of Staphylococcus aureus which had been cultivated in Brain Heart Infusion broth, milk, and brine were plated on seven isolation media. A significant difference in the growth patterns of the individual strains was found as well as a significant effect resulting from the previous cultivation history before plating. Brine and, to a lesser extent, milk were found to reduce maximal cell concentrations attained, but strains grown in brine and milk showed greater ability to withstand the selective action of the isolation media. Fibrinogen applied to the surface of five of the media allowed the formation of characteristic halos by coagulase-positive strains of S. aureus. Only half of the strains studied produced a zone of precipitation on SM110-Egg Yolk agar. The isolation medium containing cycloheximide and a high level of polymxin B was most inhibitory to the organisms. PMID:14131367

Transport and intracellular distribution of copper in a human hepatoblastoma cell line, HepG2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stockert, R.J.; Grushoff, P.S.; Morell, A.G.

1986-01-01

The uptake of radiocopper by HepG2 cells is a saturable, temperature-dependent and cellular energy-independent process with a Vmax of 7.1 +/- 0.2 pmoles min-1 mg protein-1 and an estimated Km of 3.3 +/- 0.5 microM. The rate of copper uptake is reduced at an equimolar concentration of albumin and is unaffected by zinc at a 10-fold molar excess. Approximately 70% of the newly incorporated radiocopper binds to membranes and organelles, while 30% is recovered in the cytosol. The soluble fraction can be resolved into two copper-binding protein peaks. Incubation of HepG2 with nonisotopic copper results in displacement of radiocopper associatedmore » with the proteins contained in the lower molecular weight peak. Exposure of the cells to cycloheximide inhibits the incorporation of the isotope into this fraction.« less

Enhanced isolation of Legionella species from composted material.

PubMed

McCabe, S; Brown, A; Edwards, G F S; Lindsay, D

2011-10-01

Legionella pneumophila and Legionella species were isolated from composted material when freshly prepared buffered charcoal yeast extract (BCYE) was supplemented with glycine (1.5 g/L), polymyxin B sulfate (40 000 IU/L), vancomycin hydrochloride (0.5 mg/L) and cycloheximide (40 mg/L) (GVPC medium) and Modified Wadowsky-Yee (MWY) (Oxoid, Cambridge, UK) plates were used for cultivation, but not with commercially sourced pre-poured GVPC and MWY plates (Oxoid). Legionella cincinnatiensis and pathogenic L. pneumophila serogroup (Sg) 1 Benidorm and France/Allentown were identified, as well as a non-typeable (NT) strain of L. pneumophila. As most laboratories no longer produce their own media, this may contribute to the lack of positive cultures from composted material. The antigenicity of the NT strain is discussed. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

Characterization of Zea mays endosperm C-24 sterol methyltransferase: one of two types of sterol methyltransferase in higher plants.

PubMed

Grebenok, R J; Galbraith, D W; Penna, D D

1997-08-01

We report the characterization of a higher-plant C-24 sterol methyltransferase by yeast complementation. A Zea mays endosperm expressed sequence tag (EST) was identified which, upon complete sequencing, showed 46% identity to the yeast C-24 methyltransferase gene (ERG6) and 75% and 37% amino acid identity to recently isolated higher-plant sterol methyltransferases from soybean and Arabidopsis, respectively. When placed under GALA regulation, the Z. mays cDNA functionally complemented the erg6 mutation, restoring ergosterol production and conferring resistance to cycloheximide. Complementation was both plasmid-dependent and galactose-inducible. The Z. mays cDNA clone contains an open reading frame encoding a 40 kDa protein containing motifs common to a large number of S-adenosyl-L-methionine methyltransferases (SMTs). Sequence comparisons and functional studies of the maize, soybean and Arabidopsis cDNAs indicates two types of C-24 SMTs exist in higher plants.

Relationship between Photoconvertible and Nonphotoconvertible Protochlorophyllides.

PubMed

Murray, A E; Klein, A O

1971-10-01

Two forms of protochlorophyllide are found in dark-grown bean (Phaseolus vulgaris, var. Black Velentine) leaves, one (protochlorophyllide(650)) which is directly photoconvertible to chlorophyllide and another (protochlorophyllide(632)) which is not. Dark-grown leaves placed in solutions of delta-aminolevulinic acid accumulate protochlorophyllide(632). Protochlorophyllide(650) and protochlorophyllide(632) can be partially separated on sucrose density gradients. A nitrogen atmosphere blocks chlorophyll synthesis in light or the regeneration of protochlorophyllide(650) in the dark, even in the presence of excess delta-aminolevulinic acid, except when a stockpile of protochlorophyllide(632) is present in the leaf. Under the latter conditions chlorophyll synthesis or protochlorophyllide(650) regeneration is accompanied by a decrease in protochlorophyllide(632). These experiments suggest that protochlorophyllide(632) may be converted to protochlorophyllide(650).Cycloheximide inhibited greening only after an "action-dependent" delay, requiring a predictable minimal period of illumination. This inhibition could be relieved for a time by feeding delta-aminolevulinic acid.

Quaternary ammonium salt N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride induced alterations in Saccharomyces cerevisiae physiology.

PubMed

Oblak, Ewa; Piecuch, Agata; Maciaszczyk-Dziubinska, Ewa; Wawrzycka, Donata

2016-12-01

We investigated the influence of the quaternary ammonium salt (QAS) called IM (N-(dodecyloxycarboxymethyl)- N,N,N-trimethyl ammonium chloride) on yeast cells of the parental strain and the IM-resistant mutant (EO25 IMR) growth. The phenotype of this mutant was pleiotropic. The IMR mutant exhibited resistance to ethanol, osmotic shock and oxidative stress, as well as increased sensitivity to UV. Moreover, it was noted that mutant EO25 appears to have an increased resistance to clotrimazole, ketoconazole, fluconazole, nystatin and cycloheximide. It also tolerated growth in the presence of crystal violet, DTT and metals (selenium, tin, arsenic). It was shown that the presence of IM decreased ergosterol level in mutant plasma membrane and increased its unsaturation. These results indicate changes in the cell lipid composition. Western blot analysis showed the induction of Pma1 level by IM. RT-PCR revealed an increased PMA1 expression after IM treatment.

Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae. III. Dose-response pattern of mutation induction in UV-irradiated rev2ts cells.

PubMed

Siede, W; Eckardt, F

1986-01-01

Recent studies regarding the influence of cycloheximide on the temperature-dependent increase in survival and mutation frequencies of a thermoconditional rev2 mutant lead to the suggestion that the REV2-coded mutagenic repair function is UV-inducible. In the present study we show that stationary-phase rev2ts cells are characterized by a biphasic linear-quadratic dose-dependence of mutation induction ("mutation kinetics") of ochre alleles at 23 degrees C (permissive temperature) but linear kinetics at the restrictive temperature of 36 degrees C. Mathematical analysis using a model based on Poisson statistics and a further mathematical procedure, the calculation of "apparent survival", support the assumption that the quadratic component of the reverse mutation kinetics investigated can be attributed to a UV-inducible component of mutagenic DNA repair controlled by the REV2 gene.

Mitoxantrone is More Toxic than Doxorubicin in SH-SY5Y Human Cells: A 'Chemobrain' In Vitro Study.

PubMed

Almeida, Daniela; Pinho, Rita; Correia, Verónica; Soares, Jorge; Bastos, Maria de Lourdes; Carvalho, Félix; Capela, João Paulo; Costa, Vera Marisa

2018-05-05

The potential neurotoxic effects of anticancer drugs, like doxorubicin (DOX) and mitoxantrone (MTX; also used in multiple sclerosis), are presently important reasons for concern, following epidemiological data indicating that cancer survivors submitted to chemotherapy may suffer cognitive deficits. We evaluated the in vitro neurotoxicity of two commonly used chemotherapeutic drugs, DOX and MTX, and study their underlying mechanisms in the SH-SY5Y human neuronal cell model. Undifferentiated human SH-SY5Y cells were exposed to DOX or MTX (0.13, 0.2 and 0.5 μM) for 48 h and two cytotoxicity assays were performed, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction and the neutral red (NR) incorporation assays. Phase contrast microphotographs, Hoechst, and acridine orange/ethidium bromide stains were performed. Mitochondrial membrane potential was also assessed. Moreover, putative protective drugs, namely the antioxidants N -acetyl-l-cysteine (NAC; 1 mM) and 100 μM tiron, the inhibitor of caspase-3/7, Ac-DEVD-CHO (100 μM), and a protein synthesis inhibitor, cycloheximide (CHX; 10 nM), were tested to prevent DOX- or MTX-induced toxicity. The MTT reduction assay was also done in differentiated SH-SY5Y cells following exposure to 0.2 μM DOX or MTX. MTX was more toxic than DOX in both cytotoxicity assays and according to the morphological analyses. MTX also evoked a higher number of apoptotic nuclei than DOX. Both drugs, at the 0.13 μM concentration, caused mitochondrial membrane potential depolarization after a 48-h exposure. Regarding the putative neuroprotectors, 1 mM NAC was not able to prevent the cytotoxicity caused by either drug. Notwithstanding, 100 μM tiron was capable of partially reverting MTX-induced cytotoxicity in the NR uptake assay. One hundred μM Ac-DEVD-CHO and 10 nM cycloheximide (CHX) also partially prevented the toxicity induced by DOX in the NR uptake assay. MTX was more toxic than DOX in differentiated SH-SY5Y cells, while MTX had similar toxicity in differentiated and undifferentiated SH-SY5Y cells. In fact, MTX was the most neurotoxic drug tested and the mechanisms involved seem dissimilar among drugs. Thus, its toxicity mechanisms need to be further investigated as to determine the putative neurotoxicity for multiple sclerosis and cancer patients.

Immunomodulation of glioma cells after gene therapy: induction of major histocompatibility complex class I but not class II antigen in vitro.

PubMed

Parsa, A T; Chi, J H; Hurley, P T; Jeyapalan, S A; Bruce, J N

2001-09-01

Acquired immunity has been demonstrated in Fischer rats bearing syngeneic 9L tumors after herpes simplex virus (HSV) thymidine kinase (TK) gene transfection and ganciclovir treatment. The nature of this immunity in rats and its relevance to the HSV TK/ganciclovir protocol for human subjects remain to be determined. In this study, levels of major histocompatibility complex (MHC) Class I and II antigen expression were measured before and after HSV TK transfection, in an effort to document immunomodulatory changes caused by gene therapy. Tumor cells from the 9L gliosarcoma cell line, three primary human glioma cultures, and the human glioma cell line U87 MG were transduced with HSV TK vector-containing supernatant from fibroblast-producing cells (titer of 5 x 10(6) colony-forming units/ml) and selected in G418 medium for neomycin resistance. Clones were pooled or individually selected for cell-killing assays with ganciclovir, to confirm TK expression (10(3) cells/well in a 96-well dish). Northern analyses using MHC Class I and Class II complementary deoxyribonucleic acid probes were performed on blots containing total ribonucleic acid from wild-type tumor cells and HSV TK transfectants. A beta-actin complementary deoxyribonucleic acid probe served as an internal control. Cell surface expression was confirmed with flow cytometry. The induction of MHC Class I was tested for cycloheximide and genistein sensitivity. All cell cultures exhibited increases in MHC Class I but not MHC Class II expression, as determined by Northern analysis densitometry and flow cytometry. Cycloheximide treatment did not diminish the up-regulation of MHC Class I after retroviral transfection, implicating a signal transduction pathway that does not require ongoing protein synthesis. Genistein pretreatment of cell cultures did diminish the up-regulation of MHC Class I, implicating a tyrosine kinase in the signaling cascade. Induction of MHC Class I in rat and human glioma cells after HSV TK retroviral gene therapy is a primary effect that is dependent on tyrosine kinase activity. Specific immune responses generated after transfection may represent an important general side effect of gene therapy protocols. Elucidation of the mechanism of immunomodulation after gene therapy will likely yield safer and more effective clinical protocols.

Auxin flow-mediated competition between axillary buds to restore apical dominance

PubMed Central

Balla, Jozef; Medveďová, Zuzana; Kalousek, Petr; Matiješčuková, Natálie; Friml, Jiří; Reinöhl, Vilém; Procházka, Stanislav

2016-01-01

Apical dominance is one of the fundamental developmental phenomena in plant biology, which determines the overall architecture of aerial plant parts. Here we show apex decapitation activated competition for dominance in adjacent upper and lower axillary buds. A two-nodal-bud pea (Pisum sativum L.) was used as a model system to monitor and assess auxin flow, auxin transport channels, and dormancy and initiation status of axillary buds. Auxin flow was manipulated by lateral stem wounds or chemically by auxin efflux inhibitors 2,3,5-triiodobenzoic acid (TIBA), 1-N-naphtylphtalamic acid (NPA), or protein synthesis inhibitor cycloheximide (CHX) treatments, which served to interfere with axillary bud competition. Redirecting auxin flow to different points influenced which bud formed the outgrowing and dominant shoot. The obtained results proved that competition between upper and lower axillary buds as secondary auxin sources is based on the same auxin canalization principle that operates between the shoot apex and axillary bud. PMID:27824063

The effects of drugs, other foreign compounds, and cigarette smoke on the synthesis of protein by lung slices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hellstern, K.; Curtis, C.G.; Powell, G.M.

1990-04-01

The incorporation of {sup 14}C-leucine into rabbit lung slices was monitored in the absence and presence of selected drugs and chemicals relevant to the perturbation of lung function and the development of lung disease. Known inhibitors of protein synthesis (cycloheximide and ricin) inhibited the incorporation of {sup 14}C-leucine. Marked inhibition was also recorded with the lung toxins paraquat and 4-ipomeanol. By contrast, orciprenaline, salbutamol, and terbutaline were without effect although some response was recorded with isoprenaline. The filtered gas phase of cigarette smoke and acrolein, one of its components, were inhibitory but protection was afforded by N-acetylcysteine. It is suggestedmore » that the inhibitory effects of cigarette smoke may be due to its acrolein content. It is further suggested that the use of lung slices and measurements of {sup 14}C-leucine incorporation provide valuable means for monitoring potential pulmonary toxins.« less

RNA editing produces glycine receptor alpha3(P185L), resulting in high agonist potency.

PubMed

Meier, Jochen C; Henneberger, Christian; Melnick, Igor; Racca, Claudia; Harvey, Robert J; Heinemann, Uwe; Schmieden, Volker; Grantyn, Rosemarie

2005-06-01

The function of supramedullary glycine receptors (GlyRs) is still unclear. Using Wistar rat collicular slices, we demonstrate GlyR-mediated inhibition of spike discharge elicited by low glycine (10 microM). Searching for the molecular basis of this phenomenon, we identified a new GlyR isoform. GlyR alpha3(P185L), a result of cytidine 554 deamination, confers high glycine sensitivity (EC50 approximately 5 microM) to neurons and thereby promotes the generation of sustained chloride conductances associated with tonic inhibition. The level of GlyR alpha3-C554U RNA editing is sensitive to experimentally induced brain lesion, inhibition of cytidine deamination by zebularine and inhibition of mRNA transcription by actinomycin D, but not to blockade of protein synthesis by cycloheximide. Conditional regulation of GlyR alpha3(P185L) is thus likely to be part of a post-transcriptional adaptive mechanism in neurons with enhanced excitability.

Appearance of an Alternate Pathway Cyanide-resistant during Germination of Seeds of Cicer arietinum

PubMed Central

Burguillo, Placido De La Fuente; Nicolás, Gregorio

1977-01-01

The combined action of the inhibitors antimycin A and cyanide with benzohydroxamic acid indicates the presence of a cyanide-resistant pathway of respiration in chick pea (Cicer arietinum L.) seeds. The appearance of this pathway takes place during germination. During the first 12 hours of germination, the respiration is predominantly cyanide-sensitive, showing after this time a shift to an “alternate” respiration which is sensitive to benzohydroxamic acid, reaching the maximal cyanide resistance between 72 and 96 hours of germination. The appearance of the alternate pathway is initiated by high O2 concentrations and depends on cytoplasmic protein synthesis, since its appearance is inhibited by cycloheximide but not by chloramphenicol. Actinomycin D has no effect on the appearance of the alternate pathway. Our results indicate, in agreement with other authors, that the branching point is located between the flavoproteins and cytochromes b, probably at the level of ubiquinone, but the possibility of more than one branching point of the electron flow is also considered. PMID:16660130

Prevalence of different Malassezia species in pityriasis versicolor in central India.

PubMed

Chaudhary, Rahul; Singh, Sanjay; Banerjee, Tuhina; Tilak, Ragini

2010-01-01

In the last 10 years, different studies have shown interesting geographical variations in the prevalence of different Malassezia species in pityriasis versicolor. Identification of Malassezia species isolated from patients with pityriasis versicolor. In 100 patients with pityriasis versicolor, Malassezia species were identified by culture in Sabouraud's dextrose agar containing cycloheximide with olive oil overlay and modified Dixon agar and by doing biochemical tests (catalase reaction, assimilation of glycine, and Tween utilisation tests). In 10 patients, 10% KOH smear was negative, while in 90 patients the smear showed characteristic "spaghetti and meatball" appearance. Of these 90 cases, growth was obtained on modified Dixon's agar in 87 cases. Fifty of the isolates (57.5%) were M. globosa, 15 (17.2%) were M. sympodialis, seven (8.0%) were suspected M. sympodialis, 6 (6.9%) each of the isolates were M. furfur and M. obtusa, and three (3.4%) isolates were M. restricta. M. globosa was the most common species, followed by M. sympodialis, M. furfur, M. obtusa, and M. restricta.

Regulation of L-asparaginase in a Chlamydomonas species in response to ambient concentrations of combined nitrogen.

PubMed Central

Paul, J H; Cooksey, K E

1981-01-01

Cellular levels of an L-asparaginase in a Chlamydomonas species were found to be greater in nitrogen-limited batch cultures than in batch cultures grown in ample nitrogen. Cells grown in high nitrogen medium (5 mM NH4Cl) and suspended in nitrogen-free medium showed a 2- to 3.5-fold increase in activity after 24 to 48 h. This increase in activity was inhibited by cycloheximide and by the addition of high levels of combined nitrogen (5 mM NH4Cl, NaNO3, or L-asparagine), suggesting repression by ambient nitrogen levels as the mode of regulation of this enzyme. Derepressed L-asparaginase activity did not disappear in the presence of high concentrations of medium nitrogen, indicating the absence of an asparaginase-degrading system. Derepression of asparaginase by this organism was light dependent and inhibited by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea suggesting a requirement for photosynthetic energy. PMID:7240099

Transferability and inter-laboratory variability assessment of the in vitro bovine oocyte fertilization test.

PubMed

Tessaro, Irene; Modina, Silvia C; Crotti, Gabriella; Franciosi, Federica; Colleoni, Silvia; Lodde, Valentina; Galli, Cesare; Lazzari, Giovanna; Luciano, Alberto M

2015-01-01

The dramatic increase in the number of animals required for reproductive toxicity testing imposes the validation of alternative methods to reduce the use of laboratory animals. As we previously demonstrated for in vitro maturation test of bovine oocytes, the present study describes the transferability assessment and the inter-laboratory variability of an in vitro test able to identify chemical effects during the process of bovine oocyte fertilization. Eight chemicals with well-known toxic properties (benzo[a]pyrene, busulfan, cadmium chloride, cycloheximide, diethylstilbestrol, ketoconazole, methylacetoacetate, mifepristone/RU-486) were tested in two well-trained laboratories. The statistical analysis demonstrated no differences in the EC50 values for each chemical in within (inter-runs) and in between-laboratory variability of the proposed test. We therefore conclude that the bovine in vitro fertilization test could advance toward the validation process as alternative in vitro method and become part of an integrated testing strategy in order to predict chemical hazards on mammalian fertility. Copyright © 2015 Elsevier Inc. All rights reserved.

Apoptosis induced in an early step of African swine fever virus entry into vero cells does not require virus replication.

PubMed

Carrascosa, Angel L; Bustos, María J; Nogal, María L; González de Buitrago, Gonzalo; Revilla, Yolanda

2002-03-15

Permissive Vero cells develop apoptosis, as characterized by DNA fragmentation, caspases activation, cytosolic release of mitochondrial cytochrome c, and flow cytometric analysis of DNA content, upon infection with African swine fever virus (ASFV). To determine the step in virus replication that triggers apoptosis, we used UV-inactivated virus, inhibitors of protein and nucleic acid synthesis, and lysosomotropic drugs that block virus uncoating. ASFV-induced apoptosis was accompanied by caspase-3 activation, which was detected even in the presence of either cytosine arabinoside or cycloheximide, indicating that viral DNA replication and protein synthesis were not required to activate the apoptotic process. The activation of caspase-3 was released from chloroquine inhibition 2 h after virus absorption, while the infection with UV-inactivated ASFV did not induce the activation of the caspase cascade. We conclude that ASFV induces apoptosis in the infected cell by an intracellular pathway probably triggered during the process of virus uncoating.

Chloroplast-cytoplasmic interrelations involved in chloroplast development in Chlamydomonas reinhardi y-1: effect of selective depletion of chloroplast translates

PubMed Central

1980-01-01

Chlamydomonas reinhardi y-1 cells grown in the dark in the presence of chloramphenicol (CD cells) are depleted of photosynthetic membranes and 70S translates. These cells were found to be unable to synthesize chlorophyll in the light until chloroplast protein synthesis was resumed. On the other hand, CD cells acquired the capacity to partially green in the presence of cycloheximide. This greening was characterized by the development of photosynthetic activity, as demonstrated by light- dependent oxygen evolution of whole cells and by measurements of ribulose-1,5-bisphosphate carboxylase and fluorescence kinetics. The chlorophyll synthesized de novo during greening in the absence of 80S ribosomal activity was organized in chlorophyll-protein complexes, as ascertained by low-temperature fluorescence-emission spectra. The morphology of these cells appeared to be normal. A model has been proposed as a working hypothesis, which could account for the phenomena described above and previously reported data pertaining to chloroplast development. PMID:7419587

Invasion of Human Oral Epithelial Cells by Prevotella intermedia

PubMed Central

Dorn, Brian R.; Leung, K.-P.; Progulske-Fox, Ann

1998-01-01

Invasion of oral epithelial cells by pathogenic oral bacteria may represent an important virulence factor in the progression of periodontal disease. Here we report that a clinical isolate of Prevotella intermedia, strain 17, was found to invade a human oral epithelial cell line (KB), whereas P. intermedia 27, another clinical isolate, and P. intermedia 25611, the type strain, were not found to invade the cell line. Invasion was quantified by the recovery of viable bacteria following a standard antibiotic protection assay and observed by electron microscopy. Cytochalasin D, cycloheximide, monodansylcadaverine, and low temperature (4°C) inhibited the internalization of P. intermedia 17. Antibodies raised against P. intermedia type C fimbriae and against whole cells inhibited invasion, but the anti-type-C-fimbria antibody inhibited invasion to a greater extent than the anti-whole-cell antibody. This work provides evidence that at least one strain of P. intermedia can invade an oral epithelial cell line and that the type C fimbriae and a cytoskeletal rearrangement are required for this invasion. PMID:9826397

Sustained neurotensin exposure promotes cell surface recruitment of NTS2 receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perron, Amelie; Sharif, Nadder; Gendron, Louis

2006-05-12

In this study, we investigated whether persistent agonist stimulation of NTS2 receptors gives rise to down-regulation, in light of reports that their activation induced long-lasting effects. To address this issue, we incubated COS-7 cells expressing the rat NTS2 with neurotensin (NT) for up to 24 h and measured resultant cell surface [{sup 125}I]-NT binding. We found that NTS2-expressing cells retained the same surface receptor density despite efficient internalization mechanisms. This preservation was neither due to NTS2 neosynthesis nor recycling since it was not blocked by cycloheximide or monensin. However, it appeared to involve translocation of spare receptors from internal stores,more » as NT induced NTS2 migration from trans-Golgi network to endosome-like structures. This stimulation-induced regulation of cell surface NTS2 receptors was even more striking in rat spinal cord neurons. Taken together, these results suggest that sustained NTS2 activation promotes recruitment of intracellular receptors to the cell surface, thereby preventing functional desensitization.« less

Senescense

PubMed Central

De Leo, Pietro; Sacher, Joseph A.

1970-01-01

During ripening of banana (Musa sapientum L., var. Gros Michel or Valery) acid phosphatase activity increases 13-to 26-fold in the precipitate and 2- to 4-fold in the supernatant fraction of tissue homogenates. These increases are closely correlated with the onset and peak of the climacteric. The precipitate enzyme may be extracted with Triton X-100, CaCl2 or NaCl; about 80% of it is in a 500g precipitate. Studies on effect of tonicity of the grinding medium indicate that the precipitate enzyme is desorbed from membrane or cell wall surfaces, and is not released as a result of lysis of membranes. The development of acid phosphatase during aging of tissue slices is the same as in intact fruit. Short term studies of tissue slices with cycloheximide and actinomycin D indicate that the increase in activity is owed to new enzyme synthesis, which is dependent upon synthesis of RNA. The possible effects of the increase in acid phosphatase on ripening are discussed. PMID:16657436

Streptomyces sanglieri which colonised and enhanced the growth of Elaeis guineensis Jacq. seedlings was antagonistic to Ganoderma boninense in in vitro studies.

PubMed

Nur Azura, A B; Yusoff, M; Tan, G Y A; Jegadeesh, R; Appleton, D R; Vikineswary, S

2016-04-01

Actinomycete strain AUM 00500 was 99.5 % similar to Streptomyces sanglieri NBRC 100784(T) and was evaluated for antagonistic activity towards Ganoderma boninense, the causative fungus of basal stem rot of oil palm. The strain showed strong antifungal activity towards G. boninense in in vitro and SEM analysis showed various modes of inhibition of the fungus. Ethyl acetate extracts of single culture and inhibition zone of cross-plug culture by HPLC indicated that strain AUM 00500 produced two different antibiotics of the glutarimide group namely cycloheximide and actiphenol. In greenhouse trials, oil palm seed treated with spores of S. sanglieri strain AUM 00500 at 10(9) cfu/ml showed significant (P < 0.05) increase in oil palm seedlings growth when compared to the control. Streptomyces sanglieri strain AUM 00500 successfully colonised the epidermal surface of the roots of treated oil palm seedlings and it was recovered from root fragments plated on starch casein agar.

Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kubicek, C.P.; Schreferl-Kunar, G.; Woehrer, W.

1988-03-01

Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of P/sub i/ and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added /sup 14/CO/sub 2/ was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo duringmore » the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle.« less

Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix

NASA Technical Reports Server (NTRS)

Mooney, D. J.; Langer, R.; Ingber, D. E.

1995-01-01

This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell 'shape' changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In contrast, cell spreading could be completely inhibited by combining suboptimal doses of cytochalasin D and nocodazole, suggesting that intact microtubules can stabilize cell form when the microfilament lattice is partially compromised. The physiological relevance of the cytoskeleton and cell shape in hepatocyte physiology was highlighted by the finding that a short exposure (6 hour) of cells to nocodazole resulted in production of smaller cells 42 hours later that exhibited enhanced production of a liver-specific product (albumin). These data demonstrate that spreading and flattening of the entire cell body is not driven directly by net polymerization of either microfilaments or microtubules.(ABSTRACT TRUNCATED AT 400 WORDS).

Homologous desensitization of HEL cell thrombin receptors. Distinguishable roles for proteolysis and phosphorylation.

PubMed

Brass, L F

1992-03-25

Loss of sensitivity to thrombin following an initial response is characteristic of a number of cell types, including platelets. It has recently been proposed that thrombin receptors resemble other G protein-coupled receptors, but that activation involves a novel mechanism in which thrombin cleaves the receptor, exposing a new N terminus that serves as the ligand for the receptor. Based upon this model, we have examined the mechanism of thrombin receptor desensitization by comparing the effects of thrombin with those of a peptide corresponding to the N-terminal sequence of the receptor following proteolysis by thrombin: SFLLRNPNDKYEPF or TRP42/55. Like thrombin, TRP42/55 stimulated pertussis toxin-sensitive inositol 1,4,5-trisphosphate formation, raised cytosolic Ca2+, and inhibited cAMP formation in the megakaryoblastic HEL cell line. Exposure to either thrombin or TRP42/55 desensitized the cells to both, but not to a third agonist, neuropeptide Y. The rate of recovery after desensitization depended upon the order of agonist addition. Resensitization of the cell to thrombin following a brief exposure to thrombin required up to 24 h and could be inhibited with cycloheximide. Resensitization to TRP42/55 after exposure to thrombin, or to thrombin after exposure to TRP42/55, on the other hand, was detectable within 30 min and could be inhibited by serine/threonine phosphatase inhibitors, but not by cycloheximide. Loss of responsiveness to thrombin and TRP42/55 was also observed following addition of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). However, while the protein kinase inhibitor staurosporine completely prevented the desensitization caused by TPA, it had only a limited effect on the desensitization caused by TRP42/55. These results demonstrate that the G protein-mediated effects of thrombin can be reproduced by a receptor-derived peptide and suggest that desensitization occurs by at least two mechanisms. The first, which is seen with thrombin, but not TRP42/55, involves proteolysis and requires protein synthesis for recovery. The second, which occurs with TRP42/55 and TPA, as well as with thrombin, involves phosphorylation, possibly of the receptor itself. Although protien kinase C is activated by thrombin and is presumably responsible for the desensitization caused by TPA, it does not appear to play a major role in receptor desensitization caused by thrombin and TRP42/55. This suggests that other kinases, such as those which inactivate adrenergic receptors and rhodopsin, are involved in the down-regulation of thrombin receptor function.

Progesterone potentiates the growth inhibitory effects of calcitriol in endometrial cancer via suppression of CYP24A1.

PubMed

Bokhari, Amber A; Lee, Laura R; Raboteau, Dewayne; Turbov, Jane; Rodriguez, Isabel V; Pike, John Wesley; Hamilton, Chad A; Maxwell, George Larry; Rodriguez, Gustavo C; Syed, Viqar

2016-11-22

Here, we evaluated the expression of CYP24A1, a protein that inactivates vitamin D in tissues. CYP24A1 expression was increased in advanced-stage endometrial tumors compared to normal tissues. Similarly, endometrial cancer cells expressed higher levels of CYP24A1 than immortalized endometrial epithelial cells. RT-PCR and Western blotting were used to examine CYP24A1 mRNA and protein levels in endometrial cancer cells after 8, 24, 72, and 120 h of exposure to progesterone, progestin derivatives and calcitriol, either alone or in combination. Progestins inhibited calcitriol-induced expression of CYP24A1 and splice variant CYP24SV mRNA and protein in cancer cells. Furthermore, actinomycin D, but not cycloheximide, blocked calcitriol-induced CYP24A1 splicing. siRNA-induced knockdown of CYP24A1 expression sensitized endometrial cancer cells to calcitriol-induced growth inhibition. These data suggest that CYP24A1 overexpression reduces the antitumor effects of calcitriol in cancer cells and that progestins may be beneficial for maintaining calcitriol's anti-endometrial cancer activity.

Trichostatin A down-regulates ZAP-70, LAT and SLP-76 content in Jurkat T cells.

PubMed

Januchowski, Radosław; Jagodzinski, Paweł P

2007-02-01

We exploited Jurkat leukemia T cell clone E6-1 as a model of Trichostatin A (TSA) effect on cellular levels of ZAP-70, LAT and SLP-76 molecules involved in the signal transduction pathway from T cell receptor to nucleus. Using reverse transcription real-time quantitative PCR and Western blotting analysis we observed that TSA resulted in ZAP-70, LAT and SLP-76 transcript and protein down-regulation in Jurkat leukemia T cells. We also found that TSA reduced half-life of ZAP-70, LAT and SLP-76 mRNAs from 4.8, 3.5, and 4.8 to approximately 2.3, 1.9 and 1.7 h, respectively. Employing the protein biosynthesis inhibitor cycloheximide, we demonstrated the involvement of RNase and/or mRNA stabilization protein in ZAP-70, LAT and SLP-76 mRNAs stabilization. The effect of TSA on ZAP-70, LAT and SLP-76 content in T cells confirms an immunosuppressive effect by TSA, and the usefulness of this histone deacetylase inhibitor in the treatment of autoimmune diseases.

Stimulation of nitrogen fixation in soddy-podzolic soils with fungi

NASA Astrophysics Data System (ADS)

Kurakov, A. V.; Prokhorov, I. S.; Kostina, N. V.; Makhova, E. G.; Sadykova, V. S.

2006-09-01

Stimulation of nitrogen fixation in soddy-podzolic soils is related to the hydrolytic activity of fungi decomposing plant polymers. It was found that the rate of nitrogen fixation upon the simultaneous inoculation of the strains of nitrogen-fixing bacteria Bacillus cereus var. mycoides and the cellulolytic fungus Trichoderma asperellum into a sterile soil enriched with cellulose or Jerusalem artichoke residues is two to four times higher than upon the inoculation of the strains of Bacillus cereus var. mycoides L1 only. The increase in the nitrogen fixation depended on the resistance of the substrates added into the soil to fungal hydrolysis. The biomass of the fungi decomposing plant polymers increased by two-four times. The nitrogen-fixing activity of the soil decreased when the growth of the fungi was inhibited with cycloheximide, which attested to a close correlation between the intensity of the nitrogen fixation and the decomposition of the plant polymers by fungi. The introduction of an antifungal antibiotic, together with starch or with plant residues, significantly (by 60-90%) decreased the rate of nitrogen fixation in the soll.

Catalase anabolism in yeast: loss of regulation by oxygen of catalase apoprotein synthesis after mutation.

PubMed

Berte, C; Sels, A

1979-04-17

A mutant of Saccharomyces cerevisiae which displays catalase activity when grown under strictly anaerobic conditions has been selected on solid media. Although some preformed holoenzyme has accumulated in anaerobic cells, a sharp increase of activity is still measured during adaptation to oxygen in glucose-buffer; however, a striking difference with the wild-type strain is that in the mutant, catalase formation is observed in the presence of cycloheximide that totally inhibits cytoplasmic translation. It is concluded that kat 80 mutant has lost the regulatory control by oxygen of apocatalase synthesis; the later precursor, characterized as apocatalase synthesis; the latter precursor, characterized as apocatalase T, is thought to be activated in vivo, under aerobic conditions, by inclusion of prosthetic group. Regulation of enzyme synthesis by catabolite repression (glucose erfect) persists, unmodified by reference to the wild-type parental strain. Mutation kat 80 specifically hits catalase anabolism, as no significant variations were observed for the edification of the respiratory system and (apo)cytochrome c peroxidase production. Genetic analysis shows that kat 80 phenotype, recessive in heterozygotes, results from a single nuclear mutation.

Interferon-induced Sus scrofa Mx1 blocks endocytic traffic of incoming influenza A virus particles.

PubMed

Palm, Mélanie; Garigliany, Mutien-Marie; Cornet, François; Desmecht, Daniel

2010-01-01

The interferon-induced Mx proteins of vertebrates are dynamin-like GTPases, some isoforms of which can additionally inhibit the life cycle of certain RNA viruses. Here we show that the porcine Mx1 protein (poMx1) inhibits replication of influenza A virus and we attempt to identify the step at which the viral life cycle is blocked. In infected cells expressing poMx1, the level of transcripts encoding the viral nucleoprotein is significantly lower than normal, even when secondary transcription is prevented by exposure to cycloheximide. This reveals that a pretranscriptional block participates to the anti-influenza activity. Binding and internalization of incoming virus particles are normal in the presence of poMx1 but centripetal traffic to the late endosomes is interrupted. Surprisingly but decisively, poMx1 significantly alters binding of early endosome autoantigen 1 to early endosomes and/or early endosome size and spatial distribution. This is compatible with impairment of traffic of the endocytic vesicles to the late endosomes. INRA, EDP Sciences, 2010.

Effect of different starvation conditions on the flocculation of Saccharomyces cerevisiae.

PubMed

Soares, E V; Vroman, A

2003-01-01

To study the effect of different starvation conditions on the flocculation of an ale brewing yeast of Saccharomyces cerevisiae NCYC 1195. Flocculation was assessed by a micro-flocculation technique (Soares and Mota 1997). Carbon-starved cells of a NewFlo phenotype strain did not lose flocculation during a 48 h period. Cells incubated only in the presence of fermentable carbon sources (glucose, galactose and maltose at 2%, w/v), showed a progressive flocculation loss. The incubation of cells in 4% (v/v) ethanol did not induce a flocculation loss. The simultaneous incubation of cells in the presence of 2% (w/v) glucose and 15 microg ml(-1) cycloheximide hindered flocculation loss. The presence of 0.1 mmol l(-1) PMSF or 10 mmol l-1 EDTA prevented partially or completely, respectively, the loss of flocculation in the presence of glucose. Fermentable sugars induced a flocculation loss, which seems to require de novo protein synthesis and the involvement of different proteases. The findings reported here contribute to the elucidation of the role of nutrients on the physiological control of yeast flocculation.

A low, adaptive dose of gamma-rays reduced the number and altered the spectrum of S1- mutants in human-hamster hybrid AL cells

NASA Technical Reports Server (NTRS)

Ueno, A. M.; Vannais, D. B.; Gustafson, D. L.; Wong, J. C.; Waldren, C. A.

1996-01-01

We examined the effects of a low, adaptive dose of 137Cs-gamma-irradiation (0.04 Gy) on the number and kinds of mutants induced in AL human-hamster hybrid cells by a later challenge dose of 4 Gy. The yield of S1- mutants was significantly less (by 53%) after exposure to both the adaptive and challenge doses compared to the challenge dose alone. The yield of hprt- mutants was similarly decreased. Incubation with cycloheximide (CX) or 3-aminobenzamide largely negated the decrease in mutant yield. The adaptive dose did not perturb the cell cycle, was not cytotoxic, and did not of itself increase the mutant yield above background. The adaptive dose did, however, alter the spectrum of S1- mutants from populations exposed only to the adaptive dose, as well as affecting the spectrum of S1- mutants generated by the challenge dose. The major change in both cases was a significant increase in the proportion of complex mutations compared to small mutations and simple deletions.

[Control of RNA biosynthesis in rat liver. Some features of RNA biosynthesis during prolonged protein synthesis inhibition].

PubMed

Todorov, I N; Shen, R A; Zheliabovskaia, S M; Galkin, A P

1976-10-01

A drastic inhibition of protein biosynthesis in rat liver in vivo by cycloheximide (CHI) (0.3 mg/100 g of body weight) first caused an increase of RNA synthesis (after 1 hour), which was then followed by its decrease. Partial gradual restoration of the protein synthesis level was shown to be accompanied by a repeated increase of RNA synthesis (12 hs) and its normalisation after 24 hs. The first maximum of RNA synthesis increase in the isolated nuclei system was AU-type RNA synthesis (sensitive to alpha-amanitine), the second one was due to GC-type RNA synthesis (resistant to this toxin). Purified chromatine template activity in the system with E. coli RNA polymerase (by 14%) an hour after CHI treatment, but 3 hrs later was decreased and subsequently restored (12 hrs after CHI injection). The changes of RNA biosynthesis induced by prolonged protein synthesis inhibition suggest the existence of continuous RNA synthesis control in nuclei. This control is realized by translation system using the feed back principle.

Activities of Tricarboxylic Acid Cycle Enzymes, Glyoxylate Cycle Enzymes, and Fructose Diphosphatase in Bakers' Yeast During Adaptation to Acetate Oxidation

PubMed Central

Gosling, J. P.; Duggan, P. F.

1971-01-01

Bakers' yeast oxidizes acetate at a high rate only after an adaptation period during which the capacity of the glyoxylate cycle is found to increase. There was apparently no necessity for the activity of acetyl-coenzyme A synthetase, the capacity of the tricarboxylic acid cycle, or the concentrations of the cytochromes to increase for this adaptation to occur. Elevation of fructose 1,6 diphosphatase occurred only when acetate oxidation was nearly maximal. Cycloheximide almost completely inhibited adaptation as well as increases in the activities of isocitrate lyase and aconitate hydratase, the only enzymes assayed. p-Fluorophenylalanine was partially effective and chloramphenicol did not inhibit at all. The presence of ammonium, which considerably delayed adaptation of the yeast to acetate oxidation, inhibited the increases in the activities of the glyoxylate cycle enzymes to different degrees, demonstrating noncoordinate control of these enzymes. Under the various conditions, the only enzyme activity increase consistently related to the rising oxygen uptake rate was that of isocitrate lyase which apparently limited the activity of the cycle. PMID:5557595

African Swine Fever Virus IAP Homologue Inhibits Caspase Activation and Promotes Cell Survival in Mammalian Cells

PubMed Central

Nogal, María L.; González de Buitrago, Gonzalo; Rodríguez, Clara; Cubelos, Beatriz; Carrascosa, Angel L.; Salas, María L.; Revilla, Yolanda

2001-01-01

African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis. PMID:11222676

African swine fever virus IAP homologue inhibits caspase activation and promotes cell survival in mammalian cells.

PubMed

Nogal, M L; González de Buitrago, G; Rodríguez, C; Cubelos, B; Carrascosa, A L; Salas, M L; Revilla, Y

2001-03-01

African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis.

Hyperosmolarity leads to an increase in derepressed system A activity in the renal epithelial cell line NBL-1.

PubMed Central

Soler, C; Felipe, A; Casado, F J; McGivan, J D; Pastor-Anglada, M

1993-01-01

Hyperosmolarity induced an increase in Na(+)-dependent L-alanine uptake in confluent monolayers of the established renal epithelial cell line NBL-1. This induction was attributable to system A and was only seen when the cells had been previously deprived of amino acids in the culture medium to derepress system A activity. It was additive to the adaptive regulation induction, and both were inhibited by cycloheximide. However, the hyperosmolarity effect was inhibited by colcemid (an inhibitor of microtubular function), but adaptive regulation was not. Otherwise, when cell monolayers were incubated in a control medium, basal Na(+)-dependent L-alanine uptake mediated by system B0 decreased. The results of this study show that: (i) system A activity was not induced by cell shrinkage and subsequent swelling due to extracellular hyperosmolarity when cells were incubated in control medium; (ii) previous expression of system A activity induced by amino acid starvation seems to be a prerequisite for further induction due to hyperosmolarity; and (iii) the effects of adaptive regulation and hyperosmotic stress are mediated by different mechanisms. PMID:8435065

In vitro glucose uptake activity of Aegles marmelos and Syzygium cumini by activation of Glut-4, PI3 kinase and PPARgamma in L6 myotubes.

PubMed

Anandharajan, R; Jaiganesh, S; Shankernarayanan, N P; Viswakarma, R A; Balakrishnan, A

2006-06-01

The purpose of the present study is to investigate the effect of methanolic extracts of Aegles marmelos and Syzygium cumini on a battery of targets glucose transporter (Glut-4), peroxisome proliferator activator receptor gamma (PPARgamma) and phosphatidylinositol 3' kinase (PI3 kinase) involved in glucose transport. A. marmelos and S. cumini are anti-diabetic medicinal plants being used in Indian traditional medicine. Different solvent extracts extracted sequentially were analysed for glucose uptake activity at each step and methanol extracts were found to be significantly active at 100ng/ml dose comparable with insulin and rosiglitazone. Elevation of Glut-4, PPARgamma and PI3 kinase by A. marmelos and S. cumini in association with glucose transport supported the up-regulation of glucose uptake. The inhibitory effect of cycloheximide on A. marmelos- and S. cumini-mediated glucose uptake suggested that new protein synthesis is required for the elevated glucose transport. Current observation concludes that methanolic extracts of A. marmelos and S. cumini activate glucose transport in a PI3 kinase-dependent fashion.

Induction of the SHARP-2 mRNA level by insulin is mediated by multiple signaling pathways.

PubMed

Kanai, Yukiko; Asano, Kosuke; Komatsu, Yoshiko; Takagi, Katsuhiro; Ono, Moe; Tanaka, Takashi; Tomita, Koji; Haneishi, Ayumi; Tsukada, Akiko; Yamada, Kazuya

2017-02-01

The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor which represses transcription of the rat phosphoenolpyruvate carboxykinase gene. In this study, a regulatory mechanism of the SHARP-2 mRNA level by insulin was analyzed. Insulin rapidly induced the level of SHARP-2 mRNA. This induction was blocked by inhibitors for phosphoinositide 3-kinase (PI 3-K), protein kinase C (PKC), and mammalian target of rapamycin (mTOR), actinomycin D, and cycloheximide. Whereas an adenovirus infection expressing a dominant negative form of atypical PKC lambda (aPKCλ) blocked the insulin-induction of the SHARP-2 mRNA level, insulin rapidly activated the mTOR. Insulin did not enhance transcriptional activity from a 3.7 kb upstream region of the rat SHARP-2 gene. Thus, we conclude that insulin induces the expression of the rat SHARP-2 gene at the transcription level via both a PI 3-K/aPKCλ- and a PI 3-K/mTOR- pathways and that protein synthesis is required for this induction.

[Effects of methyl tertiary butyl ether on cell cycle and cell apoptosis].

PubMed

Zhou, W; Huang, G; Zhang, H; Ye, S

2000-07-01

To explore the effects of the new gasoline additive, methyl tertiary butyl ether (MTBE) on cell cycle and cell apoptosis. Flow cytometry was used to evaluate the effect of MTBE (1, 2, 4 microl/ml, 24 h) on NIH/3T3 cell cycles; and the effect of MTBE on Hela cell apoptosis was evaluated by detecting cell survival using crystal violet staining. Flow cytometry showed that MTBE could change NIH/3T3 cell cycles, decrease the number of cells in S stage, and arrest cells at G(2) + M stage. The results suggested that MTBE could affect NIH/3T3 cell cycles and induce cell proliferation. This situation existed 48 hours after the treatment, and cell cycles came back normal 96 hours after the treatment. By detecting cell survival using crystal violet staining, we found that MTBE could inhibit the apoptosis of Hela cells which was induced by tumor necrosis factor (TNF)alpha and cycloheximide. MTBE's carcinogenicity to animals may relate to induction of cell proliferation and inhibition of cell apoptosis.

Rapid Consolidation to a radish and Protein Synthesis-Dependent Long-Term Memory after Single-Session Appetitive Olfactory Conditioning in Drosophila

PubMed Central

Krashes, Michael J.; Waddell, Scott

2008-01-01

In Drosophila, formation of aversive olfactory long-term memory (LTM) requires multiple training sessions pairing odor and electric shock punishment with rest intervals. In contrast, here we show that a single 2 min training session pairing odor with a more ethologically relevant sugar reinforcement forms long-term appetitive memory that lasts for days. Appetitive LTM has some mechanistic similarity to aversive LTM in that it can be disrupted by cycloheximide, the dCreb2-b transcriptional repressor, and the crammer and tequila LTM-specific mutations. However, appetitive LTM is completely disrupted by the radish mutation that apparently represents a distinct mechanistic phase of consolidated aversive memory. Furthermore, appetitive LTM requires activity in the dorsal paired medial neuron and mushroom body α′ β′ neuron circuit during the first hour after training and mushroom body αβ neuron output during retrieval, suggesting that appetitive middle-term memory and LTM are mechanistically linked. Last, experiments feeding and/or starving flies after training reveals a critical motivational drive that enables appetitive LTM retrieval. PMID:18354013

Translational errors as an early event in prion conversion.

PubMed

Hatin, I; Bidou, L; Cullin, C; Rousset, J P

2001-01-01

A prion is an infectious, altered form of a cellular protein which can self-propagate and affect normal phenotype. Prion conversion has been observed for mammalian and yeast proteins but molecular mechanisms that trigger this process remain unclear. Up to now, only post-translational models have been explored. In this work, we tested the hypothesis that co-translational events may be implicated in the conformation changes of the Ure2p protein of Saccharomyces cerevisiae. This protein can adopt a prion conformation leading to an [URE3] phenotype which can be easily assessed and quantified. We analyzed the effect of two antibiotics, known to affect translation, on [URE3] conversion frequency. For cells treated with G418 we observed a parallel increase of translational errors rate and frequency of [URE3] conversion. By contrast, cycloheximide which was not found to affect translational fidelity, has no influence on the induction of [URE3] phenotype. These results raise the possibility that the mechanism of prion conversion might not only involve alternative structures of strictly identical molecules but also aberrant proteins resulting from translational errors.

Ecdysteroid-stimulated synthesis and secretion of an N-acetyl-D-glucosamine-rich glycopeptide in a lepidopteran cell line derived from imaginal discs.

PubMed

Porcheron, P; Morinière, M; Coudouel, N; Oberlander, H

1991-01-01

Hormone-regulated processing of N-acetyl-D-glucosamine was studied in an insect cell line derived from imaginal wing discs of the Indian meal moth, Plodia interpunctella (Hübner). The cell line, IAL-PID2, responded to treatment with 20-hydroxyecdysone with increased incorporation of GlcNAc into glycoproteins. Cycloheximide and tunicamycin counteracted the action of the hormone. In particular, treatment with 20-hydroxyecdysone resulted in the secretion of a 5,000 dalton N-acetyl-D-glucosamine-rich glycopeptide by the IAL-PID2 cells. Accumulation of this peptide was prevented by the use of teflubenzuron, a potent chitin synthesis inhibitor. A glycopeptide of similar molecular weight was observed in imaginal discs of P. interpunctella treated with 20-hydroxyecdysone in vitro, under conditions that induce chitin synthesis. Although the function of the 5,000 dalton glycopeptide is not known, we believe that the PID2 cell line is a promising model for molecular analysis of ecdysteroid-regulated processing of aminosugars by epidermal cells during insect development.

Detoxification of corn stover and corn starch pyrolysis liquors by ligninolytic enzymes of Phanerochaete chrysosporium.

PubMed

Khiyami, Mohammad A; Pometto, Anthony L; Brown, Robert C

2005-04-20

Phanerochaete chrysosporium (ATCC 24725) shake flask culture with 3 mM veratryl alcohol addition on day 3 was able to grow and detoxify different concentrations of diluted corn stover (Dcs) and diluted corn starch (Dst) pyrolysis liquors [10, 25, and 50% (v/v)] in defined media. GC-MS analysis of reaction products showed a decrease and change in some compounds. In addition, the total phenolic assay with Dcs samples demonstrated a decrease in the phenolic compounds. A bioassay employing Lactobacillus casei growth and lactic acid production was developed to confirm the removal of toxic compounds from 10 and 25% (v/v) Dcs and Dst by the lignolytic enzymes, but not from 50% (v/v) Dcs and Dst. The removal did not occur when sodium azide or cycloheximide was added to Ph. chrysosporium culture media, confirming the participation of lignolytic enzymes in the detoxification process. A concentrated enzyme preparation decreased the phenolic compounds in 10% (v/v) corn stover and corn starch pyrolysis liquors to the same extent as the fungal cultures.

Rat Humanin is encoded and translated in mitochondria and is localized to the mitochondrial compartment where it regulates ROS production.

PubMed

Paharkova, Vladislava; Alvarez, Griselda; Nakamura, Hiromi; Cohen, Pinchas; Lee, Kuk-Wha

2015-09-15

Evidence for the putative mitochondrial origin of the Humanin (HN) peptide has been lacking, although its cytoprotective activity has been demonstrated in a variety of organismal and cellular systems. We sought to establish proof-of-principle for a mitochondria-derived peptide (MDP) in a rat-derived cellular system as the rat HN sequence is predicted to lack nuclear insertions of mitochondrial origin (NUMT). We found that the rat HN (Rattin; rHN) homologue is derived from the mitochondrial genome as evidenced by decreased production in Rho-0 cells, and that peptide translation occurs in the mitochondria as it is unaffected by cycloheximide. Rat HN localizes to the mitochondria in cellular subfractionation and immunohistochemical studies. Addition of a HN analogue to isolated mitochondria from rat INS-1 beta cells reduced hydrogen peroxide production by 55%. In summary, a locally bioactive peptide is derived and translated from an open reading frame (ORF) within rat mitochondrial DNA encoding 16S rRNA. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Mechanical stimulation of skeletal muscle increases prostaglandin F2(alpha) synthesis and cyclooxygenase activity by a pertussis toxin sensitive mechanism

NASA Technical Reports Server (NTRS)

Vandenburgh, Herman H.; Shansky, Janet; Solerssi, Rosa; Chromiak, Joseph

1992-01-01

Repetitive mechanical stimulation of differentiated skeletal muscle in tissue culture increases the production of prostaglandin F(sub 2(alpha)), an anabolic stimulator of myofiber growth. Within 4 h of initiating mechanical activity, the activity of cyclooxygenase, a regulatory enzyme in prostaglandin synthesis, was increased 82% (P is less than .005), and this increase was maintained for at least 24 h. Kinetic analysis of the stretch-activated cyclooxygenase indicated a two to three-fold decrease in the enzyme's K(sub m) with no change in V(sub max). The stretch-induced increase in enzymatic activity was not inhibited by cycloheximide, was independent of cellular electrical activity (tetrodotoxin-insensitive), but was prevented by the G protein inhibitor pertussis toxin. Pertussis toxin also inhibited the stretch-induced increases in PGF(sub 2(alpha)) production, and cell growth. It is concluded that stretch of skeletal muscle increases the synthesis of the anabolic modulator PGF(sub 2(alpha)) by a G protein-dependent process which involves activation of cyclooxygenase by a posttranslational mechanism.

The down-regulation of the mitogenic fibrinogen receptor (MFR) in serum-containing medium does not occur in defined medium.

PubMed

Levesque, J P; Hatzfeld, A; Domart, I; Hatzfeld, J

1990-02-01

Normal human hemopoietic cells such as early bone marrow progenitors, or lymphoma-derived cell lines such as Raji or JM cells, possess a low-affinity receptor specific for fibrinogen. This receptor triggers a mitogenic effect. It differs from the glycoprotein IIb-IIIa which is involved in fibrinogen-induced platelet aggregation. We demonstrate here that this mitogenic fibrinogen receptor (MFR) can be internalized or reexpressed, depending on culture conditions. Internalization was temperature-dependent. At 37 degrees C in the presence of cycloheximide or actinomycin D, the half-life of cell surface MFRs was 2 h, independent of receptor occupancy. Binding of fibrinogen to the MFR resulted in a down-regulation which was fibrinogen dose-dependent. This occurred in serum-supplemented medium but not in defined medium supplemented with fatty acids. Reexpression of MFRs could be induced in 28 to 42 h by serum removal. The down-regulation of mitogenic receptors in plasma or serum could explain why normal cells do not proliferate in the peripheral blood.

Fibroblast growth factor regulates insulin-like growth factor-binding protein production by vascular smooth muscle cells.

PubMed

Ververis, J; Ku, L; Delafontaine, P

1994-02-01

Insulin-like growth factor I is an important mitogen for vascular smooth muscle cells, and its effects are regulated by several binding proteins. Western ligand blotting of conditioned medium from rat aortic smooth muscle cells detected a 24 kDa binding protein and a 28 kDa glycosylated variant of this protein, consistent with insulin-like growth factor binding protein-4 by size. Low amounts of a glycosylated 38 to 42 kDa doublet (consistent with binding protein-3) and a 31 kDa non-glycosylated protein also were present. Basic fibroblast growth factor markedly increased secretion of the 24 kDa binding protein and its 28 kDa glycosylated variant. This effect was dose- and time-dependent and was inhibited by co-incubation with cycloheximide. Crosslinking of [125I]-insulin-like growth factor I to cell monolayers revealed no surface-associated binding proteins, either basally or after agonist treatment. Induction of binding protein production by fibroblast growth factor at sites of vascular injury may be important in vascular proliferative responses in vivo.

Maternal RNA regulates Aurora C kinase during mouse oocyte maturation in a translation-independent fashion.

PubMed

Balboula, Ahmed Z; Blengini, Cecilia S; Gentilello, Amanda S; Takahashi, Masashi; Schindler, Karen

2017-06-01

During oocyte meiotic maturation, Aurora kinase C (AURKC) is required to accomplish many critical functions including destabilizing erroneous kinetochore-microtubule (K-MT)attachments and regulating bipolar spindle assembly. How localized activity of AURKC is regulated in mammalian oocytes, however, is not fully understood. Female gametes from many species, including mouse, contain stores of maternal transcripts that are required for downstream developmental events. We show here that depletion of maternal RNA in mouse oocytes resulted in impaired meiotic progression, increased incidence of chromosome misalignment and abnormal spindle formation at metaphase I (Met I), and cytokinesis defects. Importantly, depletion of maternal RNA perturbed the localization and activity of AURKC within the chromosomal passenger complex (CPC). These perturbations were not observed when translation was inhibited by cycloheximide (CHX) treatment. These results demonstrate a translation-independent function of maternal RNA to regulate AURKC-CPC function in mouse oocytes. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Antioxidant potential of CORM-A1 and resveratrol during TNF-α/cycloheximide-induced oxidative stress and apoptosis in murine intestinal epithelial MODE-K cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Babu, Dinesh, E-mail: dinesh.babu@ugent.be; Leclercq, Georges; Goossens, Vera

2015-10-15

Targeting excessive production of reactive oxygen species (ROS) could be an effective therapeutic strategy to prevent oxidative stress-associated gastrointestinal inflammation. NADPH oxidase (NOX) and mitochondrial complexes (I and II) are the major sources of ROS production contributing to TNF-α/cycloheximide (CHX)-induced apoptosis in the mouse intestinal epithelial cell line, MODE-K. In the current study, the influence of a polyphenolic compound (resveratrol) and a water-soluble carbon monoxide (CO)-releasing molecule (CORM-A1) on the different sources of TNF-α/CHX-induced ROS production in MODE-K cells was assessed. This was compared with H{sub 2}O{sub 2}-, rotenone- or antimycin-A-induced ROS-generating systems. Intracellular total ROS, mitochondrial-derived ROS and mitochondrialmore » superoxide anion (O{sub 2}·{sup −}) production levels were assessed. Additionally, the influence on TNF-α/CHX-induced changes in mitochondrial membrane potential (Ψ{sub m}) and mitochondrial function was studied. In basal conditions, CORM-A1 did not affect intracellular total or mitochondrial ROS levels, while resveratrol increased intracellular total ROS but reduced mitochondrial ROS production. TNF-α/CHX- and H{sub 2}O{sub 2}-mediated increase in intracellular total ROS production was reduced by both resveratrol and CORM-A1, whereas only resveratrol attenuated the increase in mitochondrial ROS triggered by TNF-α/CHX. CORM-A1 decreased antimycin-A-induced mitochondrial O{sub 2}·{sup −} production without any influence on TNF-α/CHX- and rotenone-induced mitochondrial O{sub 2}·{sup −} levels, while resveratrol abolished all three effects. Finally, resveratrol greatly reduced and abolished TNF-α/CHX-induced mitochondrial depolarization and mitochondrial dysfunction, while CORM-A1 only mildly affected these parameters. These data indicate that the cytoprotective effect of resveratrol is predominantly due to mitigation of mitochondrial ROS, while CORM-A1 acts solely on NOX-derived ROS to protect MODE-K cells from TNF-α/CHX-induced cell death. This might explain the more pronounced cytoprotective effect of resveratrol. - Highlights: • In MODE-K IECs, TNF-α/CHX induces correlating ROS, mitochondrial O{sub 2}·{sup −} and cell death. • CORM-A1 does not influence basal intracellular ROS and mitochondrial O{sub 2}·{sup −} levels. • Resveratrol increases basal intracellular ROS but decreases mitochondrial O{sub 2}·{sup −} levels. • CORM-A1 acts solely on NOX-derived ROS to protect from cell death by TNF-α/CHX. • Cytoprotection by resveratrol is predominantly due to reduction of mitochondrial O{sub 2}·{sup −}.« less

Differential regulation of membrane-associated mucins in the human ocular surface epithelium.

PubMed

Hori, Yuichi; Spurr-Michaud, Sandra; Russo, Cindy Leigh; Argüeso, Pablo; Gipson, Ilene K

2004-01-01

Membrane-associated mucins present in the apical cells of the ocular surface epithelium (MUC1, -4, and -16) are believed to contribute to the maintenance of a hydrated and wet-surfaced epithelial phenotype. Serum and retinoic acid (RA) have been used to treat drying ocular surface diseases. The goal of this study was to determine whether serum or RA regulates the production of membrane-associated mucins in human conjunctival epithelial cells. A telomerase-immortalized human conjunctival epithelial cell line (HCjE) was used. Cells were cultured in serum-free medium to confluence and then cultured with either 10% calf serum or with 100 nM RA for 0 to 72 hours. Conventional RT-PCR was used to determine the expression of retinoic acid receptors (RARs) and quantitative real-time PCR was used to investigate the mRNA expression of MUC1, -4, and -16. Protein levels were assayed by immunoblot analysis, using the antibodies HMFG-2, 1G8, or OC125, which are specific to MUC1, -4 and -16, respectively. To determine whether RA-associated MUC4 mRNA induction is a direct or indirect effect, HCjE cells were treated with RA and the protein synthesis inhibitor cycloheximide (1.0 microg/mL) for 12 hours. MUC1 and -16, but not -4, mRNAs were detectable in HCjE cells grown in serum-free medium. Real-time PCR revealed that MUC4 mRNA was significantly induced by serum 3 hours after its addition, and that MUC1 and MUC16 mRNA levels were significantly upregulated at 72 hours. Western blot analysis demonstrated that the MUC1, -4, and -16 proteins increased over time after addition of serum. Conventional RT-PCR analysis demonstrated that RAR-alpha and -gamma mRNA were expressed in native human conjunctival tissue as well as in the HCjE cells. Treatment with RA upregulated the expression of both MUC4 and -16 mRNA and protein, but MUC1 was unaffected. Because the protein synthesis inhibitor cycloheximide did not prevent the RA-associated induction of MUC4 mRNA, the action of RA on the MUC4 promoter may be direct. The membrane-associated mucins of the ocular surface epithelia, MUC1, -4, and -16, are differentially regulated by serum and RA in the telomerase-immortalized human conjunctival epithelial cell line. Serum derived from vessels in the conjunctiva may play an important role in mucin regulation in the ocular surface epithelia. These data also support the clinical efficacy of autologous serum and RA application in patients with ocular surface diseases. Furthermore, the data suggest that MUC4 and -16 are particularly important hydrophilic molecules involved in maintenance of a healthy ocular surface.

[Dominating motivation in systemic memory mechanisms].

PubMed

Sudakov, K V

2005-01-01

The materials provided in the article support the key role of dominating motivation in the systemic processes of fixation and opening of memory mechanisms. The activating mechanisms of dominating motivations in the systemic architectonics of behavioural acts provide the basis for development of a multicomponent acceptor apparatus of an action outcomes broadly represented in various analysing brain sections. As result of enhancement of action outcomes on acceptors structures, molecular behaviour engrammes form within the functional systems. It is these molecular engrammes that are opened by dominating motivations in the same spatial-temporal sequence in which training takes place, and determine deliberate actions of animals. It was demonstrated that dominating motivation opens genetic information with an approximating-exploratory reaction under strong activation of early genes expression, in particular, of c-fos gene protein. Inherent motivation reactions are not blocked by inhibitors of proteins synthesis, by cycloheximide, in particular. In the process of training animals, i.e., satisfaction of the demands which are the basis of dominating motivations, expression of early genes in reduced, while expression of late genes is initiated. In this case, blockators of protein synthesis begin to produce strong inhibiting impact on behaviour of animals.

Steroid 5 alpha-reductase deficiency in a 65-year-old male pseudohermaphrodite: the natural history, ultrastructure of the testes, and evidence for inherited enzyme heterogeneity.

PubMed

Imperato-McGinley, J; Peterson, R E; Leshin, M; Griffin, J E; Cooper, G; Draghi, S; Berenyi, M; Wilson, J D

1980-01-01

We report a 65-yr-old male pseudohermaphrodite with steroid 5 alpha-reductase deficiency in whom there was no medical intervention before, during, or after puberty, enabling us to observe the natural history of this condition. The affected subject has an android build, with more facial and body hair than in previously described affected adults. Although the subject was raised as a girl, a male gender identity evolved with the events of puberty, but social factors have delayed the complete expression of a male gender role. Plasma levels of dihydrotestosterone and the in vivo conversion of radiolabeled testosterone to dihydrotestosterone were decreased. There was an elevated urinary etiocholanolone to androsterone ratio, typical of the syndrome. Characterization of 5 alpha-reductase enzyme activity in cultured genital skin fibroblasts demonstrated a pattern of enzyme activity distinctly different from three previously described families with this condition. There was decreased enzyme affinity for testosterone and NADPH. Also, the stability of the enzyme to elevated temperature was not protected by NADPH, resulting in rapid disappearance of enzyme activity after inhibition of protein synthesis with cycloheximide. Electron microscopic evaluation of the testes was carried out.

Thiazolidinediones inhibit REG I{alpha} gene transcription in gastrointestinal cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamauchi, Akiyo; Laboratory of Molecular Genetics, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai 980-8578; Department of Biochemistry, Nara Medical University, Kashihara 634-8521

2009-02-13

REG (Regenerating gene) I{alpha} protein functions as a growth factor for gastrointestinal cancer cells, and its mRNA expression is strongly associated with a poor prognosis in gastrointestinal cancer patients. We here demonstrated that PPAR{gamma}-agonist thiazolidinediones (TZDs) inhibited cell proliferation and REG I{alpha} protein/mRNA expression in gastrointestinal cancer cells. TZDs inhibited the REG I{alpha} gene promoter activity, via its cis-acting element which lacked PPAR response element and could not bind to PPAR{gamma}, in PPAR{gamma}-expressing gastrointestinal cancer cells. The inhibition was reversed by co-treatment with a specific PPAR{gamma}-antagonist GW9662. Although TZDs did not inhibit the REG I{alpha} gene promoter activity in PPAR{gamma}-non-expressingmore » cells, PPAR{gamma} overexpression in the cells recovered their inhibitory effect. Taken together, TZDs inhibit REG I{alpha} gene transcription through a PPAR{gamma}-dependent pathway. The TZD-induced REG I{alpha} mRNA reduction was abolished by cycloheximide, indicating the necessity of novel protein(s) synthesis. TZDs may therefore be a candidate for novel anti-cancer drugs for patients with gastrointestinal cancer expressing both REG I{alpha} and PPAR{gamma}.« less

Hydrogen peroxide stimulates ubiquitin-conjugating activity and expression of genes for specific E2 and E3 proteins in skeletal muscle myotubes

NASA Technical Reports Server (NTRS)

Li, Yi-Ping; Chen, Yuling; Li, Andrew S.; Reid, Michael B.

2003-01-01

Reactive oxygen species (ROS) are thought to promote muscle atrophy in chronic wasting diseases, but the underlying mechanism has not been determined. Here we show that H2O2 stimulates ubiquitin conjugation to muscle proteins through transcriptional regulation of the enzymes (E2 and E3 proteins) that conjugate ubiquitin to muscle proteins. Incubation of C2C12 myotubes with 100 microM H2O2 increased the rate of 125I-labeled ubiquitin conjugation to muscle proteins in whole cell extracts. This response required at least 4-h exposure to H2O2 and persisted for at least 24 h. Preincubating myotubes with cycloheximide or actinomycin D blocked H2O2 stimulation of ubiquitin-conjugating activity, suggesting that gene transcription is required. Northern blot analyses revealed that H2O2 upregulates expression of specific E3 and E2 proteins that are thought to regulate muscle catabolism, including atrogin1/MAFbx, MuRF1, and E214k. These results suggest that ROS stimulate protein catabolism in skeletal muscle by upregulating the ubiquitin conjugation system.

Abscisic Acid Metabolism in Salt-Stressed Cells of Dunaliella salina

PubMed Central

Cowan, A. Keith; Rose, Peter D.

1991-01-01

The interrelationship between abscisic acid (ABA) production and β-carotene accumulation was investigated in salt-stressed cells of the halotolerant green alga Dunaliella salina var bardawil. Cells were supplied with either R-[2-14C]mevalonolactone or [14C] sodium bicarbonate for 20 hours and then exposed to increased salinity (1.5 to 3.0 molar NaCl) for various lengths of time. Incorporation of label into abscisic acid and phaseic acid and the distribution of [14C]ABA between the cells and incubation media were monitored. [14C]ABA and [14C]phaseic acid were identified as products of both R-[2-14C]mevalonolactone and [14C]sodium bicarbonate metabolism. ABA metabolism was enhanced by hypersalinity stress. Actinomycin D, chloramphenicol, and cycloheximide abolished the stress-induced production of ABA, suggesting a role for gene activation in the process. Kinetic analysis of both ABA and β-carotene production demonstrated two stages of accelerated β-carotene production. In addition, ABA levels increased rapidly, and this increase occurred coincident with the early period of accelerated β-carotene production. A possible role for ABA as a regulator of carotenogenesis in cells of D. salina is therefore discussed. PMID:16668469

Cell wall invertase in tobacco crown gall cells : enzyme properties and regulation by auxin.

PubMed

Weil, M; Rausch, T

1990-12-01

The cell wall invertase from an Agrobacterium tumefaciens-transformed Nicotiana tabacum cell line (SR1-C58) was purified. The heterogeneously glycosylated enzyme has the following properties: M(r) 63,000, pH optimum at 4.7, K(m sucrose) 0.6 millimolar (at pH 4.7), pl 9.5. Enzyme activity is inhibited by micromolar concentrations of HgCl(2) but is insensitive to H(2)O(2), N-ethylmaleimide and dithiothreitol. Upon transfer of transformed cells from the stationary phase to fresh medium, a cycloheximide- and tunicamycin-sensitive de novo formation of cell wall invertase is demonstrated in the absence or presence of sucrose. While in an auxin mutant (lacking gene 1;SR1-3845) 1 micromolar 1-naphthaleneacetic acid led to a further increased activity, the wild-type transformed cell line (SR1-C58) responded with a decreased activity compared to the control. An analysis of cell wall invertase in and around tumors initiated with Agrobacterium tumefaciens (strain C58) on Nicotiana tabacum stem and Kalanchoë daigremontiana leaves revealed gradients of activity. The results indicate that the auxin-stimulated cell wall invertase is essential for the establishment of the tumor sink.

An Easy Method for Plant Polysome Profiling.

PubMed

Lecampion, Cécile; Floris, Maïna; Fantino, Jean Raphaël; Robaglia, Christophe; Laloi, Christophe

2016-08-28

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

Nitric oxide production by cultured human aortic smooth muscle cells: stimulation by fluid flow

NASA Technical Reports Server (NTRS)

Papadaki, M.; Tilton, R. G.; Eskin, S. G.; McIntire, L. V.

1998-01-01

This study demonstrated that exposure of cultured human aortic smooth muscle cells (SMC) to fluid flow resulted in nitric oxide (NO) production, monitored by nitrite and guanosine 3',5'-cyclic monophosphate production. A rapid burst in nitrite production rate was followed by a more gradual increase throughout the period of flow exposure. Neither the initial burst nor the prolonged nitrite production was dependent on the level of shear stress in the range of 1.1-25 dyn/cm2. Repeated exposure to shear stress after a 30-min static period restimulated nitrite production similar to the initial burst. Ca(2+)-calmodulin antagonists blocked the initial burst in nitrite release. An inhibitor of nitric oxide synthase (NOS) blocked nitrite production, indicating that changes in nitrite reflect NO production. Treatment with dexamethasone or cycloheximide had no effect on nitrite production. Monoclonal antibodies directed against the inducible and endothelial NOS isoforms showed no immunoreactivity on Western blots, whereas monoclonal antibodies directed against the neuronal NOS gave specific products. These findings suggest that human aortic SMC express a constitutive neuronal NOS isoform, the enzymatic activity of which is modulated by flow.

Inhibition of eukaryotic translation elongation by the antitumor natural product Mycalamide B.

PubMed

Dang, Yongjun; Schneider-Poetsch, Tilman; Eyler, Daniel E; Jewett, John C; Bhat, Shridhar; Rawal, Viresh H; Green, Rachel; Liu, Jun O

2011-08-01

Mycalamide B (MycB) is a marine sponge-derived natural product with potent antitumor activity. Although it has been shown to inhibit protein synthesis, the molecular mechanism of action by MycB remains incompletely understood. We verified the inhibition of translation elongation by in vitro HCV IRES dual luciferase assays, ribosome assembly, and in vivo [(35)S]methinione labeling experiments. Similar to cycloheximide (CHX), MycB inhibits translation elongation through blockade of eEF2-mediated translocation without affecting the eEF1A-mediated loading of tRNA onto the ribosome, AUG recognition, or dipeptide synthesis. Using chemical footprinting, we identified the MycB binding site proximal to the C3993 28S rRNA residue on the large ribosomal subunit. However, there are also subtle, but significant differences in the detailed mechanisms of action of MycB and CHX. First, MycB arrests the ribosome on the mRNA one codon ahead of CHX. Second, MycB specifically blocked tRNA binding to the E-site of the large ribosomal subunit. Moreover, they display different polysome profiles in vivo. Together, these observations shed new light on the mechanism of inhibition of translation elongation by MycB.

A novel immediate-early response gene of endothelium is induced by cytokines and encodes a secreted protein.

PubMed

Holzman, L B; Marks, R M; Dixit, V M

1990-11-01

We have previously described the cloning of a group of novel cellular immediate-early response genes whose expression in human umbilical vein endothelial cells is induced by tumor necrosis factor alpha in the presence of cycloheximide. These genes are likely to participate in mediating the response of the vascular endothelium to proinflammatory cytokines. In this study, we further characterized one of these novel gene products named B61. Sequence analysis of cDNA clones encoding B61 revealed that its protein product has no significant homology to previously described proteins. Southern analysis suggested that B61 is an evolutionarily conserved single-copy gene. B61 is primarily a hydrophilic molecule but contains both a hydrophobic N-terminal and a hydrophobic C-terminal region. The N-terminal region is typical of a signal peptide, which is consistent with the secreted nature of the protein. The mature form of the predicted protein consists of 187 amino acid residues and has a molecular weight of 22,000. Immunoprecipitation of metabolically labeled human umbilical vein endothelial cell preparations revealed that B61 is a 25-kilodalton secreted protein which is markedly induced by tumor necrosis factor.

A novel immediate-early response gene of endothelium is induced by cytokines and encodes a secreted protein.

PubMed Central

Holzman, L B; Marks, R M; Dixit, V M

1990-01-01

We have previously described the cloning of a group of novel cellular immediate-early response genes whose expression in human umbilical vein endothelial cells is induced by tumor necrosis factor alpha in the presence of cycloheximide. These genes are likely to participate in mediating the response of the vascular endothelium to proinflammatory cytokines. In this study, we further characterized one of these novel gene products named B61. Sequence analysis of cDNA clones encoding B61 revealed that its protein product has no significant homology to previously described proteins. Southern analysis suggested that B61 is an evolutionarily conserved single-copy gene. B61 is primarily a hydrophilic molecule but contains both a hydrophobic N-terminal and a hydrophobic C-terminal region. The N-terminal region is typical of a signal peptide, which is consistent with the secreted nature of the protein. The mature form of the predicted protein consists of 187 amino acid residues and has a molecular weight of 22,000. Immunoprecipitation of metabolically labeled human umbilical vein endothelial cell preparations revealed that B61 is a 25-kilodalton secreted protein which is markedly induced by tumor necrosis factor. Images PMID:2233719

Short-Term Effects of gamma-Irradiation on 1-Aminocyclopropane-1-Carboxylic Acid Metabolism in Early Climacteric Cherry Tomatoes : Comparison with Wounding.

PubMed

Larrigaudière, C; Latché, A; Pech, J C; Triantaphylidès, C

1990-03-01

gamma-Irradiation of early climacteric (breaker) cherry tomatoes (Lycopersicon pimpinellifollium L.) caused a sharp burst in ethylene production during the first hour. The extent of ethylene production was dose dependent and was maximum at about 3 kilograys. The content of 1-aminocyclopropane-1-carboxylic acid (ACC), followed the same evolution as ethylene production, while malonyl ACC increased steadily with time in irradiated fruits. The burst in ethylene production was accompanied by a sharp stimulation of ACC synthase activity which began 15 minutes after irradiation. The stimulation was completely prevented by cycloheximide, but not by actinomycin d or cordycepin. In contrast with irradiation, mechanical wounding continuously stimulated ethylene production over several hours. gamma-Irradiation and cordycepin applied to wounded tissues both caused the cessation of this continuous increase, but the initial burst was still persisting. These data suggest that gamma-irradiation, like wounding, stimulates the translation of preexisting mRNAs. It also reduces, at least temporarily, the subsequent transcription-dependent stimulation of ethylene production. gamma-Irradiation greatly inhibited the activity of ethylene-forming enzyme at doses higher than 1 kilogray. Such sensitivity is in accordance with a highly integrated membranebound enzyme.

Studies on a morphologically distinct colchicine-resistance variant of Entamoeba sp.

PubMed

Injeyan, H; Huebner, E; Meerovitch, E

1979-05-01

Colchicine has a temperature-dependent cytotoxic effect on Entamoeba sp. (Laredo isolate) that is most apparent when the drug is applied during the initiation of cultures at a concentration of 7.5 mM or higher. Continued transfer of cultures in medium containing progressively increasing concentrations of colchicine has resulted in a variant that grows prolifically in the presence of colchicine (7.5 mM) with a generation time comparable to that of the parent stock, Comparison of a number of parameters of the 2 variants revealed that colchicine resistance was accompanied by a change in cell shape, a reduced membrane permeability, which could partially be overcome by the addition of dimethyl sulfoxide (DMSO), and a reduced tolerance to osmotic stress. However, the parent strain and resistant variant were equally susceptible to cycloheximide and puromycin suggesting that the acquired colchicine resistance may not be explained on the basis of an entirely unspecific generalized reduced ability for drug uptake. Colchicine resistance and altered structure were found to be stable over a long period of time. The possible interdependence of these 2 parameters and their relation to cell motility in Entamoeba sp. are discussed.

Identification of a G protein coupled receptor induced in activated T cells.

PubMed

Kaplan, M H; Smith, D I; Sundick, R S

1993-07-15

Many genes are induced after T cell activation to make a cell competent for proliferation and ultimately, function. Many of these genes encode surface receptors for growth factors that signal a cell to proliferate. We have cloned a novel gene (clone 6H1) that codes for a member of the G protein-coupled receptor superfamily. This gene was isolated from a chicken activated T cell cDNA library by low level hybridization to mammalian IL-2 cDNA probes. The 308 amino acid open reading frame has seven hydrophobic, presumably transmembrane domains and a consensus site for interaction with G proteins. Tissue distribution studies suggest that gene expression is restricted to activated T cells. The message appears by 1 h after activation and is maintained for at least 45 h. Transcription of 6H1 is induced by a number of T cell stimuli and is inhibited by cyclosporin A, but not by cycloheximide. This is the first description of a member of this superfamily expressed specifically in activated T cells. The gene product may provide a link between T cell growth factors and G protein activation.

Pretreatment of low dose radiation reduces radiation-induced apoptosis in mouse lymphoma (EL4) cells.

PubMed

Kim, J H; Hyun, S J; Yoon, M Y; Ji, Y H; Cho, C K; Yoo, S Y

1997-06-01

Induction of an adaptive response to ionizing radiation in mouse lymphoma (EL4) cells was studied by using cell survival fraction and apoptotic nucleosomal DNA fragmentation as biological end points. Cells in early log phase were pre-exposed to low dose of gamma-rays (0.01 Gy) 4 or 20 hrs prior to high dose gamma-ray (4, 8 and 12 Gy for cell survival fraction analysis; 8 Gy for DNA fragmentation analysis) irradiation. Then cell survival fractions and the extent of DNA fragmentation were measured. Significant adaptive response, increase in cell survival fraction and decrease in the extent of DNA fragmentation were induced when low and high dose gamma-ray irradiation time interval was 4 hr. Addition of protein or RNA synthesis inhibitor, cycloheximide or 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRFB), respectively during adaptation period, the period from low dose gamma-ray irradiation to high dose gamma-ray irradiation, was able to inhibit the induction of adaptive response, which is the reduction of the extent DNA fragmentation in irradiated EL4 cells. These data suggest that the induction of adaptive response to ionizing radiation in EL4 cells required both protein and RNA synthesis.

Manganese-enhanced magnetic resonance imaging detects mossy fiber sprouting in the pilocarpine model of epilepsy

PubMed Central

Malheiros, Jackeline M.; Polli, Roberson S.; Paiva, Fernando F.; Longo, Beatriz M.; Mello, Luiz E.; Silva, Afonso C.; Tannús, Alberto; Covolan, Luciene

2012-01-01

Summary Purpose Mossy fiber sprouting (MFS) is a frequent finding following status epilepticus (SE). The present study aimed to test the feasibility of using manganese-enhanced magnetic resonance imaging (MEMRI) to detect MFS in the chronic phase of the well-established pilocarpine (Pilo) rat model of temporal lobe epilepsy (TLE). Methods To modulate MFS, cycloheximide (CHX), a protein synthesis inhibitor, was co-administered with Pilo in a sub-group of animals. In vivo MEMRI was performed 3 months after induction of SE and compared to the neo-Timm histological labeling of zinc mossy fiber terminals in the dentate gyrus (DG). Key findings Chronically epileptic rats displaying MFS as detected by neo-Timm histology had a hyperintense MEMRI signal in the DG, while chronically epileptic animals that did not display MFS had minimal MEMRI signal enhancement compared to non-epileptic control animals. A strong correlation (r = 0.81, P<0.001) was found between MEMRI signal enhancement and MFS. Significance This study shows that MEMRI is an attractive non-invasive method to detect mossy fiber sprouting in vivo and can be used as an evaluation tool in testing therapeutic approaches to manage chronic epilepsy. PMID:22642664

Trehalose accumulation induced during the oxidative stress response is independent of TPS1 mRNA levels in Candida albicans.

PubMed

Zaragoza, Oscar; González-Párraga, Pilar; Pedreño, Yolanda; Alvarez-Peral, Francisco J; Argüelles, Juan-Carlos

2003-06-01

Growing cells of the Candida albicans trehalose-deficient mutant tps1/tps1 were extremely sensitive to severe oxidative stress exposure (H2O2). However, their viability was not affected after saline stress or heat-shock treatments, being roughly equivalent to that of the parental strain. In wild-type cells, these adverse conditions induced the intracellular accumulation of trehalose together with activation of trehalose-6P synthase, whereas the endogenous trehalose content and the corresponding biosynthetic activity were barely detectable in the tps1/tps1 mutant. The addition of cycloheximide did not prevent the marked induction of trehalose-6P synthase activity. Furthermore, the presence of H2O2 decreased the level of TPS1 mRNA expression. Hence, the conspicuous trehalose accumulation in response to oxidative stress is not induced by increased transcription of TPS1. Our results are consistent with a specific requirement of trehalose in order to withstand a severe oxidative stress in C. albicans, and suggest that trehalose accumulation observed under these conditions is a complex process that most probably involves post-translational modifications of the trehalose synthase complex.

Mutualistic interaction between Salmonella enterica and Aspergillus niger and its effects on Zea mays colonization

PubMed Central

Balbontín, Roberto; Vlamakis, Hera; Kolter, Roberto

2014-01-01

Salmonella Typhimurium inhabits a variety of environments and is able to infect a broad range of hosts. Throughout its life cycle, some hosts can act as intermediates in the path to the infection of others. Aspergillus niger is a ubiquitous fungus that can often be found in soil or associated to plants and microbial consortia. Recently, S. Typhimurium was shown to establish biofilms on the hyphae of A. niger. In this work, we have found that this interaction is stable for weeks without a noticeable negative effect on either organism. Indeed, bacterial growth is promoted upon the establishment of the interaction. Moreover, bacterial biofilms protect the fungus from external insults such as the effects of the anti-fungal agent cycloheximide. Thus, the Salmonella–Aspergillus interaction can be defined as mutualistic. A tripartite gnotobiotic system involving the bacterium, the fungus and a plant revealed that co-colonization has a greater negative effect on plant growth than colonization by either organism in dividually. Strikingly, co-colonization also causes a reduction in plant invasion by S. Typhimurium. This work demonstrates that S. Typhimurium and A. niger establish a mutualistic interaction that alters bacterial colonization of plants and affects plant physiology. PMID:25351041

Alcohols inhibit translation to regulate morphogenesis in C. albicans

PubMed Central

Egbe, Nkechi E.; Paget, Caroline M.; Wang, Hui; Ashe, Mark P.

2015-01-01

Many molecules are secreted into the growth media by microorganisms to modulate the metabolic and physiological processes of the organism. For instance, alcohols like butanol, ethanol and isoamyl alcohol are produced by the human pathogenic fungus, Candida albicans and induce morphological differentiation. Here we show that these same alcohols cause a rapid inhibition of protein synthesis. More specifically, the alcohols target translation initiation, a complex stage of the gene expression process. Using molecular techniques, we have identified the likely translational target of these alcohols in C. albicans as the eukaryotic translation initiation factor 2B (eIF2B). eIF2B is the guanine nucleotide exchange factor for eIF2, which supports the exchange reaction where eIF2.GDP is converted to eIF2.GTP. Even minimal regulation at this step will lead to alterations in the levels of specific proteins that may allow the exigencies of the fungus to be realised. Indeed, similar to the effects of alcohols, a minimal inhibition of protein synthesis with cycloheximide also causes an induction of filamentous growth. These results suggest a molecular basis for the effect of various alcohols on morphological differentiation in C. albicans. PMID:25843913

Function of Oxidative Cross-Linking of Cell Wall Structural Proteins in Plant Disease Resistance.

PubMed

Brisson, L. F.; Tenhaken, R.; Lamb, C.

1994-12-01

Elicitation of soybean cells causes a rapid insolubilization of two cell wall structural proteins, p33 and p100. Likewise, a short elicitation of 30 min rendered cell walls more refractory to enzyme digestion as assayed by the yield of protoplasts released. This effect could be ascribed to protein cross-linking because of its insensitivity to inhibitors of transcription (actinomycin D) and translation (cycloheximide) and its induction by exogenous H2O2. Moreover, the induced loss of protoplasts could be prevented by preincubation with DTT, which also blocks peroxidase-mediated oxidative cross-linking. The operation of protein insolubilization in plant defense was also demonstrated by its occurrence in the incompatible interaction but not in the compatible interaction between soybean and Pseudomonas syringae pv glycinea. Likewise, protein insolubilization was observed in bean during non-host hypersensitive resistance to the tobacco pathogen P. s. pv tabaci mediated by the hypersensitive resistance and pathogenicity (Hrp) gene cluster. Our data strongly suggest that rapid protein insolubilization leads to a strengthened cell wall, and this mechanism functions as a rapid defense in the initial stages of the hypersensitive response prior to deployment of transcription-dependent defenses.

The Interplay Between Predation, Competition, and Nutrient Levels Influences the Survival of Escherichia coli in Aquatic Environments.

PubMed

Wanjugi, P; Fox, G A; Harwood, V J

2016-10-01

Nutrient levels, competition from autochthonous microorganisms, and protozoan predation may all influence survival of fecal microorganisms as they transition from the gastrointestinal tract to aquatic habitats. Although Escherichia coli is an important indicator of waterborne pathogens, the effects of environmental stressors on its survival in aquatic environments remain poorly understood. We manipulated organic nutrient, predation, and competition levels in outdoor microcosms containing natural river water, sediments, and microbial populations to determine their relative contribution to E. coli survival. The activities of predator (protozoa) and competitor (indigenous bacteria) populations were inhibited by adding cycloheximide or kanamycin. We developed a statistical model of E. coli density over time that fits with the data under all experimental conditions. Predation and competition had significant negative effects on E. coli survival, while higher nutrient levels increased survival. Among the main effects, predation accounted for the greatest variation (40 %) compared with nutrients (25 %) or competition (15 %). The highest nutrient level mitigated the effect of predation on E. coli survival. Thus, elevated organic nutrients may disproportionately enhance the survival of E. coli, and potentially that of other enteric bacteria, in aquatic habitats.

Induction of Shikimic Acid Pathway Enzymes by Light in Suspension Cultured Cells of Parsley (Petroselinum crispum) 1

PubMed Central

McCue, Kent F.; Conn, Eric E.

1990-01-01

Light treatment of suspension cultured cells of parsley (Petroselinum crispum) was shown to increase the activity of the shikimic acid pathway enzyme, 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) synthase (EC 4.1.2.15). DAHP synthase activity was assayed for two isoforms, DS-Mn and DS-Co (RJ Ganson, TA d'Amato, RA Jensen [1986] Plant Physiol 82: 203-210). Light increased the enzymatic activity of the plastidic isoform DS-Mn as much as 2-fold, averaging 1.6-fold with >95% confidence. The cytosolic isoform DS-Co was unaffected. Cycloheximide and actinomycin D, translational and transcriptional inhibitors, respectively, both reversed induction of DS-Mn by light suggesting transcriptional regulation of the gene. Chorismate mutase activity was assayed for the two isoforms CM I and CM II (BK Singh, JA Connelly, EE Conn [1985] Arch Biochem Biophys 243: 374-384). Treatment by light did not significantly affect either chorismate mutase isoform. The ratio of the two chorismate mutase isoforms changed during the growth cycle, with an increase in the ratio of plastidic to cytosolic isoforms occurring towards the end of logarithmic growth. PMID:16667741

Mode of action of the immunostimulatory effect of collagen from jellyfish.

PubMed

Nishimoto, Sogo; Goto, Yoko; Morishige, Hitoshi; Shiraishi, Ryusuke; Doi, Mikiharu; Akiyama, Koichi; Yamauchi, Satoshi; Sugahara, Takuya

2008-11-01

We have previously demonstrated that collagen from jellyfish simulated immunoglobulin and cytokine production by human-human hybridoma line HB4C5 cells and by human peripheral blood lymphocytes (hPBL). The mode of action of the collagen as an immunostimulatory factor was investigated. The expression levels of immunoglobulin mRNAs in HB4C5 cells, and those of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and transforming growth factor (TGF)-beta in hPBL were up-regulated by jellyfish collagen. In addition, this collagen activated IgM production by transcription-suppressed HB4C5 cells that had been treated with actinomycin D. This collagen also enhanced IgM production by translation-suppressed HB4C5 cells that had been treated with sodium fluoride, but was ineffective in accelerating IgM production by HB4C5 cells treated with cycloheximide. Moreover, the intracellular IgM level in HB4C5 cells treated with the post-translation inhibitor, monensin, was increased by this collagen. These results suggest that collagen from jellyfish stimulated not only the transcription activity, but also the translation activity for enhanced immunoglobulin and cytokine production.

Short-Term Effects of γ-Irradiation on 1-Aminocyclopropane-1-Carboxylic Acid Metabolism in Early Climacteric Cherry Tomatoes 1

PubMed Central

Larrigaudière, Christian; Latché, Alain; Pech, Jean Claude; Triantaphylidès, Christian

1990-01-01

γ-Irradiation of early climacteric (breaker) cherry tomatoes (Lycopersicon pimpinellifollium L.) caused a sharp burst in ethylene production during the first hour. The extent of ethylene production was dose dependent and was maximum at about 3 kilograys. The content of 1-aminocyclopropane-1-carboxylic acid (ACC), followed the same evolution as ethylene production, while malonyl ACC increased steadily with time in irradiated fruits. The burst in ethylene production was accompanied by a sharp stimulation of ACC synthase activity which began 15 minutes after irradiation. The stimulation was completely prevented by cycloheximide, but not by actinomycin d or cordycepin. In contrast with irradiation, mechanical wounding continuously stimulated ethylene production over several hours. γ-Irradiation and cordycepin applied to wounded tissues both caused the cessation of this continuous increase, but the initial burst was still persisting. These data suggest that γ-irradiation, like wounding, stimulates the translation of preexisting mRNAs. It also reduces, at least temporarily, the subsequent transcription-dependent stimulation of ethylene production. γ-Irradiation greatly inhibited the activity of ethylene-forming enzyme at doses higher than 1 kilogray. Such sensitivity is in accordance with a highly integrated membranebound enzyme. PMID:16667318

Biosynthetic requirements for the repair of sublethally injured Saccharomyces cerevisiae cells after pulsed electric fields.

PubMed

Somolinos, M; García, D; Condón, S; Mañas, P; Pagán, R

2008-07-01

The aim was to evaluate the biosynthetic requirements for the repair of sublethal membrane damages in Saccharomyces cerevisiae cells after exposure to pulsed electric fields (PEF). The partial loss of the integrity and functionality of the cytoplasmic membrane was assessed by adding sodium chloride to the recovery medium. More than 2 log(10) cycles of survivors were sublethally injured after PEF. Repair of sublethal membrane damages occurred when survivors to PEF were incubated in Sabouraud Broth for 4 h at room temperature. The addition of inhibitors, such as chloramphenicol, rifampicin, 5-fluorocytosine, nalidixic acid, cycloheximide, cerulenin, miconazol and sodium azide to the liquid repair medium showed that the repair of PEF-injured cells required energy and protein synthesis. The extent of the sublethal damages was greater in PEF-treated cells at pH 4.0 than at pH 7.0. This work confirms that membrane damage is an important event in the PEF-inactivation of yeast. The mechanism of yeast inactivation by PEF seems to differ from that of bacteria, as the repair of sublethal damages requires protein synthesis. Knowledge about the damages inflicted by PEF leads to a better description of the mechanism of yeast inactivation.

In vitro activity of chloramphenicol, florfenicol and enrofloxacin against Chlamydia pecorum isolated from koalas (Phascolarctos cinereus).

PubMed

Black, L A; Higgins, D P; Govendir, M

2015-11-01

To determine the in vitro susceptibilities of koala isolates of Chlamydia pecorum to enrofloxacin and chloramphenicol, which are frequently used to treat koalas with chlamydiosis, and florfenicol, a derivative of chloramphenicol. The in vitro susceptibilities were determined by culturing three stored isolates and seven clinical swabs of C. pecorum. Susceptibility testing was undertaken using cycloheximide-treated buffalo green monkey kidney cells in 96 well microtitre plates. The minimum inhibitory concentrations (MICs) for all isolates were 0.25-0.50 µg/mL (enrofloxacin), 1-2 µg/mL (chloramphenicol), and 1-2 µg/mL (florfenicol). Minimum bactericidal concentration (MBC) values for five isolates were also determined and were within one two-fold dilution of MICs. The MICs and MBCs of these antimicrobials were within ranges previously reported for other chlamydial species. When combined with previously published pharmacokinetic data, the in vitro susceptibility results support chloramphenicol as a more appropriate treatment option than enrofloxacin for koalas with chlamydiosis. The susceptibility results also indicate florfenicol may be an appropriate treatment option for koalas with chlamydiosis, warranting further investigation. © 2015 Australian Veterinary Association.

Time-dependent inhibition by glyceryl trinitrate of platelet aggregation caused by U46619 (a thromboxane/endoperoxide receptor agonist).

PubMed Central

Kampf, G; Ritter, J M

1994-01-01

Glyceryl trinitrate is a weak inhibitor of platelet aggregation in vitro. Its effect on platelet aggregation in response to U46619 (a thromboxane/endoperoxide receptor agonist) was studied turbidometrically in platelet-rich plasma from healthy volunteers. The object was to determine whether inhibition was influenced by a period of preincubation between preparation of platelet-rich plasma and addition of glyceryl trinitrate. Incubation was performed at 37 degrees C and 22 degrees C. Samples were removed at intervals and transferred to an aggregometer cuvette at 37 degrees C. Glyceryl trinitrate (100 microM) or an equal volume of distilled water was added 5 min before U46619 (2 microM), and aggregation recorded as change in light transmission. Inhibition by glyceryl trinitrate was markedly time and temperature dependent, with a progressive increase in inhibitory potency between 120 and 300 min preincubation at 37 degrees C but not at 22 degrees C. The explanation of this is unknown but the effect was not influenced by lipopolysaccharide or by cycloheximide, so it does not appear to be due to exposure to endotoxin or to enzyme induction in vitro. PMID:7946941

Indications for an inducible component of error-prone DNA repair in yeast.

PubMed

Siede, W; Eckardt, F

1984-01-01

In a thermoconditional mutant of mutagenic DNA repair (rev 2ts = rad 5-8) of Saccharomyces cerevisiae recovery of survival and mutation frequencies can be monitored by incubating UV-irradiated cells in growth medium at a permissive temperature (23 degrees C) before plating and a shift to restrictive temperature (36 degrees C). Inhibition of protein synthesis with cycloheximide during incubation at permissive conditions blocks this REV 2 dependent recovery process in stationary phase rev 2ts cells, whereas it can be reduced but not totally abolished in exponentially growing cells. These results indicate a strict dependence on post-irradiation protein synthesis in stationary phase cells and argue for a considerable constitutive level and only limited inducibility in logarithmic phase cells. The UV inducibility of the REV 2 coded function in stationary phase cells could be confirmed by analysis of the dose-response pattern of the his 5-2 reversion: in stationary phase rev 2ts cells, the quadratic component of the biphasic linear-quadratic induction kinetics found at 23 degrees C, which is interpreted as the consequence of induction of mutagenic repair, is eliminated at 36 degrees C.

Insulin treatment augments KCNQ1/KCNE1 currents but not KCNQ1 currents, which is associated with an increase in KCNE1 expression.

PubMed

Wu, Minghua; Obara, Yutaro; Ohshima, Shingo; Nagasawa, Yoshinobu; Ishii, Kuniaki

2017-11-04

Diabetes mellitus affects ion channel physiology. We have previously reported that acute application of insulin suppresses the KCNQ1/KCNE1 currents that play an important role in terminating ventricular action potential. In this study, we investigated the effect of long-term insulin treatment on KCNQ1/KCNE1 currents using the Xenopus oocyte expression system. Insulin treatment with a duration longer than 6 h had an opposite effect to acute insulin application, that is, it augmented the KCNQ1/KCNE1 currents. Inhibitors of PI3K, wortmannin and LY294002, and a MEK inhibitor, U0126, abolished the potentiating effect of long-term insulin treatment. The long-term treatment with insulin had no effect on KCNQ1 currents indicating an essential role of KCNE1 in the insulin effect, which is similar to the acute insulin effect. Cycloheximide, an inhibitor of protein synthesis, and brefeldin A, an inhibitor of protein transport from endoplasmic reticulum, suppressed the long-term insulin effect. Western blotting analysis combined with these pharmacological data suggest that long-term insulin treatment augments KCNQ1/KCNE1 currents by increasing KCNE1 protein expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of inhibitors of polyamine biosynthesis on gibberellin-induced internode growth in light-grown dwarf peas

NASA Technical Reports Server (NTRS)

Kaur-Sawhney, R.; Dai, Y. R.; Galston, A. W.

1986-01-01

When gibberellic acid (GA3) is sprayed on 9-day-old light-brown dwarf Progress pea (Pisum sativum) seedlings, arginine decarboxylase (ADC; EC 4.1.1.9) activity increases within 3 h and peaks at about 9 h after GA3 application. This is followed by a second lower peak at about 30 h; both peaks were higher than the corresponding peaks in the controls. In contrast, no appreciable effect of GA3 on internode length was observed until about 12 h, after which time a dramatic increase in growth rate occurred and persisted for about 12 h. Specific (DL-alpha-difluoromethylarginine) and non-specific (D-arginine and L-canavanine) inhibitors of ADC strongly inhibited ADC activity and to a lesser extent internode growth. The inhibition was reversed only slightly by the addition of polyamines. Actinomycin D and cycloheximide inhibited the rise in ADC activity induced by GA3. The half-life of the enzyme was increased by GA3 treatment. The results suggest that part of the GA3-induced increase in internode growth may result from enhanced polyamine biosynthesis through the ADC pathway. Furthermore, the GA3 induced increase in ADC activity probably requires de novo synthesis of both RNA and protein.

X-ray-related potentially lethal damage expressed by chromosome condensation and the influence of caffeine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sasaki, H.; Nishimoto, T.

1989-10-01

Caffeine has been reported to induce premature chromosome condensation (PCC) in S-phase cells in the presence of an inhibitor of DNA synthesis. We found that when S-phase cells are treated with caffeine and hydroxyurea after X irradiation, substantially more potentially lethal damage (PLD) is expressed, but the addition of cycloheximide, which inhibits PCC induction in S-phase cells, in the presence of caffeine and hydroxyurea reduces the expression of PLD to the same level as seen with caffeine alone. This can be interpreted to mean that the expression of PLD seen with caffeine in the absence of an inhibitor of DNAmore » synthesis is not associated with chromosome condensation. Evidence that PCC induction in S-phase cells and the influence of caffeine on PLD expression were suppressed by incubation at 40 degrees C of tsBN75 cells with a ts defect in ubiquitin-activating enzyme indicates the involvement of ubiquitin in these two processes. These observations as well as previous findings on ubiquitin suggest to us that caffeine induces changes in DNA-chromatin conformation, which are caused by induction of PCC or ubiquitination of chromosomal protein. Such changes occurring postirradiation would favor expression of PLD.« less

Anti-protozoal and anti-bacterial antibiotics that inhibit protein synthesis kill cancer subtypes enriched for stem cell-like properties.

PubMed

Cuyàs, Elisabet; Martin-Castillo, Begoña; Corominas-Faja, Bruna; Massaguer, Anna; Bosch-Barrera, Joaquim; Menendez, Javier A

2015-01-01

Key players in translational regulation such as ribosomes might represent powerful, but hitherto largely unexplored, targets to eliminate drug-refractory cancer stem cells (CSCs). A recent study by the Lisanti group has documented how puromycin, an old antibiotic derived from Streptomyces alboniger that inhibits ribosomal protein translation, can efficiently suppress CSC states in tumorspheres and monolayer cultures. We have used a closely related approach based on Biolog Phenotype Microarrays (PM), which contain tens of lyophilized antimicrobial drugs, to assess the chemosensitivity profiles of breast cancer cell lines enriched for stem cell-like properties. Antibiotics directly targeting active sites of the ribosome including emetine, puromycin and cycloheximide, inhibitors of ribosome biogenesis such as dactinomycin, ribotoxic stress agents such as daunorubicin, and indirect inhibitors of protein synthesis such as acriflavine, had the largest cytotoxic impact against claudin-low and basal-like breast cancer cells. Thus, biologically aggressive, treatment-resistant breast cancer subtypes enriched for stem cell-like properties exhibit exacerbated chemosensitivities to anti-protozoal and anti-bacterial antibiotics targeting protein synthesis. These results suggest that old/existing microbicides might be repurposed not only as new cancer therapeutics, but also might provide the tools and molecular understanding needed to develop second-generation inhibitors of ribosomal translation to eradicate CSC traits in tumor tissues.

Anti-protozoal and anti-bacterial antibiotics that inhibit protein synthesis kill cancer subtypes enriched for stem cell-like properties

PubMed Central

Cuyàs, Elisabet; Martin-Castillo, Begoña; Corominas-Faja, Bruna; Massaguer, Anna; Bosch-Barrera, Joaquim; Menendez, Javier A

2015-01-01

Key players in translational regulation such as ribosomes might represent powerful, but hitherto largely unexplored, targets to eliminate drug-refractory cancer stem cells (CSCs). A recent study by the Lisanti group has documented how puromycin, an old antibiotic derived from Streptomyces alboniger that inhibits ribosomal protein translation, can efficiently suppress CSC states in tumorspheres and monolayer cultures. We have used a closely related approach based on Biolog Phenotype Microarrays (PM), which contain tens of lyophilized antimicrobial drugs, to assess the chemosensitivity profiles of breast cancer cell lines enriched for stem cell-like properties. Antibiotics directly targeting active sites of the ribosome including emetine, puromycin and cycloheximide, inhibitors of ribosome biogenesis such as dactinomycin, ribotoxic stress agents such as daunorubicin, and indirect inhibitors of protein synthesis such as acriflavine, had the largest cytotoxic impact against claudin-low and basal-like breast cancer cells. Thus, biologically aggressive, treatment-resistant breast cancer subtypes enriched for stem cell-like properties exhibit exacerbated chemosensitivities to anti-protozoal and anti-bacterial antibiotics targeting protein synthesis. These results suggest that old/existing microbicides might be repurposed not only as new cancer therapeutics, but also might provide the tools and molecular understanding needed to develop second-generation inhibitors of ribosomal translation to eradicate CSC traits in tumor tissues. PMID:25970790

Regulation of atrial natriuretic peptide clearance receptors in mesangial cells by growth factors.

PubMed

Paul, R V; Wackym, P S; Budisavljevic, M; Everett, E; Norris, J S

1993-08-25

Rat mesangial cells can express both 130-kDa guanylyl cyclase-coupled and 66-kDa non-coupled atrial natriuretic peptide (ANP) receptors (ANPR-A and ANPR-C, respectively). Exposure of mesangial cells, grown in 20% fetal calf serum, to 0.1% serum for 24 h increased total ANP receptor density more than 2-fold (Bmax = 87 versus 37 fmol/mg of cell protein) without changing binding affinity (Kd = 94 versus 88 pM). Radioligand binding and cross-linking studies demonstrated that up-regulation of ANP binding after serum deprivation was entirely due to an increase in ANPR-C, with little or no change in ANPR-A. Inhibition of protein synthesis with cycloheximide blocked up-regulation after serum deprivation. Steady-state ANPR-C mRNA level was increased 15-fold by serum deprivation, as judged by Northern blotting. There was no change in ANPR-A mRNA. Platelet-derived growth factor and phorbol myristate acetate, when added to low serum medium, blocked or reversed the effect of serum deprivation on ANPR-C. We conclude that synthesis and expression of ANPR-C but not ANPR-A is suppressed by serum, platelet-derived growth factor, and phorbol myristate acetate. Suppression of ANPR-C in vivo could contribute to mesangial cell proliferative responses to growth factors.

Biochemistry of Suberization

PubMed Central

Agrawal, Vishwanath P.; Kolattukudy, P. E.

1977-01-01

A cell-free extract obtained from suberizing potato (Solanum tuberosum L.) tuber disks catalyzed the conversion of 16-hydroxy[G-3H]hexadecanoic acid to the corresponding dicarboxylic acid with NADP or NAD as the cofactor, with a slight preference for the former. This ω-hydroxyacid dehydrogenase activity, located largely in the 100,000g supernatant fraction, has a pH optimum of 9.5. It showed an apparent Km of 50 μM for 16-hydroxyhexadecanoic acid. The dehydrogenase activity was inhibited by thiol reagents, such as p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetamide, and this dehydrogenase is shown to be different from alcohol dehydrogenase. That 16-oxohexadecanoic acid was an intermediate in the conversion of 16-hydroxyhexadecanoic acid to the corresponding dicarboxylic acid was suggested by the observation that the cell-free extract also catalyzed the conversion of 16-oxohexadecanoic acid to the dicarboxylic acid, with NADP as the preferred cofactor. The time course of development of the ω-hydroxyacid dehydrogenase activity in the suberizing potato disks correlated with the rate of deposition of suberin. Experiments with actinomycin D and cycloheximide suggested that the transcriptional processes, which are directly related to suberin biosynthesis and ω-hydroxyacid dehydrogenase biosynthesis, occurred between 72 and 96 hours after wounding. These results strongly suggest that a wound-induced ω-hydroxyacid dehydrogenase is involved in suberin biosynthesis in potato disks. PMID:16659915

On the role of calcium in adrenocorticotropin-induced changes in mitochondrial pregnenolone synthesis.

PubMed

Farese, R V; Prudente, W J

1978-10-01

The importance of calcium in the ACTH-induced increase in adrenal mitochondrial pregnenolone synthesis was evaluated. In mitochondria prepared in the absence of EDTA and albumin, calcium enhanced the binding of cholesterol to cytochrome P-450 and subsequent pregnenolone synthesis. Although these effects of calcium were slightly greater in control than in ACTH-treated mitochondria, a sizeable effect of ACTH remained even at high calcium levels (500 micron). In mitochondria prepared from adrenals homogenized in fluid containing EDTA and albumin, ACTH-induced effects on pregnenolone synthesis were relatively poor unless calcium was added to the incubation mixture. High concentrations of added calcium (500 micron or greater) obviated the need for the labile protein required for ACTH-induced effects in intact mitochondria, presumably by disrupting mitochondria and allowing an "unrestrained" interaction of cholesterol with cytochrome P-450. Thus, cholesterol-rich mitochondria from ACTH plus cycloheximide-treated rats produced large amounts of pregnenolone when high (probably unphysiological) calcium concentrations were present. The present findings suggest that calcium is required at the mitochondrial level for ACTH-induced effects on pregnenolone synthesis, and the reported ACTH-induced increase in intraadrenal calcium may thus amplify the effects of ACTH on steroidogenesis. However, it seems unlikely that calcium is the agent primarily responsible for mediating the ACTH-induced steroidogenic effect at the mitochondrial level.

Interaction between glucocorticoids and glucagon in the hormonal modification of calcium retention by isolated rat liver mitochondria.

PubMed

Hughes, B P; Barritt, G J

1979-05-15

1. The administration of dexamethasone to intact fed rats by intraperitoneal injection for 3h was associated with a 6-fold increase in the time for which mitochondria subsequently isolated from the liver retain a given load of exogenous Ca2+. This effect was blocked by the co-administration of cycloheximide with dexamethasone, and partially blocked by the co-administration of puromycin. Daily administration of dexamethasone for periods of 4--7 days resulted in liver mitochondria that exhibited a decreased ability to retain exogenous Ca2+. 2. When glucagon was administered to fed adrenalectomized rats, the increase in mitochondrial Ca2+-retention time that results from the action of this hormone was reduced by 50% when compared with its effect on intact animals. The administration of dexamethasone to adrenalectomized rats partially restored the full effect of glucagon. 3. Dexamethasone did not enhance the effect of glucagon on mitochondrial Ca2+-retention time when administered to intact fed rats. 4. It is concluded that these data support the hypothesis that the hormone-induced modification of liver mitochondria, which results in an increase in the time for which exogenous Ca2+ is retained, involves a step in which new protein is synthesized.

Cytoskeleton reorganization/disorganization is a key feature of induced inaccessibility for defence to successive pathogen attacks.

PubMed

Moral, Juan; Montilla-Bascón, Gracia; Canales, Francisco J; Rubiales, Diego; Prats, Elena

2017-06-01

In this work, we investigated the involvement of the long-term dynamics of cytoskeletal reorganization on the induced inaccessibility phenomenon by which cells that successfully defend against a previous fungal attack become highly resistant to subsequent attacks. This was performed on pea through double inoculation experiments using inappropriate (Blumeria graminis f. sp. avenae, Bga) and appropriate (Erysiphe pisi, Ep) powdery mildew fungi. Pea leaves previously inoculated with Bga showed a significant reduction of later Ep infection relative to leaves inoculated only with Ep, indicating that cells had developed induced inaccessibility. This reduction in Ep infection was higher when the time interval between Bga and Ep inoculation ranged between 18 and 24 h, although increased penetration resistance in co-infected cells was observed even with time intervals of 24 days between inoculations. Interestingly, this increase in resistance to Ep following successful defence to the inappropriate Bga was associated with an increase in actin microfilament density that reached a maximum at 18-24 h after Bga inoculation and very slowly decreased afterwards. The putative role of cytoskeleton reorganization/disorganization leading to inaccessibility is supported by the suppression of the induced resistance mediated by specific actin (cytochalasin D, latrunculin B) or general protein (cycloheximide) inhibitors. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Involvement of Cytochrome P450 in Pentachlorophenol Transformation in a White Rot Fungus Phanerochaete chrysosporium

PubMed Central

Ning, Daliang; Wang, Hui

2012-01-01

The occurrence of cytochrome P450 and P450-mediated pentachlorophenol oxidation in a white rot fungus Phanerochaete chrysosporium was demonstrated in this study. The carbon monoxide difference spectra indicated induction of P450 (103±13 pmol P450 per mg protein in the microsomal fraction) by pentachlorophenol. The pentachlorophenol oxidation by the microsomal P450 was NADPH-dependent at a rate of 19.0±1.2 pmol min−1 (mg protein)−1, which led to formation of tetrachlorohydroquinone and was significantly inhibited by piperonyl butoxide (a P450 inhibitor). Tetrachlorohydroquinone was also found in the cultures, while the extracellular ligninases which were reported to be involved in tetrachlorohydroquinone formation were undetectable. The formation of tetrachlorohydroquinone was not detectable in the cultures added with either piperonyl butoxide or cycloheximide (an inhibitor of de novo protein synthesis). These results revealed the pentachlorophenol oxidation by induced P450 in the fungus, and it should be the first time that P450-mediated pentachlorophenol oxidation was demonstrated in a microorganism. Furthermore, the addition of the P450 inhibitor to the cultures led to obvious increase of pentachlorophenol, suggesting that the relationship between P450 and pentachlorophenol methylation is worthy of further research. PMID:23029295

Host DNA synthesis-suppressing factor in culture fluid of tissue cultures infected with measles virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Minagawa, T.; Nakaya, C.; Iida, H.

1974-05-01

Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated componentnent which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virusmore » as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither uv-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus. (auth)« less

Modulation of the cytosolic androgen receptor in striated muscle by sex steroids

NASA Technical Reports Server (NTRS)

Rance, N. E.; Max, S. R.

1984-01-01

The effects of orchiectomy (GDX) and of subsequent administration of testosterone propionate (TP) or 17(beta)-estradiol (E2) on the maximum binding (Bmax) and apparent Kd of the cytosolic androgen receptor in levator ani (LA) and skeletal muscles of adult male Sprague-Dawley rats are investigated experimentally. The results are presented in graphs and discussed. In LA, BMAX is found to rise from a control level of 2.5 fmol/mg protein to 280, 600, 478, and 133 percent of control at 12 h, 14 d, 30 d, and 44 d after GDX, respectively, while Kd increased only insignificantly (from 680 to 960 fM); Bmax is held at control levels for 6 h by cycloheximide given at GDX, is unaffected by TP given at 30 d, and is further increased (by 480 percent at 44 d) by administration of E2 at 30 d. Bmax in skeletal muscles is found to increase to 139, 212, 220, and 158 percent of control at 12 h, 14 d, 30 d, and 44 d, respectively; Bmax is returned to control at 44 d by TP at 30 d but is not affected by E2. The effect of E2 in LA is attributed to either induction of the cytosolic receptor or a decreased rate of receptor degradation.

Host DNA Synthesis-Suppressing Factor in Culture Fluid of Tissue Cultures Infected with Measles Virus

PubMed Central

Minagawa, Tomonori; Nakaya, Chikako; Iida, Hiroo

1974-01-01

Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated component which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virus as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither UV-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus. PMID:4207526

Competing Death Programs in Poliovirus-Infected Cells: Commitment Switch in the Middle of the Infectious Cycle

PubMed Central

Agol, Vadim I.; Belov, George A.; Bienz, Kurt; Egger, Denise; Kolesnikova, Marina S.; Romanova, Lyudmila I.; Sladkova, Larissa V.; Tolskaya, Elena A.

2000-01-01

Productive poliovirus infection of HeLa cells leads to the canonical cytopathic effect (CPE), whereas certain types of abortive infection result in apoptosis. To define the time course of commitment to the different types of poliovirus-induced death, inhibitors of viral replication (guanidine HCl) or translation (cycloheximide) were added at different times postinfection (p.i.). Early in the infection (during the first ∼2 h p.i.), predominantly proapoptotic viral function was expressed, rendering the cells committed to apoptosis, which developed several hours after viral expression was arrested. In the middle of infection, concomitantly with the onset of fast generation of viral progeny, the implementation of the viral apoptotic program was abruptly interrupted. In particular, activation of an Asp-Glu-Val-Asp (DEVD)-specific caspase(s) occurring in the apoptosis-committed cells was prevented by the ongoing productive infection. Simultaneously, the cells retaining normal or nearly normal morphology became committed to CPE, which eventually developed regardless of whether or not further viral expression was allowed to proceed. The implementation of the poliovirus-induced apoptotic program was suppressed in HeLa cells overexpressing the Bcl-2 protein, indicating that the fate of poliovirus-infected cells depends on the balance of host and viral pro- and antiapoptotic factors. PMID:10823859

Interferon Action on Parental Semliki Forest Virus Ribonucleic Acid

PubMed Central

Friedman, Robert M.; Fantes, Karl H.; Levy, Hilton B.; Carter, William B.

1967-01-01

Actinomycin D-treated chick fibroblasts were infected with purified 32P-labeled Semliki forest virus, and ribonucleic acid (RNA) was extracted after 1 or 2 hr. Within 1 hr, viral RNA forms sedimenting in sucrose gradients at 42S, 30S, and 16S were present. The 42S form corresponded to the RNA of the virion. The 16S form appeared to be a double-stranded template for the formation of new viral RNA, since nascent RNA was associated with it and the molecule could be heat-denatured and subsequently reannealed by slow cooling. Interferon treatment before infection, or puromycin (50 μg/ml) or cycloheximide (200 μg/ml) added at the time of virus infection, had no effect on the formation of the 30S RNA but inhibited the production of the 16S form. Several findings made it unlikely that these results were due to breakdown of parental RNA and reincorporation of 32P into progeny structures. The results suggested that the mechanism of interferon action involves inhibition of protein synthesis by parental viral RNA, since a specific viral RNA polymerase had previously been demonstrated to be necessary for production of 16S RNA. No protein synthesis appears necessary for formation of 30S RNA from parental virus RNA. PMID:5621488

Agmatine induces Nrf2 and protects against corticosterone effects in hippocampal neuronal cell line.

PubMed

Freitas, Andiara E; Egea, Javier; Buendía, Izaskun; Navarro, Elisa; Rada, Patricia; Cuadrado, Antonio; Rodrigues, Ana Lúcia S; López, Manuela G

2015-01-01

Hyperactivation of the hypothalamic-pituitary-adrenal axis is a common finding in major depression; this may lead to increased levels of cortisol, which are known to cause oxidative stress imbalance and apoptotic neuronal cell death, particularly in the hippocampus, a key region implicated in mood regulation. Agmatine, an endogenous metabolite of L-arginine, has been proposed for the treatment of major depression. Corticosterone induced apoptotic cell death and increased ROS production in cultured hippocampal neuronal cells, effects that were abolished in a concentration- and time-dependent manner by agmatine. Interestingly, the combination of sub-effective concentrations of agmatine with fluoxetine or imipramine afforded synergic protection. The neuroprotective effect of agmatine was abolished by yohimbine (α2-adrenoceptor antagonist), ketanserin (5-HT2A receptor antagonist), LY294002 (PI3K inhibitor), PD98059 (MEK1/2 inhibitor), SnPP (HO-1 inhibitor), and cycloheximide (protein synthesis inhibitor). Agmatine increased Akt and ERK phosphorylation and induced the transcription factor Nrf2 and the proteins HO-1 and GCLc; induction of these proteins was prevented by yohimbine, ketanserin, LY294002, and PD98059. In conclusion, agmatine affords neuroprotection against corticosterone effects by a mechanism that implicates Nrf2 induction via α2-adrenergic and 5-HT2A receptors, Akt and ERK pathways, and HO-1 and GCLc expression.

[Sporothrix globosa isolation related to a case of lymphocutaneous sporotrichosis].

PubMed

Cruz, Rodrigo; Vieille, Peggy; Oschilewski, David

2012-08-01

Sporothrix schenckii complex comprises a group of environmental dimorphic fungi that cause sporotrichosis. In Chile, isolated cases have been reported in humans, though no environmental isolates have been described. To achieve isolation of Sporothrix complex from the soil where a 75 year old patient with lymphocutaneous sporotrichosis performs horticulture work. In March and July 2011 soil and plant debris from five sectors where the patient does his work in horticulture was extracted. The soil samples were diluted and inoculated in Sabouraud agar with cycloheximide and chloramphenicol at 26 °C. The plant debris was directly inoculated in the same medium. Colonies suggestive of Sporothrix complex were reseeded in PDA agar at 26 ° C and identified as recommended by Marimon et al. Of the 10 plates from the first sampling, one colony was identified as Sporothrix globosa. In the second sampling, Sporothrix globosa grew in two plates seeded with soil, with a total of 6 colonies. There was no growth of Sporothrix complex in plant debris. The isolate from the patient was also identified as Sporothrix globosa. For the first time in Chile a species of Sporothrix complex was isolated from the environment. Sporothrix globosa was the species identified both in the ground and from the patient with sporotrichosis.

Down-modulation of receptors for phorbol ester tumor promoter in primary epidermal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Solanki, V.; Slaga, T.J.

1982-01-01

The specific (20-/sup 3/H)phorbol 12,13-dibutyrate ((/sup 3/H)PDBu) binding to intact epidermal cells displayed the phenomenon of down-modulation, i.e., the specific binding of (/sup 3/H)PDBu to its receptors on primary epidermal cells reached a maximum within 1 h and steadily declined thereafter. The apparent down-modulation of radiolabel resulted from a partial loss in the total number of receptors; the affinity of receptors for the ligand was essentially unchanged. A number of agents such as chloroquine, methylamine, or arginine which are known to prevent clustering, down-modulation, and/or internalization of several hormone receptors did not affect the down-modulation of phorbol ester receptors. Furthermore,more » cycloheximide had no effect either on down-modulation or on the binding capacity of cells. The surface binding capacity of down-modulated cells following a 90-min incubation with unlabeled ligand was almost returned to normal within 1 h. The effect of the antidepressant drug chlorpromazine, which is known to interact with calmodulin, on (/sup 3/H)PDBu binding was also investigated. Our data indicate that the effect of chlorpromazine on (/sup 3/H)PDBu binding is probably unrelated to its calmodulin-binding activity.« less

Purine Restriction Induces Pronounced Translational Upregulation of the NT1 Adenosine/Pyrimidine Nucleoside Transporter in Leishmania major

PubMed Central

Ortiz, Diana; Valdés, Raquel; Sanchez, Marco A.; Hayenga, Johanna; Elya, Carolyn; Detke, Siegfried; Landfear, Scott M.

2010-01-01

Summary Leishmania and other parasitic protozoa are unable to synthesize purines de novo and are reliant upon purine nucleoside and nucleobase transporters to import preformed purines from their hosts. To study the roles of the four purine permeases NT1-NT4 in Leishmania major, null mutants in each transporter gene were prepared and the effect of each gene deletion on purine uptake was monitored. Deletion of the NT3 purine nucleobase transporter gene or both NT3 and the NT2 nucleoside transporter gene resulted in pronounced upregulation of adenosine and uridine uptake mediated by the NT1 permease and also induced up to a 200-fold enhancement in the level of the NT1 protein but not mRNA. A similar level of upregulation of NT1 was achieved in wild type promastigotes that were transferred to medium deficient in purines. Pulse labeling and treatment of cells with the translation inhibitor cycloheximide revealed that control of NT1 expression occurs primarily at the level of translation and not protein turnover. These observations imply the existence of a translational control mechanism that enhances the ability of Leishmania parasites to import essential purines when they are present at limiting concentrations. PMID:20735779

Purine restriction induces pronounced translational upregulation of the NT1 adenosine/pyrimidine nucleoside transporter in Leishmania major.

PubMed

Ortiz, Diana; Valdés, Raquel; Sanchez, Marco A; Hayenga, Johanna; Elya, Carolyn; Detke, Siegfried; Landfear, Scott M

2010-10-01

Leishmania and other parasitic protozoa are unable to synthesize purines de novo and are reliant upon purine nucleoside and nucleobase transporters to import preformed purines from their hosts. To study the roles of the four purine permeases NT1-NT4 in Leishmania major, null mutants in each transporter gene were prepared and the effect of each gene deletion on purine uptake was monitored. Deletion of the NT3 purine nucleobase transporter gene or both NT3 and the NT2 nucleoside transporter gene resulted in pronounced upregulation of adenosine and uridine uptake mediated by the NT1 permease and also induced up to a 200-fold enhancement in the level of the NT1 protein but not mRNA. A similar level of upregulation of NT1 was achieved in wild-type promastigotes that were transferred to medium deficient in purines. Pulse labelling and treatment of cells with the translation inhibitor cycloheximide revealed that control of NT1 expression occurs primarily at the level of translation and not protein turnover. These observations imply the existence of a translational control mechanism that enhances the ability of Leishmania parasites to import essential purines when they are present at limiting concentrations. © 2010 Blackwell Publishing Ltd.

Maternal stress-associated cortisol stimulation may protect embryos from cortisol excess in zebrafish.

PubMed

Faught, Erin; Best, Carol; Vijayan, Mathilakath M

2016-02-01

Abnormal embryo cortisol level causes developmental defects and poor survival in zebrafish (Danio rerio). However, no study has demonstrated that maternal stress leads to higher embryo cortisol content in zebrafish. We tested the hypothesis that maternal stress-associated elevation in cortisol levels increases embryo cortisol content in this asynchronous breeder. Zebrafish mothers were fed cortisol-spiked food for 5 days, to mimic maternal stress, followed by daily breeding for 10 days to monitor temporal embryo cortisol content. Cortisol treatment increased mean embryo yield, but the daily fecundity was variable among the groups. Embryo cortisol content was variable in both groups over a 10-day period. A transient elevation in cortisol levels was observed in the embryos from cortisol-fed mothers only on day 3, but not on subsequent days. We tested whether excess cortisol stimulates 11βHSD2 expression in ovarian follicles as a means to regulate embryo cortisol deposition. Cortisol treatment in vitro increased 11β HSD2 levels sevenfold, and this expression was regulated by actinomycin D and cycloheximide suggesting tight regulation of cortisol levels in the ovarian follicles. We hypothesize that cortisol-induced upregulation of 11βHSD2 activity in the ovarian follicles is a mechanism restricting excess cortisol incorporation into the eggs during maternal stress.

Colonization of Legionella species in Turkish baths in hotels in Alanya, Turkey.

PubMed

Erdogan, Haluk; Arslan, Hande

2015-05-01

This study evaluated the prevalence of Legionella species in water samples collected from Turkish baths in hotels in Alanya, Turkey, from August 2003 to September 2013. Water samples were collected in 100-mL sterile containers and then concentrated by filtration. Heat treatment was used to eliminate other microorganisms from the samples, which were then spread on Legionella-selective-buffered charcoal yeast extract alpha (BCYE-α) agar and on BCYE-α agar supplemented with glycine, vancomycin, polymyxin, and cycloheximide. Cysteine-dependent colonies were identified by latex agglutination. In total, 135 samples from 52 hotels with Turkish baths were evaluated. Legionella species were identified in 11/52 (21.2%) hotels and 18/135 (13.3%) samples. The most frequently isolated species was Legionella pneumophila, with most isolates belonging to serogroups 6 (55.6%) and 1 (22.2%). The colony count was <100 colony-forming units (CFU) mL(-1) in nine samples, from 100 to 1000 CFU mL(-1) in six samples, and >1000 CFU mL(-1) in three samples. These findings suggest that the hot water systems of Turkish baths in hotels must be viewed as a possible source of travel-associated Legionnaires' disease, and preventative measures should be put in place.

Ursolic Acid Inhibits Na+/K+-ATPase Activity and Prevents TNF-α-Induced Gene Expression by Blocking Amino Acid Transport and Cellular Protein Synthesis

PubMed Central

Yokomichi, Tomonobu; Morimoto, Kyoko; Oshima, Nana; Yamada, Yuriko; Fu, Liwei; Taketani, Shigeru; Ando, Masayoshi; Kataoka, Takao

2011-01-01

Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, induce the expression of a wide variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) was identified to inhibit the cell-surface ICAM-1 expression induced by pro-inflammatory cytokines in human lung carcinoma A549 cells. Ursolic acid was found to inhibit the TNF-α-induced ICAM-1 protein expression almost completely, whereas the TNF-α-induced ICAM-1 mRNA expression and NF-κB signaling pathway were decreased only partially by ursolic acid. In line with these findings, ursolic acid prevented cellular protein synthesis as well as amino acid uptake, but did not obviously affect nucleoside uptake and the subsequent DNA/RNA syntheses. This inhibitory profile of ursolic acid was similar to that of the Na+/K+-ATPase inhibitor, ouabain, but not the translation inhibitor, cycloheximide. Consistent with this notion, ursolic acid was found to inhibit the catalytic activity of Na+/K+-ATPase. Thus, our present study reveals a novel molecular mechanism in which ursolic acid inhibits Na+/K+-ATPase activity and prevents the TNF-α-induced gene expression by blocking amino acid transport and cellular protein synthesis. PMID:24970122

Boron Nutrition of Tobacco BY-2 Cells. V. Oxidative Damage is the Major Cause of Cell Death Induced by Boron Deprivation

PubMed Central

Koshiba, Taichi; Kobayashi, Masaru; Matoh, Toru

2009-01-01

Boron (B) is an essential micronutrient for vascular plants. However, it remains unclear how B deficiency leads to various metabolic disorders and cell death. To understand this mechanism, we analyzed the physiological changes in suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells upon B deprivation. When 3-day-old cells were transferred to B-free medium, cell death was detectable as early as 12 h after treatment. The B-deprived cells accumulated more reactive oxygen species and lipid peroxides than control cells, and showed a slight but significant decrease in the cellular ascorbate pool. Supplementing the media with lipophilic antioxidants effectively suppressed the death of B-deprived cells, suggesting that the oxidative damage is the immediate and major cause of cell death under B deficiency. Dead cells in B-free culture exhibited a characteristic morphology with a shrunken cytoplasm, which is often seen in cells undergoing programmed cell death (PCD). However, they did not display other hallmarks of PCD such as internucleosomal DNA fragmentation, decreased ascorbate peroxidase expression and protection from death by cycloheximide. These results suggest that the death of tobacco cells induced by B deprivation is not likely to be a typical PCD. PMID:19054807

The effect of ethionine on ribonucleic acid synthesis in rat liver.

PubMed Central

Swann, P F

1975-01-01

1. By 1h after administration of ethionine to the female rat the appearance of newly synthesized 18SrRNA in the cytoplasm is completely inhibited. This is not caused by inhibition of RNA synthesis, for the synthesis of the large ribosomal precursor RNA (45S) and of tRNA continues. Cleavage of 45S RNA to 32S RNA also occurs, but there was no evidence for the accumulation of mature or immature rRNA in the nucleus. 2. The effect of ethionine on the maturation of rRNA was not mimicked by an inhibitor of protein synthesis (cycloheximide) or an inhibitor of polyamine synthesis [methylglyoxal bis(guanylhydrazone)]. 3. Unlike the ethionine-induced inhibition of protein synthesis, this effect was not prevented by concurrent administration of inosine. A similar effect could be induced in HeLa cells by incubation for 1h in a medium lacking methionine. The ATP concentration in these cells was normal. From these two observations it was concluded that the effect of etionine on rRNA maturation is not caused by an ethionine-induced lack of ATP. It is suggested that ethionine, by lowering the hepatic concentration of S-adenosylmethionine, prevents methylation of the ribosomal precursor. The methylation is essential for the correct maturation of the molecule; without methylation complete degradation occurs. PMID:1212195

Cultured Chinese hamster cells undergo apoptosis after exposure to cold but nonfreezing temperatures.

PubMed

Nagle, W A; Soloff, B L; Moss, A J; Henle, K J

1990-08-01

Cultured Chinese hamster V79 fibroblast cells at the transition from logarithmic to stationary growth have been shown to undergo apoptosis (programmed cell death) after cold shock [B. L. Soloff, W. A. Nagle, A. J. Moss, Jr., K. J. Henle, and J. T. Crawford, Biochem. Biophys. Res. Commun. 145, 876-883 (1987)]. In this report, we show that about 95% of the cell population was susceptible to cold-induced apoptosis, and the amount of cell killing was dependent on the duration of hypothermia. Cells treated for 0-90 min at 0 degrees C exhibited an exponential survival curve with a D0 of 32 min; thus, even short exposures to the cold (e.g., 5 min) produced measurable cell killing. The cold-induced injury was not produced by freezing, because similar results were observed at 6 degrees C, and cell killing was not influenced by the cryoprotective agent dimethyl sulfoxide. Cold-induced apoptosis was inhibited by rewarming at 23 degrees C, compared to 37 degrees C, by inhibitors of macromolecular synthesis, such as cycloheximide, and by 0.8 mM zinc sulfate. The results suggest that apoptosis represents a new manifestation of cell injury after brief exposure to 0-6 degrees C hypothermia.

Keratinophilic fungi and other moulds associated with air-dust particles from Egypt.

PubMed

Abdel-Hafez, S I; Moubasher, A H; Barakat, A

1990-01-01

One-hundred and eleven species and three species varieties belonging to 39 genera were collected from 50 dust samples on the five media used at 28 degrees C. Using the hair-baiting technique with horse hair, 10 species of Chrysosporium were isolated: C. asperatum, C. state of Arthroderma tuberculatum, C. indicum, C. inops, C. keratinophilum, C. merdarium, C. pannorum, C. queenslandicum, C. tropicum and C. xerophilum. True dermatophytes were isolated: Trichophyton verrucosum and Trichophyton sp. Also, numerous fungi tolerating high levels of cycloheximide were encountered, such as members of Acremonium, Aspergillus and Penicillium. On plates of glucose or cellulose Czapek-Dox agar (free from sucrose) the most frequent fungi were: Alternaria alternata, Aspergillus flavus, A. flavus var. columnaris, A. fumigatus, A. niger, A. ochraceus, A. sydowii, A. terreus, Chaetomium globosum, Cladosporium herbarum, Emericella nidulans, Fusarium oxysporum, Mucor hiemalis, Penicillium chrysogenum, P. oxalicum, Scopulariopsis brevicaulis and Ulocladium atrum. On plates of 50% sucrose or 10 and 20% NaCl-Czapek's agar, some interesting species were frequently encountered: Eurotium amstelodami, E. chevalieri, E. halophilicum, E. montevidensis, E. repens, E. rubrum and Scopulariopsis halophilica. The isolated fungi have been tested for osmophilicity and halophilicity, they showed different rates of growth on sucrose and sodium chloride-Czapek's medium of various osmotic potential.

Developmental reprogramming of rat GLUT-5 requires de novo mRNA and protein synthesis.

PubMed

Jiang, L; Ferraris, R P

2001-01-01

Fructose transporter (GLUT-5) expression is low in mid-weaning rat small intestine, increases normally after weaning is completed, and can be precociously induced by premature consumption of a high-fructose (HF) diet. In this study, an in vivo perfusion model was used to determine the mechanisms regulating this substrate-induced reprogramming of GLUT-5 development. HF (100 mM) but not high-glucose (HG) perfusion increased GLUT-5 activity and mRNA abundance. In contrast, HF and HG perfusion had no effect on Na(+)-dependent glucose transporter (SGLT-1) expression but increased c-fos and c-jun expression. Intraperitoneal injection of actinomycin D before intestinal perfusion blocked the HF-induced increase in fructose uptake rate and GLUT-5 mRNA abundance. Actinomycin D also prevented the perfusion-induced increase in c-fos and c-jun mRNA abundance but did not affect glucose uptake rate and SGLT-1 mRNA abundance. Cycloheximide blocked the HF-induced increase in fructose uptake rate but not the increase in GLUT-5 mRNA abundance and had no effect on glucose uptake rate and SGLT-1 mRNA abundance. In neonatal rats, the substrate-induced reprogramming of intestinal fructose transport is likely to involve transcription and translation of the GLUT-5 gene.

Methanolic extract of Momordica cymbalaria enhances glucose uptake in L6 myotubes in vitro by up-regulating PPAR-γ and GLUT-4.

PubMed

Kumar, Puttanarasaiah Mahesh; Venkataranganna, Marikunte V; Manjunath, Kirangadur; Viswanatha, Gollapalle L; Ashok, Godavarthi

2014-12-01

The present study was undertaken to evaluate the influence of the methanolic fruit extract of Momordica cymbalaria (MFMC) on PPARγ (Peroxisome Proliferator Activated Receptor gamma) and GLUT-4 (Glucose transporter-4) with respect to glucose transport. Various concentrations of MFMC ranging from 62.5 to 500 μg·mL(-1) were evaluated for glucose uptake activity in vitro using L6 myotubes, rosiglitazone was used as a reference standard. The MFMC showed significant and dose-dependent increase in glucose uptake at the tested concentrations, further, the glucose uptake activity of MFMC (500 μg·mL(-1)) was comparable with rosigilitazone. Furthermore, MFMC has shown up-regulation of GLUT-4 and PPARγ gene expressions in L6 myotubes. In addition, the MFMC when incubated along with cycloheximide (CHX), which is a protein synthesis inhibitor, has shown complete blockade of glucose uptake. This indicates that new protein synthesis is required for increased GLUT-4 translocation. In conclusion, these findings suggest that MFMC is enhancing the glucose uptake significantly and dose dependently through the enhanced expression of PPARγ and GLUT-4 in vitro. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Staurosporine induces rapid homotypic intercellular adhesion of U937 cells via multiple kinase activation

PubMed Central

Cho, Jae Youl; Katz, David R; Chain, Benjamin M

2003-01-01

Staurosporine is a broad-specificity kinase inhibitor, which has acted as lead compound for the development of some novel cytotoxic compounds for treatment of cancer. This study investigates the unexpected observation that staurosporine can also induce homotypic cellular aggregation. In this study, staurosporine is shown to activate rapid homotypic aggregation of U937 cells, at concentrations below those required to induce cell death. This activity is a particular feature of staurosporine, and is not shared by a number of other kinase inhibitors. The proaggregating activity of staurosporine is inhibited by deoxyglucose, cytochalasin B and colchicine. Staurosporine-induced aggregation can be distinguished from that induced by the phorbol 12-myristate 13-acetate by faster kinetics and insensitivity to cycloheximide. Staurosporine induces translocation of conventional and novel, but not atypical isoforms of protein kinase C (PKC). Aggregation induced by staurosporine is inhibited by a number of inhibitors of PKC isoforms, and by inhibitors of protein tyrosine kinases. Staurosporine also induces rapid phosphorylation of ERK and p38, and inhibitors of both these enzymes block aggregation. Staurosporine induces dysregulated activation of multiple kinase signaling pathways in U937 cells, and the combined activity of several of these pathways is essential for the induction of aggregation. PMID:12970105

Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast.

PubMed

Hussmann, Jeffrey A; Patchett, Stephanie; Johnson, Arlen; Sawyer, Sara; Press, William H

2015-12-01

Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics.

Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast

PubMed Central

Hussmann, Jeffrey A.; Patchett, Stephanie; Johnson, Arlen; Sawyer, Sara; Press, William H.

2015-01-01

Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics. PMID:26656907

Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2

PubMed Central

Tsabar, Michael; Eapen, Vinay V.; Mason, Jennifer M.; Memisoglu, Gonen; Waterman, David P.; Long, Marcus J.; Bishop, Douglas K.; Haber, James E.

2015-01-01

In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5′ to 3′ end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5′ to 3′ resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells. PMID:26019182

Stimulation of fibrinogen synthesis in cultured rat hepatocytes by fibrinogen degradation product fragment D

DOE Office of Scientific and Technical Information (OSTI.GOV)

LaDuca, F.M.; Tinsley, L.A.; Dang, C.V.

The direct stimulation of fibrinogen biosynthesis by fibrinogen degradation produces (FDPs) was studied in rat hepatocyte cultures. Pure rat FDP fragment D (FDP-D) (Mr 90,000) and FDP fragment E (FDP-E) (Mr 40,000) and mixtures of the two (FDP-DE) were added to rat hepatocytes cultured in serum-free hormonally defined medium. Hydrocortisone (20 microM) significantly increased synthesis of fibrinogen, as determined by incorporation of (35S)methionine. FDP-D and FDP-E did not increase fibrinogen synthesis in the presence of hydrocortisone. However, hepatocytes cultured without hydrocortisone displayed increased fibrinogen synthesis (2.0- to 2.8-fold) with FDP-D (2.6-6.7 microM) but not with FDP-E (5.7 microM). At thesemore » FDP concentrations the synthesis of albumin, haptoglobin, and transferrin was not increased. FDP-D-induced fibrinogen synthesis was inhibited (greater than 90%) by actinomycin D and cycloheximide, indicating that the increase in (35S)methionine incorporation was from de novo protein synthesis. The role of FDP-D was further substantiated by showing that FDP-D, but not FDP-E, bound to the hepatocytes. These data indicate that FDP-D, but not FDP-E, directly and specifically stimulates fibrinogen synthesis in rat hepatocytes; this stimulation does not require any additional serum or protein cofactors.« less

The role of apoptosis in LDL transport through cultured endothelial cell monolayers

PubMed Central

Cancel, Limary M.; Tarbell, John M.

2009-01-01

We have previously shown that leaky junctions associated with dying or dividing cells are the dominant pathway for LDL transport under convective conditions, accounting for more than 90% of the transport [1]. To explore the role of apoptosis in the leaky junction pathway, TNFα and cycloheximide (TNFα/CHX) were used to induce an elevated rate of apoptosis in cultured bovine aortic endothelial cell (BAEC) monolayers and the convective fluxes of LDL and water were measured. Treatment with TNFα/CHX induced a 18.3-fold increase in apoptosis and a 4.4-fold increase in LDL permeability. Increases in apoptosis and permeability were attenuated by treatment with the caspase inhibitor Z-VAD-FMK. Water flux increased by 2.7-fold after treatment with TNFα/CHX, and this increase was not attenuated by treatment with Z-VAD-FMK. Immunostaining of the tight junction protein ZO-1 showed that TNFα/CHX treatment disrupts the tight junction in addition to inducing apoptosis. This disruption is present even when Z-VAD-FMK is used to inhibit apoptosis, and likely accounts for the increase in water flux. We found a strong correlation between the rate of apoptosis and the permeability of BAEC monolayers to LDL. These results demonstrate the potential of manipulating endothelial monolayer permeability by altering the rate of apoptosis pharmacollogicaly. This has implications for the treatment of atherosclerosis. PMID:19709659

Inhibition of caspases prevents ototoxic and ongoing hair cell death

NASA Technical Reports Server (NTRS)

Matsui, Jonathan I.; Ogilvie, Judith M.; Warchol, Mark E.

2002-01-01

Sensory hair cells die after acoustic trauma or ototoxic insults, but the signal transduction pathways that mediate hair cell death are not known. Here we identify several important signaling events that regulate the death of vestibular hair cells. Chick utricles were cultured in media supplemented with the ototoxic antibiotic neomycin and selected pharmacological agents that influence signaling molecules in cell death pathways. Hair cells that were treated with neomycin exhibited classically defined apoptotic morphologies such as condensed nuclei and fragmented DNA. Inhibition of protein synthesis (via treatment with cycloheximide) increased hair cell survival after treatment with neomycin, suggesting that hair cell death requires de novo protein synthesis. Finally, the inhibition of caspases promoted hair cell survival after neomycin treatment. Sensory hair cells in avian vestibular organs also undergo continual cell death and replacement throughout mature life. It is unclear whether the loss of hair cells stimulates the proliferation of supporting cells or whether the production of new cells triggers the death of hair cells. We examined the effects of caspase inhibition on spontaneous hair cell death in the chick utricle. Caspase inhibitors reduced the amount of ongoing hair cell death and ongoing supporting cell proliferation in a dose-dependent manner. In isolated sensory epithelia, however, caspase inhibitors did not affect supporting cell proliferation directly. Our data indicate that ongoing hair cell death stimulates supporting cell proliferation in the mature utricle.

Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

PubMed

Al-Hadid, Qais; White, Jonelle; Clarke, Steven

2016-02-12

A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. Copyright © 2016 Elsevier Inc. All rights reserved.

Defects in THO/TREX-2 function cause accumulation of novel cytoplasmic mRNP granules that can be cleared by autophagy

PubMed Central

Eshleman, Nichole; Liu, Guangbo; McGrath, Kaitlyn; Parker, Roy; Buchan, J. Ross

2016-01-01

The nuclear THO and TREX-2 complexes are implicated in several steps of nuclear mRNP biogenesis, including transcription, 3′ end processing and export. In a recent genomic microscopy screen in Saccharomyces cerevisiae for mutants with constitutive stress granules, we identified that absence of THO and TREX-2 complex subunits leads to the accumulation of Pab1-GFP in cytoplasmic foci. We now show that these THO/TREX-2 mutant induced foci (“TT foci”) are not stress granules but instead are a mRNP granule containing poly(A)+ mRNA, some mRNP components also found in stress granules, as well several proteins involved in mRNA 3′ end processing and export not normally seen in stress granules. In addition, TT foci are resistant to cycloheximide-induced disassembly, suggesting the presence of mRNPs impaired for entry into translation. THO mutants also exhibit defects in normal stress granule assembly. Finally, our data also suggest that TT foci are targeted by autophagy. These observations argue that defects in nuclear THO and TREX-2 complexes can affect cytoplasmic mRNP function by producing aberrant mRNPs that are exported to cytosol, where they accumulate in TT foci and ultimately can be cleared by autophagy. This identifies a novel mechanism of quality control for aberrant mRNPs assembled in the nucleus. PMID:27251550

Chemical-genetic profile analysis of five inhibitory compounds in yeast.

PubMed

Alamgir, Md; Erukova, Veronika; Jessulat, Matthew; Azizi, Ali; Golshani, Ashkan

2010-08-06

Chemical-genetic profiling of inhibitory compounds can lead to identification of their modes of action. These profiles can help elucidate the complex interactions between small bioactive compounds and the cell machinery, and explain putative gene function(s). Colony size reduction was used to investigate the chemical-genetic profile of cycloheximide, 3-amino-1,2,4-triazole, paromomycin, streptomycin and neomycin in the yeast Saccharomyces cerevisiae. These compounds target the process of protein biosynthesis. More than 70,000 strains were analyzed from the array of gene deletion mutant yeast strains. As expected, the overall profiles of the tested compounds were similar, with deletions for genes involved in protein biosynthesis being the major category followed by metabolism. This implies that novel genes involved in protein biosynthesis could be identified from these profiles. Further investigations were carried out to assess the activity of three profiled genes in the process of protein biosynthesis using relative fitness of double mutants and other genetic assays. Chemical-genetic profiles provide insight into the molecular mechanism(s) of the examined compounds by elucidating their potential primary and secondary cellular target sites. Our follow-up investigations into the activity of three profiled genes in the process of protein biosynthesis provided further evidence concerning the usefulness of chemical-genetic analyses for annotating gene functions. We termed these genes TAE2, TAE3 and TAE4 for translation associated elements 2-4.

Genetic Analysis of Resistance to Benzimidazoles in Physarum: Differential Expression of β-Tubulin Genes

PubMed Central

Burland, Timothy G.; Schedl, Tim; Gull, Keith; Dove, William F.

1984-01-01

Physarum displays two vegetative cell types, uninucleate myxamoebae and multinucleate plasmodia. Mutant myxamoebae of Physarum resistant to the antitubulin drug methylbenzimidazole-2-yl-carbamate (MBC) were isolated. All mutants tested were cross-resistant to other benzimidazoles but not to cycloheximide or emetine. Genetic analysis showed that mutation to MBC resistance can occur at any one of four unlinked loci, benA, benB, benC or benD. MBC resistance of benB and benD mutants was expressed in plasmodia, but benA and benC mutant plasmodia were MBC sensitive, suggesting that benA and benC encode myxamoeba-specific products. Myxamoebae carrying the recessive benD210 mutation express a β-tubulin with noval electrophoretic mobility, in addition to a β-tubulin with wild-type mobility. This and other evidence indicates that benD is a structural gene for β-tubulin, and that at least two β-tubulin genes are expressed in myxamoebae. Comparisons of the β-tubulins of wildtype and benD210 strains by gel electrophoresis revealed that, of the three (or more) β-tubulin genes expressed in Physarum, one, benD, is expressed in both myxamoebae and plasmodia, one is expressed specifically in myxamoebae and one is expressed specifically in plasmodia. However, mutation in only one gene, benD, is sufficient to confer MBC resistance on both myxamoebae and plasmodia. PMID:6479584

Neutral lipid trafficking regulates alveolar type II cell surfactant phospholipid and surfactant protein expression.

PubMed

Torday, John; Rehan, Virender

2011-08-01

Adipocyte differentiation-related protein (ADRP) is a critically important protein that mediates lipid uptake, and is highly expressed in lung lipofibroblasts (LIFs). Triacylglycerol secreted from the pulmonary circulation and stored in lipid storage droplets is a robust hormonal-, growth factor-, and stretch-regulated precursor for surfactant phospholipid synthesis by alveolar type II epithelial (ATII) cells. A549 lung epithelial cells rapidly take up green fluorescent protein (GFP)-ADRP fusion protein-associated lipid droplets (LDs) in a dose-dependent manner. The LDs initially localize to the perinuclear region of the cell, followed by localization in the cytoplasm. Uptake of ADRP-LDs causes a time- and dose-dependent increase in surfactant protein-B (SP-B) expression. This mechanism can be inhibited by either actinomycin D or cycloheximide, indicating that ADRP-LDs induce newly synthesized SP-B. ADRP-LDs concomitantly stimulate saturated phosphatidylcholine (satPC) synthesis by A549 cells, which is inhibited by ADRP antibody, indicating that this is a receptor-mediated mechanism. Intravenous administration of GFP-ADRP LDs to adult rats results in dose-dependent increases in lung ADRP and SP-B expression. These data indicate that lipofibroblast-derived ADRP coordinates ATII cells' synthesis of the surfactant phospholipid-protein complex by stimulating both satPC and SP-B. The authors propose, therefore, that ADRP is the physiologic determinant for the elusive coordinated, stoichiometric synthesis of surfactant phospholipid and protein by pulmonary ATII cells.

Intracellular disposal of incompletely folded human alpha1-antitrypsin involves release from calnexin and post-translational trimming of asparagine-linked oligosaccharides.

PubMed

Liu, Y; Choudhury, P; Cabral, C M; Sifers, R N

1997-03-21

Protection of lung elastin fibers from proteolytic destruction is compromised by inefficient secretion of incompletely folded allelic variants of human alpha1-antitrypsin from hepatocytes. Pulse-chase radiolabeling with [35S]methionine and sucrose gradient sedimentation and coimmunoprecipitation techniques were employed to investigate quality control of human alpha1-antitrypsin secretion from stably transfected mouse hepatoma cells. The secretion-incompetent variant null(Hong Kong) (Sifers, R. N., Brashears-Macatee, S., Kidd, V. J., Muensch, H., and Woo, S. L. C. (1988) J. Biol. Chem. 263, 7330-7335) cannot fold into a functional conformation and was quantitatively associated with the molecular chaperone calnexin following biosynthesis. Assembly with calnexin required cotranslational trimming of glucose from asparagine-linked oligosaccharides. Intracellular disposal of pulse-radiolabeled molecules coincided with their release from calnexin. Released monomers and intracellular disposal were nonexistent in cells chased with cycloheximide, an inhibitor of protein synthesis. Post-translational trimming of asparagine-linked oligosaccharides and intracellular disposal were abrogated by 1-deoxymannojirimycin, an inhibitor of alpha-mannosidase activity, without affecting the monomer population. The data are consistent with a recently proposed quality control model (Hammond, C., Braakman, I., and Helenius, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 913-917) in which intracellular disposal requires dissociation from calnexin and post-translational trimming of mannose from asparagine-linked oligosaccharides.

Partial androgen insensitivity syndrome caused by a deep intronic mutation creating an alternative splice acceptor site of the AR gene.

PubMed

Ono, Hiroyuki; Saitsu, Hirotomo; Horikawa, Reiko; Nakashima, Shinichi; Ohkubo, Yumiko; Yanagi, Kumiko; Nakabayashi, Kazuhiko; Fukami, Maki; Fujisawa, Yasuko; Ogata, Tsutomu

2018-02-02

Although partial androgen insensitivity syndrome (PAIS) is caused by attenuated responsiveness to androgens, androgen receptor gene (AR) mutations on the coding regions and their splice sites have been identified only in <25% of patients with a diagnosis of PAIS. We performed extensive molecular studies including whole exome sequencing in a Japanese family with PAIS, identifying a deep intronic variant beyond the branch site at intron 6 of AR (NM_000044.4:c.2450-42 G > A). This variant created the splice acceptor motif that was accompanied by pyrimidine-rich sequence and two candidate branch sites. Consistent with this, reverse transcriptase (RT)-PCR experiments for cycloheximide-treated lymphoblastoid cell lines revealed a relatively large amount of aberrant mRNA produced by the newly created splice acceptor site and a relatively small amount of wildtype mRNA produced by the normal splice acceptor site. Furthermore, most of the aberrant mRNA was shown to undergo nonsense mediated decay (NMD) and, if a small amount of aberrant mRNA may have escaped NMD, such mRNA was predicted to generate a truncated AR protein missing some functional domains. These findings imply that the deep intronic mutation creating an alternative splice acceptor site resulted in the production of a relatively small amount of wildtype AR mRNA, leading to PAIS.

RuBisCO in Non-Photosynthetic Alga Euglena longa: Divergent Features, Transcriptomic Analysis and Regulation of Complex Formation.

PubMed

Záhonová, Kristína; Füssy, Zoltán; Oborník, Miroslav; Eliáš, Marek; Yurchenko, Vyacheslav

2016-01-01

Euglena longa, a close relative of the photosynthetic model alga Euglena gracilis, possesses an enigmatic non-photosynthetic plastid. Its genome has retained a gene for the large subunit of the enzyme RuBisCO (rbcL). Here we provide new data illuminating the putative role of RuBisCO in E. longa. We demonstrated that the E. longa RBCL protein sequence is extremely divergent compared to its homologs from the photosynthetic relatives, suggesting a possible functional shift upon the loss of photosynthesis. Similarly to E. gracilis, E. longa harbors a nuclear gene encoding the small subunit of RuBisCO (RBCS) as a precursor polyprotein comprising multiple RBCS repeats, but one of them is highly divergent. Both RBCL and the RBCS proteins are synthesized in E. longa, but their abundance is very low compared to E. gracilis. No RBCS monomers could be detected in E. longa, suggesting that processing of the precursor polyprotein is inefficient in this species. The abundance of RBCS is regulated post-transcriptionally. Indeed, blocking the cytoplasmic translation by cycloheximide has no immediate effect on the RBCS stability in photosynthetically grown E. gracilis, but in E. longa, the protein is rapidly degraded. Altogether, our results revealed signatures of evolutionary degradation (becoming defunct) of RuBisCO in E. longa and suggest that its biological role in this species may be rather unorthodox, if any.

Development of an in vitro test battery for assessing chemical effects on bovine germ cells under the ReProTect umbrella

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lazzari, Giovanna; Tessaro, Irene; Crotti, Gabriella

2008-12-15

Current European legislation for the registration and authorisation of chemicals (REACH) will require a dramatic increase in the use of animals for reproductive toxicity testing. Since one objective of REACH is to use vertebrates only as last resort, the development and validation of alternative methods is urgently needed. For this purpose ReProTect, an integrated research project funded by the European Union, joining together 33 partners with complementary expertise in reproductive toxicology, was designed. The study presented here describes a battery of two tests developed within ReProTect. The objective of these tests is the detection of chemical effects during the processesmore » of oocyte maturation and fertilisation in a bovine model. The corresponding toxicological endpoints are the reaching of metaphase II and the formation of the pronuclei respectively. Fifteen chemicals have been tested (Benzo[a]pyrene, Busulfan, Butylparaben, Cadmium Chloride, Carbendazim, Cycloheximide, Diethylstilbestrol, Genistein, Ionomycin, Ketoconazole, Lindane, Methylacetoacetate, Mifepristone, Nocodazole and DMSO as solvent) demonstrating high intra-laboratory reproducibility of the tests. Furthermore, the responses obtained in both tests, for several substances, had a good correlation with the available in vivo and in vitro data. These tests therefore, could predictably become part of an integrated testing strategy that combines the bovine models with additional in vitro tests, in order to predict chemical hazards on mammalian fertility.« less

SGK is a primary glucocorticoid-induced gene in the human.

PubMed

Náray-Fejes-Tóth, A; Fejes-Tóth, G; Volk, K A; Stokes, J B

2000-12-01

Serum- and glucocorticoid-induced kinase (sgk) is transcriptionally regulated by corticosteroids in several cell types. Recent findings suggest that sgk is an important gene in the early action of corticosteroids on epithelial sodium reabsorption. Surprisingly, the human sgk was reported not to be transcriptionally regulated by corticosteroids in a hepatoma cell line, and thus far no glucocorticoid response element has been identified in the human SGK gene. Since humans clearly respond to both aldosterone and glucocorticoids in cells where sgk action seems to be important, in this study we determined sgk mRNA levels following dexamethasone treatment for various duration in five human cell lines. These cell lines included epithelial cells (H441, T84 and HT29) and lymphoid/monocyte (U937 and THP-1) lines. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that sgk mRNA levels are markedly induced by glucocorticoids in all of the five cell lines studied. Time course analyses revealed that sgk mRNA levels are elevated as early as 30 min after addition of the glucocorticoid, and remain elevated for several hours. Northern analysis in H441 cells confirmed that sgk is an early induced gene. The induction of sgk by dexamethasone was unaffected by cycloheximide, indicating that it does not require de novo protein synthesis. These results indicate that the human sgk, just like its counterparts in other species, is a primary glucocorticoid-induced gene.

Obatoclax, a Pan-BCL-2 Inhibitor, Targets Cyclin D1 for Degradation to Induce Antiproliferation in Human Colorectal Carcinoma Cells.

PubMed

Or, Chi-Hung R; Chang, Yachu; Lin, Wei-Cheng; Lee, Wee-Chyan; Su, Hong-Lin; Cheung, Muk-Wing; Huang, Chang-Po; Ho, Cheesang; Chang, Chia-Che

2016-12-27

Colorectal cancer is the third most common cancer worldwide. Aberrant overexpression of antiapoptotic BCL-2 (B-cell lymphoma 2) family proteins is closely linked to tumorigenesis and poor prognosis in colorectal cancer. Obatoclax is an inhibitor targeting all antiapoptotic BCL-2 proteins. A previous study has described the antiproliferative action of obatoclax in one human colorectal cancer cell line without elucidating the underlying mechanisms. We herein reported that, in a panel of human colorectal cancer cell lines, obatoclax inhibits cell proliferation, suppresses clonogenicity, and induces G₁-phase cell cycle arrest, along with cyclin D1 downregulation. Notably, ectopic cyclin D1 overexpression abrogated clonogenicity suppression but also G₁-phase arrest elicited by obatoclax. Mechanistically, pre-treatment with the proteasome inhibitor MG-132 restored cyclin D1 levels in all obatoclax-treated cell lines. Cycloheximide chase analyses further revealed an evident reduction in the half-life of cyclin D1 protein by obatoclax, confirming that obatoclax downregulates cyclin D1 through induction of cyclin D1 proteasomal degradation. Lastly, threonine 286 phosphorylation of cyclin D1, which is essential for initiating cyclin D1 proteasomal degradation, was induced by obatoclax in one cell line but not others. Collectively, we reveal a novel anticancer mechanism of obatoclax by validating that obatoclax targets cyclin D1 for proteasomal degradation to downregulate cyclin D1 for inducing antiproliferation.

Calcineurin inhibition blocks within-, but not between-session fear extinction in mice

PubMed Central

Moulin, Thiago C.; Carneiro, Clarissa F. D.; Gonçalves, Marina M. C.; Junqueira, Lara S.; Amaral, Olavo B.

2015-01-01

Memory extinction involves the formation of a new associative memory that inhibits a previously conditioned association. Nonetheless, it could also depend on weakening of the original memory trace if extinction is assumed to have multiple components. The phosphatase calcineurin (CaN) has been described as being involved in extinction but not in the initial consolidation of fear learning. With this in mind, we set to study whether CaN could have different roles in distinct components of extinction. Systemic treatment with the CaN inhibitors cyclosporin A (CsA) or FK-506, as well as i.c.v. administration of CsA, blocked within-session, but not between-session extinction or initial learning of contextual fear conditioning. Similar effects were found in multiple-session extinction of contextual fear conditioning and in auditory fear conditioning, indicating that CaN is involved in different types of short-term extinction. Meanwhile, inhibition of protein synthesis by cycloheximide (CHX) treatment did not affect within-session extinction, but disrupted fear acquisition and slightly impaired between-session extinction. Our results point to a dissociation of within- and between-session extinction of fear conditioning, with the former being more dependent on CaN activity and the latter on protein synthesis. Moreover, the modulation of within-session extinction did not affect between-session extinction, suggesting that these components are at least partially independent. PMID:25691516

Production of fungal and bacterial growth modulating secondary metabolites is widespread among mycorrhiza-associated streptomycetes

PubMed Central

2012-01-01

Background Studies on mycorrhiza associated bacteria suggest that bacterial-fungal interactions play important roles during mycorrhiza formation and affect plant health. We surveyed Streptomyces Actinobacteria, known as antibiotic producers and antagonists of fungi, from Norway spruce mycorrhizas with predominantly Piloderma species as the fungal partner. Results Fifteen Streptomyces isolates exhibited substantial variation in inhibition of tested mycorrhizal and plant pathogenic fungi (Amanita muscaria, Fusarium oxysporum, Hebeloma cylindrosporum, Heterobasidion abietinum, Heterobasidion annosum, Laccaria bicolor, Piloderma croceum). The growth of the mycorrhiza-forming fungus Laccaria bicolor was stimulated by some of the streptomycetes, and Piloderma croceum was only moderately affected. Bacteria responded to the streptomycetes differently than the fungi. For instance the strain Streptomyces sp. AcM11, which inhibited most tested fungi, was less inhibitory to bacteria than other tested streptomycetes. The determined patterns of Streptomyces-microbe interactions were associated with distinct patterns of secondary metabolite production. Notably, potentially novel metabolites were produced by strains that were less antagonistic to fungi. Most of the identified metabolites were antibiotics (e.g. cycloheximide, actiphenol) and siderophores (e.g. ferulic acid, desferroxiamines). Plant disease resistance was activated by a single streptomycete strain only. Conclusions Mycorrhiza associated streptomycetes appear to have an important role in inhibiting the growth of fungi and bacteria. Additionally, our study indicates that the Streptomyces strains, which are not general antagonists of fungi, may produce still un-described metabolites. PMID:22852578

Nitric oxide signaling pathway regulates potassium chloride cotransporter-1 mRNA expression in vascular smooth muscle cells.

PubMed

Di Fulvio, M; Lauf, P K; Adragna, N C

2001-11-30

Rat vascular smooth muscle cells (VSMCs) express at least two mRNAs for K-Cl cotransporters (KCC): KCC1 and KCC3. cGMP-dependent protein kinase I regulates KCC3 mRNA expression in these cells. Here, we show evidence implicating the nitric oxide (NO)/cGMP signaling pathway in the expression of KCC1 mRNA, considered to be the major cell volume regulator. VSMCs, expressing soluble guanylyl cyclase (sGC) and PKG-I isoforms showed a time- and concentration-dependent increase in KCC1 mRNA levels after treatment with sodium nitroprusside as demonstrated by semiquantitative RT-PCR. sGC-dependent regulation of KCC1 mRNA expression was confirmed using YC-1, a NO-independent sGC stimulator. The sGC inhibitor LY83583 blocked the effects of sodium nitroprusside and YC-1. Moreover, 8-Br-cGMP increased KCC1 mRNA expression in a concentration- and time-dependent fashion. The 8-Br-cGMP effect was partially blocked by KT5823 but not by actinomycin D. However, actinomycin D and cycloheximide increased basal KCC1 mRNA in an additive manner, suggesting different mechanisms of action for both drugs. These findings suggest that in VSMCs, the NO/cGMP-signaling pathway participates in KCC1 mRNA regulation at the post-transcriptional level.

A liver X receptor (LXR)-{beta} alternative splicing variant (LXRBSV) acts as an RNA co-activator of LXR-{beta}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashimoto, Koshi, E-mail: khashi@med.gunma-u.ac.jp; Ishida, Emi; Matsumoto, Shunichi

2009-12-25

We report the isolation and functional characterization of a novel transcriptional co-activator, termed LXRBSV. LXRBSV is an alternative splicing variant of liver X receptor (LXR)-{beta} LXRBSV has an intronic sequence between exons 2 and 3 in the mouse LXR-{beta} gene. The LXRBSV gene is expressed in various tissues including the liver and brain. We sub-cloned LXRBSV into pSG5, a mammalian expression vector, and LXRBSV in pSG5 augmented human Sterol Response Element Binding Protein (SREBP)-1c promoter activity in HepG2 cells in a ligand (TO901317) dependent manner. The transactivation mediated by LXRBSV is selective for LXR-{beta}. The LXRBSV protein was deduced tomore » be 64 amino acids in length; however, a GAL4-LXRBSV fusion protein was not able to induce transactivation. Serial deletion constructs of LXRBSV demonstrated that the intronic sequence inserted in LXRBSV is required for its transactivation activity. An ATG mutant of LXRBSV was able to induce transactivation as wild type. Furthermore, LXRBSV functions in the presence of cycloheximide. Taken together, we have concluded that LXRBSV acts as an RNA transcript not as a protein. In the current study, we have demonstrated for the first time that an alternative splicing variant of a nuclear receptor acts as an RNA co-activator.« less

Salicylic Acid Regulation of Respiration in Higher Plants: Alternative Oxidase Expression.

PubMed Central

Rhoads, DM; McIntosh, L

1992-01-01

Alternative respiratory pathway capacity increases during the development of the thermogenic appendix of a voodoo lily inflorescence. The levels of the alternative oxidase proteins increased dramatically between D-4 (4 days prior to the day of anthesis) and D-3 and continued to increase until the day of anthesis (D-day). The level of salicylic acid (SA) in the appendix is very low early on D-1, but increases to a high level in the evening of D-1. Thermogenesis occurs after a few hours of light on D-day. Therefore, the initial accumulation of the alternative oxidase proteins precedes the increase in SA by 3 days, indicating that other regulators may be involved. A 1.6-kb transcript encoding the alternative oxidase precursor protein accumulated to a high level in the appendix tissue by D-1. Application of SA to immature appendix tissue caused an increase in alternative pathway capacity and a dramatic accumulation of the alternative oxidase proteins and the 1.6-kb transcript. Time course experiments showed that the increase in capacity, protein levels, and transcript level corresponded precisely. The response to SA was blocked by cycloheximide or actinomycin D, indicating that de novo transcription and translation are required. However, nuclear, in vitro transcription assays indicated that the accumulation of the 1.6-kb transcript did not result from a simple increase in the rate of transcription of aox1. PMID:12297672

Connexin43 synthesis, phosphorylation, and degradation in regulation of transient inhibition of gap junction intercellular communication by the phorbol ester TPA in rat liver epithelial cells.

PubMed

Rivedal, Edgar; Leithe, Edward

2005-01-15

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces transient inhibition of gap junction intercellular communication (GJIC) in several cell types. The initial block in GJIC has been attributed to protein kinase C (PKC) mediated phosphorylation of connexin gap junction proteins, including connexin43 (Cx43). Restoration of GJIC, associated with normalization of the Cx43 phosphorylation status, has been ascribed to different events, including dephosphorylation of Cx43 and de novo synthesis of Cx43 or other, non-gap junctional, proteins. The data presented suggest that restoration of GJIC during continuous TPA exposure in normal and transformed rat liver epithelial cells is dependent on synthesis of Cx43 protein, as well as the transport of already synthesized Cx43 from intracellular pools to the plasma membrane. Reactivation of inactivated Cx43 by dephosphorylation does not appear to be involved in the recovery of GJIC. Both PKC and MAP kinase is involved in TPA-induced degradation of Cx43 and inhibition of GJIC. We show that coincubation of TPA with the protein synthesis inhibitor cycloheximide or the transcription inhibitor actinomycin D results in synergistic enhancement of the level of activated ERK1/2. Together, the present data highlight Cx43 degradation and synthesis as critical determinants in TPA-induced modifications of cell-cell communication via gap junctions.

Rat Blastocysts from Nuclear Injection and Time-Lagged Enucleation and Their Commitment to Embryonic Stem Cells.

PubMed

Hara, Hiromasa; Goto, Teppei; Takizawa, Akiko; Sanbo, Makoto; Jacob, Howard J; Kobayashi, Toshihiro; Nakauchi, Hiromitsu; Hochi, Shinichi; Hirabayashi, Masumi

2016-04-01

Pronucleus-like vesicle formation following premature chromosome condensation (PCC) of the donor cell nucleus is the key event for successful generation of cloned rodents by nuclear transplantation (NT). However in rat cloning, this change is difficult to induce in enucleated recipient oocytes because of their inability to maintain maturation-promoting factor levels. In this study, intact oocytes retrieved from nuclear-visualized H2B-tdTomato knock-in rats were injected with Venus-labeled cell nuclei. Because the incidence of PCC under MG-132 treatment significantly increased with the culture period (0%, 10.8%, 36.8%, and 87.5% at 0, 0.5, 1, and 2 h postinjection, respectively), the metaphase plate of the oocyte was removed 1-2 h after the nuclear injection. The NT-derived rat zygotes (n = 748) were activated with ionomycin/cycloheximide and transferred into temporal host mothers, resulting in the harvest of three blastocysts (0.4%) with Venus fluorescence. Two blastocysts were examined for their potential to commit to NT-derived embryonic stem cells (ntESCs). One ntESC line was established successfully and found to be competent in terms of karyotype, stem cell marker expression, and pluripotency. In conclusion, time-lagged enucleation of visualized oocyte nuclei allows the PCC incidence of donor nuclei and generation of NT blastocysts, and the blastocysts can commit to germline-competent ntESCs.

Ectodomain shedding of TNF receptor 1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogura, Hirotsugu; Tsukumo, Yoshinori; Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501

2008-04-01

The transcription factor nuclear factor {kappa}B (NF-{kappa}B) plays a major role in the inducible resistance to death receptor-mediated apoptosis. It has been established that the protein synthesis inhibitor cycloheximide (CHX) sensitizes many types of cells to tumor necrosis factor (TNF)-{alpha}-induced apoptosis, mainly due to its ability to block de novo synthesis of cellular FLICE-inhibitory protein (c-FLIP). Nevertheless, we have surprisingly found that CHX, as well as its structural analogue acetoxycycloheximide (Ac-CHX), prevents TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8 in human lung carcinoma A549 cells. Both CHX and Ac-CHX reduced the expression of cell surface TNF receptor 1 (TNF-R1) in amore » dose-dependent manner, while Ac-CHX was approximately 100-fold more effective than CHX. Consistent with this observation, Ac-CHX induced the proteolytic cleavage of TNF-R1 and its release into the culture medium. CHX and Ac-CHX profoundly decreased constitutive and inducible expression of c-FLIP, whereas these compounds potentiated TNF-{alpha}-induced caspase-8 activation only when metalloprotease inhibitors were present. Thus, our results indicate that ectodomain shedding of TNF-R1 induced by protein synthesis inhibitors regulates TNF-{alpha}-mediated activation of NF-{kappa}B and caspase-8.« less

Susceptibility of pathogenic and nonpathogenic Naegleria ssp

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whiteman, L.Y.

1988-01-01

The susceptibility of four species of Naegleria amoebae to complement-mediated lysis was determined. The amoebicidal activity of normal human serum (NHS) and normal guinea pig serum (NGPS) for Naegleria amoebae was measured by an in vitro cytotoxicity assay. Release of radioactivity from amoebae labeled with {sup 3}H-uridine and visual observation with a compound microscope were used as indices of lysis. Susceptibility or resistance to complement-mediated lysis in vitro correlated with the in vivo pathogenic potential. Nonpathogenic Naegleria amoebae were lysed at a faster rate and at higher cell concentrations than were pathogenic amoebae. Electrophoretic analysis of NHS incubated with pathogenicmore » or nonpathogenic Naegleria spp. demonstrated that amoebae activate the complement cascade resulting in the production of C3 and C5 complement cleavage products. Treatment with papain or trypsin for 1 h, but not with sialidase, increase the susceptibility of highly pathogenic, mouse-passaged N. fowleri to lysis. Treatment with actinomycin D, cycloheximide or various protease inhibitors for 4 h did not increase susceptibility to lysis. Neither a repair process involving de novo protein synthesis nor a complement-inactivating protease appear to account for the increase resistance of N. fowleri amoebae to complement-mediated lysis. A binding study with {sup 125}I radiolabeled C9 indicated that the terminal complement component does not remain stably bound to the membrane of pathogenic amoebae.« less

Small molecule inhibitors of Late SV40 Factor (LSF) abrogate hepatocellular carcinoma (HCC): Evaluation using an endogenous HCC model.

PubMed

Rajasekaran, Devaraja; Siddiq, Ayesha; Willoughby, Jennifer L S; Biagi, Jessica M; Christadore, Lisa M; Yunes, Sarah A; Gredler, Rachel; Jariwala, Nidhi; Robertson, Chadia L; Akiel, Maaged A; Shen, Xue-Ning; Subler, Mark A; Windle, Jolene J; Schaus, Scott E; Fisher, Paul B; Hansen, Ulla; Sarkar, Devanand

2015-09-22

Hepatocellular carcinoma (HCC) is a lethal malignancy with high mortality and poor prognosis. Oncogenic transcription factor Late SV40 Factor (LSF) plays an important role in promoting HCC. A small molecule inhibitor of LSF, Factor Quinolinone Inhibitor 1 (FQI1), significantly inhibited human HCC xenografts in nude mice without harming normal cells. Here we evaluated the efficacy of FQI1 and another inhibitor, FQI2, in inhibiting endogenous hepatocarcinogenesis. HCC was induced in a transgenic mouse with hepatocyte-specific overexpression of c-myc (Alb/c-myc) by injecting N-nitrosodiethylamine (DEN) followed by FQI1 or FQI2 treatment after tumor development. LSF inhibitors markedly decreased tumor burden in Alb/c-myc mice with a corresponding decrease in proliferation and angiogenesis. Interestingly, in vitro treatment of human HCC cells with LSF inhibitors resulted in mitotic arrest with an accompanying increase in CyclinB1. Inhibition of CyclinB1 induction by Cycloheximide or CDK1 activity by Roscovitine significantly prevented FQI-induced mitotic arrest. A significant induction of apoptosis was also observed upon treatment with FQI. These effects of LSF inhibition, mitotic arrest and induction of apoptosis by FQI1s provide multiple avenues by which these inhibitors eliminate HCC cells. LSF inhibitors might be highly potent and effective therapeutics for HCC either alone or in combination with currently existing therapies.

Microemulsions containing long-chain oil ethyl oleate improve the oral bioavailability of piroxicam by increasing drug solubility and lymphatic transportation simultaneously.

PubMed

Xing, Qiao; Song, Jia; You, Xiuhua; Xu, Dongling; Wang, Kexin; Song, Jiaqi; Guo, Qin; Li, Pengyu; Wu, Chuanbin; Hu, Haiyan

2016-09-25

Drug solubility and lymphatic transport enhancements are two main pathways to improve drug oral bioavailability for microemulsions. However, it is not easy to have both achieved simultaneously because excipients used for improving lymphatic transport were usually insufficient in forming microemulsions and solubilizing drugs. Our research is to explore whether ethyl oleate, an oil effective in developing microemulsions with desired solubilizing capability, could increase bioavailability to a higher extent by enhancing lymphatic transport. As a long-chain oil, ethyl oleate won larger microemulsion area than short-chain tributyrin and medium-chain GTCC. In contrast, long-chain soybean oil failed to prepare microemulsions. The solubility of piroxicam in ethyl oleate microemulsions (ME-C) increased by about 30 times than in water. ME-C also won significantly higher AUC0-t compared with tributyrin microemulsions (ME-A) and GTCC microemulsions (ME-B). Oral bioavailability in ME-C decreased by 38% after lymphatic transport was blocked by cycloheximide, severer than those in ME-A and ME-B (8% and 34%). These results suggest that improving lymphatic transport and solubility simultaneously might be a novel strategy to increase drug oral bioavailability to a higher extent than increasing solubility only. Ethyl oleate is a preferred oil candidate due to its integrated advantages of high solubilizing capability, large microemulsion area and effective lymphatic transport. Copyright © 2016 Elsevier B.V. All rights reserved.

The phytoestrogen genistein enhances multidrug resistance in breast cancer cell lines by translational regulation of ABC transporters.

PubMed

Rigalli, Juan Pablo; Tocchetti, Guillermo Nicolás; Arana, Maite Rocío; Villanueva, Silvina Stella Maris; Catania, Viviana Alicia; Theile, Dirk; Ruiz, María Laura; Weiss, Johanna

2016-06-28

Breast cancer is the most frequent malignancy in women. Multidrug resistance due to overexpression of ABC drug transporters is a common cause of chemotherapy failure and disease recurrence. Genistein (GNT) is a phytoestrogen present in soybeans and hormone supplements. We investigated the effect of GNT on the expression and function of ABC transporters in MCF-7 and MDA-MB-231 breast cancer cell lines. Results demonstrated an induction at the protein level of ABCC1 and ABCG2 and of ABCC1 in MCF-7 and MDA-MB-231, respectively. MCF-7 cells showed a concomitant increase in doxorubicin and mitoxantrone efflux and resistance, dependent on ABCG2 activity. ABCC1 induction by GNT in MDA-MB-231 cells modified neither drug efflux nor chemoresistance due to simultaneous acute inhibition of the transporter activity by GNT. All inductions took place at the translational level, as no increment in mRNA was observed and protein increase was prevented by cycloheximide. miR-181a, already demonstrated to inhibit ABCG2 translation, was down-regulated by GNT, explaining translational induction. Effects were independent of classical estrogen receptors. Results suggest potential nutrient-drug interactions that could threaten chemotherapy efficacy, especially in ABCG2-expressing tumors treated with substrates of this transporter. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Heterologous expression of the yeast Tpo1p or Pdr5p membrane transporters in Arabidopsis confers plant xenobiotic tolerance.

PubMed

Remy, Estelle; Niño-González, María; Godinho, Cláudia P; Cabrito, Tânia R; Teixeira, Miguel C; Sá-Correia, Isabel; Duque, Paula

2017-07-03

Soil contamination is a major hindrance for plant growth and development. The lack of effective strategies to remove chemicals released into the environment has raised the need to increase plant resilience to soil pollutants. Here, we investigated the ability of two Saccharomyces cerevisiae plasma-membrane transporters, the Major Facilitator Superfamily (MFS) member Tpo1p and the ATP-Binding Cassette (ABC) protein Pdr5p, to confer Multiple Drug Resistance (MDR) in Arabidopsis thaliana. Transgenic plants expressing either of the yeast transporters were undistinguishable from the wild type under control conditions, but displayed tolerance when challenged with the herbicides 2,4-D and barban. Plants expressing ScTPO1 were also more resistant to the herbicides alachlor and metolachlor as well as to the fungicide mancozeb and the Co 2+ , Cu 2+ , Ni 2+ , Al 3+ and Cd 2+ cations, while ScPDR5-expressing plants exhibited tolerance to cycloheximide. Yeast mutants lacking Tpo1p or Pdr5p showed increased sensitivity to most of the agents tested in plants. Our results demonstrate that the S. cerevisiae Tpo1p and Pdr5p transporters are able to mediate resistance to a broad range of compounds of agricultural interest in yeast as well as in Arabidopsis, underscoring their potential in future biotechnological applications.

Selective effects of purine and pyrimidine analogues and of respiratory inhibitors on perithecial development and branching in sordaria.

PubMed

Lindenmayer, A; Schoen, H F

1967-08-01

The initiation of perithecia in the homothallic ascomycete Sordaria fimicola was completely suppressed, without seriously inhibiting vegetative growth, by growing the fungus on an agar medium containing one of the following additions: 1) 1 mum 5-fluorouracil, 2) 10 to 100 mum 6-azauracil, 8-azaguanine or 8-azaadenine, 3) 50 to 500 mum cyanide or azide, 4) 5% (w/v) casein hydrolysate. In contrast to the selective activity of the analogues of 3 RNA bases, whose inhibition could be reversed by the appropriate normal bases only, none of the analogues of thymine were active, neither were the thio-derivatives of RNA bases. Other inhibitors of RNA and protein synthesis, like actinomycin D, puromycin and cycloheximide, were also without selective activity, although the last of these inhibited perithecial maturation at 0.1 mum concentration but not initiation. Amino acid analogues were inactive, as were the metabolic inhibitors thiourea, 2,4-dinitrophenol and fluoride. The compounds which inhibited the formation of perithecia also lowered the branching frequency of leading hyphae, but not their linear growth rates. Consequently, the branch densities were diminished in their presence. Hypotheses to account for these findings are discussed in terms of inhibition of growth in general, of the synthesis of some specific messenger RNAs, and of RNA-mediated transport across membranes, the last of which seeming the most fruitful for further work.

Temporal and pharmacological division of fibroblast cyclooxygenase expression into transcriptional and translational phases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raz, A.; Wyche, A.; Needleman, P.

1989-03-01

The authors have recently shown that the synthesis of cyclooxygenase in human dermal fibroblasts is markedly stimulated by the cytokine interleukin 1 (IL-1). They now show that the temporal sequence of the induced synthesis of PG synthase can be separated into an early transcriptional (i.e., actinomycin D inhibitable) phase and a subsequent translational (cycloheximide but not actinomycin D inhibitable) phase and that IL-1 exerts its effect during the transcriptional phase. Phorbol 12-myristate 13-acetate also stimulates synthesis of PG synthase and, together with IL-1, produces a synergistic stimulatory effect. Inhibitors of protein kinase C activation abolished the stimulatory effect of IL-1,more » suggesting that protein kinase C activation is a critical event in the signal-transduction sequence of the IL-1-induced increase of PG synthase synthesis. The antiinflammatory glucocorticosteroids dexamethasone and triamcinolone, but not progesterone or testosterone, were potent inhibitors of PG synthase synthesis when added during the translational phase of the synthesis sequence. The glucocorticosteroid effect was blocked by RNA and protein synthesis inhibitors. This report suggests that glucocorticosteroids exert their effect via a newly synthesized protein, causing a profound translational control of PG synthase synthesis. This novel mechanism of suppression of arachidonate metabolism is distinct from any influence of steroids on phospholipase A{sub 2} activity.« less

Application of DNA Chip Scanning Technology for Automatic Detection of Chlamydia trachomatis and Chlamydia pneumoniae Inclusions

PubMed Central

Bogdanov, Anita; Endrész, Valeria; Urbán, Szabolcs; Lantos, Ildikó; Deák, Judit; Burián, Katalin; Önder, Kamil; Ayaydin, Ferhan; Balázs, Péter

2014-01-01

Chlamydiae are obligate intracellular bacteria that propagate in the inclusion, a specific niche inside the host cell. The standard method for counting chlamydiae is immunofluorescent staining and manual counting of chlamydial inclusions. High- or medium-throughput estimation of the reduction in chlamydial inclusions should be the basis of testing antichlamydial compounds and other drugs that positively or negatively influence chlamydial growth, yet low-throughput manual counting is the common approach. To overcome the time-consuming and subjective manual counting, we developed an automatic inclusion-counting system based on a commercially available DNA chip scanner. Fluorescently labeled inclusions are detected by the scanner, and the image is processed by ChlamyCount, a custom plug-in of the ImageJ software environment. ChlamyCount was able to measure the inclusion counts over a 1-log-unit dynamic range with a high correlation to the theoretical counts. ChlamyCount was capable of accurately determining the MICs of the novel antimicrobial compound PCC00213 and the already known antichlamydial antibiotics moxifloxacin and tetracycline. ChlamyCount was also able to measure the chlamydial growth-altering effect of drugs that influence host-bacterium interaction, such as gamma interferon, DEAE-dextran, and cycloheximide. ChlamyCount is an easily adaptable system for testing antichlamydial antimicrobials and other compounds that influence Chlamydia-host interactions. PMID:24189259

The rapid destabilization of p53 mRNA in immortal chicken embryo fibroblast cells.

PubMed

Kim, H; You, S; Foster, L K; Farris, J; Foster, D N

2001-08-23

The steady-state levels of p53 mRNA were dramatically lower in immortal chicken embryo fibroblast (CEF) cell lines compared to primary CEF cells. In the presence of cycloheximide (CHX), the steady-state levels of p53 mRNA markedly increased in immortal CEF cell lines, similar to levels found in primary cells. The de novo synthetic rates of p53 mRNA were relatively similar in primary and immortal cells grown in the presence or absence of CHX. Destabilization of p53 mRNA was observed in the nuclei of immortal, but not primary, CEF cells. The half-life of p53 mRNA in primary cells was found to be a relatively long 23 h compared to only 3 h in immortal cells. The expression of transfected p53 cDNA was inhibited in immortal cells, but restored upon CHX treatment. The 5'-region of the p53 mRNA was shown to be involved in the rapid p53 mRNA destabilization in immortal cells by expression analysis of 5'- and 3'-deleted p53 cDNAs as well as fusion mRNA constructs of N-terminal p53 and N-terminal deleted LacZ genes. Together, it is suggestive that the downregulation of p53 mRNA in immortal CEF cells occurs through a post-transcriptional destabilizing mechanism.

Suppressive effects of 17β-estradiol on tributyltin-induced neuronal injury via Akt activation and subsequent attenuation of oxidative stress.

PubMed

Ishihara, Yasuhiro; Fujitani, Noriko; Kawami, Tomohito; Adachi, Chika; Ishida, Atsuhiko; Yamazaki, Takeshi

2014-03-18

Neuroactive steroids are reported to protect neurons from various harmful compounds; however, the protective mechanisms remain largely unclear. In this study, we examined the suppressive effects of 17β-estradiol (E2) on tributyltin (TBT)-induced neurotoxicity. Organotypic hippocampal slices were prepared from neonatal rats and then cultured. Cell death was assayed by propidium iodide uptake. Levels of reactive oxygen species (ROS) were determined by dihydroethidium staining. Protein phosphorylation was evaluated by immunoblotting. Pretreatment of the slices with E2 dose-dependently attenuated the neuronal injury induced by TBT. An estrogen receptor antagonist, ICI182,780 abrogated these neuroprotective effects. The de novo protein synthesis inhibitors actinomycin D and cycloheximide showed no effects on the neuroprotective mechanism, indicating that a nongenomic pathway acting via the estrogen receptor may be involved in the neuroprotection conferred by E2. E2 suppressed the ROS production and lipid peroxidation induced by TBT, and these effects were almost completely canceled by ICI182,780. TBT decreased Akt phosphorylation, and this reduction was suppressed by E2. An Akt inhibitor, triciribine, attenuated the decreases in both the ROS production and neuronal injury mediated by E2. E2 enhances the phosphorylation of Akt, thereby attenuating the oxidative stress and subsequent neuronal injury induced by TBT. Copyright © 2014 Elsevier Inc. All rights reserved.

Post-transcription mediated Snail stabilization is involved in radiation exposure induced invasion and migration of hepatocarcinoma cells.

PubMed

Dong, Liyang; Zhang, Xuebang; Xiang, Wei; Ni, Junwei; Zhou, Weizhong; Li, Haiyan

2018-04-20

Increasing evidences suggested that radiotherapy can paradoxically promote tumor invasion and metastatic processes, while its detailed mechanism is not well illustrated. Our present study found that radiation can promote the migration and invasion of hepatocellular carcinoma (HCC) cells via induction of epithelial mesenchymal transition (EMT), which was evidenced by the results that radiation induced up regulation of vimentin while down regulation of E-Cadherin. As to the EMT-related transcription factors, radiation increased the expression of Snail, while not Slug, ZEB1 or TWIST. This was confirmed by the results that radiation increased the nuclear translocation of Snail in HCC cells. However, radiation had no effect on the expression or half-life of Snail mRNA. In HCC cells treated by cycloheximide (CHX, the translation inhibitor), radiation significantly increased the half-life of Snail protein, which suggested that radiation increased the expression of Snail via up regulation of its protein stability. Radiation increased the expression of COP9 signalosome 2 (CSN2), which has been reported to block the ubiquitination and degradation of Snail. Silence of CSN2/Snail can attenuate radiation induced cell migration and EMT of HCC cells. Collectively, our data suggested that radiation can promote HCC cell invasion and EMT by stabilization of Snail via CSN2 signals. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Active inhibition of herpes simplex virus type 1-induced cell fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bzik, D.J.; Person, S.; Read, G.S.

1982-01-01

Previous studies have demonstrated that syn mutant-infected cells fuse less well with nonsyncytial virus-infected cells than with uninfected cells, a phenomenon defined as function inhibition. The present study characterizes the kinetics as well as the requirements for expression of fusion inhibition. Initially, the capacity of sparse syn mutant-infected cells to fuse with uninfected surrounding cells was determined throughout infection. Of seven syn mutants examined, including representatives with alterations in two different viral genes that affect cell fusion, all showed an increase in fusion capacity up to 12 hr after infection and a decrease at later times. Fusion inhibition was examinedmore » in experiments employing sparse syn20-infected cells which had been incubated to a maximum fusion capacity; it was shown that surrounding cells infected with KOS, the parent of syn20, began to inhibit fusion by the syn20-infected cells at about 4 hr after infection, and that the maximum ability to inhibit fusion was attained at about 6 hr after infection. The metabolic blocking agents actinomycin D (RNA), cycloheximide (protein), 2-deoxyglucose, and tunicamycin (glycoslyation of glycoproteins) all showed the ability to inhibit the expression of fusion inhibition by KOS-infected cells if added shortly after infection. It is concluded that fusion inhibition is an active process that requires the synthesis of RNA, proteins, and glycoproteins. 17 references, 3 figures, 2 tables.« less

Rapid non-genomic effects of glucocorticoids on oxidative stress in a guinea pig model of asthma.

PubMed

Long, Fei; Wang, Yan; Qi, Hui-Hui; Zhou, Xin; Jin, Xian-Qiao

2008-03-01

Glucocorticoids (GC) may exert therapeutic effects in asthma by a rapid non-genomic mechanism. The lungs of asthmatic patients are exposed to oxidative stress, which is believed to be critical in the pathogenesis of asthma. The aim of this study was to investigate whether GC exert a rapid non-genomic effect on oxidative stress in asthmatic guinea pigs. The guinea pig asthma model was used to assess inhibitory effects of budesonide (BUD) on oxidative stress. BAL fluid (BALF), trolox equivalent antioxidant capacity and lung manganese superoxide dismutase (MnSOD) activity were measured by spectrophotometry. Superoxide anion production was measured by cytochrome c reduction assay. Oxidative stress occurred within minutes following antigen challenge and BUD reduced the severity of oxidative stress in asthmatic guinea pigs within 15 min. BUD significantly decreased BALF trolox equivalent antioxidant capacity and lung MnSOD activity, as compared with those of vehicle-treated asthmatic guinea pigs (P < 0.05). Additionally, BUD rapidly inhibited in vitro superoxide anion production by BALF cells and bronchi harvested from sensitized animals. These rapid effects were not blocked by the GC receptor antagonist RU486 and/or the protein synthesis inhibitor cycloheximide. BUD reduced oxidative stress in a guinea pig model of asthma by a rapid non-genomic mechanism. These data suggest new mechanisms whereby GC treatments may benefit asthma.

Dmrt1 Expression Is Regulated by Follicle-Stimulating Hormone and Phorbol Esters in Postnatal Sertoli Cells*

PubMed Central

CHEN, JIANG KAI; HECKERT, LESLIE L.

2006-01-01

Dmrt1 is a recently described gene that is expressed exclusively in the testis and is required for postnatal testis differentiation. Here we describe the expression of Dmrt1 in postnatal rat testis and Sertoli cells. RNase protection analysis was used to examine Dmrt1 messenger RNA (mRNA) levels in intact testis during postnatal development and in primary cultures of Sertoli cells under various culture conditions. We show that Dmrt1 mRNA levels rise significantly beginning approximately 10 days after birth and remain elevated until after the third postnatal week. Thereafter, mRNA levels drop coincident with the proliferation of germ cells in the testis. In freshly isolated Sertoli cells, Dmrt1 mRNA levels were robust but decreased significantly when the cells were placed in culture for 24 h. Treatment of Sertoli cells with either FSH or 8-bromo-cAMP resulted in a significant rise in Dmrt1 mRNA levels. This cAMP response was sensitive to treatment with the transcriptional inhibitor actinomycin D but not to the translational inhibitor cycloheximide. The cAMP-dependent rise in Dmrt1 mRNA also required activation of protein kinase A, as mRNA induction was sensitive to the inhibitor H89. Studies also show that Dmrt1 expression was inhibited by phorbol esters (PMA) but only modestly effected by serum. PMID:11181532

Mutualistic interaction between Salmonella enterica and Aspergillus niger and its effects on Zea mays colonization.

PubMed

Balbontín, Roberto; Vlamakis, Hera; Kolter, Roberto

2014-11-01

Salmonella Typhimurium inhabits a variety of environments and is able to infect a broad range of hosts. Throughout its life cycle, some hosts can act as intermediates in the path to the infection of others. Aspergillus niger is a ubiquitous fungus that can often be found in soil or associated to plants and microbial consortia. Recently, S. Typhimurium was shown to establish biofilms on the hyphae of A. niger. In this work, we have found that this interaction is stable for weeks without a noticeable negative effect on either organism. Indeed, bacterial growth is promoted upon the establishment of the interaction. Moreover, bacterial biofilms protect the fungus from external insults such as the effects of the anti-fungal agent cycloheximide. Thus, the Salmonella-Aspergillus interaction can be defined as mutualistic. A tripartite gnotobiotic system involving the bacterium, the fungus and a plant revealed that co-colonization has a greater negative effect on plant growth than colonization by either organism in dividually. Strikingly, co-colonization also causes a reduction in plant invasion by S. Typhimurium. This work demonstrates that S. Typhimurium and A. niger establish a mutualistic interaction that alters bacterial colonization of plants and affects plant physiology. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Cholangiopathy and tumors of the pancreas, liver, and biliary tree in boys with X-linked immunodeficiency with hyper-IgM.

PubMed

Hayward, A R; Levy, J; Facchetti, F; Notarangelo, L; Ochs, H D; Etzioni, A; Bonnefoy, J Y; Cosyns, M; Weinberg, A

1997-01-15

We report an association between X-linked immunodeficiency with hyper-IgM (XHIM) and carcinomas affecting the liver, pancreas, biliary tree, and associated neuroectodermal endocrine cells. The tumors were fatal in eight of nine cases and in most instances were preceded by chronic cholangiopathy and/or cirrhosis. An additional group of subjects with XHIM had chronic inflammation of the liver or bile ducts but no malignancy. Many patients with XHIM were infected with cryptosporidia. CD40 is normally expressed on regenerating or inflammed bile duct epithelium. A CD40+ hepatocellular carcinoma cell line, HepG2, susceptible to cryptosporidia and CMV infection became resistant when cell surface CD40 was cross-linked by a CD40 ligand fusion protein. Apoptosis was triggered in HepG2 cells if protein synthesis was blocked by cycloheximide or if the cells were infected by cryptosporidia. Ligation of CD40 on biliary epithelium may contribute to defense against infection by intracellular pathogens. We propose that the CD40 ligand mutations that cause XHIM deprive the biliary epithelium of one line of defense against intracellular pathogens and that malignant transformation in the biliary tree follows chronic infection or inflammation. The resulting tumors may then progress without check by an effective immune response. Patients with XHIM who have abnormal liver function tests should be considered at increased risk for cholangiopathy or malignancy.

Possible involvement of MAP kinase pathways in acquired metal-tolerance induced by heat in plants.

PubMed

Chen, Po-Yu; Lee, Kuo-Ting; Chi, Wen-Chang; Hirt, Heribert; Chang, Ching-Chun; Huang, Hao-Jen

2008-08-01

Cross tolerance is a phenomenon that occurs when a plant, in resisting one form of stress, develops a tolerance to another form. Pretreatment with nonlethal heat shock has been known to protect cells from metal stress. In this study, we found that the treatment of rice roots with more than 25 muM of Cu(2+) caused cell death. However, heat shock pretreatment attenuated Cu(2+)-induced cell death. The mechanisms of the cross tolerance phenomenon between heat shock and Cu(2+) stress were investigated by pretreated rice roots with the protein synthesis inhibitor cycloheximide (CHX). CHX effectively block heat shock protection, suggesting that protection of Cu(2+)-induced cell death by heat shock was dependent on de novo protein synthesis. In addition, heat pretreatment downregulated ROS production and mitogen-activated protein kinase (MAPK) activities, both of which can be greatly elicited by Cu(2+) stress in rice roots. Moreover, the addition of purified recombinant GST-OsHSP70 fusion proteins inhibited Cu(2+)-enhanced MAPK activities in an in vitro kinase assay. Furthermore, loss of heat shock protection was observed in Arabidopsis mkk2 and mpk6 but not in mpk3 mutants under Cu(2+) stress. Taken together, these results suggest that the interaction of OsHSP70 with MAPKs may contribute to the cellular protection in rice roots from excessive Cu(2+) toxicity.

Aspergillus oryzae AoSO is a novel component of stress granules upon heat stress in filamentous fungi.

PubMed

Huang, Hsiang-Ting; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

2013-01-01

Stress granules are a type of cytoplasmic messenger ribonucleoprotein (mRNP) granule formed in response to the inhibition of translation initiation, which typically occurs when cells are exposed to stress. Stress granules are conserved in eukaryotes; however, in filamentous fungi, including Aspergillus oryzae, stress granules have not yet been defined. For this reason, here we investigated the formation and localization of stress granules in A. oryzae cells exposed to various stresses using an EGFP fusion protein of AoPab1, a homolog of Saccharomyces cerevisiae Pab1p, as a stress granule marker. Localization analysis showed that AoPab1 was evenly distributed throughout the cytoplasm under normal growth conditions, and accumulated as cytoplasmic foci mainly at the hyphal tip in response to stress. AoSO, a homolog of Neurospora crassa SO, which is necessary for hyphal fusion, colocalized with stress granules in cells exposed to heat stress. The formation of cytoplasmic foci of AoSO was blocked by treatment with cycloheximide, a known inhibitor of stress granule formation. Deletion of the Aoso gene had effects on the formation and localization of stress granules in response to heat stress. Our results suggest that AoSO is a novel component of stress granules specific to filamentous fungi. The authors would specially like to thank Hiroyuki Nakano and Kei Saeki for generously providing experimental and insightful opinions.

Endothelin-1 (ET-1) stimulates carboxy terminal Smad2 phosphorylation in vascular endothelial cells by a mechanism dependent on ET receptors and de novo protein synthesis.

PubMed

Sharifat, Narges; Mohammad Zadeh, Ghorban; Ghaffari, Mohammad-Ali; Dayati, Parisa; Kamato, Danielle; Little, Peter J; Babaahmadi-Rezaei, Hossein

2017-01-01

G protein-coupled receptor (GPCR) agonists through their receptors can transactivate protein tyrosine kinase receptors such as epidermal growth factor receptor and serine/threonine kinase receptors most notably transforming growth factor (TGF)-β receptor (TβRI). This signalling mechanism represents a major expansion in the cellular outcomes attributable to GPCR signalling. This study addressed the role and mechanisms involved in GPCR agonist, endothelin-1 (ET-1)-mediated transactivation of the TβRI in bovine aortic endothelial cells (BAECs). The in-vitro model used BAECs. Signalling intermediate phospho-Smad2 in the carboxy terminal was detected and quantified by Western blotting. ET-1 treatment of BAECs resulted in a time and concentration-dependent increase in pSmad2C. Peak phosphorylation was evident with 100 nm treatment of ET-1 at 4-6 h. TβRI antagonist, SB431542 inhibited ET-1-mediated pSmad2C. In the presence of bosentan, a mixed ET A and ET B receptor antagonist ET-1-mediated pSmad2C levels were inhibited. The ET-mediated pSmad2C was blocked by the protein synthesis inhibitor, cycloheximide. In BAECs, ET-1 via the ETB receptor is involved in transactivation of the TβRI. The transactivation-dependent response is dependent upon de novo protein synthesis. © 2016 Royal Pharmaceutical Society.

Acute oral toxicity: variability, reliability, relevance and interspecies comparison of rodent LD50 data from literature surveyed for the ACuteTox project.

PubMed

Hoffmann, Sebastian; Kinsner-Ovaskainen, Agnieszka; Prieto, Pilar; Mangelsdorf, Inge; Bieler, Christian; Cole, Thomas

2010-12-01

The ACuteTox project has aimed to optimise and prevalidate an in vitro testing strategy for predicting human acute toxicity. Ninety-seven reference substances were selected and an in vivo acute toxicity database was compiled. Comprehensive statistical analyses of the in vivo LD50 data to evaluate variability and reliability, interspecies correlation, predictive capacities with regard to EU and GHS toxicity categories, and deduction of performance criteria for in vitro methods is presented. For the majority of substances variability among rodent data followed a log normal distribution where good reproducibility was found. Rat and mouse interspecies comparison of LD50 studies by ordinary regression showed high correlation, with coefficients of determination, ranging between 0.8 and 0.9. Substance specific differences were only significant for warfarin and cycloheximide. No correlation of compound LD50 range with presumed study quality rank (by assigning Klimisch reliability scores) was found. Modelling based on LD50 variability showed that with at least 90% probability ∼54% of the substances would fall into only one GHS category and ∼44% would fall within two adjacent categories. These results could form the basis for deriving a predictive capacity that should be expected from alternative approaches to the conventional in vivo acute oral toxicity test. Copyright © 2010 Elsevier Inc. All rights reserved.

Viability of ligaments after freezing: an experimental study in a rabbit model.

PubMed

Frank, C; Edwards, P; McDonald, D; Bodie, D; Sabiston, P

1988-01-01

Our purpose in this study was to assess ligament fibroblast viability after freezing by quantifying the subsequent ability of fibroblasts to synthesize collagen in vitro. Both medial collateral ligament (MCL) complexes from 40 adolescent rabbits were studied. Collagen production was determined by in vitro incubation of ligaments in 3H-proline (a collagen precursor) and subsequent analysis of 3H-hydroxyproline (a marker of newly synthesized collagen). Autoradiographs determined the distributions of ligament cell activity. All right MCL complexes served as fresh controls, providing a baseline of collagen production. Each left MCL was assigned to an experimental group and was either incubated fresh (10 animals); "killed" by drying, multiple freeze thawing, or cycloheximide (six animals); or slowly frozen at -70 degrees C without cryoprotection (24 animals). Collagen production of rapidly thawed ligaments was studied by proline incubation at 1 day, 9 days, or 6 weeks after freezing and was compared with that of contralateral fresh controls. Results demonstrate that some cells in the substance of these rabbit ligaments retained the ability to synthesize collagen in vitro after being frozen for up to 6 weeks. Mean collagen production of frozen ligaments was decreased, but tests of mean and median values as well as ratios were statistically similar to fresh contralateral ligaments in all animals. This postfreezing ligament cell survival and collagen production after -70 degrees C storage may have implications for ligament transplantation.

Soluble interleukin 2 receptors are released from activated human lymphoid cells in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rubin, L.A.; Kurman, C.C.; Fritz, M.E.

1985-11-01

With the use of an enzyme-linked immunoabsorbent assay to measure soluble human interleukin 2 receptors (IL 2R), certain human T cell leukemia virus I (HTLV I)-positive T cell lines were found to spontaneously release large quantities of IL 2R into culture supernatants. This was not found with HTLV I-negative and IL 2 independent T cell lines, and only one of seven B cell-derived lines examined produced small amounts of IL 2R. In addition to this constitutive production of soluble IL 2R by certain cell lines, normal human peripheral blood mononuclear cells (PBMC) could be induced to release soluble IL 2Rmore » by plant lectins, the murine monoclonal antibody OKT3, tetanus toxoid, and allogeneic cells. Such activated cells also expressed cellular IL 2R measurable in detergent solubilized cell extracts. The generation of cellular and supernatant IL 2R was: dependent on cellular activation, rapid, radioresistant (3000 rad), and inhibited by cycloheximide treatment. NaDodSO4-polyacrylamide gel electrophoresis analysis of soluble IL 2R demonstrated molecules of apparent Mr = 35,000 to 40,000, and 45,000 to 50,000, respectively, somewhat smaller than the mature surface receptor on these cells. The release of soluble IL 2R appears to be a characteristic marker of T lymphocyte activation and might serve an immunoregulatory function during both normal and abnormal cell growth and differentiation.« less

Mechanical Force-induced TGFB1 Increases Expression of SOST/POSTN by hPDL Cells.

PubMed

Manokawinchoke, J; Limjeerajarus, N; Limjeerajarus, C; Sastravaha, P; Everts, V; Pavasant, P

2015-07-01

The aim of this study was to investigate the response of human periodontal ligament (hPDL) fibroblasts to an intermittent compressive force and its effect on the expression of SOST, POSTN, and TGFB1. A computerized cell compressive force loading apparatus was introduced, and hPDL cells were subjected to intermittent compressive force. The changes in messenger RNA (mRNA) and protein expression were monitored by real-time polymerase chain reaction and Western blot analysis, respectively. An increased expression of SOST, POSTN, and TGFB1 was observed in a time-dependent fashion. Addition of cycloheximide, a transforming growth factor (TGF)-β inhibitor (SB431542), or a neutralizing antibody against TGF-β1 attenuated the force-induced expression of SOST and POSTN as well as sclerostin and periostin, indicating a role of TGF-β1 in the pressure-induced expression of these proteins. Enzyme-linked immunosorbent assay analysis revealed an increased level of TGF-β1 in the cell extracts but not in the medium, suggesting that intermittent compressive force promoted the accumulation of TGF-β1 in the cells or their surrounding matrix. In conclusion, an intermittent compressive force regulates SOST/POSTN expression by hPDL cells via the TGF-β1 signaling pathway. Since these proteins play important roles in the homeostasis of the periodontal tissue, our results indicate the importance of masticatory forces in this process. © International & American Associations for Dental Research 2015.

Adjuvant-induced joint inflammation causes very rapid transcription of beta-preprotachykinin and alpha-CGRP genes in innervating sensory ganglia.

PubMed

Bulling, D G; Kelly, D; Bond, S; McQueen, D S; Seckl, J R

2001-04-01

Neuropeptides synthesized in dorsal root ganglia (DRG) have been implicated in neurogenic inflammation and nociception in experimental and clinical inflammatory arthritis. We examined the very early changes in response to adjuvant injection in a rat model of unilateral tibio-tarsal joint inflammation and subsequent monoarthritis. Within 30 min of adjuvant injection ipsilateral swelling and hyperalgesia were apparent, and marked increases in beta-preprotachykinin-A (beta-PPT-A) and alpha-calcitonin gene-related peptide (CGRP)-encoding mRNAs were observed in small-diameter L5 DRG neurones innervating the affected joint. This response was augmented by recruitment of additional small-diameter DRG neurones expressing beta-PPT-A and CGRP transcripts. The increased mRNA was paralleled by initial increases in L5 DRG content of the protein products, substance P and calcitonin gene-related peptide. Within 15 min of adjuvant injection there were increases in electrical activity in sensory nerves innervating a joint. Blockade of this activity prevented the rapid induction in beta-PPT-A and CGRP mRNA expression in DRG neurones. Increased expression of heteronuclear (intron E) beta-PPT-A RNA suggests that increases in beta-PPT-A mRNA levels were, at least in part, due to transcription. Pre-treatment with the protein synthesis inhibitor cycloheximide had no effect upon the early rise in neuropeptide mRNAS: This and the rapid time course of these changes suggest that increased sensory neural discharge and activation of a latent modulator of transcription are involved.

Vincristine- and cisplatin-induced apoptosis in human retinoblastoma. Potentiation by sodium butyrate.

PubMed

Conway, R M; Madigan, M C; Billson, F A; Penfold, P L

1998-10-01

Chemotherapy alone has largely been unsuccessful in controlling retinoblastoma growth, and has traditionally been limited in use as an alternative to irradiation for the treatment of retinoblastoma. Recently, clinical studies combining chemotherapy with local therapies, including radiotherapy, laser therapy or cryotherapy and in some cases, cyclosporine A, have been effective in treating retinoblastoma. Differentiating agents may also be combined with chemotherapy to enhance the action of cytotoxic drugs on tumor cell growth, although this approach has not been fully investigated in retinoblastoma. In this study, we evaluated the cytotoxic response of human retinoblastoma cell lines (Y79 and WERI-Rb1) to two chemotherapy agents commonly used in treating retinoblastoma, vincristine (VCR) and cisplatin (CDDP). Retinoblastoma cells have been shown to be sensitive to the differentiating agent sodium butyrate, and cell lines were also treated with a combination of VCR or CDDP with sodium butyrate, and the effects on retinoblastoma viability assessed. Both VCR and CDDP induced dose-dependent death of Y79 and WERI-Rb1 cells, accompanied by nuclear and cytoplasmic condensation and DNA laddering, features characteristic of apoptosis. Inhibitors of macromolecular synthesis, cycloheximide and actinomycin-D, significantly reduced VCR- and CDDP-induced apoptosis, although putative endonuclease inhibitors zinc sulphate and aurintricarboxylic acid had no apparent effect. Treatment with 0.5 mM or 1 mM sodium butyrate combined with VCR or CDDP significantly increased induction of apoptosis by these agents. This augmentation of chemotherapy-induced apoptosis may have implications for retinoblastoma therapy.

Verocytotoxin-induced apoptosis of human microvascular endothelial cells.

PubMed

Pijpers, A H; van Setten, P A; van den Heuvel, L P; Assmann, K J; Dijkman, H B; Pennings, A H; Monnens, L A; van Hinsbergh, V W

2001-04-01

The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.

The Dynamics and Regulatory Mechanism of Pronuclear H3k9me2 Asymmetry in Mouse Zygotes

PubMed Central

Ma, Xue-Shan; Chao, Shi-Bin; Huang, Xian-Ju; Lin, Fei; Qin, Ling; Wang, Xu-Guang; Meng, Tie-Gang; Zhu, Cheng-Cheng; Schatten, Heide; Liu, Hong-Lin; Sun, Qing-Yuan

2015-01-01

H3K9 methylation is an important histone modification that is correlated with gene transcription repression. The asymmetric H3K9 dimethylation (H3K9me2) pattern between paternal and maternal genomes is generated soon after fertilization. In the present study, we carefully determined the dynamics of H3K9me2 changes in mouse zygotes, and investigated the regulatory mechanisms. The results indicated that histone methyltransferase G9a, but not GLP, was involved in the regulation of asymmetric H3K9me2, and G9a was the methyltransferase that induced the appearance of H3K9me2 in the male pronucleus of the zygote treated with cycloheximide. We found that there were two distinct mechanisms that regulate H3K9me2 in the male pronucleus. Before 8 h of in vitro fertilization (IVF), a mechanism exists that inhibits the association of G9a with the H3K9 sites. After 10 h of IVF the inhibition of G9a activity depends on yet unknown novel protein(s) synthesis. The two mechanisms of transfer take place between 8–10 h of IVF, and the novel protein failed to inhibit G9a activity in time, resulting in the appearance of a low level de novo H3K9me2 in the male pronucleus. PMID:26639638

Acylation of keratinocyte transglutaminase by palmitic and myristic acids in the membrane anchorage region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakravarty, R.; Rice, R.H.

1989-01-05

The membrane-bound form of keratinocyte transglutaminase was found to be labeled by addition of (/sup 3/H) acetic, (/sup 3/H)myristic, or (/sup 3/H)palmitic acids to the culture medium of human epidermal cells. Acid methanolysis and high performance liquid chromatography analysis of palmitate-labeled transglutaminase yielded only methyl palmitate. In contrast, analysis of the myristate-labeled protein yielded approximately 40% methyl myristate and 60% methyl palmitate. Incorporation of neither label was significantly affected by cycloheximide inhibition of protein synthesis. The importance of the fatty acid moiety for membrane anchorage was demonstrated in three ways. First, the enzyme was solubilized from the particulate fraction ofmore » cell extracts by treatment with neutral 1 M hydroxylamine, which was sufficient to release the fatty acid label. Second, solubilization of active enzyme from the particulate fraction upon mild trypsin treatment resulted in a reduction in size by approximately 10 kDa and removal of the fatty acid radiolabels. Third, the small fraction of soluble transglutaminase in cell extracts was found almost completely to lack fatty acid labeling. Keratinocyte transglutaminase translated from poly(A+) RNA in a reticulocyte cell-free system was indistinguishable in size from the native enzyme, suggesting anchorage requires only minor post-translational processing. Thus, the data are highly compatible with membrane anchorage by means of fatty acid acylation within 10 kDa of the NH/sub 2/ or COOH terminus.« less

New use for CETSA: monitoring innate immune receptor stability via post-translational modification by OGT.

PubMed

Drake, Walter R; Hou, Ching-Wen; Zachara, Natasha E; Grimes, Catherine Leimkuhler

2018-06-01

O-GlcNAcylation is a dynamic and functionally diverse post-translational modification shown to affect thousands of proteins, including the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (Nod2). Mutations of Nod2 (R702W, G908R and 1007 fs) are associated with Crohn's disease and have lower stabilities compared to wild type. Cycloheximide (CHX)-chase half-life assays have been used to show that O-GlcNAcylation increases the stability and response of both wild type and Crohn's variant Nod2, R702W. A more rapid method to assess stability afforded by post-translational modifications is necessary to fully comprehend the correlation between NLR stability and O-GlcNAcylation. Here, a recently developed cellular thermal shift assay (CETSA) that is typically used to demonstrate protein-ligand binding was adapted to detect shifts in protein stabilization upon increasing O-GlcNAcylation levels in Nod2. This assay was used as a method to predict if other Crohn's associated Nod2 variants were O-GlcNAcylated, and also identified the modification on another NLR, Nod1. Classical immunoprecipitations and NF-κB transcriptional assays were used to confirm the presence and effect of this modification on these proteins. The results presented here demonstrate that CETSA is a convenient method that can be used to detect the stability effect of O-GlcNAcylation on O-GlcNAc-transferase (OGT) client proteins and will be a powerful tool in studying post-translational modification.

Induction of heat-shock response and alterations of protein phosphorylation by a novel topoisomerase II inhibitor, withangulatin A, in 9L rat brain tumor cells.

PubMed

Lee, W C; Lin, K Y; Chen, C M; Chen, Z T; Liu, H J; Lai, Y K

1991-10-01

Withangulatin A is a newly identified in vitro topoisomerase II inhibitor isolated from the Chinese antitumor herb Physalis angulata. In vivo, it was found to be cytotoxic, capable of suppressing general protein synthesis and of inducing the synthesis of a small set of proteins including those generated by heat-shock treatment. The 70 kDa protein generated by withangulatin A was unequivocally identified as the heat-shock protein 70 (HSP70) since both proteins migrated to the same position on two-dimensional polyacrylamide gels, could be recognized by a monoclonal antibody to human HSP70, and exhibited identical peptide maps. The induction of protein synthesis by withangulatin A was regulated at the transcriptional level since it was aborted in cells pre-treated with actinomycin D. However, the initiation of this process did not require de novo protein synthesis since it was not affected by cycloheximide. Other cellular effect of withangulatin A was alterations of protein phosphorylation including an enhancement of phosphorylation of a 65 kDa protein which was also detected in the heat-shocked cells. Moreover, this process was observed within 7.5 min after the initial heat treatment which is much faster than the onset of HSP synthesis. Therefore, increased phosphorylation of the 65 kDa protein may represent one of the earliest signals generated by both heat-shock and withangluatin A and may be involved in the upstream regulation of heat-shock response in cells.

Induction of autocrine factor inhibiting cell motility from murine B16-BL6 melanoma cells by alpha-melanocyte stimulating hormone.

PubMed

Murata, J; Ayukawa, K; Ogasawara, M; Watanabe, H; Saiki, I

1999-03-15

We have previously reported that neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) successfully inhibited Matrigel invasion and haptotactic migration of B16-BL6 melanoma cells towards both fibronectin and laminin without affecting their growth. In the present study, we investigated the inhibitory mechanism of tumor cell motility by alpha-MSH. Alpha-MSH significantly blocked the autocrine motility factor (AMF)-enhanced cell motility. However, alpha-MSH did neither prevent the secretion of AMF from B16-BL6 cells nor alter the expression level of AMF receptor (gp78). On the other hand, alpha-MSH induced the secretion of the motility inhibitory factor(s) from B16-BL6 cells in a concentration- and time-dependent manner. The induction of the motility inhibitor(s) was proportional to increasing levels of intracellular cAMP induced by alpha-MSH as well as forskolin, and the activity was abolished by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA). The motility-inhibiting activity in conditioned medium (CM) from alpha-MSH-treated B16-BL6 cells was found to have a m.w. below 3 kDa after fractionation. This activity was abolished by boiling but insensitive to trypsin. The treatment of tumor cells with cycloheximide reduced the activity in alpha-MSH-stimulated CM. Our results suggest that alpha-MSH inhibited the motility of B16-BL6 cells through induction of autocrine factor(s).

Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

PubMed

Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

1995-02-10

Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

Forskolin stimulation promotes urea transporter UT-A1 ubiquitination, endocytosis, and degradation in MDCK cells

PubMed Central

Su, Hua; Carter, Conner B.; Laur, Oskar; Sands, Jeff M.

2012-01-01

The adenylyl cyclase stimulator forskolin (FSK) stimulates UT-A1 phosphorylation, membrane trafficking, and urea transport activity. Here, we found that FSK stimulation induces UT-A1 ubiquitination in UT-A1 Madin-Darby canine kidney (MDCK) cells. This suggests that phosphorylation by FSK also triggers the protein degradation machinery for UT-A1. UT-A1-MDCK cells were treated with 100 μg/ml cycloheximide to inhibit protein synthesis, with or without 10 μM FSK. Total UT-A1 protein abundance was significantly reduced after FSK treatment, concomitantly ubiquitinated UT-A1 was increased. We then specifically investigated the effect of FSK on UT-A1 expressed on the cell plasma membrane. FSK treatment accelerated UT-A1 removal from the cell plasma membrane by increasing UT-A1 endocytosis as judged by biotinylation/MesNa treatment and confocal microscopy. We further found that inhibition of the clathrin-mediated endocytic pathway, but not the caveolin-mediated endocytic pathway, significantly blocks FSK-stimulated UT-A1 endocytosis. The PKA inhibitor H89 and the proteasome inhibitors MG132 and lactacystin reduced FSK-induced membrane UT-A1 reduction. Our study shows that FSK activates the UT-A1 urea transporter and the activation/phosphorylation subsequently triggers the downregulation of UT-A1, which represents an important mechanism for the cell to return to the basal conditions after vasopressin stimulation. PMID:22914781

Estrogens Induce Rapid Cytoskeleton Re-Organization in Human Dermal Fibroblasts via the Non-Classical Receptor GPR30

PubMed Central

Carnesecchi, Julie; Malbouyres, Marilyne; de Mets, Richard; Balland, Martial; Beauchef, Gallic; Vié, Katell; Chamot, Christophe; Lionnet, Claire; Ruggiero, Florence; Vanacker, Jean-Marc

2015-01-01

The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2) addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation. PMID:25781607

Estrogens induce rapid cytoskeleton re-organization in human dermal fibroblasts via the non-classical receptor GPR30.

PubMed

Carnesecchi, Julie; Malbouyres, Marilyne; de Mets, Richard; Balland, Martial; Beauchef, Gallic; Vié, Katell; Chamot, Christophe; Lionnet, Claire; Ruggiero, Florence; Vanacker, Jean-Marc

2015-01-01

The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2) addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation.

A comparison of the intoxication pathways of tumor necrosis factor and diphtheria toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, M.P.

1988-01-01

The mechanism by which tumor necrosis factor-alpha (TNF) initiates tumor cell destruction is unknown. We have approached this problem by comparing the biological properties of TNF with diphtheria toxin (DTx), a well-characterized cytotoxin. Initial studies with human U937 cells revealed that a transient exposure to low pH enhances the cytotoxic activity of TNF. Detailed studies on the interaction of TNF with pure lipid vesicles revealed that the acid-enhanced cytolytic activity of this cytokine is correlated with the acquisition of membrane binding and insertion properties. Significantly, an increase in target membrane stabilization was observed in the presence of TNF; hence, TNFmore » is not directly lytic for membranes. In susceptible target cells, DTx induces the release of {sup 51}Cr- and {sup 75}Se-labeled proteins within 7 h. Although DTx-triggered cell death has generally been accepted as a straightforward effect of translation inhibition, little or no cell lysis was observed over a 20-30 h period when target cells were exposed to cycloheximide, amino acid deficient medium or metabolic poisons even though protein synthesis was inhibited to levels observed with DTx. The protein synthesis inhibition and cytolytic activities of DTx showed similar dose-dependencies, target cell specificities, and sensitivities to NH{sub 4}Cl inhibition. DTx-induced DNA fragmentation preceded cells lysis and did not occur in cells that were treated with the other protein synthesis inhibitors.« less

Cytological characteristics and classification of spindle inhibitors according to their effects on segmentation mitoses.

PubMed

Sentein, P; Ates, Y

1978-01-01

The effects of spindle inhibitors and of protein synthesis inhibitors on segmentation mitoses allow us to classify them into six groups : 1. Colchicine type : destruction of the whole achromatic apparatus and centrospheres without storing of dense bodies; 2. Quinoline type : same effect on the achromatic apparatus, but blocked centrospheres with accumulation of dense bodies; 3. Chloralhydrate type : Incomplete destruction of achromatic apparatus, spindle residue which maintains the chromosomes in a star shape, inactive centrospheres sequestered by the reticulum, but without accumulation of dense bodies; 4. Phenylurethane type : Incomplete and reversible action, which leads to easy production of pluripolar mitoses; 5. Carboxylic acid type : dissociation of the spindle, sometimes with blocking of the centrosphere, together with profound chromosome changes without primitive breaks; the intensity and quality of their action is related to the number of carbon atoms in the acid considered; 6. Protein synthesis inhibitor type : (cycloheximide, pederin) characterized by a stop of the nuclear cycle at telo-prophase when the action is sufficient, chromosome abnormalities, sometimes, reduced to strings of beads, and freeing of asters; at weaker concentrations mitosis is possible, but the congression of chromosomes at the equator is abnormal because of functional disturbance of the kinetochores. The nature and grading of these effects, their association (or non - association) to chromosome damage, the soundness of the spindle when only the chromosomes are affected (nitrogen mustard) make this one of the tests which gives the most specific data about the action of antimitotic substances.

Piperine inhibits ABCA1 degradation and promotes cholesterol efflux from THP‐1‐derived macrophages

PubMed Central

Wang, Limei; Palme, Veronika; Rotter, Susanne; Schilcher, Nicole; Cukaj, Malsor; Wang, Dongdong; Ladurner, Angela; Heiss, Elke H.; Stangl, Herbert; Dirsch, Verena M.

2016-01-01

1 Scope Increased macrophage cholesterol efflux (ChE) is considered to have anti‐atherosclerotic effect counteracting cardiovascular disease. The principle pungent ingredient of the fruits of Piper nigrum, piperine, is identified in this study as a ChE inducer in THP‐1‐derived macrophages, and mechanisms underlying this effect are explored. 2 Methods and results Without affecting cell viability, piperine concentration‐dependently enhances ChE in THP‐1‐derived macrophages from 25 to 100 μM. The expression level of the key cholesterol transporter protein ATP‐binding cassette transporter A1 (ABCA1) is significantly upregulated by piperine, as revealed by western blot analyses. However, two other ChE‐mediating transporter proteins, ATP‐binding cassette transporter G1 (ABCG1) and scavenger receptor class B member 1 (SR‐B1), remain unaffected. Piperine exerts no influence on ABCA1 mRNA levels, but significantly inhibits the degradation of ABCA1, as evident by an increased half‐life of the protein in the presence of cycloheximide. Furthermore, it is found that piperine likely interferes with the calpain‐mediated ABCA1 degradation pathway and exhibits significant inhibition of calpain activity. 3 Conclusion Our findings suggest that piperine promotes ChE in THP‐1‐derived macrophages by upregulation of ABCA1, which might be mediated by inhibition of calpain activity. This novel bioactivity makes the dietary constituent piperine a good candidate to be further explored for therapeutic or preventive applications in the context of atherosclerosis. PMID:27862930

Global Transcriptome Analysis of Primary Cerebrocortical Cells: Identification of Genes Regulated by Triiodothyronine in Specific Cell Types.

PubMed

Gil-Ibañez, Pilar; García-García, Francisco; Dopazo, Joaquín; Bernal, Juan; Morte, Beatriz

2017-01-01

Thyroid hormones, thyroxine, and triiodothyronine (T3) are crucial for cerebral cortex development acting through regulation of gene expression. To define the transcriptional program under T3 regulation, we have performed RNA-Seq of T3-treated and untreated primary mouse cerebrocortical cells. The expression of 1145 genes or 7.7% of expressed genes was changed upon T3 addition, of which 371 responded to T3 in the presence of cycloheximide indicating direct transcriptional regulation. The results were compared with available transcriptomic datasets of defined cellular types. In this way, we could identify targets of T3 within genes enriched in astrocytes and neurons, in specific layers including the subplate, and in specific neurons such as prepronociceptin, cholecystokinin, or cortistatin neurons. The subplate and the prepronociceptin neurons appear as potentially major targets of T3 action. T3 upregulates mostly genes related to cell membrane events, such as G-protein signaling, neurotransmission, and ion transport and downregulates genes involved in nuclear events associated with the M phase of cell cycle, such as chromosome organization and segregation. Remarkably, the transcriptomic changes induced by T3 sustain the transition from fetal to adult patterns of gene expression. The results allow defining in molecular terms the elusive role of thyroid hormones on neocortical development. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Expression of bitter taste receptors of the T2R family in the gastrointestinal tract and enteroendocrine STC-1 cells.

PubMed

Wu, S Vincent; Rozengurt, Nora; Yang, Moon; Young, Steven H; Sinnett-Smith, James; Rozengurt, Enrique

2002-02-19

Although a role for the gastric and intestinal mucosa in molecular sensing has been known for decades, the initial molecular recognition events that sense the chemical composition of the luminal contents has remained elusive. Here we identified putative taste receptor gene transcripts in the gastrointestinal tract. Our results, using reverse transcriptase-PCR, demonstrate the presence of transcripts corresponding to multiple members of the T2R family of bitter taste receptors in the antral and fundic gastric mucosa as well as in the lining of the duodenum. In addition, cDNA clones of T2R receptors were detected in a rat gastric endocrine cell cDNA library, suggesting that these receptors are expressed, at least partly, in enteroendocrine cells. Accordingly, expression of multiple T2R receptors also was found in STC-1 cells, an enteroendocrine cell line. The expression of alpha subunits of G proteins implicated in intracellular taste signal transduction, namely Galpha(gust), and Galpha(t)-(2), also was demonstrated in the gastrointestinal mucosa as well as in STC-1 cells, as revealed by reverse transcriptase-PCR and DNA sequencing, immunohistochemistry, and Western blotting. Furthermore, addition of compounds widely used in bitter taste signaling (e.g., denatonium, phenylthiocarbamide, 6-n-propil-2-thiouracil, and cycloheximide) to STC-1 cells promoted a rapid increase in intracellular Ca(2+) concentration. These results demonstrate the expression of bitter taste receptors of the T2R family in the mouse and rat gastrointestinal tract.

Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells.

PubMed

Zhang, Jing; Lauf, Peter K; Adragna, Norma C

2003-03-01

Platelet-derived growth factor (PDGF), a potent serum mitogen for vascular smooth muscle cells (VSMCs), plays an important role in membrane transport regulation and in atherosclerosis. K-Cl cotransport (K-Cl COT/KCC), the coupled-movement of K and Cl, is involved in ion homeostasis. VSMCs possess K-Cl COT activity and the KCC1 and KCC3 isoforms. Here, we report on the effect of PDGF on K-Cl COT activity and mRNA expression in primary cultures of rat VSMCs. K-Cl COT was determined as the Cl-dependent Rb influx and mRNA expression by semiquantitative RT-PCR. Twenty four-hour serum deprivation inhibited basal K-Cl COT activity. Addition of PDGF increased total protein content and K-Cl COT activity in a time-dependent manner. PDGF activated K-Cl COT in a dose-dependent manner, both acutely (10 min) and chronically (12 h). AG-1296, a selective inhibitor of the PDGF receptor tyrosine kinase, abolished these effects. Actinomycin D and cycloheximide had no effect on the acute PDGF activation of K-Cl COT, suggesting posttranslational regulation by the drug. Furthermore, PDGF increased KCC1 and decreased KCC3 mRNA expression in a time-dependent manner. These results indicate that chronic activation of K-Cl COT activity by PDGF may involve regulation of the two KCC mRNA isoforms, with KCC1 playing a dominant role in the mechanism of PDGF-mediated activation.

mTOR signaling regulates myotube hypertrophy by modulating protein synthesis, rDNA transcription, and chromatin remodeling.

PubMed

von Walden, Ferdinand; Liu, Chang; Aurigemma, Nicole; Nader, Gustavo A

2016-10-01

Ribosome production is an early event during skeletal muscle hypertrophy and precedes muscle protein accretion. Signaling via mTOR is crucial for ribosome production and hypertrophy; however, the mechanisms by which it regulates these processes remain to be identified. Herein, we investigated the activation of mTOR signaling in hypertrophying myotubes and determined that mTOR coordinates various aspects of gene expression important for ribosome production. First, inhibition of translation with cycloheximide had a more potent effect on protein synthesis than rapamycin indicating that mTOR function during hypertrophy is not on general, but rather on specific protein synthesis. Second, blocking Pol II transcription had a similar effect as Rapamycin and, unexpectedly, revealed the necessity of Pol II transcription for Pol I transcription, suggesting that mTOR may regulate ribosome production also by controlling Class II genes at the transcriptional level. Third, Pol I activity is essential for rDNA transcription and, surprisingly, for protein synthesis as selective Pol I inhibition blunted rDNA transcription, protein synthesis, and the hypertrophic response of myotubes. Finally, mTOR has nuclear localization in muscle, which is not sensitive to rapamycin. Inhibition of mTOR signaling by rapamycin disrupted mTOR-rDNA promoter interaction and resulted in altered histone marks indicative of repressed transcription and formation of higher-order chromatin structure. Thus mTOR signaling appears to regulate muscle hypertrophy by affecting protein synthesis, Class I and II gene expression, and chromatin remodeling. Copyright © 2016 the American Physiological Society.

Granulocyte macrophage-colony stimulating factor and interleukin-3 increase expression of type II tumour necrosis factor receptor, increasing susceptibility to tumour necrosis factor-induced apoptosis. Control of leukaemia cell life/death switching.

PubMed

Rae, C; MacEwan, D J

2004-12-01

Tumour necrosis factor (TNF) induces apoptosis in a range of cell types via its two receptors, TNFR1 and TNFR2. Here, we demonstrate that proliferation and TNFR2 expression was increased in human leukaemic TF-1 cells by granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), with TNFR1 expression unaffected. Consequently, they switch from a proliferative to a TNF-induced apoptotic phenotype. Raised TNFR2 expression and susceptibility to TNF-induced apoptosis was not a general effect of proliferation as IL-1beta and IFN-gamma both proliferated TF-1 cells with no effect on TNFR expression or apoptosis. Although raised TNFR2 expression correlated with the apoptotic phenotype, stimulation of apoptosis in GM-CSF-pretreated cells was mediated by TNFR1, with stimulation of TNFR2 alone insufficient to initiate cell death. However, TNFR2 did play a role in apoptotic and proliferative responses as they were blocked by the presence of an antagonistic TNFR2 antibody. Additionally, coincubation with cycloheximide blocked the mitotic effects of GM-CSF or IL-3, allowing only the apoptotic responses of TNF to persist. TNF life/death was also observed in K562, but not MOLT-4 and HL-60 human leukaemic cell types. These findings show a cooperative role of TNFR2 in the TNF life/death switching phenomenon.

Superoxide Dismutase as an Anaerobic Polypeptide 1

PubMed Central

Monk, Lorna S.; Fagerstedt, Kurt V.; Crawford, Robert M. M.

1987-01-01

The perennating organ, the rhizome, was chosen for examination of response to anoxia in the species Iris pseudacorus L., Iris germanica L. var Quechei, and Glyceria maxima (Hartm.) Holmberg. These monocots are known to differ in their tolerance of anoxia. Intact rhizomes were subjected to periods of prolonged anoxia of up to 28 days and superoxide dismutase (SOD) activity was determined in a 48 hour postanoxic recovery phase. Tests were performed to ensure the accuracy of the measured enzyme activities. In the most anoxia tolerant species, I. pseudacorus, SOD activity rose continuously during the period of imposed anoxia, and levels were maintained in the postanoxic recovery phases: 28 days brought about a 13-fold increase to 1576 U SOD per milligram protein. Small increases were found in the less anoxia tolerant I. germanica during anoxic/postanoxic phases, while a drop in activity was recorded in the least anoxia tolerant G. maxima. However, initial levels in G. maxima were more than twice as high as in the other two species. Experiments applying cycloheximide to anoxic rhizome slices of I. pseudacorus inhibited the increase in SOD activity. This indicates that SOD is, paradoxically, induced under anoxia and we suggest that in this species SOD is one of the enzymes identified as anaerobic polypeptides. The significance of the induction of an `oxygen-protecting' enzyme during complete oxygen deprivation is discussed with regard to a possible critical role during recovery from anoxic stress. PMID:16665795

Differential cellular internalization of anti-CD19 and -CD22 immunotoxins results in different cytotoxic activity.

PubMed

Du, Xing; Beers, Richard; Fitzgerald, David J; Pastan, Ira

2008-08-01

B-cell malignancies routinely express surface antigens CD19 and CD22. Immunotoxins against both antigens have been evaluated, and the immunotoxins targeting CD22 are more active. To understand this disparity in cytotoxicity and guide the screening of therapeutic targets, we compared two immunotoxins, FMC63(Fv)-PE38-targeting CD19 and RFB4(Fv)-PE38 (BL22)-targeting CD22. Six lymphoma cell lines have 4- to 9-fold more binding sites per cell for CD19 than for CD22, but BL22 is 4- to 140-fold more active than FMC63(Fv)-PE38, although they have a similar cell binding affinity (Kd, approximately 7 nmol/L). In 1 hour, large amounts of BL22 are internalized (2- to 3-fold more than the number of CD22 molecules on the cell surface), whereas only 5.2% to 16.6% of surface-bound FMC63(Fv)-PE38 is internalized. The intracellular reservoir of CD22 decreases greatly after immunotoxin internalization, indicating that it contributes to the uptake of BL22. Treatment of cells with cycloheximide does not reduce the internalization of BL22. Both internalized immunotoxins are located in the same vesicles. Our results show that the rapid internalization of large amounts of BL22 bound to CD22 makes CD22 a better therapeutic target than CD19 for immunotoxins and probably for other immunoconjugates that act inside cells.

Bitter-responsive brainstem neurons: characteristics and functions.

PubMed

Travers, Susan P; Geran, Laura C

2009-07-14

The sensation that humans describe as "bitter" is evoked by a large group of chemically diverse ligands. Bitter stimuli are avoided by a range of species and elicit reflex rejection, behaviors considered adaptations to the toxicity of many of these compounds. We review novel evidence for neurons that are narrowly tuned to bitter ligands at the initial stages of central processing. These "B-best" neurons in the nucleus of the solitary tract (NST) and parabrachial nucleus (PBN) respond to multiple types of bitter stimuli and exhibit average responses to bitter tastants that are 6-8 times larger than to moderate concentrations of compounds representing other qualities. However, in the PBN B-best units are appreciably activated by intense salt and acid. Neurons broadly sensitive to salts and acids ("AN" neurons) also responded to bitter stimuli. This sensitivity appeared restricted to stronger intensities of ionic bitters, as cycloheximide remained ineffective across concentrations. In addition to chemosensitive profile, B-best neurons were also distinctive with regard to their posterior receptive fields, long latencies, slow firing rates and projection status. Compared to B-best NST cells, those in the PBN received increased convergence from anterior and posterior receptive fields and responded to a greater number of bitter stimuli. We conclude that B-best neurons likely contribute to pathways underlying gaping, aversive hedonic quality and taste coding. The differential responsiveness of B-best and AN neurons to ionic and nonionic bitter ligands also suggests a potential substrate for discrimination within this quality.

Lower growth temperature increases alternative pathway capacity and alternative oxidase protein in tobacco.

PubMed

Vanlerberghe, G C; McIntosh, L

1992-09-01

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30 degrees C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18 degrees C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30 degrees C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18 degrees C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein.

Cytochrome and Alternative Pathway Respiration in Tobacco (Effects of Salicylic Acid).

PubMed

Rhoads, D. M.; McIntosh, L.

1993-11-01

In suspension cultures of NT1 tobacco (Nicotiana tabacum L. cv Bright Yellow) cells the cytochrome pathway capacity increased between d 3 and d 4 following subculturing and reached the highest level observed on d 7. The capacity decreased significantly by d 10 and was at the same level on d 14. Both alternative pathway capacity and the amount of the 35-kD alternative oxidase protein increased significantly between d 5 and d 6, reached the highest point observed on d 7, remained constant until d 10, and decreased by d 14. The highest capacities of the alternative and cytochrome pathways and the highest amount of the 35-kD protein were attained on the day that cell cultures reached a stationary phase of growth. Addition of salicylic acid to cell cultures on d 4 caused a significant increase in alternative pathway capacity and a dramatic accumulation of the 35-kD protein by 12 h. The alternative pathway capacity and the protein level reached the highest level observed by 16 h after salicylic acid addition, and the cytochrome pathway capacity was at about the same level at each time point. The accumulation of the 35-kD alternative oxidase protein was significantly decreased by addition of actinomycin D 1 h before salicylic acid and was blocked by addition of cycloheximide. These results indicate that de novo transcription and translation were necessary for salicylic acid to cause the maximum accumulation of the 35-kD protein.

Cytochrome and Alternative Pathway Respiration in Tobacco (Effects of Salicylic Acid).

PubMed Central

Rhoads, D. M.; McIntosh, L.

1993-01-01

In suspension cultures of NT1 tobacco (Nicotiana tabacum L. cv Bright Yellow) cells the cytochrome pathway capacity increased between d 3 and d 4 following subculturing and reached the highest level observed on d 7. The capacity decreased significantly by d 10 and was at the same level on d 14. Both alternative pathway capacity and the amount of the 35-kD alternative oxidase protein increased significantly between d 5 and d 6, reached the highest point observed on d 7, remained constant until d 10, and decreased by d 14. The highest capacities of the alternative and cytochrome pathways and the highest amount of the 35-kD protein were attained on the day that cell cultures reached a stationary phase of growth. Addition of salicylic acid to cell cultures on d 4 caused a significant increase in alternative pathway capacity and a dramatic accumulation of the 35-kD protein by 12 h. The alternative pathway capacity and the protein level reached the highest level observed by 16 h after salicylic acid addition, and the cytochrome pathway capacity was at about the same level at each time point. The accumulation of the 35-kD alternative oxidase protein was significantly decreased by addition of actinomycin D 1 h before salicylic acid and was blocked by addition of cycloheximide. These results indicate that de novo transcription and translation were necessary for salicylic acid to cause the maximum accumulation of the 35-kD protein. PMID:12231986

Cyclooxygenase-2-dependent bronchoconstriction in perfused rat lungs exposed to endotoxin.

PubMed

Uhlig, S; Nüsing, R; von Bethmann, A; Featherstone, R L; Klein, T; Brasch, F; Müller, K M; Ullrich, V; Wendel, A

1996-05-01

Lipopolysaccharides (LPS), widely used to study the mechanisms of gram-negative sepsis, increase airway resistance by constriction of terminal bronchioles. The role of the cyclooxygenase (COX) isoenzymes and their prostanoid metabolites in this process was studied. Pulmonary resistance, the release of thromboxane (TX) and the expression of COX-2 mRNA were measured in isolated blood-free perfused rat lungs exposed to LPS. LPS induced the release of TX and caused increased airway resistance after about 30 min. Both TX formation and LPS-induced bronchoconstriction were prevented by treatment with the unspecific COX inhibitor acetyl salicylic acid, the specific COX-2 inhibitor CGP-28238, dexamethasone, actinomycin D, or cycloheximide. LPS-induced bronchoconstriction was also inhibited by the TX receptor antagonist BM-13177. The TX-mimetic compound, U-46619, increased airway resistance predominantly by constricting terminal bronchioles. COX-2-specific mRNA in lung tissue was elevated after LPS exposure, and this increase was attenuated by addition of dexamethasone or of actinomycin D. In contrast to LPS, platelet-activating factor (PAF) induced immediate TX release and bronchoconstriction that was prevented by acetyl salicylic acid, but not by CGP-28238. LPS elicits the following biochemical and functional changes in rat lungs: (i) induction of COX-2; (ii) formation of prostaglandins and TX; (iii) activation of the TX receptor on airway smooth muscle cells; (iv) constriction of terminal bronchioles; and (v) increased airway resistance. In contrast to LPS, the PAF-induced TX release is likely to depend on COX-1.

Selective Effects of Purine and Pyrimidine Analogues and of Respiratory Inhibitors on Perithecial Development and Branching in Sordaria 12

PubMed Central

Lindenmayer, Aristid; Schoen, Howard F.

1967-01-01

The initiation of perithecia in the homothallic ascomycete Sordaria fimicola was completely suppressed, without seriously inhibiting vegetative growth, by growing the fungus on an agar medium containing one of the following additions: 1) 1 μm 5-fluorouracil, 2) 10 to 100 μm 6-azauracil, 8-azaguanine or 8-azaadenine, 3) 50 to 500 μm cyanide or azide, 4) 5% (w/v) casein hydrolysate. In contrast to the selective activity of the analogues of 3 RNA bases, whose inhibition could be reversed by the appropriate normal bases only, none of the analogues of thymine were active, neither were the thio-derivatives of RNA bases. Other inhibitors of RNA and protein synthesis, like actinomycin D, puromycin and cycloheximide, were also without selective activity, although the last of these inhibited perithecial maturation at 0.1 μm concentration but not initiation. Amino acid analogues were inactive, as were the metabolic inhibitors thiourea, 2,4-dinitrophenol and fluoride. The compounds which inhibited the formation of perithecia also lowered the branching frequency of leading hyphae, but not their linear growth rates. Consequently, the branch densities were diminished in their presence. Hypotheses to account for these findings are discussed in terms of inhibition of growth in general, of the synthesis of some specific messenger RNAs, and of RNA-mediated transport across membranes, the last of which seeming the most fruitful for further work. PMID:16656614

Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice

PubMed Central

De Domenico, Ivana; Zhang, Tian Y.; Koening, Curry L.; Branch, Ryan W.; London, Nyall; Lo, Eric; Daynes, Raymond A.; Kushner, James P.; Li, Dean; Ward, Diane M.; Kaplan, Jerry

2010-01-01

Hepcidin is a peptide hormone that regulates iron homeostasis and acts as an antimicrobial peptide. It is expressed and secreted by a variety of cell types in response to iron loading and inflammation. Hepcidin mediates iron homeostasis by binding to the iron exporter ferroportin, inducing its internalization and degradation via activation of the protein kinase Jak2 and the subsequent phosphorylation of ferroportin. Here we have shown that hepcidin-activated Jak2 also phosphorylates the transcription factor Stat3, resulting in a transcriptional response. Hepcidin treatment of ferroportin-expressing mouse macrophages showed changes in mRNA expression levels of a wide variety of genes. The changes in transcript levels for half of these genes were a direct effect of hepcidin, as shown by cycloheximide insensitivity, and dependent on the presence of Stat3. Hepcidin-mediated transcriptional changes modulated LPS-induced transcription in both cultured macrophages and in vivo mouse models, as demonstrated by suppression of IL-6 and TNF-α transcript and secreted protein. Hepcidin-mediated transcription in mice also suppressed toxicity and morbidity due to single doses of LPS, poly(I:C), and turpentine, which is used to model chronic inflammatory disease. Most notably, we demonstrated that hepcidin pretreatment protected mice from a lethal dose of LPS and that hepcidin-knockout mice could be rescued from LPS toxicity by injection of hepcidin. The results of our study suggest a new function for hepcidin in modulating acute inflammatory responses. PMID:20530874

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woloshuk, C.P.; Kolattukudy, P.E.

Spores of the phytopathogenic fungus Fusarium solani f. sp. pisi were shown to produce the extracellular enzyme, cutinase, only when cutin or cutin hydrolysate was added to the spore suspension. Dihydroxy-C/sub 16/ acid and trihydroxy-C/sub 18/ acid, which are unique cutin monomers, showed the greatest cutinase-inducing activity. Experiments with several compounds structurally related to these fatty acids suggested that both a omega-hydroxyl and a midchain hydroxyl are required for cutinase-inducing activity. Cutinase appeared in the medium 30-45 min after the addition of the inducers to the spore suspension, and the activity level increased for 6 hr. Addition of cycloheximide (5more » ..mu..g/ml) completely inhibited cutinase production, suggesting that protein synthesis was involved in the increase of cutinase activity. Immunoblot analysis with rabbit antibodies prepared against cutinase showed that cutinase protein increased in parallel with the increase in enzyme activity. Measurement of cutinase-specific RNA levels by dot-blot hybridization with /sup 32/P-labeled cutinase cDNA showed that the cutinase gene transcripts could be detected within 15 min after addition of the inducers. Addition of exogenous cutinase greatly enhanced the level of cutinase gene transcripts induced by cutin. These results strongly suggest that the fungal spore senses that it is in contact with the plant by the production of small amounts of cutin monomers catalyzed by the low level of cutinase carried by the spore and that these monomers induce the synthesis of cutinase needed for penetration of the fungus into the plant.« less

Methanolic leaf extract of Gymnema sylvestre augments glucose uptake and ameliorates insulin resistance by upregulating glucose transporter-4, peroxisome proliferator-activated receptor-gamma, adiponectin, and leptin levels in vitro.

PubMed

Kumar, Puttanarasaiah Mahesh; Venkataranganna, Marikunte V; Manjunath, Kirangadur; Viswanatha, Gollapalle L; Ashok, Godavarthi

2016-01-01

The present study was undertaken to evaluate the effect of methanolic leaf extract of Gymnema sylvestre (MLGS) on glucose transport (GLUT) and insulin resistance in vitro. Peroxisome proliferator-activated receptor-gamma (PPAR-γ) and GLUT-4 expression were assessed in L6 myotubes for concluding the GLUT activity, and adiponectin and leptin expression was studied in 3T3 L1 murine adipocyte cell line to determine the effect of MLGS (250-750 μg/ml) on insulin resistance. The findings of the experiments have demonstrated a significant and dose-dependent increase in glucose uptake in all the tested concentrations of MLGS, further the glucose uptake activity of MLGS (750 μg/ml) was at par with rosiglitazone (50 μg/ml). Concomitantly, MLGS has shown enhanced GLUT-4 and PPAR-γ gene expressions in L6 myotubes. Furthermore, cycloheximide (CHX) had completely abolished the glucose uptake activity of MLGS when co-incubated, which further confirmed that glucose uptake activity of MLGS was linked to enhanced expression of GLUT-4 and PPAR-γ. In addition, in another experimental set, MLGS showed enhanced expression of adiponectin and leptin, thus confirms the ameliorative effect of MLGS on insulin resistance. These findings suggest that MLGS has an enhanced glucose uptake activity in L6 myotubes, and ameliorate the insulin resistance in 3T3 L1 murine adipocyte cell line in vitro.

Nitric oxide regulation of leaf phosphoenolpyruvate carboxylase-kinase activity: implication in sorghum responses to salinity.

PubMed

Monreal, José A; Arias-Baldrich, Cirenia; Tossi, Vanesa; Feria, Ana B; Rubio-Casal, Alfredo; García-Mata, Carlos; Lamattina, Lorenzo; García-Mauriño, Sofía

2013-11-01

Nitric oxide (NO) is a signaling molecule that mediates many plant responses to biotic and abiotic stresses, including salt stress. Interestingly, salinity increases NO production selectively in mesophyll cells of sorghum leaves, where photosynthetic C₄ phosphoenolpyruvate carboxylase (C₄ PEPCase) is located. PEPCase is regulated by a phosphoenolpyruvate carboxylase-kinase (PEPCase-k), which levels are greatly enhanced by salinity in sorghum. This work investigated whether NO is involved in this effect. NO donors (SNP, SNAP), the inhibitor of NO synthesis NNA, and the NO scavenger cPTIO were used for long- and short-term treatments. Long-term treatments had multifaceted consequences on both PPCK gene expression and PEPCase-k activity, and they also decreased photosynthetic gas-exchange parameters and plant growth. Nonetheless, it could be observed that SNP increased PEPCase-k activity, resembling salinity effect. Short-term treatments with NO donors, which did not change photosynthetic gas-exchange parameters and PPCK gene expression, increased PEPCase-k activity both in illuminated leaves and in leaves kept at dark. At least in part, these effects were independent on protein synthesis. PEPCase-k activity was not decreased by short-term treatment with cycloheximide in NaCl-treated plants; on the contrary, it was decreased by cPTIO. In summary, NO donors mimicked salt effect on PEPCase-k activity, and scavenging of NO abolished it. Collectively, these results indicate that NO is involved in the complex control of PEPCase-k activity, and it may mediate some of the plant responses to salinity.

Effect of 2,4-Dichlorophenoxyacetic Acid on Glucosylation of Scopoletin to Scopolin in Tobacco Tissue Culture

PubMed Central

Hino, Fumitsugu; Okazaki, Mitsuo; Miura, Yoshiharu

1982-01-01

2,4-Dichlorophenoxyacetic acid (2,4-D) stimulated the formation of scopoletin and scopolin in tobacco (Nicotiana tabacum L. `Bright Yellow') cell culture. It especially stimulated the uptake of scopoletin from culture medium into the cells and the glucosylation of scopoletin to its monoglucoside, scopolin. This phenomenon is peculiar to 2,4-D, in contrast to other plant hormones. 2,4-D (1 μg/ml) stimulated the glucosylation of scopoletin to scopolin by enhancing UDP-glucose:scopoletin glucosyltransferase (SGTase) activity. The enhancement of SGTase activity caused by treatment with 2,4-D was observed when the syntheses of RNA and protein were inhibited by either actinomycin-D and/or cycloheximide. However, the stimulatory effect of 2,4-D was inhibited by treatment with dinitrophenol. Furthermore, SGTase with or without treatment by 2,4-D in vivo for 24 hours, was isolated from cultured tobacco cells. The enzymes were purified about 200-fold by precipitation with (NH4)2SO4 and chromatography with Sephadex G-100, DEAE-cellulose, and hydroxyapatite. The specific activity of 2,4-D-treated SGTase was 10 times higher than that of untreated SGTase even in the purified fraction, which showed one protein band under electrophoresis. These results suggest that the enhancement of SGTase activity by 2,4-D is due to the energy-dependent activation of the enzyme already present, but not due to the de novo synthesis of the enzyme. Images PMID:16662301

Stimulation of progesterone production by phorbol-12-myristate 13-acetate (PMA) in cultured Leydig tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chaudhary, L.R.; Raju, V.S.; Stocco, D.M.

1987-05-01

It has been shown that addition of hCG or c-AMP to cultured Leydig tumor cells (MA-10) increases synthesis of progesterone as the major steroid. To investigate the possible involvement of protein kinase C (PK-C) in the regulation of steroid synthesis, the authors have studied the effect of PMA, an activator of PK-C, on progesterone production in MA-10 cells. The addition of PMA (100 ng/ml) stimulated steroid production whereas 4 -phorbol-12,13-didecanoate, an inactive phorbol ester, did not have any effects. Like hCG and c-AMP, PMA-stimulated progesterone production was inhibited by cycloheximide. hCG-stimulated steroid synthesis was inhibited by PMA. The addition ofmore » PMA to MA-10 Leydig cells further increased the c-AMP-stimulated progesterone production. To determine whether c-AMP has a obligatory role in the regulation of steroid production, the effect of adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (TFA), was studied on progesterone production in the presence of hCG. At lower dose (17 ng/ml) hCG-stimulated intracellular c-AMP levels and steroid production were inhibited by TFA (300 M). At higher dose of hCG (34 ng/ml) TFA did not inhibit the hCG-stimulated intracellular c-AMP levels, however, progesterone production was inhibited. Results suggest that the action of hCG, c-AMP and PMA in controlling steroidogenesis might be regulated by similar but different mechanisms.« less

Infusions of allopregnanolone into the hippocampus and amygdala, but not into the nucleus accumbens and medial prefrontal cortex, produce antidepressant effects on the learned helplessness rats.

PubMed

Shirayama, Yukihiko; Muneoka, Katsumasa; Fukumoto, Makoto; Tadokoro, Shigenori; Fukami, Goro; Hashimoto, Kenji; Iyo, Masaomi

2011-10-01

Patients with depression showed a decrease in plasma and cerebrospinal fluid allopregnanolone (ALLO). But antidepressants increased the contents of ALLO in the rat brain. We examined the antidepressant-like effects of infusion of ALLO into the cerebral ventricle, hippocampus, amygdala, nucleus accumbens, or prefrontal cortex of learned helplessness (LH) rats (an animal model of depression). Of these regions, infusions of ALLO into the cerebral ventricle, the CA3 region of hippocampus, or the central region of amygdala exerted antidepressant-like effects. Infusion of ALLO into the hippocampal CA3 region or the central amygdala did not produce memory deficits or locomotor activation in the passive avoidance and open field tests. It is well documented that ALLO exerts its effects through GABA receptors. Therefore, we examined the antagonistic effects of flumazenil (a GABA receptor antagonist) on the antidepressant-like effects of ALLO. Coinfusion of flumazenil with ALLO into the hippocampal CA3 region, but not into the central amygdala, blocked the antidepressant-like effects of ALLO. However, coinfusion of (+)MK801 (an NMDA receptor antagonist), but not cycloheximide (a protein synthesis inhibitor), blocked the antidepressant-like effects of ALLO in the central amygdala. These results suggest that ALLO exerts antidepressant-like effects in the CA3 region of hippocampus through the GABA system and in the central region of amygdala, dependently on the activation of the glutamatergic mechanisms. Copyright © 2010 Wiley-Liss, Inc.

Interferon-γ Inhibits Ebola Virus Infection.

PubMed

Rhein, Bethany A; Powers, Linda S; Rogers, Kai; Anantpadma, Manu; Singh, Brajesh K; Sakurai, Yasuteru; Bair, Thomas; Miller-Hunt, Catherine; Sinn, Patrick; Davey, Robert A; Monick, Martha M; Maury, Wendy

2015-01-01

Ebola virus outbreaks, such as the 2014 Makona epidemic in West Africa, are episodic and deadly. Filovirus antivirals are currently not clinically available. Our findings suggest interferon gamma, an FDA-approved drug, may serve as a novel and effective prophylactic or treatment option. Using mouse-adapted Ebola virus, we found that murine interferon gamma administered 24 hours before or after infection robustly protects lethally-challenged mice and reduces morbidity and serum viral titers. Furthermore, we demonstrated that interferon gamma profoundly inhibits Ebola virus infection of macrophages, an early cellular target of infection. As early as six hours following in vitro infection, Ebola virus RNA levels in interferon gamma-treated macrophages were lower than in infected, untreated cells. Addition of the protein synthesis inhibitor, cycloheximide, to interferon gamma-treated macrophages did not further reduce viral RNA levels, suggesting that interferon gamma blocks life cycle events that require protein synthesis such as virus replication. Microarray studies with interferon gamma-treated human macrophages identified more than 160 interferon-stimulated genes. Ectopic expression of a select group of these genes inhibited Ebola virus infection. These studies provide new potential avenues for antiviral targeting as these genes that have not previously appreciated to inhibit negative strand RNA viruses and specifically Ebola virus infection. As treatment of interferon gamma robustly protects mice from lethal Ebola virus infection, we propose that interferon gamma should be further evaluated for its efficacy as a prophylactic and/or therapeutic strategy against filoviruses. Use of this FDA-approved drug could rapidly be deployed during future outbreaks.

A novel hNIS/tdTomato fusion reporter for visualizing the relationship between the cellular localization of sodium iodide symporter and its iodine uptake function under heat shock treatment.

PubMed

Yeom, Chan Joo; Chung, Taemoon; Youn, Hyewon; Kang, Keon Wook; Lee, Dong Soo; Chung, June-Key

2015-01-01

The function of membrane-localized sodium iodide symporter (NIS) determines the efficacy of radioiodine therapy in thyroid cancer. Here, we describe a dual mode reporter fused with human NIS (hNIS) and a red fluorescent protein named tandem dimeric Tomato (tdTomato) for the in vitro and in vivo imaging of hNIS protein expression, localization, and iodide uptake function. Human cervical epithelial adenocarcinoma cell line (HeLa)-hNIS/tdTomato cells were established by transducing a fusion gene expressing hNIS/tdTomato under the control of a cytomegalovirus promoter. Fluorescence imaging, confocal microscopy, and an 125I uptake assay were performed to validate the integrity of the fusion protein. Actinomycin D and cycloheximide were used to block newly synthesized hNIS proteins. In vivo images were acquired using a gamma camera and a Maestro fluorescence imaging device. The fluorescence intensity of membrane-localized hNIS and 125I uptake both were increased after heat shock. Scintigraphy and fluorescence imaging indicated specific accumulation of the hNIS/tdTomato fusion protein in xenografted tumors, supporting the utility of this system for in vivo monitoring of hNIS expression and activity. We developed a novel hNIS/tdTomato dual mode reporter that enables visualization of the expression, localization, and iodine uptake function of hNIS in vitro and in vivo.

Both coding exons of the c-myc gene contribute to its posttranscriptional regulation in the quiescent liver and regenerating liver and after protein synthesis inhibition.

PubMed Central

Lavenu, A; Pistoi, S; Pournin, S; Babinet, C; Morello, D

1995-01-01

In vivo, the steady-state level of c-myc mRNA is mainly controlled by posttranscriptional mechanisms. Using a panel of transgenic mice in which various versions of the human c-myc proto-oncogene were under the control of major histocompatibility complex H-2Kb class I regulatory sequences, we have shown that the 5' and the 3' noncoding sequences are dispensable for obtaining a regulated expression of the transgene in adult quiescent tissues, at the start of liver regeneration, and after inhibition of protein synthesis. These results indicated that the coding sequences were sufficient to ensure a regulated c-myc expression. In the present study, we have pursued this analysis with transgenes containing one or the other of the two c-myc coding exons either alone or in association with the c-myc 3' untranslated region. We demonstrate that each of the exons contains determinants which control c-myc mRNA expression. Moreover, we show that in the liver, c-myc exon 2 sequences are able to down-regulate an otherwise stable H-2K mRNA when embedded within it and to induce its transient accumulation after cycloheximide treatment and soon after liver ablation. Finally, the use of transgenes with different coding capacities has allowed us to postulate that the primary mRNA sequence itself and not c-Myc peptides is an important component of c-myc posttranscriptional regulation. PMID:7623834

Changes in Oscillatory Dynamics in the Cell Cycle of Early Xenopus laevis Embryos

PubMed Central

Tsai, Tony Y.-C.; Theriot, Julie A.; Ferrell, James E.

2014-01-01

During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min) and the subsequent 11 cycles are short (∼30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development. PMID:24523664

CD36 signaling inhibits the translation of heat shock protein 70 induced by oxidized low density lipoprotein through activation of peroxisome proliferators-activated receptor γ

PubMed Central

Lee, Kyoung-Jin; Ha, Eun-Soo; Kim, Min-Kyoung; Lee, Sang-Hoon; Suh, Jae Sung; Lee, Sun-Hee; Park, Kyeong Han; Park, Jeong Hyun; Kim, Dae Joong; Kang, Dongmin; Kim, Byung-Chul; Jeoung, Dooil; Kim, Young-Kyoun; Kim, Ho-Dirk

2008-01-01

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, induces the expression of heat shock proteins (Hsp) in a variety of cells. In this study, we investigated the role of CD36, an OxLDL receptor, and peroxisome proliferator-activated receptor γ (PPARγ) in OxLDL-induced Hsp70 expression. Overexpression of dominant-negative forms of CD36 or knockdown of CD36 by siRNA transfection increased OxLDL-induced Hsp70 protein expression in human monocytic U937 cells, suggesting that CD36 signaling inhibits Hsp70 expression. Similar results were obtained by the inhibition of PPARγ activity or knockdown of PPARγ expression. In contrast, overexpression of CD36, which is induced by treatment of MCF-7 cells with troglitazone, decreased Hsp70 protein expression induced by OxLDL. Interestingly, activation of PPARγ through a synthetic ligand, ciglitazone or troglitazone, decreased the expression levels of Hsp70 protein in OxLDL-treated U937 cells. However, major changes in Hsp70 mRNA levels were not observed. Cycloheximide studies demonstrate that troglitazone attenuates Hsp70 translation but not Hsp70 protein stability. PPARγ siRNA transfection reversed the inhibitory effects of troglitazone on Hsp70 translation. These results suggest that CD36 signaling may inhibit stress-induced gene expression by suppressing translation via activation of PPARγ in monocytes. These findings reveal a new molecular basis for the anti-inflammatory effects of PPARγ. PMID:19116451

Onychomycosis due to nondermatophytic molds.

PubMed

Hwang, Sung Min; Suh, Moo Kyu; Ha, Gyoung Yim

2012-05-01

Although there have been many studies about onychomycosis due to nondermatophytic molds (NDM), few studies about etiologic agents including NDM in onychomycosis have been reported in Korea. This study investigated onychomycosis due to NDM in the Gyeongju area of Korea. In the 10-year period from 1999~2009, we reviewed 59 patients with onychomycosis due to NDM. The etiologic agents were identified by cultures on Sabouraud's Dextrose agar with and without cycloheximide. In some cases, internal transcribed spacer sequence analysis was done. NDM isolated considered pathogens when the presence of fungal elements was identified by direct microscopy observation and in follow-up cultures yielding the same fungi. Onychomycosis due to NDM comprised 2.3% of all onychomycosis. Of the 59 patients with onychomycosis due to NDM, 84.7% were toenail onychomycosis and 15.3% were fingernail onychomycosis. The incidence rate was highest in the fifth decade (27.1%). The ratio of male to female patients was 1:1.6. The frequency of associated diseases, in descending order, was hypertension, diabetes mellitus, and cerebral hematoma. Distal and lateral subungual onychomycosis (86.4%) was the most common clinical type of onychomycosis. Aspergillus spp. was the most frequently isolated etiologic agent of onychomycosis due to NDM (83.0%). Other causative agents were Scopulariopsis brevicaulis (10.2%), Acremonium spp. (3.4%), Fusarium solani (1.7%), and Chaetomium globosum (1.7%). Because of the increase in onychomycosis due to NDM, we suggest the need of a careful mycological examination in patients with onychomycosis.

The sucrose-trehalose 6-phosphate (Tre6P) nexus: specificity and mechanisms of sucrose signalling by Tre6P.

PubMed

Yadav, Umesh Prasad; Ivakov, Alexander; Feil, Regina; Duan, Guang You; Walther, Dirk; Giavalisco, Patrick; Piques, Maria; Carillo, Petronia; Hubberten, Hans-Michael; Stitt, Mark; Lunn, John Edward

2014-03-01

Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, has a profound influence on plant metabolism, growth, and development. It has been proposed that Tre6P acts as a signal of sugar availability and is possibly specific for sucrose status. Short-term sugar-feeding experiments were carried out with carbon-starved Arabidopsis thaliana seedlings grown in axenic shaking liquid cultures. Tre6P increased when seedlings were exogenously supplied with sucrose, or with hexoses that can be metabolized to sucrose, such as glucose and fructose. Conditional correlation analysis and inhibitor experiments indicated that the hexose-induced increase in Tre6P was an indirect response dependent on conversion of the hexose sugars to sucrose. Tre6P content was affected by changes in nitrogen status, but this response was also attributable to parallel changes in sucrose. The sucrose-induced rise in Tre6P was unaffected by cordycepin but almost completely blocked by cycloheximide, indicating that de novo protein synthesis is necessary for the response. There was a strong correlation between Tre6P and sucrose even in lines that constitutively express heterologous trehalose-phosphate synthase or trehalose-phosphate phosphatase, although the Tre6P:sucrose ratio was shifted higher or lower, respectively. It is proposed that the Tre6P:sucrose ratio is a critical parameter for the plant and forms part of a homeostatic mechanism to maintain sucrose levels within a range that is appropriate for the cell type and developmental stage of the plant.

Magnolol stimulates steroidogenesis in rat adrenal cells

PubMed Central

Wang, Seu-Mei; Lee, Li-Jen; Huang, Yu-Tsung; Chen, Jian-Jiun; Chen, Yuh-Lien

2000-01-01

This study investigated the effect of magnolol, a compound purified from Magnolia officinalis, on glucocorticoid production by primary adrenal cell culture.Magnolol increased corticosterone secretion in a dose-dependent manner, this effect being maximal at 40 μM. A similar effect was seen in a minced adrenal gland system.In magnolol-treated cells, the number and total area of cytoplasmic lipid droplets were reduced, suggesting a high utilization rate of cholesterol esters stored in lipid droplets. In control cells, the capsule of the lipid droplet was clearly delineated by immunostaining with antibody A2, whereas capsular staining was discontinuous or undetectable following magnolol treatment. The percentage of decapsulated cells increased significantly from 20% in the control group to 80% in the magnolol-treated group.Magnolol-induced steroidogenesis was not mediated either via the traditional ACTH-cyclic AMP-protein kinase A pathway or by protein kinase C, since the intracellular cyclic AMP level did not change and inhibition of protein kinase A or C did not block the action of magnolol. Furthermore, calcium/calmodulin-dependent protein kinase II was not involved in magnolol-induced steroidogenesis.The stimulatory effect of magnolol on steroidogenesis apparently requires new protein synthesis, since cycloheximide inhibited magnolol-induced corticosterone production by 50%.Although other studies have shown that high concentrations of magnolol inhibit acyl-CoA: cholesterol acyltransferase and 11β-hydroxysteroid dehydrogenase in a cell-free system, our data show that, in adrenal cell cultures, low concentrations of magnolol have a stimulatory effect on steroidogenesis, and the glucocorticoid produced may explain the effective control of asthma by Magnolia officinalis. PMID:11082125

Presenilin expression during induced differentiation of the human neuroblastoma SH-SY5Y cell line.

PubMed

Flood, Fiona; Sundström, Erik; Samuelsson, Eva-Britt; Wiehager, Birgitta; Seiger, Ake; Johnston, Janet A; Cowburn, Richard F

2004-06-01

Human neuroblastoma SH-SY5Y cells stably transfected with both wild-type and exon-9 deleted (deltaE9) presenilin constructs were used to study the role of the presenilin proteins during differentiation. Cells transfected with either wild-type or deltaE9 PS1, of which the latter abolishes normal endoproteolytic cleavage of the protein, showed no obvious differences in their ability to differentiate to a neuronal-like phenotype upon treatment with retinoic acid (RA). A defined pattern of PS1 expression was observed during differentiation with both RA and the phorbol ester TPA. Full-length PS1 was shown to increase dramatically within 5-24 h of RA treatment. TPA gave an earlier and longer lasting increase in full-length PS1 levels. The intracellular distribution pattern of PS1 was markedly altered following RA treatment. Within 24h PS1 was highly up-regulated throughout the cell body around the nucleus. Between 2 and 4 weeks PS1 staining appeared punctate and also localised to the nucleus. Increases in PS1 expression upon treatment with RA and TPA were blocked by treatment with cycloheximide, indicating a role of de-novo protein synthesis in this effect. PS2 expression remained unchanged during differentiation. Levels of full-length PS1 were also seen to increase during neurogenesis and neuronal differentiation in the forebrain of first trimester human foetuses between 6.5 and 11 weeks. These combined observations support the idea that PS1 is involved in neuronal differentiation by a mechanism likely independent of endoproteolysis of the protein.

MicroRNA-142-3p and let-7g Negatively Regulates Augmented IL-6 Production in Neonatal Polymorphonuclear Leukocytes

PubMed Central

Huang, Hsin-Chun; Yu, Hong-Ren; Hsu, Te-Yao; Chen, I-Lun; Huang, Hui-Chen; Chang, Jen-Chieh; Yang, Kuender D.

2017-01-01

Neonatal PMN are qualitatively impaired in functions, yet they frequently reveal augmented inflammatory reactions during sepsis. Here, we hypothesized that PMN from newborns produce more IL-6 than those from adults under LPS stimulation, in which transcriptional or posttranscriptional regulation is involved in the altered expression. We found that neonatal PMN produced significantly higher IL-6 mRNA and protein than adult PMN. The higher IL-6 expression was not related to transcriptional but posttranscriptional regulation as the IL-6 expression was affected by the addition of cycloheximide but not actinomycin. To examine whether miRNA was involved in the IL-6 regulation of neonatal PMN, we surveyed differential displays of miRNAs that could potentially regulate IL-6 expression before and after LPS stimulation. Four miRNAs: hsa-miR-26a, hsa-miR-26b, hsa-miR-142-3p and hsa-let 7g decreased or increased after LPS treatment for 4 h. Further validation by qRT-PCR identified miR-26b, miR-142-3p and let-7g significantly changed in neonatal PMN after LPS stimulation. The functional verification by transfection of miR-142-3p and let-7g precursors into neonatal PMN significantly repressed the IL-6 mRNA and protein expression, suggesting that miR-142-3p and let-7g negatively regulate IL-6 expression in neonatal PMN. Modulation of miRNA expression may be used to regulate IL-6 production in newborns with altered inflammatory reactions. PMID:28655995

The clinical implication and molecular mechanism of preferential IL-4 production by modified glycolipid-stimulated NKT cells

PubMed Central

Oki, Shinji; Chiba, Asako; Yamamura, Takashi; Miyake, Sachiko

2004-01-01

OCH, a sphingosine-truncated analog of α-galactosylceramide (αGC), is a potential therapeutic reagent for a variety of Th1-mediated autoimmune diseases through its selective induction of Th2 cytokines from natural killer T (NKT) cells. We demonstrate here that the NKT cell production of IFN-γ is more susceptible to the sphingosine length of glycolipid ligand than that of IL-4 and that the length of the sphingosine chain determines the duration of NKT cell stimulation by CD1d-associated glycolipids. Furthermore, IFN-γ production by NKT cells requires longer T cell receptor stimulation than is required for IL-4 production by NKT cells stimulated either with immobilized mAb to CD3 or with immobilized “αGC-loaded” CD1d molecules. Interestingly, transcription of IFN-γ but not that of IL-4 was sensitive to cycloheximide treatment, indicating the intrinsic involvement of de novo protein synthesis for IFN-γ production by NKT cells. Finally, we determined c-Rel was preferentially transcribed in αGC-stimulated but not in OCH-stimulated NKT cells and was essential for IFN-γ production by activated NKT cells. Given the dominant immune regulation by the remarkable cytokine production of ligand-stimulated NKT cells in vivo, in comparison with that of (antigen-specific) T cells or NK cells, the current study confirms OCH as a likely therapeutic reagent for use against Th1-mediated autoimmune diseases and provides a novel clue for the design of drugs targeting NKT cells. PMID:15173890

Thiopental Inhibits Global Protein Synthesis by Repression of Eukaryotic Elongation Factor 2 and Protects from Hypoxic Neuronal Cell Death

PubMed Central

Schwer, Christian I.; Lehane, Cornelius; Guelzow, Timo; Zenker, Simone; Strosing, Karl M.; Spassov, Sashko; Erxleben, Anika; Heimrich, Bernd; Buerkle, Hartmut; Humar, Matjaz

2013-01-01

Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited. PMID:24167567

Block of ATP-Binding Cassette B19 Ion Channel Activity by 5-Nitro-2-(3-Phenylpropylamino)-Benzoic Acid Impairs Polar Auxin Transport and Root Gravitropism1[OPEN

PubMed Central

Cho, Misuk; Henry, Elizabeth M.; Lewis, Daniel R.; Wu, Guosheng; Muday, Gloria K.

2014-01-01

Polar transport of the hormone auxin through tissues and organs depends on membrane proteins, including some B-subgroup members of the ATP-binding cassette (ABC) transporter family. The messenger RNA level of at least one B-subgroup ABCB gene in Arabidopsis (Arabidopsis thaliana), ABCB19, increases upon treatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), possibly to compensate for an inhibitory effect of the drug on ABCB19 activity. Consistent with this hypothesis, NPPB blocked ion channel activity associated with ABCB19 expressed in human embryonic kidney cells as measured by patch-clamp electrophysiology. NPPB inhibited polar auxin transport through Arabidopsis seedling roots similarly to abcb19 mutations. NPPB also inhibited shootward auxin transport, which depends on the related ABCB4 protein. NPPB substantially decreased ABCB4 and ABCB19 protein levels when cycloheximide concomitantly inhibited new protein synthesis, indicating that blockage by NPPB enhances the degradation of ABCB transporters. Impairing the principal auxin transport streams in roots with NPPB caused aberrant patterns of auxin signaling reporters in root apices. Formation of the auxin-signaling gradient across the tips of gravity-stimulated roots, and its developmental consequence (gravitropism), were inhibited by micromolar concentrations of NPPB that did not affect growth rate. These results identify ion channel activity of ABCB19 that is blocked by NPPB, a compound that can now be considered an inhibitor of polar auxin transport with a defined molecular target. PMID:25324509

Oral delivery system prolongs blood circulation of docetaxel nanocapsules via lymphatic absorption

PubMed Central

Attili-Qadri, Suha; Karra, Nour; Nemirovski, Alina; Schwob, Ouri; Talmon, Yeshayahu; Nassar, Taher; Benita, Simon

2013-01-01

An original oral formulation of docetaxel nanocapsules (NCs) embedded in microparticles elicited in rats a higher bioavailability compared with the i.v. administration of the commercial docetaxel solution, Taxotere. In the present study, various animal studies were designed to elucidate the absorption process of docetaxel from such a delivery system. Again, the docetaxel NC formulation elicited a marked enhanced absorption compared with oral Taxotere in minipigs, resulting in relative bioavailability and Cmax values 10- and 8.4-fold higher, respectively, confirming the previous rat study results. It was revealed that orally absorbed NCs altered the elimination and distribution of docetaxel, as shown in the organ biodistribution rat study, due to their reinforced coating, while transiting through the enterocytes by surface adsorption of apoproteins and phospholipids. These findings were demonstrated by the cryogenic-temperature transmission electron microscopy results and confirmed by the use of a chylomicron flow blocker, cycloheximide, that prevented the oral absorption of docetaxel from the NC formulation in an independent pharmacokinetic study. The lipoproteinated NCs reduced the docetaxel release in plasma and its distribution among the organs. The improved anticancer activity compared with i.v. Taxotere, observed in the metastatic lung cancer model in Severe Combined Immune Deficiency-beige (SCID-bg) mice, should be attributed to the extravasation effect, leading to the lipoproteinated NC accumulation in lung tumors, where they exert a significant therapeutic action. To the best of our knowledge, no study has reported that the absorption of NCs was mediated by a lymphatic process and reinforced during their transit. PMID:24101508

The role of apoptosis in LDL transport through cultured endothelial cell monolayers.

PubMed

Cancel, Limary M; Tarbell, John M

2010-02-01

We have previously shown that leaky junctions associated with dying or dividing cells are the dominant pathway for low density lipoprotein (LDL) transport under convective conditions, accounting for more than 90% of the transport [Cancel LM, Fitting A, Tarbell JM. In vitro study of LDL transport under pressurized (convective) conditions. Am J Physiol Heart Circ Physiol 2007;293:H126-32]. To explore the role of apoptosis in the leaky junction pathway, TNFalpha and cycloheximide (TNFalpha/CHX) were used to induce an elevated rate of apoptosis in cultured bovine aortic endothelial cell (BAEC) monolayers and the convective fluxes of LDL and water were measured. Treatment with TNFalpha/CHX induced a 18.3-fold increase in apoptosis and a 4.4-fold increase in LDL permeability. Increases in apoptosis and permeability were attenuated by treatment with the caspase inhibitor Z-VAD-FMK. Water flux increased by 2.7-fold after treatment with TNFalpha/CHX, and this increase was not attenuated by treatment with Z-VAD-FMK. Immunostaining of the tight junction protein ZO-1 showed that TNFalpha/CHX treatment disrupts the tight junction in addition to inducing apoptosis. This disruption is present even when Z-VAD-FMK is used to inhibit apoptosis, and likely accounts for the increase in water flux. We found a strong correlation between the rate of apoptosis and the permeability of BAEC monolayers to LDL. These results demonstrate the potential of manipulating endothelial monolayer permeability by altering the rate of apoptosis pharmacollogicaly. This has implications for the treatment of atherosclerosis. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

Role of cytoskeleton in regulating fusion of nucleoli: a study using the activated mouse oocyte model.

PubMed

Lian, Hua-Yu; Jiao, Guang-Zhong; Wang, Hui-Li; Tan, Xiu-Wen; Wang, Tian-Yang; Zheng, Liang-Liang; Kong, Qiao-Qiao; Tan, Jing-He

2014-09-01

Although fusion of nucleoli was observed during pronuclear development of zygotes and the behavior of nucleoli in pronuclei has been suggested as an indicator of embryonic developmental potential, the mechanism for nucleolar fusion is unclear. Although both cytoskeleton and the nucleolus are important cellular entities, there are no special reports on the relationship between the two. Role of cytoskeleton in regulating fusion of nucleoli was studied using the activated mouse oocyte model. Mouse oocytes were cultured for 6 h in activating medium (Ca²⁺-free CZB medium containing 10 mM SrCl₂) supplemented with or without inhibitors for cytoskeleton or protein synthesis before pronuclear formation, nucleolar fusion, and the activity of maturation-promoting factor (MPF) were examined. Whereas treatment with microfilament inhibitor cytochalasin D or B or intermediate filament inhibitor acrylamide suppressed nucleolar fusion efficiently, treatment with microtubule inhibitor demecolcine or nocodazole or protein synthesis inhibitor cycloheximide had no effect. The cytochalasin D- or acrylamide-sensitive temporal window coincided well with the reported temporal window for nucleolar fusion in activated oocytes. Whereas a continuous incubation with demecolcine prevented pronuclear formation, pronuclei formed normally when demecolcine was excluded during the first hour of activation treatment when the MPF activity dropped dramatically. The results suggest that 1) microfilaments and intermediate filaments but not microtubules support nucleolar fusion, 2) proteins required for nucleolar fusion including microfilaments and intermediate filaments are not de novo synthesized, and 3) microtubule disruption prevents pronuclear formation by activating MPF. © 2014 by the Society for the Study of Reproduction, Inc.

Hypoxic adaptation engages the CBP/CREST-induced coactivator complex of Creb-HIF-1α in transactivating murine neuroblastic glucose transporter

PubMed Central

Thamotharan, Shanthie; Raychaudhuri, Nupur; Tomi, Masatoshi; Shin, Bo-Chul

2013-01-01

We have shown in vitro a hypoxia-induced time-dependent increase in facilitative glucose transporter isoform 3 (GLUT3) expression in N2A murine neuroblasts. This increase in GLUT3 expression is partially reliant on a transcriptional increase noted in actinomycin D and cycloheximide pretreatment experiments. Transient transfection assays in N2A neuroblasts using murine glut3-luciferase reporter constructs mapped the hypoxia-induced enhancer activities to −857- to −573-bp and −203- to −177-bp regions. Hypoxia-exposed N2A nuclear extracts demonstrated an increase in HIF-1α and p-Creb binding to HRE (−828 to −824 bp) and AP-1 (−187 to −180 bp) cis-elements, respectively, in electromobility shift and supershift assays, which was confirmed by chromatin immunoprecipitation assays. In addition, the interaction of CBP with Creb and HIF-1α and CREST with CBP in hypoxia was detected by coimmunoprecipitation. Furthermore, small interference (si)RNA targeting Creb in these cells decreased endogenous Creb concentrations that reduced by twofold hypoxia-induced glut3 gene transcription. Thus, in N2A neuroblasts, phosphorylated HIF-1α and Creb mediated the hypoxia-induced increase in glut3 transcription. Coactivation by the Ca++-dependent CREST and CBP proteins may enhance cross-talk between p-Creb-AP-1 and HIF-1α/HRE of the glut3 gene. Collectively, these processes can facilitate an adaptive response to hypoxic energy depletion targeted at enhancing glucose transport and minimizing injury while fueling the proliferative potential of neuroblasts. PMID:23321477

Exogenously induced expression of ethylene biosynthesis, ethylene perception, phospholipase D, and Rboh-oxidase genes in broccoli seedlings

PubMed Central

Jakubowicz, Małgorzata; Gałgańska, Hanna; Nowak, Witold; Sadowski, Jan

2010-01-01

In higher plants, copper ions, hydrogen peroxide, and cycloheximide have been recognized as very effective inducers of the transcriptional activity of genes encoding the enzymes of the ethylene biosynthesis pathway. In this report, the transcriptional patterns of genes encoding the 1-aminocyclopropane-1-carboxylate synthases (ACSs), 1-aminocyclopropane-1-carboxylate oxidases (ACOs), ETR1, ETR2, and ERS1 ethylene receptors, phospholipase D (PLD)-α1, -α2, -γ1, and -δ, and respiratory burst oxidase homologue (Rboh)-NADPH oxidase-D and -F in response to these inducers in Brassica oleracea etiolated seedlings are shown. ACS1, ACO1, ETR2, PLD-γ1, and RbohD represent genes whose expression was considerably affected by all of the inducers used. The investigations were performed on the seedlings with (i) ethylene insensitivity and (ii) a reduced level of the PLD-derived phosphatidic acid (PA). The general conclusion is that the expression of ACS1, -3, -4, -5, -7, and -11, ACO1, ETR1, ERS1, and ETR2, PLD-γ 1, and RbohD and F genes is undoubtedly under the reciprocal cross-talk of the ethylene and PAPLD signalling routes; both signals affect it in concerted or opposite ways depending on the gene or the type of stimuli. The results of these studies on broccoli seedlings are in agreement with the hypothesis that PA may directly affect the ethylene signal transduction pathway via an inhibitory effect on CTR1 (constitutive triple response 1) activity. PMID:20581125

Detection of dermatophytes in healthy companion dogs and cats in eastern India.

PubMed

Debnath, C; Mitra, T; Kumar, A; Samanta, I

2016-01-01

In recent times increasing occurrence of dermatophytosis, especially among the school children in eastern India was evidenced along with increased tendency of keeping companion animals such as dogs and cats. This study was undertaken to detect the occurrence of dermatophytes with antifungal susceptibility among the companion animals. A total of 1501 healthy companion animals comprising 1209 dogs and 292 cats belonged to individual owners in and around Kolkata (West Bengal, India) were examined for the evidence of dermatophytosis during 2011-2013. The collected samples were subjected to direct examination by standard KOH mount technique. The samples were inoculated into both Sabouraud dextrose agar (SDA) with 0.05% chloramphenicol and 0.5% cycloheximide and dermatophyte test medium (DTM). Each of the fungal isolate was identified based upon its colony characteristics and hyphal and conidial cells it produced. Antifungal susceptibility of the isolates was tested by broth micro dilution assay using fluconazole, ketoconazole, itraconazole, miconazole, griseofulvin and amphotericin-B antifungals. Among the 1209 samples from dogs and 292 samples from cats, 253 (20.93%) and 109 (37.33%) samples were positive for dermatophytes by direct examination. Three identified species of dermatophytes with predominant occurrence were Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes. Ketoconazole (0.06-0.5 µgm/ml), itraconazole (0.03-0.5 µgm/ml) and amphotericin-B (0.03-0.5 µgm/ml) showed lowest MIC values against M. canis, T. mentagrophytes and M. gypseum, respectively. This is the first systemic report of dermatophytes in healthy companion animals with large numbers of samples in India.

Gravity-stimulated changes in auxin and invertase gene expression in maize pulvinal cells

NASA Technical Reports Server (NTRS)

Long, Joanne C.; Zhao, Wei; Rashotte, Aaron M.; Muday, Gloria K.; Huber, Steven C.; Brown, C. S. (Principal Investigator)

2002-01-01

Maize (Zea mays) stem gravitropism involves differential elongation of cells within a highly specialized region, the stem internodal pulvinus. In the present study, we investigated factors that control gravitropic responses in this system. In the graviresponding pulvinus, hexose sugars (D-Glc and D-Fru) accumulated asymmetrically across the pulvinus. This correlated well with an asymmetric increase in acid invertase activity across the pulvinus. Northern analyses revealed asymmetric induction of one maize acid invertase gene, Ivr2, consistent with transcriptional regulation by gravistimulation. Several lines of evidence indicated that auxin redistribution, as a result of polar auxin transport, is necessary for gravity-stimulated Ivr2 transcript accumulation and differential cell elongation across the maize pulvinus. First, the auxin transport inhibitor, N-1-naphthylphthalamic acid, inhibited gravistimulated curvature and Ivr2 transcript accumulation. Second, a transient gradient of free indole-3-acetic acid (IAA) across the pulvinus was apparent shortly after initiation of gravistimulation. This temporarily free IAA gradient appears to be important for differential cell elongation and Ivr2 transcript accumulation. This is based on the observation that N-1-naphthylphthalamic acid will not inhibit gravitropic responses when applied to pulvinus tissue after the free IAA gradient peak has occurred. Third, IAA alone can stimulate Ivr2 transcript accumulation in non-gravistimulated pulvini. The gravity- and IAA-stimulated increase in Ivr2 transcripts was sensitive to the protein synthesis inhibitor, cycloheximide. Based on these results, a two-phase model describing possible relationships between gravitropic curvature, IAA redistribution, and Ivr2 expression is presented.

Mechanisms involved in p53 downregulation by leptin in trophoblastic cells.

PubMed

Toro, Ayelén Rayen; Pérez-Pérez, Antonio; Corrales Gutiérrez, Isabel; Sánchez-Margalet, Víctor; Varone, Cecilia Laura

2015-11-01

Leptin, a 16-kDa polypeptide hormone, is produced by the adipocyte and can also be synthesized by placenta. We previously demonstrated that leptin promotes proliferation and survival in placenta, in part mediated by the p53 pathway. In this work, we investigated the mechanisms involved in leptin down-regulation of p53 level. The human first trimester cytotrophoblastic Swan-71 cell line and human placental explants at term were used. In order to study the late phase of apoptosis, triggered by serum deprivation, experiments of DNA fragmentation were carried out. Exogenous leptin added to human placental explants, showed a decrease on DNA ladder formation and MAPK pathway is involved in this leptin effect. We also found that under serum deprivation condition, leptin decreases p53 levels and the inhibitory leptin effect is lost when cells were pretreated with 50 μM PD98059 or 10 μM LY29004; or were transfected with dominant negative mutants of intermediates of these pathways, suggesting that MAPK and PI3K signaling pathways are necessaries for leptin action. Additionally, leptin diminished Ser-46 p53 phosphorylation and this effect in placental explants was mediated by the activation of MAPK and PI3K pathways. Finally, in order to assess leptin effect on p53 half-life experiments with cycloheximide were performed and MDM-2 expression was analyzed. Leptin diminished p53 half-life and up-regulated MDM-2 expression. In summary, we provided evidence suggesting that leptin anti-apoptotic effect is mediated by MAPK and PI3K pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

Combination of erlotinib and EGCG induces apoptosis of head and neck cancers through posttranscriptional regulation of Bim and Bcl-2.

PubMed

Haque, Abedul; Rahman, Mohammad Aminur; Chen, Zhuo Georgia; Saba, Nabil F; Khuri, Fadlo R; Shin, Dong M; Ruhul Amin, A R M

2015-07-01

Combinatorial approaches using two or more compounds are gaining increasing attention for cancer therapy. We have previously reported that the combination of the EGFR-TKI erlotinib and epigallocatechin-3-gallate (EGCG) exhibited synergistic chemopreventive effects in head and neck cancers by inducing the expression of Bim, p21, p27, and by inhibiting the phosphorylation of ERK and AKT and expression of Bcl-2. In the current study, we further investigated the mechanism of regulation of Bim, Bcl-2, p21 and p27, and their role in apoptosis. shRNA-mediated silencing of Bim significantly inhibited apoptosis induced by the combination of erlotinib and EGCG (p = 0.005). On the other hand, overexpression of Bcl-2 markedly protected cells from apoptosis (p = 0.003), whereas overexpression of constitutively active AKT only minimally protected cells from apoptosis induced by the combination of the two compounds. Analysis of mRNA expression by RT-PCR revealed that erlotinib, EGCG and their combination had no significant effects on the mRNA expression of Bim, p21, p27 or Bcl-2 suggesting the post-transcriptional regulation of these molecules. Furthermore, we found that erlotinib or the combination of EGCG and erlotinib inhibited the phosphorylation of Bim and stabilized Bim after inhibition of protein translation by cycloheximide. Taken together, our results strongly suggest that the combination of erlotinib and EGCG induces apoptosis of SCCHN cells by regulating Bim and Bcl-2 at the posttranscriptional level.

Iridovirus CARD Protein Inhibits Apoptosis through Intrinsic and Extrinsic Pathways

PubMed Central

Chen, Chien-Wen; Wu, Ming-Shan; Huang, Yi-Jen; Lin, Pei-Wen; Shih, Chueh-Ju; Lin, Fu-Pang; Chang, Chi-Yao

2015-01-01

Grouper iridovirus (GIV) belongs to the genus Ranavirus of the family Iridoviridae; the genomes of such viruses contain an anti-apoptotic caspase recruitment domain (CARD) gene. The GIV-CARD gene encodes a protein of 91 amino acids with a molecular mass of 10,505 Daltons, and shows high similarity to other viral CARD genes and human ICEBERG. In this study, we used Northern blot to demonstrate that GIV-CARD transcription begins at 4 h post-infection; furthermore, we report that its transcription is completely inhibited by cycloheximide but not by aphidicolin, indicating that GIV-CARD is an early gene. GIV-CARD-EGFP and GIV-CARD-FLAG recombinant proteins were observed to translocate from the cytoplasm into the nucleus, but no obvious nuclear localization sequence was observed within GIV-CARD. RNA interference-mediated knockdown of GIV-CARD in GK cells infected with GIV inhibited expression of GIV-CARD and five other viral genes during the early stages of infection, and also reduced GIV infection ability. Immunostaining was performed to show that apoptosis was effectively inhibited in cells expressing GIV-CARD. HeLa cells irradiated with UV or treated with anti-Fas antibody will undergo apoptosis through the intrinsic and extrinsic pathways, respectively. However, over-expression of recombinant GIV-CARD protein in HeLa cells inhibited apoptosis induced by mitochondrial and death receptor signaling. Finally, we report that expression of GIV-CARD in HeLa cells significantly reduced the activities of caspase-8 and -9 following apoptosis triggered by anti-Fas antibody. Taken together, these results demonstrate that GIV-CARD inhibits apoptosis through both intrinsic and extrinsic pathways. PMID:26047333

Phytotoxic effects of trichothecenes on the growth and morphology of Arabidopsis thaliana.

PubMed

Masuda, Daisuke; Ishida, Mamoru; Yamaguchi, Kazuo; Yamaguchi, Isamu; Kimura, Makoto; Nishiuchi, Takumi

2007-01-01

Non-volatile sesquiterpenoids, a trichothecene family of phytotoxins such as deoxynivalenol (DON) and T-2 toxin, contain numerous molecular species and are synthesized by phytopathogenic Fusarium species. Although trichothecene chemotypes might play a role in the virulence of individual Fusarium strains, the phytotoxic action of individual trichothecenes has not been systematically studied. To perform a comparative analysis of the phytotoxic action of representative trichothecenes, the growth and morphology of Arabidopsis thaliana growing on media containing these compounds was investigated. Both DON and diacetoxyscirpenol (DAS) preferentially inhibited root elongation. DON-treated roots were less organized compared with control roots. Moreover, preferential inhibition of root growth by DON was also observed in wheat plants. In addition, T-2 toxin-treated seedlings exhibited dwarfism with aberrant morphological changes (e.g. petiole shortening, curled dark-green leaves, and reduced cell size). These results imply that the phytotoxic action of trichothecenes differed among their molecular species. Cycloheximide (CHX)-treated seedlings displayed neither feature, although it is known that trichothecenes inhibit translation in eukaryotic ribosomes. Microarray analyses suggested that T-2 toxin caused a defence response, the inactivation of brassinosteroid (BR), and the generation of reactive oxygen species in Arabidopsis. This observation is in agreement with our previous reports in which trichothecenes such as T-2 toxin have an elicitor-like activity when infiltrated into the leaves of Arabidopsis. Since it has been reported that BR plays an important role in a broad range of disease resistance in tobacco and rice, inactivation of BR might affect pathogenicity during the infection of host plants by trichothecene-producing fungi.

Fungi, bacteria and soil pH: the oxalate-carbonate pathway as a model for metabolic interaction.

PubMed

Martin, Gaëtan; Guggiari, Matteo; Bravo, Daniel; Zopfi, Jakob; Cailleau, Guillaume; Aragno, Michel; Job, Daniel; Verrecchia, Eric; Junier, Pilar

2012-11-01

The oxalate-carbonate pathway involves the oxidation of calcium oxalate to low-magnesium calcite and represents a potential long-term terrestrial sink for atmospheric CO(2). In this pathway, bacterial oxalate degradation is associated with a strong local alkalinization and subsequent carbonate precipitation. In order to test whether this process occurs in soil, the role of bacteria, fungi and calcium oxalate amendments was studied using microcosms. In a model system with sterile soil amended with laboratory cultures of oxalotrophic bacteria and fungi, the addition of calcium oxalate induced a distinct pH shift and led to the final precipitation of calcite. However, the simultaneous presence of bacteria and fungi was essential to drive this pH shift. Growth of both oxalotrophic bacteria and fungi was confirmed by qPCR on the frc (oxalotrophic bacteria) and 16S rRNA genes, and the quantification of ergosterol (active fungal biomass) respectively. The experiment was replicated in microcosms with non-sterilized soil. In this case, the bacterial and fungal contribution to oxalate degradation was evaluated by treatments with specific biocides (cycloheximide and bronopol). Results showed that the autochthonous microflora oxidized calcium oxalate and induced a significant soil alkalinization. Moreover, data confirmed the results from the model soil showing that bacteria are essentially responsible for the pH shift, but require the presence of fungi for their oxalotrophic activity. The combined results highlight that the interaction between bacteria and fungi is essential to drive metabolic processes in complex environments such as soil. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Pollen selection under acid rain stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Y.

To investigate whether acid rain stress induces pollen selection in nature, three different approaches were used, based on the assumption that the response of pollen grains to acid rain is controlled by an acid sensitive gene product. Germination of pollen from homozygous and heterozygous individuals under acid rain stress was examined to detect any differences in rate of germination between populations of homogeneous and heterogeneous pollen grains. In vitro and in vivo bulked segregant analysis using RAPDs was used to search for differences in DNA constitution between the survivors of acid rain stressed and non-acid rain stressed pollen populations inmore » vitro and between the progenies of acid rain stressed and non-acid rain stressed populations during pollination, respectively. No evidence for the pollen selection under acid rain stress was obtained in any of the test systems. Inhibition of protein synthesis using cycloheximide led to significant reduction of tube elongation at 4 hr and had no effect on pollen germination at any time interval tested. Total proteins extracted from control and acid rain stressed pollen grain populations exhibited no differences. The reduction of corn pollen germination in vitro under acid rain stress was mainly due to pollen rupture. The present data indicates the reduction of pollen germination and tube growth under acid rain stress may be a physiological response rather than a genetic response. A simple, nontoxic, and effective method to separate germinated from ungerminated pollen grains has been developed using pollen from corn (Zea mays, L. cv. Pioneer 3747). The separated germinated pollen grains retained viability and continued tube growth when placed in culture medium.« less

Rab11-dependent Recycling of the Human Ether-a-go-go-related Gene (hERG) Channel*

PubMed Central

Chen, Jeffery; Guo, Jun; Yang, Tonghua; Li, Wentao; Lamothe, Shawn M.; Kang, Yudi; Szendrey, John A.; Zhang, Shetuan

2015-01-01

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr). A reduction in the hERG current causes long QT syndrome, which predisposes affected individuals to ventricular arrhythmias and sudden death. We reported previously that hERG channels in the plasma membrane undergo vigorous internalization under low K+ conditions. In the present study, we addressed whether hERG internalization occurs under normal K+ conditions and whether/how internalized channels are recycled back to the plasma membrane. Using patch clamp, Western blot, and confocal imaging analyses, we demonstrated that internalized hERG channels can effectively recycle back to the plasma membrane. Low K+-enhanced hERG internalization is accompanied by an increased rate of hERG recovery in the plasma membrane upon reculture following proteinase K-mediated clearance of cell-surface proteins. The increased recovery rate is not due to enhanced protein synthesis, as hERG mRNA expression was not altered by low K+ exposure, and the increased recovery was observed in the presence of the protein biosynthesis inhibitor cycloheximide. GTPase Rab11, but not Rab4, is involved in the recycling of hERG channels. Interfering with Rab11 function not only delayed hERG recovery in cells after exposure to low K+ medium but also decreased hERG expression and function in cells under normal culture conditions. We concluded that the recycling pathway plays an important role in the homeostasis of plasma membrane-bound hERG channels. PMID:26152716

Triiodothyronine (T3) induces HIF1A and TGFA expression in MCF7 cells by activating PI3K.

PubMed

Moretto, Fernanda Cristina Fontes; De Sibio, Maria Teresa; Luvizon, Aline Carbonera; Olimpio, Regiane Marques Castro; de Oliveira, Miriane; Alves, Carlos Augusto Barnabe; Conde, Sandro José; Nogueira, Célia Regina

2016-06-01

High expression levels of hypoxia inducing factor 1 alpha are related to mammary carcinogenesis. In previous studies, we demonstrated that expression of transforming growth factor alpha increases upon treatment with triiodothyronine, but this expression does not occur in cellular models that do not express the estrogen receptor, or when cells are co-treated with the anti-estrogen, tamoxifen. The aim of this study was to determine the effect of the hormone triiodothyronine on the expression of the genes HIF1A and TGFA in the breast cancer cell line MCF7. The cell line was subjected to treatment with triiodothyronine at the supraphysiological dose of 10(-8)M for 10min, 30min, 1h, and 4h in the presence or absence of actinomycin D, the gene expression inhibitor, cycloheximide, the protein synthesis inhibitor, and LY294002, the phosphoinositide 3 kinase inhibitor. HIF1A and TGFA mRNA expression was analyzed by reverse transcription polymerase chain reaction. For data analysis, we used analysis of variance complemented by Tukey test and an adopted minimum of 5% significance. We found that HIF1A and TGFA expression increased in the presence of triiodothyronine at all times studied. HIF1A expression decreased in triiodothyronine-treated cells when gene transcription was also inhibited; however, TGFA expression decreased after 10 and 30min of treatment even when transcription was not inhibited. We found that activation of PI3K was necessary for triiodothyronine to modulate HIF1A and TGFA expression. Copyright © 2016 Elsevier Inc. All rights reserved.

Biomechanical, biochemical, and morphological mechanisms of heat shock-mediated germination in Carica papaya seed

PubMed Central

Webster, Rachel E.; Waterworth, Wanda M.; Stuppy, Wolfgang; West, Christopher E.; Ennos, Roland; Bray, Clifford M.; Pritchard, Hugh W.

2016-01-01

Carica papaya (papaya) seed germinate readily fresh from the fruit, but desiccation induces a dormant state. Dormancy can be released by exposure of the hydrated seed to a pulse of elevated temperature, typical of that encountered in its tropical habitat. Carica papaya is one of only a few species known to germinate in response to heat shock (HS) and we know little of the mechanisms that control germination in tropical ecosystems. Here we investigate the mechanisms that mediate HS-induced stimulation of germination in pre-dried and re-imbibed papaya seed. Exogenous gibberellic acid (GA3 ≥250 µM) overcame the requirement for HS to initiate germination. However, HS did not sensitise seeds to GA3, indicative that it may act independently of GA biosynthesis. Seed coat removal also overcame desiccation-imposed dormancy, indicative that resistance to radicle emergence is coat-imposed. Morphological and biomechanical studies identified that neither desiccation nor HS alter the physical structure or the mechanical strength of the seed coat. However, cycloheximide prevented both seed coat weakening and germination, implicating a requirement for de novo protein synthesis in both processes. The germination antagonist abscisic acid prevented radicle emergence but had no effect on papaya seed coat weakening. Desiccation therefore appears to reduce embryo growth potential, which is reversed by HS, without physically altering the mechanical properties of the seed coat. The ability to germinate in response to a HS may confer a competitive advantage to C. papaya, an opportunistic pioneer species, through detection of canopy removal in tropical forests. PMID:27811004

Evidence for a relief of repression mechanism for activation of the human telomerase reverse transcriptase promoter.

PubMed

Wang, Shuwen; Zhu, Jiyue

2003-05-23

The transcriptional activation of human telomerase reverse transcriptase (hTERT) is an important step during cellular immortalization and tumorigenesis. To study how this activation occurs during immortalization, we have established a set of genetically related pre-crisis cells and their immortal progeny. As expected, hTERT mRNA was detected in our telomerase-positive immortal cells but not in pre-crisis cells or telomerase-negative immortal cells. However, transiently transfected luciferase reporters controlled by hTERT promoter sequences exhibited similar levels of luciferase activity in both telomerase-positive and -negative cells, suggesting that the endogenous chromatin context is likely required for hTERT regulation. Analysis of chromatin susceptibility to DNase I digestion consistently identified a DNase I hypersensitivity site (DHS) near the hTERT transcription initiation site in telomerase-positive cells. In addition, the histone deacetylase inhibitor trichostatin A (TSA) induced hTERT transcription and also a general increase in chromatin sensitivity to DNase treatment in telomerase-negative cells. The TSA-induced hTERT transcription in pre-crisis cells was accompanied by the formation of a DHS at the hTERT promoter. Furthermore, the TSA-induced hTERT transcription and chromatin alterations were not blocked by cycloheximide, suggesting that this induction does not require de novo protein synthesis and that TSA induces hTERT expression through the inhibition of histone deacetylation at the hTERT promoter. Taken together, our results suggest that the endogenous chromatin environment plays a critical role in the regulation of hTERT expression during cellular immortalization.

Karanjin from Pongamia pinnata induces GLUT4 translocation in skeletal muscle cells in a phosphatidylinositol-3-kinase-independent manner.

PubMed

Jaiswal, Natasha; Yadav, Prem P; Maurya, Rakesh; Srivastava, Arvind K; Tamrakar, Akhilesh K

2011-11-16

Insulin-stimulated glucose uptake in skeletal muscle is decreased in type 2 diabetes due to impaired translocation of insulin-sensitive glucose transporter 4 (GLUT4) from intracellular pool to plasma membrane. Augmenting glucose uptake into this tissue may help in management of type 2 diabetes. Here, the effects of an identified antihyperglycemic molecule, karanjin, isolated from the fruits of Pongamia pinnata were investigated on glucose uptake and GLUT4 translocation in skeletal muscle cells. Treatment of L6-GLUT4myc myotubes with karanjin caused a substantial increase in the glucose uptake and GLUT4 translocation to the cell surface, in a concentration-dependent fashion, without changing the total amount of GLUT4 protein and GLUT4 mRNA. This effect was associated with increased activity of AMP-activated protein kinase (AMPK). Cycloheximide treatment inhibited the effect of karanjin on GLUT4 translocation suggesting the requirement of de novo synthesis of protein. Karanjin-induced GLUT4 translocation was further enhanced with insulin and the effect is completely protected in the presence of wortmannin. Moreover, karanjin did not affect the phosphorylation of AKT (Ser-473) and did not alter the expression of the key molecules of insulin signaling cascade. We conclude that karanjin-induced increase in glucose uptake in L6 myotubes is the result of an increased translocation of GLUT4 to plasma membrane associated with activation of AMPK pathway, in a PI-3-K/AKT-independent manner. Copyright © 2011 Elsevier B.V. All rights reserved.

Block of ATP-binding cassette B19 ion channel activity by 5-nitro-2-(3-phenylpropylamino)-benzoic acid impairs polar auxin transport and root gravitropism.

PubMed

Cho, Misuk; Henry, Elizabeth M; Lewis, Daniel R; Wu, Guosheng; Muday, Gloria K; Spalding, Edgar P

2014-12-01

Polar transport of the hormone auxin through tissues and organs depends on membrane proteins, including some B-subgroup members of the ATP-binding cassette (ABC) transporter family. The messenger RNA level of at least one B-subgroup ABCB gene in Arabidopsis (Arabidopsis thaliana), ABCB19, increases upon treatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), possibly to compensate for an inhibitory effect of the drug on ABCB19 activity. Consistent with this hypothesis, NPPB blocked ion channel activity associated with ABCB19 expressed in human embryonic kidney cells as measured by patch-clamp electrophysiology. NPPB inhibited polar auxin transport through Arabidopsis seedling roots similarly to abcb19 mutations. NPPB also inhibited shootward auxin transport, which depends on the related ABCB4 protein. NPPB substantially decreased ABCB4 and ABCB19 protein levels when cycloheximide concomitantly inhibited new protein synthesis, indicating that blockage by NPPB enhances the degradation of ABCB transporters. Impairing the principal auxin transport streams in roots with NPPB caused aberrant patterns of auxin signaling reporters in root apices. Formation of the auxin-signaling gradient across the tips of gravity-stimulated roots, and its developmental consequence (gravitropism), were inhibited by micromolar concentrations of NPPB that did not affect growth rate. These results identify ion channel activity of ABCB19 that is blocked by NPPB, a compound that can now be considered an inhibitor of polar auxin transport with a defined molecular target. © 2014 American Society of Plant Biologists. All Rights Reserved.

Norepinephrine is coreleased with serotonin in mouse taste buds.

PubMed

Huang, Yijen A; Maruyama, Yutaka; Roper, Stephen D

2008-12-03

ATP and serotonin (5-HT) are neurotransmitters secreted from taste bud receptor (type II) and presynaptic (type III) cells, respectively. Norepinephrine (NE) has also been proposed to be a neurotransmitter or paracrine hormone in taste buds. Yet, to date, the specific stimulus for NE release in taste buds is not well understood, and the identity of the taste cells that secrete NE is not known. Chinese hamster ovary cells were transfected with alpha(1A) adrenoceptors and loaded with fura-2 ("biosensors") to detect NE secreted from isolated mouse taste buds and taste cells. Biosensors responded to low concentrations of NE (>or=10 nm) with a reliable fura-2 signal. NE biosensors did not respond to stimulation with KCl or taste compounds. However, we recorded robust responses from NE biosensors when they were positioned against mouse circumvallate taste buds and the taste buds were stimulated with KCl (50 mm) or a mixture of taste compounds (cycloheximide, 10 microm; saccharin, 2 mm; denatonium, 1 mm; SC45647, 100 microm). NE biosensor responses evoked by stimulating taste buds were reversibly blocked by prazosin, an alpha(1A) receptor antagonist. Together, these findings indicate that taste bud cells secrete NE when they are stimulated. We isolated individual taste bud cells to identify the origin of NE release. NE was secreted only from presynaptic (type III) taste cells and not receptor (type II) cells. Stimulus-evoked NE release depended on Ca(2+) in the bathing medium. Using dual biosensors (sensitive to 5-HT and NE), we found all presynaptic cells secrete 5-HT and 33% corelease NE with 5-HT.

Mapping of epitopes and structural analysis of antigenic sites in the nucleoprotein of rabies virus.

PubMed

Goto, H; Minamoto, N; Ito, H; Ito, N; Sugiyama, M; Kinjo, T; Kawai, A

2000-01-01

Linear epitopes on the rabies virus nucleoprotein (N) recognized by six MAbs raised against antigenic sites I (MAbs 6-4, 12-2 and 13-27) and IV (MAbs 6-9, 7-12 and 8-1) were investigated. Based on our previous studies on sites I and IV, 24 consecutively overlapping octapeptides and N- and C-terminal-deleted mutant N proteins were prepared. Results showed that all three site I epitopes studied and two site IV epitopes (for MAbs 8-1 and 6-9) mapped to aa 358-367, and that the other site IV epitope of MAb 7-12 mapped to aa 375-383. Tests using chimeric and truncated proteins showed that MAb 8-1 also requires the N-terminal sequence of the N protein to recognize its binding region more efficiently. Immunofluorescence studies demonstrated that all three site I-specific MAbs and one site IV-specific MAb (7-12) stained the N antigen that was diffusely distributed in the whole cytoplasm; the other two site IV-specific MAbs (6-9 and 8-1) detected only the N antigen in the cytoplasmic inclusion bodies (CIB). An antigenic site II-specific MAb (6-17) also detected CIB-associated N antigen alone. Furthermore, the level of diffuse N antigens decreased after treatment of infected cells with cycloheximide. These results suggest that epitopes at site I are expressed on the immature form of the N protein, but epitope structures of site IV MAbs 6-9 and 8-1 are created and/or exposed only after maturation of the N protein.

Physiological and ultrastructural characterisation of a desiccation-tolerant filmy fern, Hymenophyllum caudiculatum: Influence of translational regulation and ABA on recovery.

PubMed

Garcés, M; Ulloa, M; Miranda, A; Bravo, L A

2018-03-01

The filmy fern Hymenophyllum caudiculatum can lose 60% of its relative water content, remain dry for some time and recover 88% of photochemical efficiency after 30 min of rehydration. Little is known about the protective strategies and regulation of the cellular rehydration process in this filmy fern species. The aim of this study was to characterise the filmy fern ultrastructure during a desiccation-rehydration cycle, and measure the physiological effects of transcription/translation inhibitors and ABA during desiccation recovery. Confocal and transmission electron microscopy were used to compare changes in structure during fast or slow desiccation. Transcription (actinomycin D) and translation (cycloheximide) inhibitors and ABA were used to compare photochemical efficiency during desiccation recovery. Cell structure was conserved during slow desiccation and rehydration, constitutive properties of the cell wall, allowing invagination and folding of the membranes and an important change in chloroplast size. The use of a translational inhibitor impeded recovery of photochemical efficiency during the first 80 min of rehydration, but the transcriptional inhibitor had no effect. Exogenous ABA delayed photochemical inactivation, and endogenous ABA levels decreased during desiccation and rehydration. Frond curling and chloroplast movements are possible strategies to avoid photodamage. Constitutive membrane plasticity and rapid cellular repair can be adaptations evolved to tolerate a rapid recovery during rehydration. Further research is required to explore the importance of existing mRNAs during the first minutes of recovery, and ABA function during desiccation of H. caudiculatum. © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.

Pyrogenicity of interferon and its inducer in rabbits.

PubMed

Won, S J; Lin, M T

1988-03-01

The effects of intracerebral administration of interferon (IFN) or its inducer polyriboinosinic acid-polyribocytidylic acid (poly I:C) on thermoregulatory responses were assessed in conscious rabbits. Administration of IFN (10(2)-10(6) IU) or poly I:C (0.012-12 micrograms) into the preoptic anterior hypothalamus or the third cerebral ventricle caused a dose-dependent fever in rabbits at three ambient temperatures (Ta) tested. In the cold (Ta = 8 degrees C), the fever was due to increased metabolism, whereas in the heat (Ta = 32 degrees C) the fever was due to a reduction in respiratory evaporative heat loss and ear skin blood flow. At the moderate environmental temperature (Ta = 22 degrees C), the fever was due to increased metabolism and cutaneous vasoconstriction. Compared with the febrile responses induced by cerebroventricular route injection of IFN or poly I:C, the hypothalamic route of injection required a much lower dose of IFN or poly I:C to produce a similar fever. Furthermore, the fever induced by intrahypothalamic injection of IFN or poly I:C was reduced by pretreatment of animals with a systemic dose of indomethacin (an inhibitor of all prostaglandins formation) or cycloheximide (an inhibitor of protein synthesis). The data indicate that IFN or its inducer may act through the endogenous release of a prostaglandin or a protein factor of an unknown chemical nature in the preoptic anterior hypothalamic region to induce fever in rabbits. The fever induced by IFN or its inducer is brought about by a decrease in heat loss and/or an increase in heat production in rabbits.

Parathyroid hormone induces c-fos and c-jun messenger RNA in rat osteoblastic cells

NASA Technical Reports Server (NTRS)

Clohisy, J. C.; Scott, D. K.; Brakenhoff, K. D.; Quinn, C. O.; Partridge, N. C.

1992-01-01

PTH is a potent regulator of osteoblast gene expression, yet the nuclear events that mediate PTH action are poorly understood. We were interested in identifying immediate early genes which may regulate PTH-altered gene expression in the osteoblast. Therefore, we examined the effects of PTH on c-fos and c-jun gene expression in a rat osteoblastic cell line (UMR 106-01). Under control conditions, c-fos and c-jun mRNAs were present at low basal levels. After PTH treatment, c-fos mRNA abundance dramatically increased, with a maximal and transient response at 30 min. PTH also stimulated an increase in c-jun mRNA, but in a biphasic manner, with maximal levels at 30 min and 2 h. These responses were dose dependent, not altered by cotreatment with the protein synthesis inhibitor cycloheximide, and preceded PTH-induced expression of matrix metallo-proteinase-1 mRNA. Nuclear run-on assays demonstrated an increased rate of c-fos and c-jun transcription after PTH exposure. To determine the signal transduction pathways involved, second messenger analogs were tested for their ability to mimic the effects of PTH. 8-Bromo-cAMP and phorbol 12-myristate 13-acetate (PMA) caused increases in the abundance of c-fos and c-jun transcripts. Ionomycin had no effect on the expression of these genes. Pretreatment of the cells with PMA resulted in a decrease in basal c-jun expression, but did not alter the PTH-mediated increase in c-fos, c-jun, or matrix metalloproteinase-1 mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS).

Interferon-γ Reduces the Proliferation of Primed Human Renal Tubular Cells.

PubMed

García-Sánchez, Omar; López-Novoa, José Miguel; López-Hernández, Francisco J

2014-01-01

Chronic kidney disease (CKD) is a progressive deterioration of the kidney function, which may eventually lead to renal failure and the need for dialysis or kidney transplant. Whether initiated in the glomeruli or the tubuli, CKD is characterized by progressive nephron loss, for which the process of tubular deletion is of key importance. Tubular deletion results from tubular epithelial cell death and defective repair, leading to scarring of the renal parenchyma. Several cytokines and signaling pathways, including transforming growth factor-β (TGF-β) and the Fas pathway, have been shown to participate in vivo in tubular cell death. However, there is some controversy about their mode of action, since a direct effect on normal tubular cells has not been demonstrated. We hypothesized that epithelial cells would require specific priming to become sensitive to TGF-β or Fas stimulation and that this priming would be brought about by specific mediators found in the pathological scenario. Herein we studied whether the combined effect of several stimuli known to take part in CKD progression, namely TGF-β, tumor necrosis factor-α, interferon-γ (IFN-γ), and Fas stimulation, on primed resistant human tubular cells caused cell death or reduced proliferation. We demonstrate that these cytokines have no synergistic effect on the proliferation or viability of human kidney (HK2) cells. We also demonstrate that IFN-γ, but not the other stimuli, reduces the proliferation of cycloheximide-primed HK2 cells without affecting their viability. Our results point at a potentially important role of IFN-γ in defective repair, leading to nephron loss during CKD.

Interferon-γ Reduces the Proliferation of Primed Human Renal Tubular Cells

PubMed Central

García-Sánchez, Omar; López-Novoa, José Miguel; López-Hernández, Francisco J.

2014-01-01

Background/Aims Chronic kidney disease (CKD) is a progressive deterioration of the kidney function, which may eventually lead to renal failure and the need for dialysis or kidney transplant. Whether initiated in the glomeruli or the tubuli, CKD is characterized by progressive nephron loss, for which the process of tubular deletion is of key importance. Tubular deletion results from tubular epithelial cell death and defective repair, leading to scarring of the renal parenchyma. Several cytokines and signaling pathways, including transforming growth factor-β (TGF-β) and the Fas pathway, have been shown to participate in vivo in tubular cell death. However, there is some controversy about their mode of action, since a direct effect on normal tubular cells has not been demonstrated. We hypothesized that epithelial cells would require specific priming to become sensitive to TGF-β or Fas stimulation and that this priming would be brought about by specific mediators found in the pathological scenario. Methods Herein we studied whether the combined effect of several stimuli known to take part in CKD progression, namely TGF-β, tumor necrosis factor-α, interferon-γ (IFN-γ), and Fas stimulation, on primed resistant human tubular cells caused cell death or reduced proliferation. Results We demonstrate that these cytokines have no synergistic effect on the proliferation or viability of human kidney (HK2) cells. We also demonstrate that IFN-γ, but not the other stimuli, reduces the proliferation of cycloheximide-primed HK2 cells without affecting their viability. Conclusion Our results point at a potentially important role of IFN-γ in defective repair, leading to nephron loss during CKD. PMID:24575118

Cold Induction of Arabidopsis CBF Genes Involves Multiple ICE (Inducer of CBF Expression) Promoter Elements and a Cold-Regulatory Circuit That Is Desensitized by Low Temperature1

PubMed Central

Zarka, Daniel G.; Vogel, Jonathan T.; Cook, Daniel; Thomashow, Michael F.

2003-01-01

The Arabidopsis CBF1, 2, and 3 genes (also known as DREB1b, c, and a, respectively) encode transcriptional activators that have a central role in cold tolerance. CBF1-3 are rapidly induced upon exposing plants to low temperature, followed by expression of CBF-targeted genes, the CBF regulon, resulting in an increase in plant freezing tolerance. At present, little is known about the cold-sensing mechanism that controls CBF expression. Results presented here indicate that this mechanism does not require a cold shock to bring about the accumulation of CBF transcripts, but instead, absolute temperature is monitored with a greater degree of input, i.e. lower temperature, resulting in a greater output, i.e. higher levels of CBF transcripts. Temperature-shift experiments also indicate that the cold-sensing mechanism becomes desensitized to a given low temperature, such as 4°C, and that resensitization to that temperature requires between 8 and 24 h at warm temperature. Gene fusion experiments identified a 125-bp section of the CBF2 promoter that is sufficient to impart cold-responsive gene expression. Mutational analysis of this cold-responsive region identified two promoter segments that work in concert to impart robust cold-regulated gene expression. These sequences, designated ICEr1 and ICEr2 (induction of CBF expression region 1 or 2), were also shown to stimulate transcription in response to mechanical agitation and the protein synthesis inhibitor, cycloheximide. PMID:14500791

Lower Growth Temperature Increases Alternative Pathway Capacity and Alternative Oxidase Protein in Tobacco 1

PubMed Central

Vanlerberghe, Greg C.; McIntosh, Lee

1992-01-01

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30°C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18°C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30°C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18°C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein. Images Figure 3 Figure 4 PMID:16652932

Induction of antiviral genes, Mx and vig-1, by dsRNA and Chum salmon reovirus in rainbow trout monocyte/macrophage and fibroblast cell lines.

PubMed

DeWitte-Orr, Stephanie J; Leong, Jo-Ann C; Bols, Niels C

2007-09-01

The expression of potential antiviral genes, Mx1, Mx2, Mx3 and vig-1, was studied in two rainbow trout cell lines: monocyte/macrophage RTS11 and fibroblast-like RTG-2. Transcripts were monitored by RT-PCR; Mx protein by Western blotting. In unstimulated cultures Mx1 and vig-1 transcripts were seen occasionally in RTS11 but rarely in RTG-2. A low level of Mx protein was seen in unstimulated RTS11 but not in RTG-2. In both cell lines, Mx and vig-1 transcripts were induced by a dsRNA, poly inosinic: poly cytidylic acid (poly IC), and by Chum salmon reovirus (CSV). Medium conditioned by cells previously exposed to poly IC or CSV and assumed to contain interferon (IFN) induced the antiviral genes in RTS11. However, RTG-2 responded only to medium conditioned by RTG-2 exposed previously to CSV. In both cell lines, poly IC and CSV induced Mx transcripts in the presence of cycloheximide, suggesting a direct induction mechanism, independent of IFN, was also possible. For CSV, ribavirin blocked induction in RTS11 but not in RTG-2, suggesting viral RNA synthesis was required for induction only in RTS11. In both RTS11 and RTG-2 cultures, Mx protein showed enhanced accumulation by 24h after exposure to poly IC and CSV, but subsequently Mx protein levels declined back to control levels in RTS11 but not in RTG-2. These results suggest that Mx can be regulated differently in macrophages and fibroblasts.

Cinematographic observations of growth cycles of Chlamydia trachomatis in primary cultures of human amniotic cells.

PubMed Central

Neeper, I D; Patton, D L; Kuo, C C

1990-01-01

Time-lapse cinematography was used to study the growth cycle of Chlamydia trachomatis in primary cell cultures of human amnion. Twelve preterm and twelve term placentas were obtained within 8 h of delivery, and epithelial cells were dissociated from the amniotic membranes by trypsinization and grown in Rose chambers. The epithelial nature of the cultured cells was documented by morphology and by immunofluorescence staining for cytoskeletal proteins, which matched the staining of intact amnion. With regular feedings, uninfected cultures remained healthy for up to 30 days. Confluent cultures (7 to 10 days) were infected with a genital strain (E/UW-5/CX) of C. trachomatis at 10(5) infectious units per chamber. Infections were done in culture medium without cycloheximide, which is often used to induce susceptibility of the cells. Between 66 and 90% of the cells were infected. Intracytoplasmic inclusions were visible by 18 h post infection (p.i.) and grew larger as the organisms inside multiplied. By 72 h p.i., the inclusions occupied the entire cytoplasm of the host cells. Further growth of the inclusions overdistended and ruptured the host cells on days 3 to 7. Cells not infected by the original inoculum became infected on day 5 or 6 p.i. by the chlamydial particles released from the ruptured cells. No amniotic cell was ever observed to survive the infection. The data presented support the hypothesis that amniotic epithelium is susceptible to infection and damage by C. trachomatis. This culture system provided detailed and dynamic observations of chlamydial infection under conditions more nearly physiologic than previously reported. Images PMID:2365450

Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants.

PubMed

Krug, Anne K; Balmer, Nina V; Matt, Florian; Schönenberger, Felix; Merhof, Dorit; Leist, Marcel

2013-12-01

Organ-specific in vitro toxicity assays are often highly sensitive, but they lack specificity. We evaluated here examples of assay features that can affect test specificity, and some general procedures are suggested on how positive hits in complex biological assays may be defined. Differentiating human LUHMES cells were used as potential model for developmental neurotoxicity testing. Forty candidate toxicants were screened, and several hits were obtained and confirmed. Although the cells had a definitive neuronal phenotype, the use of a general cell death endpoint in these cultures did not allow specific identification of neurotoxicants. As alternative approach, neurite growth was measured as an organ-specific functional endpoint. We found that neurite extension of developing LUHMES was specifically inhibited by diverse compounds such as colchicine, vincristine, narciclasine, rotenone, cycloheximide, or diquat. These compounds reduced neurite growth at concentrations that did not compromise cell viability, and neurite growth was affected more potently than the integrity of developed neurites of mature neurons. A ratio of the EC50 values of neurite growth inhibition and cell death of >4 provided a robust classifier for compounds associated with a developmental neurotoxic hazard. Screening of unspecific toxicants in the test system always yielded ratios <4. The assay identified also compounds that accelerated neurite growth, such as the rho kinase pathway modifiers blebbistatin or thiazovivin. The negative effects of colchicine or rotenone were completely inhibited by a rho kinase inhibitor. In summary, we suggest that assays using functional endpoints (neurite growth) can specifically identify and characterize (developmental) neurotoxicants.

Methanolic leaf extract of Gymnema sylvestre augments glucose uptake and ameliorates insulin resistance by upregulating glucose transporter-4, peroxisome proliferator-activated receptor-gamma, adiponectin, and leptin levels in vitro

PubMed Central

Kumar, Puttanarasaiah Mahesh; Venkataranganna, Marikunte V.; Manjunath, Kirangadur; Viswanatha, Gollapalle L.; Ashok, Godavarthi

2016-01-01

Aims: The present study was undertaken to evaluate the effect of methanolic leaf extract of Gymnema sylvestre (MLGS) on glucose transport (GLUT) and insulin resistance in vitro. Materials and Methods: Peroxisome proliferator-activated receptor-gamma (PPAR-γ) and GLUT-4 expression were assessed in L6 myotubes for concluding the GLUT activity, and adiponectin and leptin expression was studied in 3T3 L1 murine adipocyte cell line to determine the effect of MLGS (250-750 μg/ml) on insulin resistance. Results: The findings of the experiments have demonstrated a significant and dose-dependent increase in glucose uptake in all the tested concentrations of MLGS, further the glucose uptake activity of MLGS (750 μg/ml) was at par with rosiglitazone (50 μg/ml). Concomitantly, MLGS has shown enhanced GLUT-4 and PPAR-γ gene expressions in L6 myotubes. Furthermore, cycloheximide (CHX) had completely abolished the glucose uptake activity of MLGS when co-incubated, which further confirmed that glucose uptake activity of MLGS was linked to enhanced expression of GLUT-4 and PPAR-γ. In addition, in another experimental set, MLGS showed enhanced expression of adiponectin and leptin, thus confirms the ameliorative effect of MLGS on insulin resistance. Conclusion: These findings suggest that MLGS has an enhanced glucose uptake activity in L6 myotubes, and ameliorate the insulin resistance in 3T3 L1 murine adipocyte cell line in vitro. PMID:27104035

Mechanical force-mediated pathological cartilage thinning is regulated by necroptosis and apoptosis.

PubMed

Zhang, C; Lin, S; Li, T; Jiang, Y; Huang, Z; Wen, J; Cheng, W; Li, H

2017-08-01

This study aimed to identify the mechanisms underlying mandibular chondrocyte cell death and cartilage thinning in response to mechanical force. An in vivo model (compressive mechanical force) and an in vitro model (TNF-α+cycloheximide) were used to induce mandibular chondrocyte necroptosis. Hematoxylin and eosin staining and transmission electron microscopy were used to assess histological and subcellular changes in mandibular chondrocyte. Immunohistochemistry, western blotting, and real-time PCR were performed to evaluate changes in necroptotic protein markers. Cell activity, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were examined in vitro. The expression of RIP1, RIP3 and Caspase-8 in mandibular chondrocytes significantly increased after 4 days of compressive mechanical force. Furthermore, the inhibition of necroptosis by Necrostatin-1 (Nec-1) or the inhibition of apoptosis by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD) partially restored mechanical force-mediated mandibular cartilage thinning and chondrocyte death. Moreover, a synergistic effect on cell death inhibition and mandibular cartilage thickness restoration were found when treated with Nec-1+Z-VAD. The results of the in vitro model were in line with the in vivo ones, indicating that the changes in MMP and ROS generation contributed to mandibular chondrocyte apoptosis and necroptosis. In addition to apoptosis, necroptosis also plays critical roles in pathological changes in mandibular cartilage after compressive mechanical force stimulation, implying RIP1, a master protein that mediates both necroptosis and apoptosis, as a potential therapeutic target in temporal mandibular osteoarthritis. Copyright © 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Molecular typing of environmental Cryptococcus neoformans/C. gattii species complex isolates from Manaus, Amazonas, Brazil.

PubMed

Alves, Gleica Soyan Barbosa; Freire, Ana Karla Lima; Bentes, Amaury Dos Santos; Pinheiro, José Felipe de Souza; de Souza, João Vicente Braga; Wanke, Bodo; Matsuura, Takeshi; Jackisch-Matsuura, Ani Beatriz

2016-08-01

Cryptococcus neoformans and Cryptococcus gattii are the main causative agents of cryptococcosis, a systemic fungal disease that affects internal organs and skin, and which is acquired by inhalation of spores or encapsulated yeasts. It is currently known that the C. neoformans/C. gattii species complex has a worldwide distribution, however, some molecular types seem to prevail in certain regions. Few environmental studies of Cryptococcus have been conducted in the Brazilian Amazon. This is the first ecological study of the pathogenic fungi C. neoformans/C. gattii species complex in the urban area of Manaus, Amazonas, Brazil. A total of 506 samples from pigeon droppings (n = 191), captive bird droppings (n = 60) and tree hollows (n = 255) were collected from June 2012 to January 2014 at schools and public buildings, squares, pet shops, households, the zoo and the bus station. Samples were plated on niger seed agar (NSA) medium supplemented with chloramphenicol and incubated at 25°C for 5 days. Dark-brown colonies were isolated and tested for thermotolerance at 37°C, cycloheximide resistance and growth on canavanine-glycine-bromothymol blue agar. Molecular typing was done by PCR-RFLP. Susceptibility to the antifungal drugs amphotericin B, fluconazole, itraconazole and ketoconazole was tested using Etest(®) strips. In total, 13 positive samples were obtained: one tree hollow (C. gattiiVGII), nine pigeon droppings (C. neoformansVNI) and three captive bird droppings (C. neoformansVNI). The environmental cryptococcal isolates found in this study were of the same molecular types as those responsible for infections in Manaus. © 2016 Blackwell Verlag GmbH.

Photon emissions from rice cells elicited by N-acetylchitooligosaccharide are generated through phospholipid signaling in close association with the production of reactive oxygen species.

PubMed

Kageyama, C; Kato, K; Iyozumi, H; Inagaki, H; Yamaguchi, A; Furuse, K; Baba, K

2006-01-01

Biophotons are ultraweak light emissions from biochemical reactions in a living body. They increase in suspension-cultured rice (Oryza sativa L.) cells when elicited by N-acetylchitooligosaccharide. Biochemical analyses were undertaken to investigate the relationship between disease response and biophotons in order to clarify the emission mechanism of biophotons caused by this elicitor. Photon emissions induced by N-acetylchitohexaose were suppressed when cells were pretreated with the reactive oxygen species (ROS)-generating inhibitors: pyrocatechol-3,5-disulfonic acid disodium salt (Tiron); diphenylene iodonium (DPI); and salicylhydroxamic acid (SHAM). Conversely, exogenously applied ROS (superoxide and hydrogen peroxide) were able to induce photon emissions. The effects of protein phosphorylation (K-252a) and the Ca(2+) signaling inhibitors, ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and LaCl(3), caused photon emissions to decrease. It is clear that photon emissions from rice cells elicited by N-acetylchitohexaose are closely associated with the ROS-generating system, and are regulated by Ca(2+) signaling and protein phosphorylation. Exogenously applied phosphatidic acid (PA), the second messenger in the signal transduction of disease response, raised photon emissions in rice cells. Comparisons of photon emissions from PA and N-acetylchitohexaose regarding time courses, spectral compositions, and the inhibition ratios of several inhibitors, as well as a loss- and gain-of-function assay using the protein synthesis inhibitor cycloheximide (CHX) and PA, showed the possibility that photon emissions from rice cells elicited by N-acetylchitooligosaccharide were generated through PA, an intermediate of phospholipid signaling.

Haloanaerobium salsugo sp. nov., a moderately halophilic, anaerobic bacterium from a subterranean brine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhupathiraju, V.K.; Sharma, P.K.; Tanner, R.S.

A strictly anaerobic, moderately halophilic, gram-negative bacterium was isolated from a highly saline oil field brine. The bacterium was a non-spore-forming, nonmotile rod, appearing singly, in pairs, or occasionally as long chains, and measured 0.3 to 0.4 by 2.6 to 4 {micro}m. The bacterium had a specific requirement for NaCl and grew at NaCl concentrations of between 6 and 24%, with optimal growth at 9% NaCl. The isolate grew at temperatures of between 22 and 51 C and pH values of between 5.6 and 8.0. The doubling time in a complex medium containing 10% NaCl was 9 h. Growth wasmore » inhibited by chloramphenicol, tetracycline, and penicillin but not by cycloheximide or azide. Fermentable substrates were predominantly carbohydrates. The end products of glucose fermentation were acetate, ethanol, CO{sub 2}, and H{sub 2}. The major components of the cellular fatty acids were C{sub 14:0}, C{sub 16:0}, C{sub 16:1}, and C{sub 17:0 cyc} acids. The DNA base composition of the isolate was 34 mol% G+C. Oligonucleotide catalog and sequence analyses of the 16S rRNA showed that strain VS-752{sup T} was most closely related to Haloanaerobium praevalens GSL{sup T} (ATCC 33744), the sole member of the genus Haloanaerobium. The authors propose that strain VS-752 (ATCC 51327) by established as the type strain of a new species, Haloanaerobium salsugo, in the genus Haloanaerobium. 40 refs., 3 figs, 5 tabs.« less

Hyperaccumulation of zinc by zinc-depleted Candida utilis grown in chemostat culture.

PubMed

Lawford, H G; Pik, J R; Lawford, G R; Williams, T; Kligerman, A

1980-01-01

The steady-state levels of zinc in Candida utilis yeast grown in continuous culture under conditions of zinc limitations are <1nmol Zn2+/mg dry weight of cells. Unlike carbon-limited cells, zinc-depleted cells from a zinc-limited chemostat possess the capacity to accumulate and store zinc at levels far in excess of the steady-state level of 4 nmol/mg dry biomass observed in carbon-limited chemostat cultures. Zinc uptake is energy-dependent and apparently undirectional since accumulated 65Zn neither exists from preloaded cells nor exchanges with cold Zn2+. The transport system exhibits a high affinity for Zn2+ (Km =.36micrM) with a Vmaxof 2.2 nmol per minute per milligram dry weight of cells. Growth during the period of the uptake assay is responsible for the apparent plateau level of 35 nmol Zn2+/mg dry weight of cells achieved after 20-30 min in the presence of 65Zn at pH 4.5 and 30 degrees C. Inhibition of growth during the uptake assay by cycloheximide results in a biphasic linear pattern of zinc accumulation where the cellular zinc is about 60 nmol/mg dry weight after 1 h. The enhanced level of accumulated zinc is not inhibtory to growth. Zinc-depleted C. utilis contains elevated amounts of polyphosphate and this anionic evidence does not allow discrimination between possible regulation of zinc homestasis either by inhibitions of zinc efflux through control of the membrane carrier or by control of the synthesis of a cytoplasmic zinc-sequestering macromolecule.

Chronic hypoxia up-regulates expression of adenosine A1 receptors in DDT1-MF2 cells.

PubMed

Hammond, Lucy C; Bonnet, Claire; Kemp, Paul J; Yates, Michael S; Bowmer, Christopher J

2004-02-01

As the first step to understand how chronic hypoxia might regulate smooth muscle function in health and disease, we have employed an established immortalised cell model of smooth muscle, DDT1-MF2 cells, to address the hypothesis that adenosine A1 receptor density is modulated by O2 availability. Maximal specific binding (Bmax) of the selective adenosine A1 receptor antagonist, [3H]-DPCPX, to cell membranes increased 3.5-fold from 0.48 +/- 0.02 pmol/mg to 1.7 +/- 0.5 pmol/mg protein after 16 hr of hypoxia and this effect was not accompanied by any statistically significant changes in either binding affinity (0.84 +/- 0.2 nM vs. 1.2 +/- 0.3 nM) or Hill coefficient (1.1 +/- 0.1 vs. 0.99 +/- 0.03). Hypoxia-evoked increases in membrane receptor density were paralleled in intact DDT1-MF2 cells. In addition, the increase in [3H]-DPCPX binding to intact cells was inhibited by co-incubation during hypoxia with the translational inhibitor cycloheximide, the transcriptional blocker actinomycin D and the NFkappaB inhibitor sulphasalazine. Together, these data show that adenosine A1 receptor density is modulated, at least in part, by O2-dependent activation of the transcription factor NFkappaB and adds to the list of processes dynamically regulated by ambient oxygen availability. Since hypoxia is an initiating factor in acute renal failure, similar changes in transcription may account for up-regulation of adenosine A1 receptors noted previously in the renal vasculature of rats with acute renal failure.

Overproduction of lactimidomycin by cross-overexpression of genes encoding Streptomyces antibiotic regulatory proteins.

PubMed

Zhang, Bo; Yang, Dong; Yan, Yijun; Pan, Guohui; Xiang, Wensheng; Shen, Ben

2016-03-01

The glutarimide-containing polyketides represent a fascinating class of natural products that exhibit a multitude of biological activities. We have recently cloned and sequenced the biosynthetic gene clusters for three members of the glutarimide-containing polyketides-iso-migrastatin (iso-MGS) from Streptomyces platensis NRRL 18993, lactimidomycin (LTM) from Streptomyces amphibiosporus ATCC 53964, and cycloheximide (CHX) from Streptomyces sp. YIM56141. Comparative analysis of the three clusters identified mgsA and chxA, from the mgs and chx gene clusters, respectively, that were predicted to encode the PimR-like Streptomyces antibiotic regulatory proteins (SARPs) but failed to reveal any regulatory gene from the ltm gene cluster. Overexpression of mgsA or chxA in S. platensis NRRL 18993, Streptomyces sp. YIM56141 or SB11024, and a recombinant strain of Streptomyces coelicolor M145 carrying the intact mgs gene cluster has no significant effect on iso-MGS or CHX production, suggesting that MgsA or ChxA regulation may not be rate-limiting for iso-MGS and CHX production in these producers. In contrast, overexpression of mgsA or chxA in S. amphibiosporus ATCC 53964 resulted in a significant increase in LTM production, with LTM titer reaching 106 mg/L, which is five-fold higher than that of the wild-type strain. These results support MgsA and ChxA as members of the SARP family of positive regulators for the iso-MGS and CHX biosynthetic machinery and demonstrate the feasibility to improve glutarimide-containing polyketide production in Streptomyces strains by exploiting common regulators.

Activation of IFN-beta element by IRF-1 requires a posttranslational event in addition to IRF-1 synthesis.

PubMed Central

Watanabe, N; Sakakibara, J; Hovanessian, A G; Taniguchi, T; Fujita, T

1991-01-01

Expression of the Type I IFN (i.e., IFN-alpha s and IFN-beta) genes is efficiently induced by viruses at the transcriptional level. This induction is mediated by at least two types of positive regulatory elements located in the human IFN-beta gene promoter: (1) the repeated elements which bind both the transcriptional activator IRF-1 and the repressor IRF-2 (IRF-elements; IRF-Es), and (2) the kappa B element (kappa B-E), which binds NF kappa B and is located between the IRF-Es and the TATA box. In this study we demonstrate that a promoter containing synthetic IRF-E, which displays high affinity for the IRFs can be efficiently activated by Newcastle disease virus (NDV). In contrast, such activation was either very weak or nil when cells were treated by IFN-beta or tumor necrosis factor-alpha (TNF-alpha), despite the fact they both efficiently induce de novo synthesis of the short-lived IRF-1 in L929 cells. In fact, efficient activation of the IRF-E apparently requires an event in addition to de novo IRF-1 induction, which can be elicited by NDV even in the presence of protein synthesis inhibitor, cycloheximide. Moreover, efficient activation of the IRF-E by NDV is specifically inhibited by the protein kinase inhibitor, Staurosporin. Hence our results suggest the importance of IRF-1 synthesis and post-translational modification event(s), possibly phosphorylation for the efficient activation of IRF-Es, which are otherwise under negative regulation by IRF-2. Images PMID:1886766

Induction of ceruloplasmin synthesis by IFN-gamma in human monocytic cells

NASA Technical Reports Server (NTRS)

Mazumder, B.; Mukhopadhyay, C. K.; Prok, A.; Cathcart, M. K.; Fox, P. L.

1997-01-01

Ceruloplasmin is a 132-kDa glycoprotein abundant in human plasma. It has multiple in vitro activities, including copper transport, lipid pro- and antioxidant activity, and oxidation of ferrous ion and aromatic amines; however, its physiologic role is uncertain. Although ceruloplasmin is synthesized primarily by the liver in adult humans, production by cells of monocytic origin has been reported. We here show that IFN-gamma is a potent inducer of ceruloplasmin synthesis by monocytic cells. Activation of human monoblastic leukemia U937 cells with IFN-gamma increased the production of ceruloplasmin by at least 20-fold. The identity of the protein was confirmed by plasmin fingerprinting. IFN-gamma also increased ceruloplasmin mRNA. Induction followed a 2- to 4-h lag and was partially blocked by cycloheximide, indicating a requirement for newly synthesized factors. Ceruloplasmin induction in monocytic cells was agonist specific, as IL-1, IL-4, IL-6, IFN-alpha, IFN-beta, TNF-alpha, and LPS were completely ineffective. The induction was also cell type specific, as IFN-gamma did not induce ceruloplasmin synthesis in endothelial or smooth muscle cells. In contrast, IFN-gamma was stimulatory in other monocytic cells, including THP-1 cells and human peripheral blood monocytes, and also in HepG2 cells. Ceruloplasmin secreted by IFN-gamma-stimulated U937 cells had ferroxidase activity and was, in fact, the only secreted protein with this activity. Monocytic cell-derived ceruloplasmin may contribute to defense responses via its ferroxidase activity, which may drive iron homeostasis in a direction unfavorable to invasive organisms.

Ceruloplasmin ferroxidase activity stimulates cellular iron uptake by a trivalent cation-specific transport mechanism

NASA Technical Reports Server (NTRS)

Attieh, Z. K.; Mukhopadhyay, C. K.; Seshadri, V.; Tripoulas, N. A.; Fox, P. L.

1999-01-01

The balance required to maintain appropriate cellular and tissue iron levels has led to the evolution of multiple mechanisms to precisely regulate iron uptake from transferrin and low molecular weight iron chelates. A role for ceruloplasmin (Cp) in vertebrate iron metabolism is suggested by its potent ferroxidase activity catalyzing conversion of Fe2+ to Fe3+, by identification of yeast copper oxidases homologous to Cp that facilitate high affinity iron uptake, and by studies of "aceruloplasminemic" patients who have extensive iron deposits in multiple tissues. We have recently shown that Cp increases iron uptake by cultured HepG2 cells. In this report, we investigated the mechanism by which Cp stimulates cellular iron uptake. Cp stimulated the rate of non-transferrin 55Fe uptake by iron-deficient K562 cells by 2-3-fold, using a transferrin receptor-independent pathway. Induction of Cp-stimulated iron uptake by iron deficiency was blocked by actinomycin D and cycloheximide, consistent with a transcriptionally induced or regulated transporter. Cp-stimulated iron uptake was completely blocked by unlabeled Fe3+ and by other trivalent cations including Al3+, Ga3+, and Cr3+, but not by divalent cations. These results indicate that Cp utilizes a trivalent cation-specific transporter. Cp ferroxidase activity was required for iron uptake as shown by the ineffectiveness of two ferroxidase-deficient Cp preparations, copper-deficient Cp and thiomolybdate-treated Cp. We propose a model in which iron reduction and subsequent re-oxidation by Cp are essential for an iron uptake pathway with high ion specificity.

Proteolytic activities in yeast after UV irradiation. II. Variation in proteinase levels in mutants blocked in DNA-repair pathways.

PubMed

Schwencke, J; Moustacchi, E

1982-01-01

When the levels of three common yeast proteinases in exponentially growing cells of mutants blocked in different repair pathways are compared to that of isogenic wild-type cells, it can be seen that the level of proteinase B is enhanced in the mutants whereas the levels of leucin aminopeptidase (Leu.AP) and lysine aminopeptidase (Lys.AP) are similar in all strains. As in its corresponding wild type, the level of proteinase B activity is further enhanced after UV-irradiation in a mutant blocked in excision-repair (rad1-3). In contrast, following the same treatment the level of proteinase B remains almost constant in a mutant blocked in a general error-prone repair system (rad6-1) and in a mutant defective in a more specific mutagenic repair pathway (pso2-1). Cycloheximide, an inhibitor of protein synthesis, blocks the post-UV enhancement in proteinase B activity observed in rad1-3 indicating that, as in the wild-type cells, an inducible process is involved. The levels of Lys.AP and Leu.AP are, respectively, either unaffected or only moderately increased following UV-treatment of the repair defective mutants, as in wild-type strains. It is obvious that the induction of protease B activity following UV-treatment in Saccharomyces cannot be equated to the induction of the recA protein in Escherichia coli. However the correlation found between the block in mutagenic repair and the lack of UV-induction of protease B activity leads to questions on the possible role of certain protease activities in mutagenic repair in eucaryotic cells.

Lactoferrin release and interleukin-1, interleukin-6, and tumor necrosis factor production by human polymorphonuclear cells stimulated by various lipopolysaccharides: relationship to growth inhibition of Candida albicans.

PubMed

Palma, C; Cassone, A; Serbousek, D; Pearson, C A; Djeu, J Y

1992-11-01

Lipopolysaccharides (LPSs) from Escherichia coli, Serratia marcescens, and Salmonella typhimurium, at doses from 1 to 100 ng/ml, strongly enhanced growth inhibition of Candida albicans by human polymorphonuclear leukocytes (PMN) in vitro. Flow cytometry analysis demonstrated that LPS markedly augmented phagocytosis of Candida cells by increasing the number of yeasts ingested per neutrophil as well as the number of neutrophils capable of ingesting fungal cells. LPS activation caused augmented release of lactoferrin, an iron-binding protein which itself could inhibit the growth of C. albicans in vitro. Antibodies against lactoferrin effectively and specifically reduced the anti-C. albicans activity of both LPS-stimulated and unstimulated PMN. Northern (RNA blot) analysis showed enhanced production of mRNAs for interleukin-1 beta, tumor necrosis factor alpha, and interleukin-6 and in neutrophils within 1 h of stimulation with LPS. The cytokines were also detected in the supernatant of the activated PMN, and their synthesis was prevented by pretreatment of LPS-stimulated PMN with protein synthesis inhibitors, such as emetine and cycloheximide. These inhibitors, however, did not block either lactoferrin release or the anti-Candida activity of LPS-stimulated PMN. These results demonstrate the ability of various bacterial LPSs to augment neutrophil function against C. albicans and suggest that the release of a candidastatic, iron-binding protein, lactoferrin, may contribute to the antifungal effect of PMN. Moreover, the ability to produce cytokines upon stimulation by ubiquitous microbial products such as the endotoxins points to an extraphagocytic, immunomodulatory role of PMN during infection.

Induction of human spermine oxidase SMO(PAOh1) is regulated at the levels of new mRNA synthesis, mRNA stabilization and newly synthesized protein

PubMed Central

2004-01-01

The oxidation of polyamines induced by antitumour polyamine analogues has been associated with tumour response to specific agents. The human spermine oxidase, SMO(PAOh1), is one enzyme that may play a direct role in the cellular response to the antitumour polyamine analogues. In the present study, the induction of SMO(PAOh1) enzyme activity by CPENSpm [N1-ethyl-N11-(cyclopropyl)methyl-4,8,diazaundecane] is demonstrated to be a result of newly synthesized mRNA and protein. Inhibition of new RNA synthesis by actinomycin D inhibits both the appearance of SMO(PAOh1) mRNA and enzyme activity. Similarly, inhibition of newly synthesized protein with cycloheximide prevents analogue-induced enzyme activity. Half-life determinations indicate that stabilization of SMO(PAOh1) protein does not play a significant role in analogue-induced activity. However, half-life experiments using actinomycin D indicate that CPENSpm treatment not only increases mRNA expression, but also leads to a significant increase in mRNA half-life (17.1 and 8.8 h for CPENSpm-treated cells and control respectively). Using reporter constructs encompassing the SMO(PAOh1) promoter region, a 30–90% increase in transcription is observed after exposure to CPENSpm. The present results are consistent with the hypothesis that analogue-induced expression of SMO(PAOh1) is a result of increased transcription and stabilization of SMO(PAOh1) mRNA, leading to increased protein production and enzyme activity. These data indicate that the major level of control of SMO(PAOh1) expression in response to polyamine analogues exposure is at the level of mRNA. PMID:15496143

Activation of l-arginine transport by protein kinase C in rabbit, rat and mouse alveolar macrophages

PubMed Central

Racké, Kurt; Hey, Claudia; Mössner, Jutta; Hammermann, Rainer; Stichnote, Christina; Wessler, Ignaz

1998-01-01

The role of protein kinase C in controlling L-arginine transport in alveolar macrophages was investigated. L-[3H]Arginine uptake in rabbit alveolar macrophages declined by 80 % after 20 h in culture. 4β-Phorbol 12-myristate 13-acetate (PMA), but not 4α-phorbol 12-myristate 13-acetate (α-PMA), present during 20 h culture, enhanced L-[3H]arginine uptake more than 10-fold. Staurosporine and chelerythrine opposed this effect. L-[3H]Arginine uptake was saturable and blockable by L-lysine. After PMA treatment Vmax was increased more than 5-fold and Km was reduced from 0.65 to 0.32 mM. Time course experiments showed that PMA increased L-[3H]arginine uptake almost maximally within 2 h. This short-term effect was not affected by cycloheximide or actinomycin D. L-[3H]Arginine uptake and its stimulation by PMA was also observed in sodium-free medium. L-Leucine (0.1 mM) inhibited L-[3H]arginine uptake by 50 % in sodium-containing medium, but not in sodium-free medium. At 1 mM, L-leucine caused significant inhibition in sodium-free medium also. L-Leucine showed similar effects on PMA-treated cells. N-Ethylmaleimide (200 μm, 10 min) reduced L-[3H]arginine uptake by 70 % in control cells, but had no effect on PMA-treated (20 or 2 h) cells. In alveolar macrophages, multiple transport systems are involved in L-arginine uptake, which is markedly stimulated by protein kinase C, probably by modulation of the activity of already expressed cationic amino acid transporters. PMID:9714862

Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels

PubMed Central

Ferrero, Rut; Torres, Magdalena

2002-01-01

Background Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. Results Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β1 subunit protein levels. Both sGC activity and β1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β1 subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β1 subunits. Conclusions These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β1 subunit stability. PMID:12350235

The D153del Mutation in GNB3 Gene Causes Tissue Specific Signalling Patterns and an Abnormal Renal Morphology in Rge Chickens

PubMed Central

Tummala, Hemanth; Fleming, Stewart; Hocking, Paul M.; Wehner, Daniel; Naseem, Zahid; Ali, Manir; Inglehearn, Christopher F.; Zhelev, Nikolai; Lester, Douglas H.

2011-01-01

Background The GNB3 gene is expressed in cone but not rod photoreceptors of vertebrates, where it acts as the β transducin subunit in the colour visual transduction process. A naturally occurring mutation ‘D153del’ in the GNB3 gene causes the recessively inherited blinding phenotype retinopathy globe enlarged (rge) disease in chickens. GNB3 is however also expressed in most other vertebrate tissues suggesting that the D153del mutation may exert pathological effects that outlie from eye. Principal Findings Recombinant studies in COS-7 cells that were transfected with normal and mutant recombinant GNB3 constructs and subjected to cycloheximide chase showed that the mutant GNB3d protein had a much shorter half life compared to normal GNB3. GNB3 codes for the Gβ3 protein subunit that, together with different Gγ and Gα subunits, activates and regulates phosphorylation cascades in different tissues. As expected, the relative levels of cGMP and cAMP secondary messengers and their activated kinases such as MAPK, AKT and GRK2 were also found to be altered significantly in a tissue specific manner in rge chickens. Histochemical analysis on kidney tissue sections, from rge homozygous affected chickens, showed the chickens had enlargement of the glomerular capsule, causing glomerulomegaly and tubulointerstitial inflammation whereas other tissues (brain, heart, liver, pancreas) were unaffected. Significance These findings confirm that the D153del mutation in GNB3 gene targets GNB3 protein to early degradation. Lack of GNB3 signalling causes reduced phosphorylation activity of ERK2 and AKT leading to severe pathological phenotypes such as blindness and renal abnormalities in rge chickens. PMID:21887213

6-Mercaptopurine, an activator of Nur77, enhances transcriptional activity of HIF-1alpha resulting in new vessel formation.

PubMed

Yoo, Y-G; Na, T-Y; Yang, W-K; Kim, H-J; Lee, I-K; Kong, G; Chung, J-H; Lee, M-O

2007-05-31

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays a central role in oxygen homeostasis. Previously, we reported that the orphan nuclear receptor Nur77 functions in stabilizing HIF-1alpha. Here, we demonstrate that 6-mercaptopurine (6-MP), an activator of the NR4A family members, enhances transcriptional activity of HIF-1. 6-MP enhanced the protein-level of HIF-1alpha as well as vascular endothelial growth factor (VEGF) in a dose- and time-dependent manner. The induction of HIF-1alpha was abolished by the transfection of either a dominant-negative Nur77 mutant or si-Nur77, indicating a critical role of Nur77 in the 6-MP action. The HIF-1alpha protein level remained up to 60 min in the presence of 6-MP when de novo protein synthesis was blocked by cycloheximide, suggesting that 6-MP induces stabilization of the HIF-1alpha protein. The fact that 6-MP decreased the association of HIF-1alpha with von Hippel-Lindau protein and the acetylation of HIF-1alpha, may explain how 6-MP induced stability of HIF-1alpha. Further, 6-MP induced the transactivation function of HIF-1alpha by recruiting co-activator cyclic-AMP-response-element-binding protein. Finally, 6-MP enhanced the expression of HIF-1alpha and VEGF, and the formation of capillary tubes in human umbilical vascular endothelial cells. Together, our results provide a new insight for 6-MP action in the stabilization of HIF-1alpha and imply a potential application of 6-MP in hypoxia-associated human vascular diseases.

Pressure-induced endocytic degradation of the Saccharomyces cerevisiae low-affinity tryptophan permease Tat1 is mediated by Rsp5 ubiquitin ligase and functionally redundant PPxY motif proteins.

PubMed

Suzuki, Asaha; Mochizuki, Takahiro; Uemura, Satoshi; Hiraki, Toshiki; Abe, Fumiyoshi

2013-07-01

Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1(K29R-K31R)-GFP remained. The HPG1-1 (Rsp5(P514T)) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.

Plastocyanin Controls the Stabilization of the Thylakoid Cu-transporting P-type ATPase PAA2/HMA8 in Response to Low Copper in Arabidopsis*

PubMed Central

Tapken, Wiebke; Ravet, Karl; Pilon, Marinus

2012-01-01

PAA2/HMA8 (P-type ATPase of Arabidopsis/Heavy-metal-associated 8) is a thylakoid located copper (Cu)-transporter in Arabidopsis thaliana. In tandem with PAA1/HMA6, which is located in the inner chloroplast envelope, it supplies Cu to plastocyanin (PC), an essential cuproenzyme of the photosynthetic machinery. We investigated whether the chloroplast Cu transporters are affected by Cu addition to the growth media. Immunoblots showed that PAA2 protein abundance decreased significantly and specifically when Cu in the media was increased, while PAA1 remained unaffected. The function of SPL7, the transcriptional regulator of Cu homeostasis, was not required for this regulation of PAA2 protein abundance and Cu addition did not affect PAA2 transcript levels, as determined by qRT-PCR. We used the translational inhibitor cycloheximide to analyze turnover and observed that the stability of the PAA2 protein was decreased in plants grown with elevated Cu. Interestingly, PAA2 protein abundance was significantly increased in paa1 mutants, in which the Cu content in the chloroplast is half of that of the wild-type, due to impaired Cu import into the organelle. In contrast in a pc2 insertion mutant, which has strongly reduced plastocyanin expression, the PAA2 protein levels were low regardless of Cu addition to the growth media. Together, these data indicate that plastid Cu levels control PAA2 stability and that plastocyanin, which is the target of PAA2 mediated Cu delivery in thylakoids, is a major determinant of this regulatory mechanism. PMID:22493454

Regulation of S-adenosylmethionine decarboxylase by polyamines in Ehrlich ascites-carcinoma cells grown in culture

PubMed Central

Alhonen-Hongisto, Leena

1980-01-01

1. The mechanism of stimulation of S-adenosylmethionine decarboxylase (EC 4.1.1.50) activity by inhibitors of ornithine decarboxylase (EC 4.1.1.17), namely dl-α-difluoromethylornithine, 1,3-diaminopropane and 1,3-diaminopropan-2-ol, was studied in Ehrlich ascites-tumour cells grown in suspension cultures. 2. Difluoromethylornithine and diaminopropane, although decreasing the content of putrescine and spermidine, markedly stimulated adenosylmethionine decarboxylase activity after exposure of the cells to the drugs for 8h, whereas the effect of diaminopropanol only became apparent many hours later. In tumour cells exposed to any of the inhibitors, a close negative correlation existed between the activity of adenosylmethionine decarboxylase and the intracellular concentration of spermidine and/or spermidine plus spermine, suggesting that a depletion of higher polyamines triggered enhancement of adenosylmethionine decarboxylase activity. 3. The mechanism of difluoromethylornithine- and diaminopropane-induced stimulation of adenosylmethionine decarboxylase involved (a) a marked increase in the apparent half-life of the enzyme and (b) an induction of enhanced enzyme synthesis. Diaminopropanol seemed to act solely via an induction mechanism. 4. The increased adenosylmethionine decarboxylase activity elicited by difluoromethylornithine could be restored to control values by micromolar concentrations of exogenous spermidine and spermine in 4h and by putrescine in 22h. In addition to the natural polyamines, elevated adenosylmethionine decarboxylase activity could be repressed by 3,3′-iminodipropylamine, a close analogue of spermidine, but not by non-physiological diamines. 5. Addition of spermidine and actinomycin D to cultures treated with difluoromethylornithine produced a comparable decay of enhanced adenosylmethionine decarboxylase activity (with an apparent half-life of about 2.5h), whereas the effect of cycloheximide was much more rapid. The present results suggest that polyamines may regulate adenosylmethionine decarboxylase at the transcriptional level of gene expression. PMID:6781485

The peptidase inhibitor CGS-26303 increases endothelin converting enzyme-1 expression in endothelial cells through accumulation of big endothelin-1

PubMed Central

Raoch, V; Martinez-Miguel, P; Arribas-Gomez, I; Rodriguez-Puyol, M; Rodriguez-Puyol, D; Lopez-Ongil, S

2007-01-01

Background and purpose: CGS-26303 inhibits endothelin converting enzyme (ECE)-1 more specifically than phosphoramidon. We have studied the effect of CGS-26303 on ECE-1 expression in bovine aortic endothelial cells. Methods: ECE-1 activity and big endothelin (ET)-1 levels were measured by ELISA, ECE-1 expression using western and northern blot and promoter activity using transfection assays. Key results: ECE-1 activity was completely inhibited by CGS-26303 25 μ M and phosphoramidon 100 μ M. CGS-26303 and phosphoramidon, though not thiorphan, a neutral endopeptidase (NEP) inhibitor, stimulated ECE-1 expression in cells (maximal effect at 16 h, 25 μ M). Cycloheximide abolished that effect. CGS-26303 induced ECE-1 mRNA expression and ECE-1 promoter activity. CGS-35066, a selective ECE-1 inhibitor, mimicked the effects of CGS-26303, suggesting that the effect was specific to ECE-1 inhibition. Big ET-1 accumulated in the cells and in the supernatants after CGS-26303 treatment. Neither exogenously added ET-1 nor the blockade of their receptors with bosentan modified ECE-1 protein. When big ET-1 was added to cells, significant increases in ECE-1 protein content and ECE-1 promoter activity were found. Bosentan did not block those effects. CGS-26303 did not modify prepro-ET-1 expression. CGS-26303 and big ET-1 induced the same effects in human endothelial cells, at lower doses. Conclusions: These results suggest that the accumulation of big ET-1 is responsible for the effects of CGS-26303 on ECE-1 and they did not depend on NEP blockade. Changes in ECE-1 protein after the administration of CGS-26303 could lead to a decreased response in long-term treatments. PMID:17643133

Induction of apoptosis in PA-1 ovarian cancer cells by vitamin K2 is associated with an increase in the level of TR3/Nur77 and its accumulation in mitochondria and nuclei.

PubMed

Sibayama-Imazu, Toshiko; Fujisawa, Yukari; Masuda, Yutaka; Aiuchi, Toshihiro; Nakajo, Shigeo; Itabe, Hiroyuki; Nakaya, Kazuyasu

2008-07-01

We examined the growth-inhibitory and apoptosis-inducing effects of vitamin K(2) (VK(2); menaquinone-4) on various lines of human ovarian cancer cells to study the mechanism of induction of apoptosis by VK(2). Cell proliferation was determined by XTT method, and apoptotic cells were detected by Hoechst staining. TR3, also known as Nur77 and NGFI-B, was detected by immunoblotting and immunofluorescence analysis. Role of TR3 on induction of apoptosis was examined by a siRNA experiment. We found that PA-1 cells were the most sensitive to VK(2) (IC(50) = 5.0 +/- 0.7 microM), while SK-OV-3 cells were resistant to VK(2). Immunoblotting and immunofluorescence analyses indicated that levels of TR3 were elevated in cell lysates 48 h after the start of treatment with 30 microM VK(2). In the VK(2)-treated cells, TR3 accumulated at significant levels in mitochondria, as well as in the nuclei of PA-1 cells. No similar changes were observed in SK-OV-3 cells under the same conditions. Treatment of PA-1 cells with small interfering RNA (siRNA) directed against TR3, and with cycloheximide or SP600125 (an inhibitor of c-jun N-terminal kinase; JNK), separately, inhibited the VK(2)-induced synthesis of TR3 and apoptosis. From these results, we can conclude that an increase in the synthesis of TR3 and the accumulation of TR3 in mitochondria and in nuclei might be involved in the induction of apoptosis by VK(2) and that the synthesis of TR3 might be regulated through a JNK signaling pathway.

Changes in translation rate modulate stress-induced damage of diverse proteins

PubMed Central

Kim, Heejung

2013-01-01

Proteostasis is the maintenance of the proper function of cellular proteins. Hypertonic stress disrupts proteostasis and causes rapid and widespread protein aggregation and misfolding in the nematode Caenorhabditis elegans. Optimal survival in hypertonic environments requires degradation of damaged proteins. Inhibition of protein synthesis occurs in response to diverse environmental stressors and may function in part to minimize stress-induced protein damage. We recently tested this idea directly and demonstrated that translation inhibition by acute exposure to cycloheximide suppresses hypertonicity-induced aggregation of polyglutamine::YFP (Q35::YFP) in body wall muscle cells. In this article, we further characterized the relationship between protein synthesis and hypertonic stress-induced protein damage. We demonstrate that inhibition of translation reduces hypertonic stress-induced formation and growth of Q35::YFP, Q44::YFP, and α-synuclein aggregates; misfolding of paramyosin and ras GTPase; and aggregation of multiple endogenous proteins expressed in diverse cell types. Activation of general control nonderepressible-2 (GCN-2) kinase signaling during hypertonic stress inhibits protein synthesis via phosphorylation of eukaryotic initiation factor-2α (eIF-2α). Inhibition of GCN-2 activation prevents the reduction in translation rate and greatly exacerbates the formation and growth of Q35::YFP aggregates and the aggregation of endogenous proteins. The current studies together with our previous work provide the first direct demonstration that hypertonic stress-induced reduction in protein synthesis minimizes protein aggregation and misfolding. Reduction in translation rate also serves as a signal that activates osmoprotective gene expression. The cellular proteostasis network thus plays a critical role in minimizing hypertonic stress-induced protein damage, in degrading stress-damaged proteins, and in cellular osmosensing and signaling. PMID:24153430

Human T cell activation. III. Induction of an early activation antigen, EA 1 by TPA, mitogens and antigens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hara, T.; Jung, L.K.L.; FU, S.M.

1986-03-01

With human T cells activated for 12 hours by 12-o-tetradecanoyl phorbol-13-acetate (TPA) as immunogen, an IgG/sub 2a/ monoclonal antibody, mAb Ea 1, has been generated to a 60KD phosphorylated protein with 32KD and 28KD subunits. The antigen, Ea 1, is readily detected on 60% of isolated thymocytes by indirect immunofluorescence. A low level of Ea 1 expression is detectable on 2-6% of blood lymphocytes. Isolated T cells have been induced to express Ea 1 by TPA, mitogens and anitgens. TPA activated T cells express Ea 1 as early as 1 hour after activation. By 4 hours, greater than 95% ofmore » the T cells stain with mAb Ea 1. About 50% of the PHA or Con A activated T cells express Ea 1 with a similar kinetics. Ea 1 expression proceeds that of IL-2 receptor in these activation processes. T cells activated by soluble antigens (tetanus toxoid and PPD) and alloantigens in MLR also express Ea 1 after a long incubation. About 20% of the T cells stain for Ea 1 at day 6. Ea 1 expression is not limited to activated T cells. B cells activated by TPA or anti-IgM Ab plus B cell growth factor express Ea 1. The kinetics of Ea 1 expression is slower and the staining is less intense. Repeated attempts to detect Ea 1 on resting and activated monocytes and granulocytes have not been successful. Ea 1 expression is due to de novo synthesis for its induction is blocked by cycloheximide and actinomycin D. Ea 1 is the earliest activation antigen detectable to-date.« less

Co-induction of p75NTR and p75NTR-associated death executor in neurons after zinc exposure in cortical culture or transient ischemia in the rat.

PubMed

Park, J A; Lee, J Y; Sato, T A; Koh, J Y

2000-12-15

Recently, a 22 kDa protein termed p75(NTR)-associated death executor (NADE) was discovered to be a necessary factor for p75(NTR)-mediated apoptosis in certain cells. However, the possible role for p75(NTR)/NADE in pathological neuronal death has yet been undetermined. In the present study, we have examined this possibility in vivo and in vitro. Exposure of cortical cultures to zinc induced both p75(NTR) and NADE in neurons, whereas exposure to NMDA, ionomycin, iron, or H(2)O(2) induced neither. In addition, zinc exposure increased neuronal NGF expression and its release into the medium. A function-blocking antibody of p75(NTR) (REX) inhibited association between p75(NTR) and NADE as well as neuronal death induced by zinc. Conversely, NGF augmented zinc-induced neuronal death. Caspase inhibitors reduced zinc-induced neuronal death, indicating that caspases were involved. Because reduction of NADE expression with cycloheximide or NADE antisense oligonucleotides attenuated zinc-induced neuronal death, NADE appears to contribute to p75(NTR)-induced cortical neuronal death as shown in other cells. Because zinc neurotoxicity may be a key mechanism of neuronal death after transient forebrain ischemia, we next examined this model. After ischemia, p75(NTR) and NADE were induced in degenerating rat hippocampal CA1 neurons. There was a close correlation between zinc accumulation and p75(NTR)/NADE induction. Suggesting the role of zinc here, injection of a metal chelator, CaEDTA, into the lateral ventricle completely blocked the induction of p75(NTR) and NADE. Our results suggest that co-induction of p75(NTR) and NADE plays a role in zinc-triggered neuronal death in vitro and in vivo.

Nature and origin of the calcium asymmetry-arising during gravitropic response in etiolated pea epicotyls

DOE Office of Scientific and Technical Information (OSTI.GOV)

Migliaccio, F.; Galston, A.W.

1987-10-01

Seven day old etiolated pea epicotyls were loaded symmetrically with /sup 3/H-indole 3-acetic acid (IAA) or /sup 45/Ca/sup 2 +/, then subjected to 1.5 hours of 1g gravistimulation. Epidermal peels taken from top and bottom surfaces after 90 minutes showed an increase in IAA on the lower side and of Ca/sup 2 +/ on the upper side. Inhibitors of IAA movement (TIBA, 9-hydroxyfluorene carboxylic acid) block the development of both IAA and Ca/sup 2 +/ asymmetries, but substances known to interfere with normal Ca/sup 2 +/ transport do not significantly alter either IAA or Ca/sup 2 +/ asymmetries. These substances,more » however, are active in modifying both Ca/sup 2 +/ uptake and efflux through oat and pea leaf protoplast membranes. The authors conclude that the /sup 45/Ca/sup 2 +/ fed to pea epicotyls occurs largely in the cell wall, and that auxin movement is primary and Ca/sup 2 +/ movement secondary in gravitropism. They hypothesize that apoplastic Ca/sup 2 +/ changes during the graviresponse because it is displaced by H/sup +/ secreted through auxin-induced proton release. This proposed mechanism is supported by localized pH experiments, in which filter paper soaked in various buffers was applied to one side of a carborundum-abraded epicotyls. Buffer at pH 3 increased calcium loss from the side to which it is applied, whereas pH 7 buffer decreases it. Moreover, 10 micromolar IAA and 1 micromolar fusicoccin, which promote H/sup +/ efflux, increase Ca/sup 2 +/ release from pea epicotyl segments, whereas cycloheximide, which inhibits H/sup +/ efflux, has the reverse effect.« less

Grouper translationally controlled tumor protein prevents cell death and inhibits the replication of Singapore grouper iridovirus (SGIV).

PubMed

Wei, Jingguang; Guo, Minglan; Ji, Huasong; Yan, Yang; Ouyang, Zhengliang; Huang, Xiaohong; Hang, Youhua; Qin, Qiwei

2012-10-01

Translationally controlled tumor protein (TCTP) is an important molecule involved in multiple biological processes, such as cell growth, cell cycle progression, malignant transformation, and enhancement of the anti-apoptotic activity. In this study, the TCTP from orange-spotted grouper Epinephelus coioides (Ec-TCTP) was cloned and characterized. The full-length cDNA of Ec-TCTP was comprised of 1057 bp with a 510 bp open reading frame that encodes a putative protein of 170 amino acids. Recombinant Ec-TCTP (rEc-TCTP) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-Ec-TCTP serum preparation. The rEc-TCTP fusion protein was demonstrated to possess antioxidant activity, which conferred resistance to H(2)O(2) damage. Quantitative real-time PCR analysis revealed that Ec-TCTP mRNA is predominately expressed in the liver, and the expression was up-regulated in the liver of grouper after viral challenge with Singapore grouper iridovirus (SGIV). Intracellular localization revealed that Ec-TCTP expression was distributed predominantly in the cytoplasm. Although human TCTP has a role in apoptosis regulation, it is not known if grouper TCTP has any role in apoptosis regulation. Strikingly, grouper TCTP, when overexpressed in fathead minnow (FHM) cells, protected them from cell death induced by cycloheximide (CHX). In addition, overexpressed Ec-TCTP in grouper spleen (GS) cells inhibited the replication of SGIV. These results suggest that Ec-TCTP may play a critical role in their response to SGIV infection, through regulation of a cell death pathway that is common to fish and humans. Copyright © 2012 Elsevier Ltd. All rights reserved.

Dynamic proteomics emphasizes the importance of selective mRNA translation and protein turnover during Arabidopsis seed germination.

PubMed

Galland, Marc; Huguet, Romain; Arc, Erwann; Cueff, Gwendal; Job, Dominique; Rajjou, Loïc

2014-01-01

During seed germination, the transition from a quiescent metabolic state in a dry mature seed to a proliferative metabolic state in a vigorous seedling is crucial for plant propagation as well as for optimizing crop yield. This work provides a detailed description of the dynamics of protein synthesis during the time course of germination, demonstrating that mRNA translation is both sequential and selective during this process. The complete inhibition of the germination process in the presence of the translation inhibitor cycloheximide established that mRNA translation is critical for Arabidopsis seed germination. However, the dynamics of protein turnover and the selectivity of protein synthesis (mRNA translation) during Arabidopsis seed germination have not been addressed yet. Based on our detailed knowledge of the Arabidopsis seed proteome, we have deepened our understanding of seed mRNA translation during germination by combining two-dimensional gel-based proteomics with dynamic radiolabeled proteomics using a radiolabeled amino acid precursor, namely [(35)S]-methionine, in order to highlight de novo protein synthesis, stability, and turnover. Our data confirm that during early imbibition, the Arabidopsis translatome keeps reflecting an embryonic maturation program until a certain developmental checkpoint. Furthermore, by dividing the seed germination time lapse into discrete time windows, we highlight precise and specific patterns of protein synthesis. These data refine and deepen our knowledge of the three classical phases of seed germination based on seed water uptake during imbibition and reveal that selective mRNA translation is a key feature of seed germination. Beyond the quantitative control of translational activity, both the selectivity of mRNA translation and protein turnover appear as specific regulatory systems, critical for timing the molecular events leading to successful germination and seedling establishment.

Tissue-specific expression and regulation of the alternatively-spliced forms of lysyl hydroxylase 2 (LH2) in human kidney cells and skin fibroblasts.

PubMed

Walker, Linda C; Overstreet, Mayra A; Yeowell, Heather N

2005-01-01

Lysyl hydroxylases 1, 2, and 3 catalyse the hydroxylation of specific lysines in collagen. A small percentage of these hydroxylysine residues are precursors for the cross-link formation essential for the tensile strength of collagen. Lysyl hydroxylase 2 (LH2) exists as two alternatively-spliced forms; the long transcript (the major ubiquitously-expressed form) includes a 63 bp exon (13A) that is spliced out in the short form (expressed, together with the long form, in human kidney, spleen, liver, and placenta). This study shows that this alternative splicing event can be regulated by both cell density and cycloheximide (CHX). Although only the long form of LH2 is detected in untreated confluent human skin fibroblasts, after 24 h treatment with CHX the short LH2 transcript is also expressed. In kidney cells, in which both LH2 transcripts are equally expressed, the long LH2 transcript is significantly decreased after 24 h CHX treatment, whereas expression of the short transcript is slightly increased. This suggests that, in kidney cells, the splicing mechanism for the inclusion of exon 13A in LH2 requires a newly-synthesized protein factor that is suppressed by CHX, whereas, in skin fibroblasts in which levels of LH2 (long) are unaffected, CHX appears to suppress a factor that inhibits exclusion of exon 13A, thereby promoting expression of LH2 (short). As these alternate transcripts of LH2 may have specificity for hydroxylation of lysines in either telopeptide or helical collagen domains, their relative expression determines the type of cross-links formed, thereby affecting collagen strength. Therefore, any perturbation of the regulation of LH2 splicing could influence the stability of the extracellular matrix and contribute to specific connective tissue disorders.

Anatomical and functional evidence for trace amines as unique modulators of locomotor function in the mammalian spinal cord

PubMed Central

Gozal, Elizabeth A.; O'Neill, Brannan E.; Sawchuk, Michael A.; Zhu, Hong; Halder, Mallika; Chou, Ching-Chieh; Hochman, Shawn

2014-01-01

The trace amines (TAs), tryptamine, tyramine, and β-phenylethylamine, are synthesized from precursor amino acids via aromatic-L-amino acid decarboxylase (AADC). We explored their role in the neuromodulation of neonatal rat spinal cord motor circuits. We first showed that the spinal cord contains the substrates for TA biosynthesis (AADC) and for receptor-mediated actions via trace amine-associated receptors (TAARs) 1 and 4. We next examined the actions of the TAs on motor activity using the in vitro isolated neonatal rat spinal cord. Tyramine and tryptamine most consistently increased motor activity with prominent direct actions on motoneurons. In the presence of N-methyl-D-aspartate, all applied TAs supported expression of a locomotor-like activity (LLA) that was indistinguishable from that ordinarily observed with serotonin, suggesting that the TAs act on common central pattern generating neurons. The TAs also generated distinctive complex rhythms characterized by episodic bouts of LLA. TA actions on locomotor circuits did not require interaction with descending monoaminergic projections since evoked LLA was maintained following block of all Na+-dependent monoamine transporters or the vesicular monoamine transporter. Instead, TA (tryptamine and tyramine) actions depended on intracellular uptake via pentamidine-sensitive Na+-independent membrane transporters. Requirement for intracellular transport is consistent with the TAs having much slower LLA onset than serotonin and for activation of intracellular TAARs. To test for endogenous actions following biosynthesis, we increased intracellular amino acid levels with cycloheximide. LLA emerged and included distinctive TA-like episodic bouts. In summary, we provided anatomical and functional evidence of the TAs as an intrinsic spinal monoaminergic modulatory system capable of promoting recruitment of locomotor circuits independent of the descending monoamines. These actions support their known sympathomimetic function. PMID:25426030

Anatomical and functional evidence for trace amines as unique modulators of locomotor function in the mammalian spinal cord.

PubMed

Gozal, Elizabeth A; O'Neill, Brannan E; Sawchuk, Michael A; Zhu, Hong; Halder, Mallika; Chou, Ching-Chieh; Hochman, Shawn

2014-01-01

The trace amines (TAs), tryptamine, tyramine, and β-phenylethylamine, are synthesized from precursor amino acids via aromatic-L-amino acid decarboxylase (AADC). We explored their role in the neuromodulation of neonatal rat spinal cord motor circuits. We first showed that the spinal cord contains the substrates for TA biosynthesis (AADC) and for receptor-mediated actions via trace amine-associated receptors (TAARs) 1 and 4. We next examined the actions of the TAs on motor activity using the in vitro isolated neonatal rat spinal cord. Tyramine and tryptamine most consistently increased motor activity with prominent direct actions on motoneurons. In the presence of N-methyl-D-aspartate, all applied TAs supported expression of a locomotor-like activity (LLA) that was indistinguishable from that ordinarily observed with serotonin, suggesting that the TAs act on common central pattern generating neurons. The TAs also generated distinctive complex rhythms characterized by episodic bouts of LLA. TA actions on locomotor circuits did not require interaction with descending monoaminergic projections since evoked LLA was maintained following block of all Na(+)-dependent monoamine transporters or the vesicular monoamine transporter. Instead, TA (tryptamine and tyramine) actions depended on intracellular uptake via pentamidine-sensitive Na(+)-independent membrane transporters. Requirement for intracellular transport is consistent with the TAs having much slower LLA onset than serotonin and for activation of intracellular TAARs. To test for endogenous actions following biosynthesis, we increased intracellular amino acid levels with cycloheximide. LLA emerged and included distinctive TA-like episodic bouts. In summary, we provided anatomical and functional evidence of the TAs as an intrinsic spinal monoaminergic modulatory system capable of promoting recruitment of locomotor circuits independent of the descending monoamines. These actions support their known sympathomimetic function.

Assessment of the Incidence of Posttreatment Endodontic Flare-ups in Patients undergoing Single-sitting Root Canal Therapies: A Clinical Study.

PubMed

Priyank, Harsh; Devi, T M Chaitra; Goel, Pallavi; Sahu, Nivedita; Nihalani, Shweta; Shandilya, Ashutosh

2016-10-01

Endodontic therapy is one of the commonly used procedures for treating the teeth affected by various pathologies. One of the major problems for endodontists despite the advancements in the root canal procedures is the posttreatment endodontic flare-ups. Much debate exists regarding the completion of endodontic therapy in a single sitting or multiple sittings. Hence, we assessed the incidence of endodontic flare-ups in patients undergoing single-sitting root canal therapies. The present study included 200 patients who underwent single-sitting endodontic therapy. Clinical details and conditions of each and every tooth of every patient were recorded before and after the completion of endodontic therapy. Irrigation during the root canal procedures was done by 2.5% NaOCl solution in most of the cases while others were irrigated with various combinations of ethylenediaminetetraacetic acid (EDTA) and cycloheximide (CHX) solutions. Follow-up records and readings of the patents were noted and were subjected to statistical analysis. Four groups were formed which divided the patients equally on the basis of their age. Out of 50 patients in the age group of 21 to 30 years, only 4 showed posttreatment endodontic flare-ups, while no endodontic flare-up was recorded in patients with age group of 31 to 50 years. Only two male and four females showed flare-ups postoperatively. A nonsignificant correlation was obtained when flare-up cases were compared on the basis of type of irrigation solution used during canal preparation. Single-sitting endodontic therapy appears to be a successful procedure with good prognosis and minimal posttreatment flare-up results, even in patients with periapical pathologies. Single-sitting root canal procedures can be successfully carried in patients with vital or nonvital pulp tissues and also in patients with periapical lesions.

Increased mature interleukin-1beta (IL-1beta) secretion from THP-1 cells induced by nigericin is a result of activation of p45 IL-1beta-converting enzyme processing.

PubMed

Cheneval, D; Ramage, P; Kastelic, T; Szelestenyi, T; Niggli, H; Hemmig, R; Bachmann, M; MacKenzie, A

1998-07-10

Perregaux and Gabel (Perregaux, D., and Gabel, C. A. (1994) J. Biol. Chem. 269, 15195-15203) reported that potassium depletion of lipopolysaccharide-stimulated mouse macrophages induced by the potassium ionophore, nigericin, leads to the rapid release of mature interleukin-1beta (IL-1beta). We have now shown a similar phenomenon in lipopolysaccharide-stimulated human monocytic leukemia THP-1 cells. Rapid secretion of mature, 17-kDa IL-1beta occurred, in the presence of nigericin (4-16 microM). No effects on the release of tumor necrosis factor-alpha, IL-6, or proIL-1beta were seen. Addition of the irreversible interleukin-1beta-converting enzyme (ICE) inhibitor, Z-Val-Ala-Asp-dichlorobenzoate, or a radicicol analog, inhibited nigericin-induced mature IL-1beta release and activation of p45 ICE precursor. The radicicol analog itself did not inhibit ICE, but markedly, and very rapidly depleted intracellular levels of 31-kDa proIL-1beta. By contrast, dexamethasone, cycloheximide, and the Na+/H+ antiporter inhibitor, 5-(N-ethyl-N-isopropyl)amiloride, had no effect on nigericin-induced release of IL-1beta. We have therefore shown conclusively, for the first time, that nigericin-induced release of IL-1beta is dependent upon activation of p45 ICE processing. So far, the mechanism by which reduced intracellular potassium ion concentration triggers p45 ICE processing is not known, but further investigation in this area could lead to the discovery of novel molecular targets whereby control of IL-1beta production might be effected.

Protein kinase-A-dependent osteoprotegerin production on interleukin-1 stimulation in human gingival fibroblasts is distinct from periodontal ligament fibroblasts

PubMed Central

Hormdee, D; Nagasawa, T; Kiji, M; Yashiro, R; Kobayashi, H; Koshy, G; Noguchi, K; Nitta, H; Ishikawa, I

2005-01-01

Periodontitis, a chronic inflammatory disease, is characterized by increased expression of interleukin (IL)-1 and other inflammatory mediators resulting in extensive osteoclast formation and bone loss. Expression of receptor activator of nuclear factor kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), by osteoblasts is important to regulate osteoclast differentiation. The aim of the present study was to investigate the regulatory effects of IL-1 on RANKL and OPG production by mesenchymal fibroblasts in periodontal tissue. Human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDL) were stimulated with IL-1α with or without protein synthesis inhibitor cycloheximide (CHX), protein kinase A (PKA) inhibitors, protein kinase C (PKC) inhibitors and prostaglandin E2 (PGE2) inhibitor. In some experiments, the cultured cells were directly stimulated with either PKA or PKC activators. In HGF, IL-1α-stimulated OPG mRNA expression was high and could be reduced by CHX. PKA inhibitor completely abrogated IL-1α-induced OPG mRNA expression and OPG production. Endogenous PGE2 further enhanced IL-1α-induced OPG production in HGF. In PDL, RANKL mRNA expression was greatly augmented by IL-1α. IL-1α induced OPG mRNA expression and protein production. PKC inhibitor partially reduced IL-1α-induced OPG production and PKC activator enhanced OPG production in PDL. The IL-1α-stimulated OPG mRNA expression in HGF was greater than PDL. These results provide new evidence for the possible osteoclastogenesis-inhibitory function of HGF through PKA activity pathway. PDL utilized PKC for OPG production. Thus, we emphasize that HGF and PDL have different characteristics of host defence mechanism against inflammatory process. PMID:16297161

Temporal characteristics of gustatory responses in rat parabrachial neurons vary by stimulus and chemosensitive neuron type.

PubMed

Geran, Laura; Travers, Susan

2013-01-01

It has been demonstrated that temporal features of spike trains can increase the amount of information available for gustatory processing. However, the nature of these temporal characteristics and their relationship to different taste qualities and neuron types are not well-defined. The present study analyzed the time course of taste responses from parabrachial (PBN) neurons elicited by multiple applications of "sweet" (sucrose), "salty" (NaCl), "sour" (citric acid), and "bitter" (quinine and cycloheximide) stimuli in an acute preparation. Time course varied significantly by taste stimulus and best-stimulus classification. Across neurons, the ensemble code for the three electrolytes was similar initially but quinine diverged from NaCl and acid during the second 500 ms of stimulation and all four qualities became distinct just after 1s. This temporal evolution was reflected in significantly broader tuning during the initial response. Metric space analyses of quality discrimination by individual neurons showed that increases in information (H) afforded by temporal factors was usually explained by differences in rate envelope, which had a greater impact during the initial 2s (22.5% increase in H) compared to the later response (9.5%). Moreover, timing had a differential impact according to cell type, with between-quality discrimination in neurons activated maximally by NaCl or citric acid most affected. Timing was also found to dramatically improve within-quality discrimination (80% increase in H) in neurons that responded optimally to bitter stimuli (B-best). Spikes from B-best neurons were also more likely to occur in bursts. These findings suggest that among PBN taste neurons, time-dependent increases in mutual information can arise from stimulus- and neuron-specific differences in response envelope during the initial dynamic period. A stable rate code predominates in later epochs.

Temporal regulation of expression of immediate early and second phase transcripts by endothelin-1 in cardiomyocytes

PubMed Central

Cullingford, Timothy E; Markou, Thomais; Fuller, Stephen J; Giraldo, Alejandro; Pikkarainen, Sampsa; Zoumpoulidou, Georgia; Alsafi, Ali; Ekere, Collins; Kemp, Timothy J; Dennis, Jayne L; Game, Laurence; Sugden, Peter H; Clerk, Angela

2008-01-01

Background Endothelin-1 stimulates Gq protein-coupled receptors to promote proliferation in dividing cells or hypertrophy in terminally differentiated cardiomyocytes. In cardiomyocytes, endothelin-1 rapidly (within minutes) stimulates protein kinase signaling, including extracellular-signal regulated kinases 1/2 (ERK1/2; though not ERK5), with phenotypic/physiological changes developing from approximately 12 h. Hypertrophy is associated with changes in mRNA/protein expression, presumably consequent to protein kinase signaling, but the connections between early, transient signaling events and developed hypertrophy are unknown. Results Using microarrays, we defined the early transcriptional responses of neonatal rat cardiomyocytes to endothelin-1 over 4 h, differentiating between immediate early gene (IEG) and second phase RNAs with cycloheximide. IEGs exhibited differential temporal and transient regulation, with expression of second phase RNAs within 1 h. Of transcripts upregulated at 30 minutes encoding established proteins, 28 were inhibited >50% by U0126 (which inhibits ERK1/2/5 signaling), with 9 inhibited 25-50%. Expression of only four transcripts was not inhibited. At 1 h, most RNAs (approximately 67%) were equally changed in total and polysomal RNA with approximately 17% of transcripts increased to a greater extent in polysomes. Thus, changes in expression of most protein-coding RNAs should be reflected in protein synthesis. However, approximately 16% of transcripts were essentially excluded from the polysomes, including some protein-coding mRNAs, presumably inefficiently translated. Conclusion The phasic, temporal regulation of early transcriptional responses induced by endothelin-1 in cardiomyocytes indicates that, even in terminally differentiated cells, signals are propagated beyond the primary signaling pathways through transcriptional networks leading to phenotypic changes (that is, hypertrophy). Furthermore, ERK1/2 signaling plays a major role in this response. PMID:18275597

Platelet-rich plasma inhibits the apoptosis of highly adipogenic homogeneous preadipocytes in an in vitro culture system.

PubMed

Fukaya, Yoshitaka; Kuroda, Masayuki; Aoyagi, Yasuyuki; Asada, Sakiyo; Kubota, Yoshitaka; Okamoto, Yoshitaka; Nakayama, Toshinori; Saito, Yasushi; Satoh, Kaneshige; Bujo, Hideaki

2012-05-31

Auto-transplantation of adipose tissue is commonly used for the treatment of tissue defects in plastic surgery. The survival of the transplanted adipose tissue is not always constant, and one of reasons is the accelerated apoptosis of the implanted preadipocytes. We have recently established highly homogeneous preadipocytes, named ccdPAs. The aim of the current study was to evaluate the regulation of the potency of platelet-rich plasma (PRP) on the apoptosis of ccdPAs in vitro. PRP stimulated the proliferation of the preadipocytes in a dose-dependent manner, and the stimulatory activity of 2% PRP was significantly higher than that of 2% FBS or 2% platelet-poor plasma (PPP). The presence of 2% PRP significantly inhibited serum starvation- or TNF-α/cycloheximide-induced apoptosis in comparison to 2% FBS or 2% PPP. DAPK1 and Bcl-2-interacting mediator of cell death (BIM) mRNAs were reduced in the preadipocytes cultured with 2% PRP in comparison to those cultured in 2% FBS. The gene expression levels were significantly higher in cells cultured without serum in comparison to cells cultured with 2% FBS, and the levels in the cells with 2% PRP were reduced to 5-10% of those in the cells without serum. These results indicated that ccdPAs exhibit anti- apoptotic activities, in addition to increased proliferation, when cultured in 2% PRP in comparison to the same concentration of FBS, and that this was accompanied with reduced levels of DAPK1 and BIM mRNA expression in in vitro culture. PRP may improve the outcome of transplantation of adipose tissue by enhancing the anti-apoptotic activities of the implanted preadipocytes.

MFS Transporters and GABA Metabolism Are Involved in the Self-Defense Against DON in Fusarium graminearum

PubMed Central

Wang, Qinhu; Chen, Daipeng; Wu, Mengchun; Zhu, Jindong; Jiang, Cong; Xu, Jin-Rong; Liu, Huiquan

2018-01-01

Trichothecene mycotoxins, such as deoxynivalenol (DON) produced by the fungal pathogen, Fusarium graminearum, are not only important for plant infection but are also harmful to human and animal health. Trichothecene targets the ribosomal protein Rpl3 that is conserved in eukaryotes. Hence, a self-defense mechanism must exist in DON-producing fungi. It is reported that TRI (trichothecene biosynthesis) 101 and TRI12 are two genes responsible for self-defense against trichothecene toxins in Fusarium. In this study, however, we found that simultaneous disruption of TRI101 and TRI12 has no obvious influence on DON resistance upon exogenous DON treatment in F. graminearum, suggesting that other mechanisms may be involved in self-defense. By using RNA-seq, we identified 253 genes specifically induced in DON-treated cultures compared with samples from cultures treated or untreated with cycloheximide, a commonly used inhibitor of eukaryotic protein synthesis. We found that transporter genes are significantly enriched in this group of DON-induced genes. Of those genes, 15 encode major facilitator superfamily transporters likely involved in mycotoxin efflux. Significantly, we found that genes involved in the metabolism of gamma-aminobutyric acid (GABA), a known inducer of DON production in F. graminearum, are significantly enriched among the DON-induced genes. The GABA biosynthesis gene PROLINE UTILIZATION 2-2 (PUT2-2) is downregulated, while GABA degradation genes are upregulated at least twofold upon treatment with DON, resulting in decreased levels of GABA. Taken together, our results suggest that transporters influencing DON efflux are important for self-defense and that GABA mediates the balance of DON production and self-defense in F. graminearum. PMID:29706976

MFS Transporters and GABA Metabolism Are Involved in the Self-Defense Against DON in Fusarium graminearum.

PubMed

Wang, Qinhu; Chen, Daipeng; Wu, Mengchun; Zhu, Jindong; Jiang, Cong; Xu, Jin-Rong; Liu, Huiquan

2018-01-01

Trichothecene mycotoxins, such as deoxynivalenol (DON) produced by the fungal pathogen, Fusarium graminearum , are not only important for plant infection but are also harmful to human and animal health. Trichothecene targets the ribosomal protein Rpl3 that is conserved in eukaryotes. Hence, a self-defense mechanism must exist in DON-producing fungi. It is reported that TRI (trichothecene biosynthesis) 101 and TRI12 are two genes responsible for self-defense against trichothecene toxins in Fusarium . In this study, however, we found that simultaneous disruption of TRI101 and TRI12 has no obvious influence on DON resistance upon exogenous DON treatment in F. graminearum , suggesting that other mechanisms may be involved in self-defense. By using RNA-seq, we identified 253 genes specifically induced in DON-treated cultures compared with samples from cultures treated or untreated with cycloheximide, a commonly used inhibitor of eukaryotic protein synthesis. We found that transporter genes are significantly enriched in this group of DON-induced genes. Of those genes, 15 encode major facilitator superfamily transporters likely involved in mycotoxin efflux. Significantly, we found that genes involved in the metabolism of gamma-aminobutyric acid (GABA), a known inducer of DON production in F. graminearum , are significantly enriched among the DON-induced genes. The GABA biosynthesis gene PROLINE UTILIZATION 2-2 ( PUT2-2 ) is downregulated, while GABA degradation genes are upregulated at least twofold upon treatment with DON, resulting in decreased levels of GABA. Taken together, our results suggest that transporters influencing DON efflux are important for self-defense and that GABA mediates the balance of DON production and self-defense in F. graminearum .

Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bouhelal, R.; Bockaert, J.; Mermet-Bouvier, R.

1987-06-25

We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC/sup 3/H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, /sup 3/H, /sup 13/C, /sup 15/N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher massmore » (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme. Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control. After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value.« less

Synthesis, antibacterial activity, synergistic effect, cytotoxicity, docking and molecular dynamics of benzimidazole analogues.

PubMed

Srivastava, Ritika; Gupta, Sunil K; Naaz, Farha; Singh, Anuradha; Singh, Vishal K; Verma, Rajesh; Singh, Nidhi; Singh, Ramendra K

2018-05-24

A series of 2-Cl-benzimidazole derivatives was synthesized and assessed for antibacterial activity. Antibacterial results indicated that compounds 2d, 2e, 3a, 3b, 3c, 4d and 4e showed promising activity against B. cerus, S. aureus and P. aeruginosa (MIC: 6.2 μg/mL) and excellent efficacy against E. coli (MIC: 3.1 μg/mL). Furthermore, compounds 3d and 3e displayed better activity (MIC: 3.1 μg/mL) than the reference drugs chloramphenicol and cycloheximide against gram positive and gram negative bacterial strains. The compounds 3d-e also showed better activity than the reference drug paromomycin against B. cerus and P. aeruginosa and showed similar inhibition pattern against S. aureus and E. coli. (MIC: 3.1 μg/mL). Studies on fractional inhibitory concentration (FIC) determination of compounds 1a-e, 2a-c, 4a-c and the reference antibiotic via combination approach revealed a synergistic effect as the MIC values were lowered up to 1 / 8 th to 1 / 33 rd of the original MIC. In-vitro cytotoxicity study indicated that 2-Cl-benzimidazole derivatives showed less toxicity than the reference used against PBM, CEM and Vero cell lines. Docking studies and MD simulations of compounds on bacterial protein (eubacterial ribosomal decoding A site, PDB: 1j7t) have been conducted to find the possible mode of action of the molecules. In silico ADMET evaluations of compounds 3d and 3e showed promising results comparable to the reference drugs used in this study. Copyright © 2018 Elsevier Ltd. All rights reserved.

Palmitoylation of caveolin-1 in endothelial cells is post-translational but irreversible

NASA Technical Reports Server (NTRS)

Parat, M. O.; Fox, P. L.

2001-01-01

Caveolin-1 is a palmitoylated protein involved in assembly of signaling molecules in plasma membrane subdomains termed caveolae and in intracellular cholesterol transport. Three cysteine residues in the C terminus of caveolin-1 are subject to palmitoylation, which is not necessary for caveolar targeting of caveolin-1. Protein palmitoylation is a post-translational and reversible modification that may be regulated and that in turn may regulate conformation, membrane association, protein-protein interactions, and intracellular localization of the target protein. We have undertaken a detailed analysis of [(3)H]palmitate incorporation into caveolin-1 in aortic endothelial cells. The linkage of palmitate to caveolin-1 was hydroxylamine-sensitive and thus presumably a thioester bond. However, contrary to expectations, palmitate incorporation was blocked completely by the protein synthesis inhibitors cycloheximide and puromycin. In parallel experiments to show specificity, palmitoylation of aortic endothelial cell-specific nitric-oxide synthase was unaffected by these reagents. Inhibitors of protein trafficking, brefeldin A and monensin, blocked caveolin-1 palmitoylation, indicating that the modification was not cotranslational but rather required caveolin-1 transport from the endoplasmic reticulum and Golgi to the plasma membrane. In addition, immunophilin chaperones that form complexes with caveolin-1, i.e. FK506-binding protein 52, cyclophilin A, and cyclophilin 40, were not necessary for caveolin-1 palmitoylation because agents that bind immunophilins did not inhibit palmitoylation. Pulse-chase experiments showed that caveolin-1 palmitoylation is essentially irreversible because the release of [(3)H]palmitate was not significant even after 24 h. These results show that [(3)H]palmitate incorporation is limited to newly synthesized caveolin-1, not because incorporation only occurs during synthesis but because the continuous presence of palmitate on caveolin-1 prevents subsequent repalmitoylation.

Cooling towers--a potential environmental source of slow-growing mycobacterial species.

PubMed

Black, Walter C; Berk, Sharon G

2003-01-01

Over the last decade a rise in the frequency of disease caused by nontuberculous mycobacteria (NTM) has occurred, especially among AIDS patients. The lack of evidence for person-to-person transmission indicates the environment is a source of infection. The ecology and environmental sources of NTMs are poorly understood, and many pathogenic strains have not been observed outside of clinical cases. Several species of NTMs have been reported from treated water distribution systems; however, one type of manmade environment that has not been examined for mycobacteria is that of cooling towers of air-conditioning systems. Such environments not only harbor a variety of microbial species, they also disseminate them in aerosols. The present investigation examined nine cooling towers from various locations in the United States. Cooling tower water was concentrated, treated with cetylpyridinium chloride, and plated onto Middlebrook 7H10 agar supplemented with OADC and cycloheximide. Colonies presumed to be mycobacterial species were isolated and acid-fast stained. Identification was made by amplifying and sequencing 1450 bp fragments of the 16S rRNA gene in both directions, and comparing resulting sequences with those in GenBank. Results showed that at least 75% of tower samples contained NTMs, and most of the isolates closely matched known mycobacterial pathogens. Isolates most closely matched the following GenBank sequences: Mycobacterium intracellulare, M. szulgai, M. bohemicum, M. gordonae, M. nonchromogenicum, and M. n. sp. "Fuerth 1999." This is the first report of specific NTMs in cooling tower water, and the first report of M. n. sp. "Fuerth 1999" from any environmental sample. Although cooling towers have a relatively high pH, they may favor the growth and dissemination of such potential pathogens, and future epidemiologic investigations should consider cooling towers as possible environmental sources of mycobacteria.

The pnk/pnl gene (ORF 86) of Autographa californica nucleopolyhedrovirus is a non-essential, immediate early gene.

PubMed

Durantel, D; Croizier, L; Ayres, M D; Croizier, G; Possee, R D; López-Ferber, M

1998-03-01

Autographa californica nucleopolyhedrovirus (AcMNPV) ORF 86, located within the HindIII C fragment, potentially encodes a protein which shares sequence similarity with two T4 bacteriophage gene products, RNA ligase and polynucleotide kinase. This AcMNPV gene has been designated pnk/pnl but has yet to be assigned a function in virus replication. It has been classified as an immediate early virus gene, since the promoter was active in uninfected insect cells and mRNA transcripts were detectable from 4 to 48 h post-infection and in the presence of cycloheximide or aphidicolin in virus-infected cells. The extremities of the transcript have been mapped by primer extension and 3' RACE-PCR to positions -18 from the translational start codon and +15 downstream of the stop codon. The function of pnk/pnl was investigated by producing a recombinant virus (Acdel86lacZ) with the coding region replaced with that of lacZ. This virus replicated normally in Spodoptera frugiperda (Sf 21) cells, indicating that pnk/pnl is not essential for propagation in these cells. Virus protein production in Acdel86lacZ-infected Sf 21 cells also appeared to be unaffected, with normal synthesis of the IE-1, GP64, VP39 and polyhedrin proteins. Shut-down of host protein synthesis was not abolished in recombinant infection. When other baculovirus genomes were examined for the presence of pnk/pnl by restriction enzyme digestion and PCR, a deletion was found in AcMNPV 1.2, Galleria mellonella NPV (GmMNPV) and Bombyx mori NPV (BmNPV), suggesting that in many isolates this gene has either never been acquired or has been lost during genome evolution. This is one of the first baculovirus immediate early genes that appears to be nonessential for virus survival.

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor.

PubMed

Stahl, Sebastian; da Silva Mateus Seidl, Ana Rita; Ducret, Axel; Kux van Geijtenbeek, Sabine; Michel, Sven; Racek, Tomas; Birzele, Fabian; Haas, Alexander K; Rueger, Ruediger; Gerg, Michael; Niederfellner, Gerhard; Pastan, Ira; Brinkmann, Ulrich

2015-08-25

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1-DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.

Comparative induction of nitrate reductase by nitrate and nitrite in barley leaves

NASA Technical Reports Server (NTRS)

Aslam, M.; Rosichan, J. L.; Huffaker, R. C.

1987-01-01

The comparative induction of nitrate reductase (NR) by ambient NO3- and NO2- as a function of influx, reduction (as NR was induced) and accumulation in detached leaves of 8-day-old barley (Hordeum vulgare L.) seedlings was determined. The dynamic interaction of NO3- influx, reduction and accumulation on NR induction was shown. The activity of NR, as it was induced, influenced its further induction by affecting the internal concentration of NO3-. As the ambient concentration of NO3- increased, the relative influences imposed by influx and reduction on NO3- accumulation changed with influx becoming a more predominant regulant. Significant levels of NO3- accumulated in NO2(-)-fed leaves. When the leaves were supplied cycloheximide or tungstate along with NO2-, about 60% more NO3- accumulated in the leaves than in the absence of the inhibitors. In NO3(-)-supplied leaves NR induction was observed at an ambient concentration of as low as 0.02 mM. No NR induction occurred in leaves supplied with NO2- until the ambient NO2- concentration was 0.5 mM. In fact, NR induction from NO2- solutions was not seen until NO3- was detected in the leaves. The amount of NO3- accumulating in NO2(-)-fed leaves induced similar levels of NR as did equivalent amounts of NO3- accumulating from NO3(-)-fed leaves. In all cases the internal concentration of NO3-, but not NO2-, was highly correlated with the amount of NR induced. The evidence indicated that NO3- was a more likely inducer of NR than was NO2-.

Biomechanical, biochemical, and morphological mechanisms of heat shock-mediated germination in Carica papaya seed.

PubMed

Webster, Rachel E; Waterworth, Wanda M; Stuppy, Wolfgang; West, Christopher E; Ennos, Roland; Bray, Clifford M; Pritchard, Hugh W

2016-12-01

Carica papaya (papaya) seed germinate readily fresh from the fruit, but desiccation induces a dormant state. Dormancy can be released by exposure of the hydrated seed to a pulse of elevated temperature, typical of that encountered in its tropical habitat. Carica papaya is one of only a few species known to germinate in response to heat shock (HS) and we know little of the mechanisms that control germination in tropical ecosystems. Here we investigate the mechanisms that mediate HS-induced stimulation of germination in pre-dried and re-imbibed papaya seed. Exogenous gibberellic acid (GA 3 ≥250 µM) overcame the requirement for HS to initiate germination. However, HS did not sensitise seeds to GA 3 , indicative that it may act independently of GA biosynthesis. Seed coat removal also overcame desiccation-imposed dormancy, indicative that resistance to radicle emergence is coat-imposed. Morphological and biomechanical studies identified that neither desiccation nor HS alter the physical structure or the mechanical strength of the seed coat. However, cycloheximide prevented both seed coat weakening and germination, implicating a requirement for de novo protein synthesis in both processes. The germination antagonist abscisic acid prevented radicle emergence but had no effect on papaya seed coat weakening. Desiccation therefore appears to reduce embryo growth potential, which is reversed by HS, without physically altering the mechanical properties of the seed coat. The ability to germinate in response to a HS may confer a competitive advantage to C. papaya, an opportunistic pioneer species, through detection of canopy removal in tropical forests. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Herpes simplex virus 2 modulates apoptosis and stimulates NF-{kappa}B nuclear translocation during infection in human epithelial HEp-2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yedowitz, Jamie C.; Blaho, John A.

2005-11-25

Virus-mediated apoptosis is well documented in various systems, including herpes simplex virus 1 (HSV-1). HSV-2 is closely related to HSV-1 but its apoptotic potential during infection has not been extensively scrutinized. We report that (i) HEp-2 cells infected with HSV-2(G) triggered apoptosis, assessed by apoptotic cellular morphologies, oligosomal DNA laddering, chromatin condensation, and death factor processing when a translational inhibitor (CHX) was added at 3 hpi. Thus, HSV-2 induced apoptosis but was unable to prevent the process from killing cells. (ii) Results from a time course of CHX addition experiment indicated that infected cell protein produced between 3 and 5more » hpi, termed the apoptosis prevention window, are required for blocking virus-induced apoptosis. This corresponds to the same prevention time frame as reported for HSV-1. (iii) Importantly, CHX addition prior to 3 hpi led to less apoptosis than that at 3 hpi. This suggests that proteins produced immediately upon infection are needed for efficient apoptosis induction by HSV-2. This finding is different from that observed previously with HSV-1. (iv) Infected cell factors produced during the HSV-2(G) prevention window inhibited apoptosis induced by external TNF{alpha} plus cycloheximide treatment. (v) NF-{kappa}B translocated to nuclei and its presence in nuclei correlated with apoptosis prevention during HSV-2(G) infection. (vi) Finally, clinical HSV-2 isolates induced and prevented apoptosis in HEp-2 cells in a manner similar to that of laboratory strains. Thus, while laboratory and clinical HSV-2 strains are capable of modulating apoptosis in human HEp-2 cells, the mechanism of HSV-2 induction of apoptosis differs from that of HSV-1.« less

CDKN1C mutation affecting the PCNA-binding domain as a cause of familial Russell Silver syndrome.

PubMed

Brioude, F; Oliver-Petit, I; Blaise, A; Praz, F; Rossignol, S; Le Jule, M; Thibaud, N; Faussat, A-M; Tauber, M; Le Bouc, Y; Netchine, I

2013-12-01

Russell Silver syndrome (RSS) leads to prenatal and postnatal growth retardation. About 55% of RSS patients present a loss-of-methylation of the paternal ICR1 domain on chromosome 11p15. CDKN1C is a cell proliferation inhibitor encoded by an imprinted gene in the 11p15 ICR2 domain. CDKN1C mutations lead to Beckwith Wiedemann syndrome (BWS, overgrowth syndrome) and in IMAGe syndrome which associates growth retardation and adrenal insufficiency. We searched for CDKN1C mutations in a cohort of clinically diagnosed RSS patients with no molecular anomaly. The coding sequence and intron-exon boundaries of CDKN1C were analysed in 97 RSS patients. The impact of CDKN1C variants on the cell cycle in vitro were determined by flow cytometry. Stability of CDKN1C was studied by western immunoblotting after inhibition of translation with cycloheximide. We identified the novel c.836G>[G;T] (p.Arg279Leu) mutation in a familial case of intrauterine growth retardation (IUGR) with RSS phenotype and no evidence of IMAGe. All the RSS patients inherited this mutation from their mothers (consistent with monoallelic expression from the maternal allele of the gene). A mutation of this amino acid (p.Arg279Pro) has been reported in cases of IMAGe. Functional analysis showed that Arg279Leu (RSS) did not affect the cell cycle, whereas the Arg279Pro mutation (IMAGe) led to a gain of function. Arg279Leu (RSS) led to an increased stability which could explain an increased activity of CDKN1C. CDKN1C mutations cause dominant maternally transmitted RSS, completing the molecular mirror with BWS. CDKN1C should be investigated in cases with family history of RSS.

Restriction point control of cell growth by a labile protein: evidence for increased stability in transformed cells.

PubMed Central

Campisi, J; Medrano, E E; Morreo, G; Pardee, A B

1982-01-01

It has been proposed that animal cells must accumulate a labile protein(s) before they can pass the restriction (R) point in the G1 phase of the cell cycle [Rossow, P. W., Riddle, V. G. H. & Pardee, A. B. (1979) Proc. Natl. Acad. Sci. USA 76, 4446--4450]. Here, we present evidence that this R protein acquires increased stability in transformed 3T3 cells, thereby allowing these cells to continue growth under conditions that arrest untransformed cells. Low doses of cycloheximide or histidinol drastically reduced the rate at which normal 3T3 (A31) fibroblasts in early G1 could enter DNA synthesis. These drugs had less effect on entry of two tumorigenic A31 derivatives, BPA31 and SVA31, in S, although measurement of [3H]leucine incorporation showed that the inhibitors were equally effective in the three cell lines. The hypothesis is that the transformed lines are less sensitive because moderate inhibition of their R protein synthesis is compensated by lower rates of protein degradation. To test this idea, we completely inhibited cytoplasmic protein synthesis for several hours shortly before A31 and BPA31 cells had reached the R point. After removal of inhibitor, A31 cells showed delays in the onset of S that were in excess of the inhibitor pulse, consistent with decay of labile protein during the pulse. BPA31 cells showed no excess delays, suggesting a much more stable R protein. The half-life of the R protein was estimated as 2.5 hr in A31 cells, indicating that, in these cells, R protein synthesis starts at the beginning of G1. In the BPA31 cells the R protein showed no signs of decay for at least 8 hr. PMID:6952194

Induction of hepatic and renal ornithine decarboxylase by cobalt and other metal ions in rats.

PubMed Central

Yoshida, T; Numazawa, S; Kuroiwa, Y

1986-01-01

We previously showed that Cd2+ is able to induce hepatic and renal ornithine decarboxylase (ODC). In addition to Cd2+, the administration of Co2+ and other metal ions such as Se2+, Zn2+ and Cr2+ produced a significant increase of hepatic and/or renal ODC activity. Of the metal ions used in this study, Co2+ produced the greatest increase of ODC activity. The maximum increases in hepatic and renal ODC activity, to respectively 70 and 14 times the control values in male rats, were observed 6 h after the administration of Co2+. A similar response was seen in the liver, but not in the kidney, of female rats. Thereafter, ODC activity gradually returned to control values in the liver, but it was profoundly decreased to 7% of the control value at 24 h in the kidney. The pretreatment of animals with either actinomycin D or cycloheximide almost completely blocked the Co2+-mediated increase of ODC activity. Co2+ complexed with either cysteine or glutathione (GSH) failed to induce ODC. Depletion of hepatic GSH content by treatment of rats with diethyl maleate greatly enhanced the inducing effect of Co2+ on ODC. The inhibitors of ODC, 1,3-diaminopropane and alpha-difluoromethylornithine, were able to inhibit the induction of the enzyme, without affecting the induction of haem oxygenase by Co2+. Methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, significantly inhibited the Co2+-mediated induction of both ODC and haem oxygenase. It is suggested that the inducing effects of Co2+ on ODC and haem oxygenase are brought about in a similar manner. PMID:3754136

Ethylene regulates phosphorus remobilization and expression of a phosphate transporter (PhPT1) during petunia corolla senescence

PubMed Central

Chapin, Laura J.; Jones, Michelle L.

2009-01-01

The programmed degradation of macromolecules during petal senescence allows the plant to remobilize nutrients from dying to developing tissues. Ethylene is involved in regulating the timing of nucleic acid degradation in petunia, but it is not clear if ethylene has a role in the remobilization of phosphorus during petal senescence. To investigate ethylene's role in nutrient remobilization, the P content of petals (collectively called the corolla) during early development and senescence was compared in ethylene-sensitive wild type Petunia×hybrida ‘Mitchell Diploid’ (MD) and transgenic petunias with reduced sensitivity to ethylene (35S::etr1-1). When compared to the total P content of corollas on the day of flower opening (the early non-senescing stage), P in MD corollas had decreased 74% by the late stage of senescence (advanced wilting). By contrast, P levels were only reduced by an average of 32% during etr1-1 corolla (lines 44568 and Z00-35-10) senescence. A high-affinity phosphate transporter, PhPT1 (PhPht1;1), was cloned from senescing petunia corollas by RT-PCR. PhPT1 expression was up-regulated during MD corolla senescence and a much smaller increase was detected during the senescence of etr1-1 petunia corollas. PhPT1 mRNA levels showed a rapid increase in detached corollas (treated at 1 d after flower opening) following treatment with low levels of ethylene (0.1 μl l-1). Transcripts accumulated in the presence of the protein synthesis inhibitor, cycloheximide, indicating that PhPT1 is a primary ethylene response gene. PhPT1 is a putative phosphate transporter that may function in Pi translocation during senescence. PMID:19380421

Cloning and regulation of rat tissue inhibitor of metalloproteinases-2 in osteoblastic cells

NASA Technical Reports Server (NTRS)

Cook, T. F.; Burke, J. S.; Bergman, K. D.; Quinn, C. O.; Jeffrey, J. J.; Partridge, N. C.

1994-01-01

Rat tissue inhibitor of metalloproteinases-2 (TIMP-2) was cloned from a UMR 106-01 rat osteoblastic osteosarcoma cDNA library. The 969-bp full-length clone demonstrates 98 and 86% sequence identity to human TIMP-2 at the amino acid and nucleic acid levels, respectively. Parathyroid hormone (PTH), at 10(-8) M, stimulates an approximately twofold increase in both the 4.2- and 1.0-kb transcripts over basal levels in UMR cells after 24 h of exposure. The PTH stimulation of TIMP-2 transcripts was not affected by the inhibitor of protein synthesis, cycloheximide (10(-5) M), suggesting a primary effect of the hormone. This is in contradistinction to regulation of interstitial collagenase (matrix metalloproteinase-1) by PTH in these same cells. Nuclear run-on assays demonstrate that PTH causes an increase in TIMP-2 transcription that parallels the increase in message levels. Parathyroid hormone, in its stimulation of TIMP-2 mRNA, appears to act through a signal transduction pathway involving protein kinase A (PKA) since the increase in TIMP-2 mRNA is reproduced by treatment with the cAMP analogue, 8-bromo-cAMP (5 x 10(-3) M). The protein kinase C and calcium pathways do not appear to be involved due to the lack of effect of phorbol 12-myristate 13-acetate (2.6 x 10(-6) M) and the calcium ionophore, ionomycin (10(-7) M), on TIMP-2 transcript abundance. In this respect, regulation of TIMP-2 and collagenase in osteoblastic cells by PTH are similar. However, we conclude that since stimulation of TIMP-2 transcription is a primary event, the PKA pathway must be responsible for a direct increase in transcription of this gene.

Dynamin-dependent amino acid endocytosis activates mechanistic target of rapamycin complex 1 (mTORC1).

PubMed

Shibutani, Shusaku; Okazaki, Hana; Iwata, Hiroyuki

2017-11-03

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of protein synthesis and potential target for modifying cellular metabolism in various conditions, including cancer and aging. mTORC1 activity is tightly regulated by the availability of extracellular amino acids, and previous studies have revealed that amino acids in the extracellular fluid are transported to the lysosomal lumen. There, amino acids induce recruitment of cytoplasmic mTORC1 to the lysosome by the Rag GTPases, followed by mTORC1 activation by the small GTPase Ras homolog enriched in brain (Rheb). However, how the extracellular amino acids reach the lysosomal lumen and activate mTORC1 remains unclear. Here, we show that amino acid uptake by dynamin-dependent endocytosis plays a critical role in mTORC1 activation. We found that mTORC1 is inactivated when endocytosis is inhibited by overexpression of a dominant-negative form of dynamin 2 or by pharmacological inhibition of dynamin or clathrin. Consistently, the recruitment of mTORC1 to the lysosome was suppressed by the dynamin inhibition. The activity and lysosomal recruitment of mTORC1 were rescued by increasing intracellular amino acids via cycloheximide exposure or by Rag overexpression, indicating that amino acid deprivation is the main cause of mTORC1 inactivation via the dynamin inhibition. We further show that endocytosis inhibition does not induce autophagy even though mTORC1 inactivation is known to strongly induce autophagy. These findings open new perspectives for the use of endocytosis inhibitors as potential agents that can effectively inhibit nutrient utilization and shut down the upstream signals that activate mTORC1. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Metabotropic glutamate receptor I (mGluR1) antagonism impairs cocaine-induced conditioned place preference via inhibition of protein synthesis.

PubMed

Yu, Fei; Zhong, Peng; Liu, Xiaojie; Sun, Dalong; Gao, Hai-Qing; Liu, Qing-Song

2013-06-01

Antagonism of group I metabotropic glutamate receptors (mGluR1 and mGluR5) reduces behavioral effects of drugs of abuse, including cocaine. However, the underlying mechanisms remain poorly understood. Activation of mGluR5 increases protein synthesis at synapses. Although mGluR5-induced excessive protein synthesis has been implicated in the pathology of fragile X syndrome, it remains unknown whether group I mGluR-mediated protein synthesis is involved in any behavioral effects of drugs of abuse. We report that group I mGluR agonist DHPG induced more pronounced initial depression of inhibitory postsynaptic currents (IPSCs) followed by modest long-term depression (I-LTD) in dopamine neurons of rat ventral tegmental area (VTA) through the activation of mGluR1. The early component of DHPG-induced depression of IPSCs was mediated by the cannabinoid CB1 receptors, while DHPG-induced I-LTD was dependent on protein synthesis. Western blotting analysis indicates that mGluR1 was coupled to extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) signaling pathways to increase translation. We also show that cocaine conditioning activated translation machinery in the VTA via an mGluR1-dependent mechanism. Furthermore, intra-VTA microinjections of mGluR1 antagonist JNJ16259685 and protein synthesis inhibitor cycloheximide significantly attenuated or blocked the acquisition of cocaine-induced conditioned place preference (CPP) and activation of translation elongation factors. Taken together, these results suggest that mGluR1 antagonism inhibits de novo protein synthesis; this effect may block the formation of cocaine-cue associations and thus provide a mechanism for the reduction in CPP to cocaine.

Effects of deoxynivalenol on content of chloroplast pigments in barley leaf tissues.

PubMed

Bushnell, W R; Perkins-Veazie, P; Russo, V M; Collins, J; Seeland, T M

2010-01-01

To understand further the role of deoxynivalenol (DON) in development of Fusarium head blight (FHB), we investigated effects of the toxin on uninfected barley tissues. Leaf segments, 1 to 1.2 cm long, partially stripped of epidermis were floated with exposed mesophyll in contact with DON solutions. In initial experiments with the leaf segments incubated in light, DON at 30 to 90 ppm turned portions of stripped tissues white after 48 to 96 h. The bleaching effect was greatly enhanced by addition of 1 to 10 mM Ca(2+), so that DON at 10 to 30 ppm turned virtually all stripped tissues white within 48 h. Content of chlorophylls a and b and of total carotenoid pigment was reduced. Loss of electrolytes and uptake of Evans blue indicated that DON had a toxic effect, damaging plasmalemmas in treated tissues before chloroplasts began to lose pigment. When incubated in the dark, leaf segments also lost electrolytes, indicating DON was toxic although the tissues remained green. Thus, loss of chlorophyll in light was due to photobleaching and was a secondary effect of DON, not required for toxicity. In contrast to bleaching effects, some DON treatments that were not toxic kept tissues green without bleaching or other signs of injury, indicating senescence was delayed compared with slow yellowing of untreated leaf segments. Cycloheximide, which like DON, inhibits protein synthesis, also bleached some tissues and delayed senescence of others. Thus, the effects of DON probably relate to its ability to inhibit protein synthesis. With respect to FHB, the results suggest DON may have multiple roles in host cells of infected head tissues, including delayed senescence in early stages of infection and contributing to bleaching and death of cells in later stages.

Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila.

PubMed

Köhler, R; Bubert, A; Goebel, W; Steinert, M; Hacker, J; Bubert, B

2000-01-01

The gene encoding the green fluorescent protein (GFP) was used as a reporter gene in Legionella pneumophila. To analyze GFP expression in Legionella, transcriptional fusions of gfp with the Legionella-specific mip (Macrophage Infectivity Potentiator) promoter (P(mip)) and the sod (SuperOxide Dismutase) promoter (P(sod)) derived from Listeria monocytogenes were constructed. Following transformation into the virulent L. pneumophila strain JR 32, strong GFP-mediated fluorescence was detected with both plasmids, although the sod promoter was associated with a 1ten-fold higher intensity. No fluorescence was observed in L. pneumophila transformed with the promoterless gfp gene. Comparison of fluorescence yields between various L. pneumophila strains that differ in their virulence characteristics and were transformed with the P(mip)-gfp carrying plasmid revealed no differences in GFP expression. Infection studies using Acanthamoeba castellanii as host and recombinant L. pneumophila strains carrying the P(mip)-gfp and P(sod)-gfp fusions indicated that the mip promoter was expressed when the bacteria replicated intracellularly. GFP expression was also used to monitor, in infected A. castellanii cells, the intracellular survival of, and incidence of host-cell killing by. L. pneumophila strains that vary in their virulence properties. As quantified by flow cytometry the highly virulent L. pneumophila strain Corby was twice as infectious to A. castellanii as the Philadelphia strain JR 32. Using the avirulent Philadelphia derivative 25D invasion but no intracellular multiplication was observed. In addition, we examined by flow cytometry the influence of cytochalasin D, cycloheximide, and methylamine on the uptake of Legionella by A. castellanii. In conclusion, gfp appears to be a convenient reporter gene whose expression in Legionella can be followed in real time and allows analysis of promoter activities in Legionella and monitoring of the infection process.

Modulation by glycyrrhetinic acid derivatives of TPA-induced mouse ear oedema.

PubMed Central

Inoue, H.; Mori, T.; Shibata, S.; Koshihara, Y.

1989-01-01

1. The anti-inflammatory effects of glycyrrhetinic acid and its derivatives on TPA (12-O-tetradecanoylphorbol-13-acetate)-induced mouse ear oedema were studied. The mechanisms of TPA-induced ear oedema were first investigated with respect to the chemical mediators. 2. The formation of ear oedema reached a maximum 5 h after TPA application (2 micrograms per ear) and the prostaglandin E2 (PGE2) production of mouse ear increased with the oedema formation. 3. TPA-induced ear oedema was prevented by actinomycin D and cycloheximide (0.1 mg per ear, respectively) when applied during 60 min after TPA treatment. 4. Of glycyrrhetinic acid derivatives examined, dihemiphthalate derivatives (IIe, IIe', IIIa, IIIa', IVa, IVa') most strongly inhibited ear oedema on both topical (ID50, 1.6 mg per ear for IIe, 2.0 mg per ear for IIIa and 1.6 mg per ear for IVa) and oral (ID50, 88 mg kg-1 for IIe', 130 mg kg-1 for IIIa' and 92 mg kg-1 for IVa') administration. 5. Glycyrrhetinic acid (Ia) and its derivatives applied 30 min before TPA treatment were much more effective in inhibiting oedema than when applied 30 min after TPA. A dihemiphthalate of triterpenoid compound IVa completely inhibited oedema, even when applied 3 h before TPA treatment. 6. Glycyrrhetinic acid (Ia) and deoxoglycyrrhetol (IIa), the parent compounds, produced little inhibition by oral administration at less than 200 mg kg-1. 7. These results suggest that the dihemiphthalate derivatives of triterpenes derived from glycyrrhetinic acid by chemical modification are useful for the treatment of skin inflammation by both topical and oral application. PMID:2924072

Herpes simplex virus interferes with amyloid precursor protein processing.

PubMed

Shipley, Suzanne J; Parkin, Edward T; Itzhaki, Ruth F; Dobson, Curtis B

2005-08-18

The early events underlying Alzheimer's disease (AD) remain uncertain, although environmental factors may be involved. Work in this laboratory has shown that the combination of herpes simplex virus type 1 (HSV1) in brain and carriage of the APOE-epsilon4 allele of the APOE gene strongly increases the risk of developing AD. The development of AD is thought to involve abnormal aggregation or deposition of a 39-43 amino acid protein--beta amyloid (Abeta)--within the brain. This is cleaved from the much larger transmembranal protein 'amyloid precursor protein' (APP). Any agent able to interfere directly with Abeta or APP metabolism may therefore have the capacity to contribute towards AD. One recent report showed that certain HSV1 glycoprotein peptides may aggregate like Abeta; a second study described a role for APP in transport of virus in squid axons. However to date the effects of acute herpesvirus infection on metabolism of APP in human neuronal-type cells have not been investigated. In order to find if HSV1 directly affects APP and its degradation, we have examined this protein from human neuroblastoma cells (normal and transfected with APP 695) infected with the virus, using Western blotting. We have found that acute HSV1 (and also HSV2) infection rapidly reduces full length APP levels--as might be expected--yet surprisingly markedly increases levels of a novel C-terminal fragment of APP of about 55 kDa. This band was not increased in cells treated with the protein synthesis inhibitor cycloheximide Herpes virus infection leads to rapid loss of full length APP from cells, yet also causes increased levels of a novel 55 kDa C-terminal APP fragment. These data suggest that infection can directly alter the processing of a transmembranal protein intimately linked to the aetiology of AD.

Effects of drugs affecting endogenous amines or cyclic nucleotides on ethanol withdrawal head twitches in mice.

PubMed Central

Collier, H O; Hammond, M D; Schneider, C

1976-01-01

1 Twenty-four hours after ethanol withdrawal, dependent mice exhibited frequent head twitching. Naive mice exhibited similar twitching 15 min after treatment with 5-hydroxytryptophan (5-HTP) or 6 h after alpha-methyl-p-tyrosine (AMPT). Ethanol lessened the incidence of head twitches induced by any of these treatments. 5-HTP and AMPT each increased the incidence of head twitches induced by withdrawal of ethanol from dependent mice. 2 Drugs that affect the amount or activity of endogenous amines or cyclic nucleotides modified the incidence of head twitches. Nearly all drugs acted in the same direction on twitching elicited by any of these three treatments. 3 The incidence was lessened by: (a) methysergide, methergoline, MA 1420, p-chlorophenylalanine and p-chloroamphetamine; (b) dopamine, noradrenaline, L-DOPA, amphetamine and apomorphine; (c) hyoscine and nicotine; and (d) adenosine triphosphate, dibutyryl cyclic adenosine-3',5'-monophosphate (db cyclic AMP) and prostaglandins E1 and E2. 4 The incidence was increased by: (a) acetylcholine, carbachol and physostigmine; and (b) guanosine triphosphate, dibutyryl cyclic guanosine monophosphate (db cyclic GMP), theophylline and 3-isobutyl-1-methyl-xanthine. 5 These findings suggest that head twitching induced by these three treatments arises from a common biochemical mechanism, which may ultimately be a change in favour of cyclic GMP of the balance between this nucleotide and cyclic AMP within appropriate neurones. This imbalance appears to be elicited or increased by 5-hydroxytryptamine and acetylcholine and to be decreased by dopamine, noradrenaline and E prostaglandins. 6 Neither actinomycin D nor cycloheximide, given during the induction of ethanol dependence, altered the incidence of head twitches after ethanol withdrawal. PMID:987821

FAP-1-mediated activation of NF-kappaB induces resistance of head and neck cancer to Fas-induced apoptosis.

PubMed

Wieckowski, Eva; Atarashi, Yoshinari; Stanson, Joanna; Sato, Taka-Aki; Whiteside, Theresa L

2007-01-01

Molecular mechanisms responsible for tumor resistance to apoptosis often involve the Fas/FasL pathway. While squamous cell carcinomas of the head and neck (SCCHN) express both Fas and FasL, their resistance to self-induced apoptosis or apoptosis mediated by Fas agonistic antibody (CH-11Ab) was independent of the level of Fas surface expression or the presence of soluble Fas in supernatants of primary or metastatic SCCHN cell lines. By in vitro immunoselection, using PCI-15A cell line treated with successive cycles of CH-11 Ab, Fas-resistant sublines with the parental genotype were selected. Such sublines failed to cleave caspase-8 upon Fas engagement and were resistant to CH-11 Ab, although they remained sensitive to VP-16 or staurosporin. In the presence of cycloheximide, the selected SCCHN sublines become susceptible to CH-11 Ab, and showed cleavage of caspase-8, suggesting that apoptosis resistance was mediated by an inhibitory protein(s) acting upstream of caspase-8. Overexpression of Fas-associated phosphatase 1 (FAP-1), but not cellular FLICE-inhibitory protein (cFLIP) in SCCHN sublines was documented by Western blots and RT-PCR analyses. The FAP-1+ selected sublines also downregulated cell surface Fas. A high phosphorylation level of IkappaB kappa, NFkappaB activation and upregulation of Bcl-2 expression were observed in the FAP-1+ sublines. Treatment with the phosphatase inhibitor, orthovanadate, or silencing of FAP-1 with siRNA abolished their resistance to apoptosis, suggesting that FAP-1 phosphatase activity could be responsible for NF-kappaB activation and resistance of SCCHN cells to Fas-mediated apoptosis. 2006 Wiley-Liss, Inc.

Chemical coding and chemosensory properties of cholinergic brush cells in the mouse gastrointestinal and biliary tract.

PubMed

Schütz, Burkhard; Jurastow, Innokentij; Bader, Sandra; Ringer, Cornelia; von Engelhardt, Jakob; Chubanov, Vladimir; Gudermann, Thomas; Diener, Martin; Kummer, Wolfgang; Krasteva-Christ, Gabriela; Weihe, Eberhard

2015-01-01

The mouse gastro-intestinal and biliary tract mucosal epithelia harbor choline acetyltransferase (ChAT)-positive brush cells with taste cell-like traits. With the aid of two transgenic mouse lines that express green fluorescent protein (EGFP) under the control of the ChAT promoter (EGFP (ChAT) ) and by using in situ hybridization and immunohistochemistry we found that EGFP (ChAT) cells were clustered in the epithelium lining the gastric groove. EGFP (ChAT) cells were numerous in the gall bladder and bile duct, and found scattered as solitary cells along the small and large intestine. While all EGFP (ChAT) cells were also ChAT-positive, expression of the high-affinity choline transporter (ChT1) was never detected. Except for the proximal colon, EGFP (ChAT) cells also lacked detectable expression of the vesicular acetylcholine transporter (VAChT). EGFP (ChAT) cells were found to be separate from enteroendocrine cells, however they were all immunoreactive for cytokeratin 18 (CK18), transient receptor potential melastatin-like subtype 5 channel (TRPM5), and for cyclooxygenases 1 (COX1) and 2 (COX2). The ex vivo stimulation of colonic EGFP (ChAT) cells with the bitter substance denatonium resulted in a strong increase in intracellular calcium, while in other epithelial cells such an increase was significantly weaker and also timely delayed. Subsequent stimulation with cycloheximide was ineffective in both cell populations. Given their chemical coding and chemosensory properties, EGFP (ChAT) brush cells thus may have integrative functions and participate in induction of protective reflexes and inflammatory events by utilizing ACh and prostaglandins for paracrine signaling.

The Cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae

PubMed Central

Poklepovich, Tomas J.; Rinaldi, Mauro A.; Tomazic, Mariela L.; Favale, Nicolas O.; Turkewitz, Aaron P.; Nudel, Clara B.; Nusblat, Alejandro D.

2012-01-01

Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b5, Cyt b5 reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes. PMID:22982564

Rapid transient isoform-specific neuregulin1 transcription in motor neurons is regulated by neurotrophic factors and axon-target interactions.

PubMed

Wang, Jiajing; Hmadcha, Abdelkrim; Zakarian, Vaagn; Song, Fei; Loeb, Jeffrey A

2015-09-01

The neuregulins (NRGs) are a family of alternatively spliced factors that play important roles in nervous system development and disease. In motor neurons, NRG1 expression is regulated by activity and neurotrophic factors, however, little is known about what controls isoform-specific transcription. Here we show that NRG1 expression in the chick embryo increases in motor neurons that have extended their axons and that limb bud ablation before motor axon outgrowth prevents this induction, suggesting a trophic role from the developing limb. Consistently, NRG1 induction after limb bud ablation can be rescued by adding back the neurotrophic factors BDNF and GDNF. Mechanistically, BDNF induces a rapid and transient increase in type I and type III NRG1 mRNAs that peak at 4h in rat embryonic ventral spinal cord cultures. Blocking MAPK or PI3K signaling or blocking transcription with Actinomycin D blocks BDNF induced NRG1 gene induction. BDNF had no effect on mRNA degradation, suggesting that transcriptional activation rather than message stability is important. Furthermore, BDNF activates a reporter construct that includes 700bp upstream of the type I NRG1 start site. Protein synthesis is also required for type I NRG1 mRNA transcription as cycloheximide produced a super-induction of type I, but not type III NRG1 mRNA, possibly through a mechanism involving sustained activation of MAPK and PI3K. These results reveal the existence of highly responsive, transient transcriptional regulatory mechanisms that differentially modulate NRG1 isoform expression as a function of extracellular and intracellular signaling cascades and mediated by neurotrophic factors and axon-target interactions. Copyright © 2015 Elsevier Inc. All rights reserved.

Substance P induced preprotachykinin-a mRNA, neutral endopeptidase mRNA and substance P in cultured normal fibroblasts.

PubMed

Bae, Sang-Jae; Matsunaga, Yoshitaka; Takenaka, Motoi; Tanaka, Yoichi; Hamazaki, Yoichiro; Shimizu, Kazuhiro; Katayama, Ichiro

2002-04-01

In certain skin diseases, stress can modulate the induction and/or progression of cutaneous manifestations. However, little is known about the circuit in neuroendocrine and in the immune systems of the skin. To address this question, we have analyzed the regulatory mechanisms of autocrine induction of substance P (SP) by cultured normal human fibroblasts that compose the major population of the skin and might augment stress-induced skin inflammatory responses. In nonstimulated conditions, normal fibroblasts express a moderate amount of preprotachykinin-A (PPT-A), a precursor of SP mRNA, and exogenous SP significantly upregulated PPT-A mRNA expression. Maximum response of SP peptide and SP mRNA in fibroblasts was observed 1-3 h after stimulation with SP. In contrast, the expression of neutral endopeptidase (NEP), a cell surface peptide with hydrolyzing activity of SP, was increased in fibroblasts stimulated with SP after 24 h. The administration of NEP inhibitor (phosphoramidon) to the fibroblasts induced higher SP production. In addition, the neurokinin (NK) receptor antagonists (spantide, FK224 and FK888) and protein synthesis inhibitor (cycloheximide) inhibited SP production by 30-40% of control response. In immunostaining study, specific cytoplasmic staining of SP was observed in fibroblasts stimulated with SP. Finally, we confirmed that the nucleotide sequence of the PPT-A expressed in fibroblasts perfectly corresponded to the gene bank human PPT-A cDNA. This is the first report that SP mRNA, NEP mRNA and SP peptide can be induced by normal human skin fibroblasts in response to exogenous SP, and that fibroblast-derived SP might play an important role in the induction and acceleration of certain cutaneous diseases. Copyright 2002 S. Karger AG, Basel

Opposite effects of flurbiprofen and the nitroxybutyl ester of flurbiprofen on apoptosis in cultured guinea-pig gastric mucous cells

PubMed Central

Johal, Kamaljit; Hanson, Peter J

2000-01-01

The nitric oxide (NO)-donating nitroxybutyl ester of flurbiprofen (NO-flurbiprofen), shows reduced gastro-intestinal toxicity relative to flurbiprofen. NO may exert either pro- or anti-apoptotic effects, while non-steroidal anti-inflammatory drugs may induce apoptosis. The aim of the present work was therefore to compare the effects of flurbiprofen and NO-flurbiprofen on apoptosis in guinea-pig gastric mucous cells. Apoptotic activity was assessed by assay of caspase activity and from the fragmentation and condensation of nuclei. Incubation with flurbiprofen for 24 h produced a concentration-dependent induction of apoptosis in cells attached to the culture plate (caspase 3-like activity increased by 257% at 500 μM), while NO-flurbiprofen inhibited basal apoptosis (caspase 3-like activity decreased by 71% at 500 μM). Caspase activity and nuclear fragmentation were substantially increased in cells that had spontaneously detached from the culture plate. NO-flurbiprofen inhibited caspase activity (55% at 500 μM) but not nuclear fragmentation in these detached cells. NO flurbiprofen inhibited the activation of apoptosis by 25 μM C6-ceramide in cells attached to the culture plate. Inhibition of caspase activity by NO-flurbiprofen was detectable after 6 h of incubation with intact cells, but by contrast with the NO-donor S-nitrosyl-N-acetyl-penicillamine, was not demonstrable with cell homogenates. Activation of caspase 3-like activity by flurbiprofen was slow (>6 h incubation needed) and was inhibited by cycloheximide. The presence of a nitroxybutyl ester moiety on flurbiprofen prevents the pro-apoptotic activity of the parent compound and may contribute to the reduced gastro-intestinal toxicity of NO-flurbiprofen. PMID:10864887

Intracellular Proliferation of Legionella pneumophila in Hartmannella vermiformis in Aquatic Biofilms Grown on Plasticized Polyvinyl Chloride

PubMed Central

Kuiper, Melanie W.; Wullings, Bart A.; Akkermans, Antoon D. L.; Beumer, Rijkelt R.; van der Kooij, Dick

2004-01-01

The need for protozoa for the proliferation of Legionella pneumophila in aquatic habitats is still not fully understood and is even questioned by some investigators. This study shows the in vivo growth of L. pneumophila in protozoa in aquatic biofilms developing at high concentrations on plasticized polyvinyl chloride in a batch system with autoclaved tap water. The inoculum, a mixed microbial community including indigenous L. pneumophila originating from a tap water system, was added in an unfiltered as well as filtered (cellulose nitrate, 3.0-μm pore size) state. Both the attached and suspended biomasses were examined for their total amounts of ATP, for culturable L. pneumophila, and for their concentrations of protozoa. L. pneumophila grew to high numbers (6.3 log CFU/cm2) only in flasks with an unfiltered inoculum. Filtration obviously removed the growth-supporting factor, but it did not affect biofilm formation, as determined by measuring ATP. Cultivation, direct counting, and 18S ribosomal DNA-targeted PCR with subsequent sequencing revealed the presence of Hartmannella vermiformis in all flasks in which L. pneumophila multiplied and also when cycloheximide had been added. Fluorescent in situ hybridization clearly demonstrated the intracellular growth of L. pneumophila in trophozoites of H. vermiformis, with 25.9% ± 10.5% of the trophozoites containing L. pneumophila on day 10 and >90% containing L. pneumophila on day 14. Calculations confirmed that intracellular growth was most likely the only way for L. pneumophila to proliferate within the biofilm. Higher biofilm concentrations, measured as amounts of ATP, gave higher L. pneumophila concentrations, and therefore the growth of L. pneumophila within engineered water systems can be limited by controlling biofilm formation. PMID:15528550

Insulin stimulates the expression of the SHARP-1 gene via multiple signaling pathways.

PubMed

Takagi, K; Asano, K; Haneishi, A; Ono, M; Komatsu, Y; Yamamoto, T; Tanaka, T; Ueno, H; Ogawa, W; Tomita, K; Noguchi, T; Yamada, K

2014-06-01

The rat enhancer of split- and hairy-related protein-1 (SHARP-1) is a basic helix-loop-helix transcription factor. An issue of whether SHARP-1 is an insulin-inducible transcription factor was examined. Insulin rapidly increased the level of SHARP-1 mRNA both in vivo and in vitro. Then, signaling pathways involved with the increase of SHARP-1 mRNA by insulin were determined in H4IIE rat hepatoma cells. Pretreatments with LY294002, wortmannin, and staurosporine completely blocked the induction effect, suggesting the involvement of both phosphoinositide 3-kinase (PI 3-K) and protein kinase C (PKC) pathways. In fact, overexpression of a dominant negative form of atypical protein kinase C lambda (aPKCλ) significantly decreased the induction of the SHARP-1 mRNA. In addition, inhibitors for the small GTPase Rac or Jun N-terminal kinase (JNK) also blocked the induction of SHARP-1 mRNA by insulin. Overexpression of a dominant negative form of Rac1 prevented the activation by insulin. Furthermore, actinomycin D and cycloheximide completely blocked the induction of SHARP-1 mRNA by insulin. Finally, when a SHARP-1 expression plasmid was transiently transfected with various reporter plasmids into H4IIE cells, the promoter activity of PEPCK reporter plasmid was specifically decreased. Thus, we conclude that insulin induces the SHARP-1 gene expression at the transcription level via a both PI 3-K/aPKCλ/JNK- and a PI 3-K/Rac/JNK-signaling pathway; protein synthesis is required for this induction; and that SHARP-1 is a potential repressor of the PEPCK gene expression. © Georg Thieme Verlag KG Stuttgart · New York.

Na+-independent phosphate transport in Caco2BBE cells

PubMed Central

Candeal, Eduardo; Caldas, Yupanqui A.; Guillén, Natalia; Levi, Moshe

2014-01-01

Pi transport in epithelia has both Na+-dependent and Na+-independent components, but so far only Na+-dependent transporters have been characterized in detail and molecularly identified. Consequently, in the present study, we initiated the characterization and analysis of intestinal Na+-independent Pi transport using an in vitro model, Caco2BBE cells. Only Na+-independent Pi uptake was observed in these cells, and Pi uptake was dramatically increased when cells were incubated in high-Pi DMEM (4 mM) from 1 day to several days. No response to low-Pi medium was observed. The increased Pi transport was mainly caused by Vmax changes, and it was prevented by actinomycin D and cycloheximide. Pi transport in cells grown in 1 mM Pi (basal DMEM) decreased at pH > 7.5, and it was inhibited with proton ionophores. Pi transport in cells incubated with 4 mM Pi increased with alkaline pH, suggesting a preference for divalent phosphate. Pi uptake in cells in 1 mM Pi was completely inhibited only by Pi and partially inhibited by phosphonoformate, oxalate, DIDS, SITS, SO42−, HCO3−, and arsenate. This inhibition pattern suggests that more than one Pi transporter is active in cells maintained with 1 mM Pi. Phosphate transport from cells maintained at 4 mM Pi was only partially inhibited by phosphonoformate, oxalate, and arsenate. Attempts to identify the responsible transporters showed that multifunctional anion exchangers of the Slc26 family as well as members of Slc17, Slc20, and Slc37 and the Pi exporter xenotropic and polytropic retrovirus receptor 1 are not involved. PMID:25298422

Development of a pilot-scale 1 for Legionella elimination in biofilm in hot water network: heat shock treatment evaluation.

PubMed

Farhat, M; Trouilhé, M-C; Briand, E; Moletta-Denat, M; Robine, E; Frère, J

2010-03-01

(i) To develop an analytical tool in order to evaluate the effectiveness of anti-Legionella treatment in biofilm and (ii) study the fate of Legionella populations in water and biofilm after applying a heat shock treatment. A pilot-scale unit simulating a hot water system was built and designed by the Scientific and Technical Building Centre (CSTB, France). At the end of the contamination period, a stable cultivable Legionella spp. concentration of 5x10(5) CFU l(-1) was obtained. Two heat shock treatments (70 degrees C for 30 min) were applied. The results showed that the first treatment had a transitional effect on the abatement of Legionella concentrations, while the second treatment had no detectable effect on Legionella populations in water and biofilm. The DAPI (4',6'-diamidino-2-phenylindole), Legionella PCR and GVPC (glycocolle vancomycin pyrophosphate cycloheximide) counts measured in the dead leg water of the Test Loop were 1, 2 and 2 log units higher than results found in the Test Loop water. Moreover, Legionella spp. count in tap water was about 10(4) GU l(-1). These analyses revealed that they are responsible for the rapid recolonization as well as the uncomplete destroyed biofilm. In addition, a resistance test was conducted and showed that Legionella in the second heat shock treatment was not thermo-resistant but thermo-acclimated. Thermal disinfection does not seem to be efficient enough to eliminate Legionella when it is used as a curative treatment. This work could help water managers for a better management of water network and for a better control of Legionella.

Differential Function of N-Cadherin and Cadherin-7 in the Control of Embryonic Cell Motility

PubMed Central

Dufour, Sylvie; Beauvais-Jouneau, Alice; Delouvée, Annie; Thiery, Jean Paul

1999-01-01

Similar amounts of N-cadherin and cadherin-7, the prototypes of type I and type II cadherin, induced cell-cell adhesion in murine sarcoma 180 transfectants, Ncad-1 and cad7-29, respectively. However, in the initial phase of aggregation, Ncad-1 cells aggregated more rapidly than cad7-29 cells. Isolated Ncad-1 and cad7-29 cells adhered and spread in a similar manner on fibronectin (FN), whereas aggregated cad7-29 cells were more motile and dispersed than aggregated Ncad-1 cells. cad7-29 cells established transient contacts with their neighbors which were stabilized if FN-cell interactions were perturbed. In contrast, Ncad-1 cells remained in close contact when they migrated on FN. Both β-catenin and cadherin were more rapidly downregulated in cad7-29 than in Ncad-1 cells treated with cycloheximide, suggesting a higher turnover rate for cadherin-7–mediated cell-cell contacts than for those mediated by N-cadherin. The extent of FN-dependent focal adhesion kinase phosphorylation was much lower if the cells had initiated N-cadherin–mediated rather than cadherin-7–mediated cell adhesion before plating. On grafting into the embryo, Ncad-1 cells did not migrate and remained at or close to the graft site, even after 48 h, whereas grafted cad7-29 cells dispersed efficiently into embryonic structures. Thus, the adhesive phenotype of cadherin-7–expressing cells is regulated by the nature of the extracellular matrix environment which also controls the migratory behavior of the cells. In addition, adhesions mediated by different cadherins differentially regulate FN-dependent signaling. The transient contacts specifically observed in cadherin- 7–expressing cells may also be important in the control of cell motility. PMID:10427101

Multiple interactions between maternally-activated signalling pathways control Xenopus nodal-related genes.

PubMed

Rex, Maria; Hilton, Emma; Old, Robert

2002-03-01

We have investigated the induction of the six Xenopus nodal-related genes, Xnr1-Xnr6, by maternal determinants. The beta-catenin pathway was modelled by stimulation using Xwnt8, activin-like signalling was modelled by activin, and VegT action was studied by overexpression in animal cap explants. Combinations of factors were examined, and previously unrecognised interactions were revealed in animal caps and whole embryos. For the induction of Xnr5 and Xnr6 in whole embryos, using a beta-catenin antisense morpholino oligonucleotide or a dominant negative XTcf3, we have demonstrated an absolute permissive requirement for the beta-catenin/Tcf pathway, in addition to the requirement for VegT action. In animal caps Xnr5 and Xnr6 are induced in response to VegT overexpression, and this induction is dependent upon the concomitant activation of the beta-catenin pathway that VegT initiates in animal caps. For the induction of Xnr3, VegT interacts negatively so as to inhibit the induction otherwise observed with wnt-signalling alone. The negative effect of VegT is not the result of a general inhibition of wnt-signalling, and does not result from an inhibition of wnt-induced siamois expression. A 294 bp proximal promoter fragment of the Xnr3 gene is sufficient to mediate the negative effect of VegT. Further experiments, employing cycloheximide to examine the dependence of Xnr gene expression upon proteins translated after the mid-blastula stage, demonstrated that Xnrs 4, 5 and 6 are 'primary' Xnr genes whose expression in the late blastula is solely dependent upon factors present before the mid-blastula stage.

Detection of seasonal asymptomatic dermatophytes in Van cats.

PubMed

Ilhan, Ziya; Karaca, Mehmet; Ekin, Ismail Hakki; Solmaz, Hasan; Akkan, Hasan Altan; Tutuncu, Mehmet

2016-01-01

The Van cat is a domestic landrace found in the Van province of eastern Turkey. In this study, we aimed to determine the seasonal carriage of dermatophytes in Van cats without clinical lesions. A total of 264 hair specimens were collected from clinically healthy cats in and around the Van Province. Of these samples, 30.3% were obtained in spring, 30.6% in summer, 16.6% in autumn, and 22.3% in winter; 45.1% of samples were from male cats and the rest from female ones. Of the studied cats, 118 were younger than 1 year, 78 were 1-3 years old, and 68 were older than 3 years. The specimens were subjected to direct microscopic examination with 15% potassium hydroxide and cultured on Sabouraud dextrose agar and dermatophyte test medium supplemented with cycloheximide and chloramphenicol. Dermatophyte identification was carried out based on macroscopic and microscopic colony morphology, urease activities, in vitro hair perforation test, growth at 37°C, and pigmentation on corn meal agar. Dermatophytes were isolated from 19 (7.1%) of the 264 specimens examined. The most frequently isolated fungi were Trichophyton terrestre (4.1%), followed by Microsporum gypseum (1.1%), M. nanum (1.1%), and T. mentagrophytes (0.7%), and these fungi may represent a health risk for humans in contact with clinically healthy Van cats. M. canis was not isolated from any of the specimens. Our results show no significant (p>0.05) association between carriage of dermatophytes and the gender of cats. The carriage rate of dermatophytes was high in spring and winter, and the only possible risk factor for infection was age of the animal. Copyright © 2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

New Saccharomyces cerevisiae baker's yeast displaying enhanced resistance to freezing.

PubMed

Codón, Antonio C; Rincón, Ana M; Moreno-Mateos, Miguel A; Delgado-Jarana, Jesús; Rey, Manuel; Limón, Carmen; Rosado, Ivan V; Cubero, Beatriz; Peñate, Xenia; Castrejón, Francisco; Benítez, Tahía

2003-01-15

Three procedures were used to obtain new Saccharomyces cerevisiae baker's yeasts with increased storage stability at -20, 4, 22, and 30 degrees C. The first used mitochondria from highly ethanol-tolerant wine yeast, which were transferred to baker's strains. Viability of the heteroplasmons was improved shortly after freezing. However, after prolonged storage, viability dramatically decreased and was accompanied by an increase in the frequency of respiratory-deficient (petite) mutant formation. This indicated that mitochondria were not stable and were incompatible with the nucleus. The strains tested regained their original resistance to freezing after recovering their own mitochondria. The second procedure used hybrid formation after protoplast fusion and isolation on selective media of fusants from baker's yeast meiotic products resistant to parafluorphenylalanine and cycloheximide, respectively. No hybrids were obtained when using the parentals, probably due to the high ploidy of the baker's strains. Hybrids obtained from nonisogenic strains manifested in all cases a resistance to freezing intermediate between those of their parental strains. Hybrids from crosses between meiotic products of the same strain were always more sensitive than their parentals. The third method was used to develop baker's yeast mutants resistant to 2-deoxy-d-glucose (DOG) and deregulated for maltose and sucrose metabolism. Mutant DOG21 displayed a slight increase in trehalose content and viability both in frozen doughs and during storage at 4 and 22 degrees C. This mutant also displayed a capacity to ferment, under laboratory conditions, both lean and sweet fresh and frozen doughs. For industrial uses, fermented lean and sweet bakery products, both from fresh and frozen doughs obtained with mutant DOG21, were of better quality with regard to volume, texture, and organoleptic properties than those produced by the wild type.

Chloroplast and reactive oxygen species involvement in apoptotic-like programmed cell death in Arabidopsis suspension cultures

PubMed Central

Doyle, Siamsa M.; Diamond, Mark; McCabe, Paul F.

2010-01-01

Chloroplasts produce reactive oxygen species (ROS) during cellular stress. ROS are known to act as regulators of programmed cell death (PCD) in plant and animal cells, so it is possible that chloroplasts have a role in regulating PCD in green tissue. Arabidopsis thaliana cell suspension cultures are model systems in which to test this, as here it is shown that their cells contain well-developed, functional chloroplasts when grown in the light, but not when grown in the dark. Heat treatment at 55 °C induced apoptotic-like (AL)-PCD in the cultures, but light-grown cultures responded with significantly less AL-PCD than dark-grown cultures. Chloroplast-free light-grown cultures were established using norflurazon, spectinomycin, and lincomycin and these cultures responded to heat treatment with increased AL-PCD, demonstrating that chloroplasts affect AL-PCD induction in light-grown cultures. Antioxidant treatment of light-grown cultures also resulted in increased AL-PCD induction, suggesting that chloroplast-produced ROS may be involved in AL-PCD regulation. Cycloheximide treatment of light-grown cultures prolonged cell viability and attenuated AL-PCD induction; however, this effect was less pronounced in dark-grown cultures, and did not occur in antioxidant-treated light-grown cultures. This suggests that a complex interplay between light, chloroplasts, ROS, and nuclear protein synthesis occurs during plant AL-PCD. The results of this study highlight the importance of taking into account the time-point at which cells are observed and whether the cells are light-grown and chloroplast-containing or not, for any study on plant AL-PCD, as it appears that chloroplasts can play a significant role in AL-PCD regulation. PMID:19933317

A Highly-Conserved Single-Stranded DNA-Binding Protein in Xanthomonas Functions as a Harpin-Like Protein to Trigger Plant Immunity

PubMed Central

Che, Yi-Zhou; Zou, Li-Fang; Zakria, Muhammad; Zou, Hua-Song; Chen, Gong-You

2013-01-01

Harpins are produced by Gram-negative phytopathogenic bacteria and typically elicit hypersensitive response (HR) in non-host plants. The characterization of harpins in Xanthomonas species is largely unexplored. Here we demonstrate that Xanthomonas produce a highly conserved single-stranded DNA-binding protein (SSBX) that elicits HR in tobacco as by harpin Hpa1. SSBX, like Hpa1, is an acidic, glycine-rich, heat-stable protein that lacks cysteine residues. SSBX-triggered HR in tobacco, as by Hpa1, is characterized by the oxidative burst, the expression of HR markers (HIN1, HSR203J), pathogenesis-related genes, and callose deposition. Both SSBX- and Hpa1-induced HRs can be inhibited by general metabolism inhibitors actinomycin D, cycloheximide, and lanthanum chloride. Furthermore, those HRs activate the expression of BAK1 and BIK1 genes that are essential for induction of mitogen-activated protein kinase (MAPK) and salicylic acid pathways. Once applied to plants, SSBX induces resistance to the fungal pathogen Alternaria alternata and enhances plant growth. When ssbX was deleted in X. oryzae pv. oryzicola, the causal agent of bacterial leaf streak in rice, the resulting ssbXoc mutant was reduced in virulence and bacterial growth in planta, but retained its ability to trigger HR in tobacco. Interestingly, ssbXoc contains an imperfect PIP-box (plant-inducible promoter) and the expression of ssbXoc is regulated by HrpX, which belongs to the AraC family of transcriptional activators. Immunoblotting evidence showed that SSBx secretion requires a functional type-III secretion system as Hpa1 does. This is the first report demonstrating that Xanthomonas produce a highly-conserved SSBX that functions as a harpin-like protein for plant immunity. PMID:23418541

Suppressed translation and ULK1 degradation as potential mechanisms of autophagy limitation under prolonged starvation.

PubMed

Allavena, Giulia; Boyd, Caroline; Oo, Kyaw Soe; Maellaro, Emilia; Zhivotovsky, Boris; Kaminskyy, Vitaliy O

2016-11-01

Macroautophagy/autophagy is a well-organized process of intracellular degradation, which is rapidly activated under starvation conditions. Recent data demonstrate a transcriptional upregulation of several autophagy genes as a mechanism that controls autophagy in response to starvation. Here we report that despite the significant upregulation of mRNA of the essential autophagy initiation gene ULK1, its protein level is rapidly reduced under starvation. Although both autophagic and proteasomal systems contribute to the degradation of ULK1, under prolonged nitrogen deprivation, its level was still reduced in ATG7 knockout cells, and only initially stabilized in cells treated with the lysosomal or proteasomal inhibitors. We demonstrate that under starvation, protein translation is rapidly diminished and, similar to treatments with the proteosynthesis inhibitors cycloheximide or anisomycin, is associated with a significant reduction of ULK1. Furthermore, it was found that inhibition of the mitochondrial respiratory complexes or the mitochondrial ATP synthase function that could also take place in the absence of substrates, promote upregulation of ULK1 mRNA and protein expression in an AMPK-dependent manner in U1810 lung cancer cells growing in complete culture medium. These inhibitors could also drastically increase the ULK1 protein in U1810 cells with knockout of ATG13, where the ULK1 expression is significantly diminished. However, such upregulation of ULK1 protein is negligible under starvation conditions, further signifying the contribution of translation and suggesting that transcriptional upregulation of ULK1 protein will be diminished under such conditions. Thus, we propose a model where inhibition of protein translation, together with the degradation systems, limit autophagy during starvation.

Temporal Characteristics of Gustatory Responses in Rat Parabrachial Neurons Vary by Stimulus and Chemosensitive Neuron Type

PubMed Central

Geran, Laura; Travers, Susan

2013-01-01

It has been demonstrated that temporal features of spike trains can increase the amount of information available for gustatory processing. However, the nature of these temporal characteristics and their relationship to different taste qualities and neuron types are not well-defined. The present study analyzed the time course of taste responses from parabrachial (PBN) neurons elicited by multiple applications of “sweet” (sucrose), “salty” (NaCl), “sour” (citric acid), and “bitter” (quinine and cycloheximide) stimuli in an acute preparation. Time course varied significantly by taste stimulus and best-stimulus classification. Across neurons, the ensemble code for the three electrolytes was similar initially but quinine diverged from NaCl and acid during the second 500ms of stimulation and all four qualities became distinct just after 1s. This temporal evolution was reflected in significantly broader tuning during the initial response. Metric space analyses of quality discrimination by individual neurons showed that increases in information (H) afforded by temporal factors was usually explained by differences in rate envelope, which had a greater impact during the initial 2s (22.5% increase in H) compared to the later response (9.5%). Moreover, timing had a differential impact according to cell type, with between-quality discrimination in neurons activated maximally by NaCl or citric acid most affected. Timing was also found to dramatically improve within-quality discrimination (80% increase in H) in neurons that responded optimally to bitter stimuli (B-best). Spikes from B-best neurons were also more likely to occur in bursts. These findings suggest that among PBN taste neurons, time-dependent increases in mutual information can arise from stimulus- and neuron-specific differences in response envelope during the initial dynamic period. A stable rate code predominates in later epochs. PMID:24124597

Stimulation of IFN-γ production by garlic lectin in mouse spleen cells: involvement of IL-12 via activation of p38 MAPK and ERK in macrophages.

PubMed

Dong, Qing; Sugiura, Tsutomu; Toyohira, Yumiko; Yoshida, Yasuhiro; Yanagihara, Nobuyuki; Karasaki, Yuji

2011-02-15

Several lectins, present in beans and edible plant products, have immuno-potentiating and anti-tumor activities. We here report the effects of garlic lectin purified from garlic bulbs on the production of cytokines such as interleukin-12 (IL-12) and interferon-γ (IFN-γ) in the mouse. Garlic lectin induced IFN-γ production in spleen cells in a bell-shaped time (24-60 h)- and concentration (0.25-2.0 mg/ml)-dependent manner. The maximal enhancement was observed at 36 h with 0.5 mg/ml of garlic lectin. The stimulatory effect of garlic lectin on IFN-γ production was completely inhibited by both actinomycin D and cycloheximide, an inhibitor of ribosomal protein synthesis and DNA-dependent RNA polymerase, respectively, and was associated with an increase in IFN-γ mRNA level. Garlic lectin also induced IL-12 production in mouse peritoneal macrophages in a concentration (0.25-1.0 mg/ml)- and bell-shaped time (3-24 h)-dependent manner. The lectin increased the phosphorylation of extracellular signal-regulated kinases (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) in macrophages. Furthermore, specific pharmacological inhibitors of ERK kinase (U0126) and p38 MAPK (SB203580) also suppressed the production of IL-12 induced by garlic lectin. The present findings suggest that garlic lectin induces IL-12 production via activation of p38 MAPK and ERK in mouse macrophages, which, in turn, stimulates IFN-γ production through an increase in IFN-γ mRNA in the spleen cells. Copyright © 2010 Elsevier GmbH. All rights reserved.

Relative Contribution of nirK- and nirS- Bacterial Denitrifiers as Well as Fungal Denitrifiers to Nitrous Oxide Production from Dairy Manure Compost.

PubMed

Maeda, Koki; Toyoda, Sakae; Philippot, Laurent; Hattori, Shohei; Nakajima, Keiichi; Ito, Yumi; Yoshida, Naohiro

2017-12-19

The relative contribution of fungi, bacteria, and nirS and nirK denirifiers to nitrous oxide (N 2 O) emission with unknown isotopic signature from dairy manure compost was examined by selective inhibition techniques. Chloramphenicol (CHP), cycloheximide (CYH), and diethyl dithiocarbamate (DDTC) were used to suppress the activity of bacteria, fungi, and nirK-possessing denitrifiers, respectively. Produced N 2 O were surveyed to isotopocule analysis, and its 15 N site preference (SP) and δ 18 O values were compared. Bacteria, fungi, nirS, and nirK gene abundances were compared by qPCR. The results showed that N 2 O production was strongly inhibited by CHP addition in surface pile samples (82.2%) as well as in nitrite-amended core samples (98.4%), while CYH addition did not inhibit the N 2 O production. N 2 O with unknown isotopic signature (SP = 15.3-16.2‰), accompanied by δ 18 O (19.0-26.8‰) values which were close to bacterial denitrification, was also suppressed by CHP and DDTC addition (95.3%) indicating that nirK denitrifiers were responsible for this N 2 O production despite being less abundant than nirS denitrifiers. Altogether, our results suggest that bacteria are important for N 2 O production with different SP values both from compost surface and pile core. However, further work is required to decipher whether N 2 O with unknown isotopic signature is mostly due to nirK denitrifiers that are taxonomically different from the SP-characterized strains and therefore have different SP values rather than also being interwoven with the contribution of the NO-detoxifying pathway and/or of co-denitrification.

Inbred mouse strains C57BL/6J and DBA/2J vary in sensitivity to a subset of bitter stimuli

PubMed Central

Boughter, John D; Raghow, Sandeep; Nelson, Theodore M; Munger, Steven D

2005-01-01

Background Common inbred mouse strains are genotypically diverse, but it is still poorly understood how this diversity relates to specific differences in behavior. To identify quantitative trait genes that influence taste behavior differences, it is critical to utilize assays that exclusively measure the contribution of orosensory cues. With a few exceptions, previous characterizations of behavioral taste sensitivity in inbred mouse strains have generally measured consumption, which can be confounded by post-ingestive effects. Here, we used a taste-salient brief-access procedure to measure taste sensitivity to eight stimuli characterized as bitter or aversive in C57BL/6J (B6) and DBA/2J (D2) mice. Results B6 mice were more sensitive than D2 mice to a subset of bitter stimuli, including quinine hydrochloride (QHCl), 6-n-propylthiouracil (PROP), and MgCl2. D2 mice were more sensitive than B6 mice to the bitter stimulus raffinose undecaacetate (RUA). These strains did not differ in sensitivity to cycloheximide (CYX), denatonium benzoate (DB), KCl or HCl. Conclusion B6-D2 taste sensitivity differences indicate that differences in consumption of QHCl, PROP, MgCl2 and RUA are based on immediate orosensory cues, not post-ingestive effects. The absence of a strain difference for CYX suggests that polymorphisms in a T2R-type taste receptor shown to be differentially sensitive to CYX in vitro are unlikely to differentially contribute to the CYX behavioral response in vivo. The results of these studies point to the utility of these common mouse strains and their associated resources for investigation into the genetic mechanisms of taste. PMID:15967025

Regulation of L-phenylalanine ammonia-lyase from Rhizoctonia solani.

PubMed Central

Kalghatgi, K K; Subba Rao, P V

1976-01-01

Maximal levels of L-henylalanine ammonia-lyase activity were observed when the mycelial felts of Rhizoctonia solani were grown for 4.5 days on Byrde synthetic medium containing 3.5% glucose and 0.3% L-phenylalanine, Differential centrifugation studies have indicated that the enzyme is localized in the soluble fraction. The time course of induction of L-phenylalanine ammonia-lyase activity by L-phenylalanine showed a lag period of 1 to 1.5 h and reached a maximum around 4 to 6 h after the addition of the inducer to the medium. L-Phenylalanine, L-tyrosine, and L-tryptophan were nearly equally efficient inducers of the enzyme. D-Phenylalanine was as efficient as the L-isomer, whereas D-tyrosine was a poor inducer. Light, gibberellic acid, indole 3-acetic acid, and kinetin had no effect on the induction of L-phenylalanine ammonia-lyase activity. Cycloheximide did not inhibit the uptake of amino acids by the mycelia but completely blocked the incorporation of radioactive amino acids into soluble proteins and the development of L-phenylalanine ammonia-lyase activity. Actinomycin D inhibited both the incorporation of 32P into ribonucleic acid and the enzyme activity. Conclusive evidence for de novo synthesis of L-phenylalanine ammonia-lyase was obtained by the incorporation of radioactive amino acids into the enzyme. Electrophoretic analysis of the purified preparation showed a single protein band that coincided with radioactivity and L-phenylalanine ammonia-lyase activity. Glucose and intermediates of the tricarboxylic acid cycle, like citric acid, alpha-ketoglutaric acid, and succinic acid, and the metabolites of L-phenylalanine, like o-coumaric acid, o-hydroxyphenylacetic acid, and protocatechuic acid, significantly repressed L-phenylalanine ammonia-lyase activity. The observed repression was not relieved by cyclic adenosine 5'-triphosphate. Images PMID:1262311

Apoptotic death in cerebral hemisphere cells is density dependent and modulated by transient oxygen and glucose deprivation.

PubMed

Yavin, E; Billia, D M

1997-03-01

Flow cytometry, light and fluorescence microscopy, and designated biochemical techniques were used to examine the type of death which occurs in cerebral cortex cells when grown under crowded vs. sparse conditions or after brief anoxia/hypoglycemia. A 4 hr episode of anoxia combined with glucose deprivation enhanced apoptotic cell death as assessed by 4',6-diamidino-2-phenylindole (DAPI) staining and reduced neutral red eye uptake. An additional form of cell death involving exclusion of the nucleus was recorded by time lapse cinematography and DAPI stain. The presence of the endonuclease inhibitor aurintricarboxylic acid (0.1 mM) reduced cell death by 56.6%, while the protein and RNA synthesis inhibitors actinomycin D and cycloheximide (each at 5 micrograms/ml) effectively decreased cell death by 83.3% and 90.6%, respectively. In contrast, 5 mM glutamate had no effect on cell death in accord with the immature state of the cells. Growth of cells under crowded conditions improved cell survival; after 2 h or 4 days in culture, cells seeded at high density (34 microgram cellular DNA/cm2) showed a nearly 3-fold decline in the amount of cell death in comparison to cells seeded at low density (5 micrograms cellular DNA/cm2). At high cell density, anoxic episodes enhanced cell death most likely by preventing a cell density-mediated rescue. Neutral red dye uptake, an index for cell viability, was enhanced with increasing cell density and in vitro maturation, but was reduced in dense cultures exposed to anoxic/hypoglycemic conditions. The data suggest that cell density may play a critical role in brain organogenesis and that anoxic stress is more deleterious in dense than sparse cell assemblies.

Impact of capsaicin, an active component of chili pepper, on pathogenic chlamydial growth (Chlamydia trachomatis and Chlamydia pneumoniae) in immortal human epithelial HeLa cells.

PubMed

Yamakawa, Kazuya; Matsuo, Junji; Okubo, Torahiko; Nakamura, Shinji; Yamaguchi, Hiroyuki

2018-02-01

Chlamydia trachomatis is the leading cause of sexually transmitted infections worldwide. Capsaicin, a component of chili pepper, which can stimulate actin remodeling via capsaicin receptor TRPV1 (transient receptor potential vanilloid 1) and anti-inflammatory effects via PPARγ (peroxisome proliferator-activated receptor-γ) and LXRα (liver X receptor α), is a potential candidate to control chlamydial growth in host cells. We examined whether capsaicin could inhibit C. trachomatis growth in immortal human epithelial HeLa cells. Inclusion forming unit and quantitative PCR assays showed that capsaicin significantly inhibited bacterial growth in cells in a dose-dependent manner, even in the presence of cycloheximide, a eukaryotic protein synthesis inhibitor. Confocal microscopic and transmission electron microscopic observations revealed an obvious decrease in bacterial numbers to inclusions bodies formed in the cells. Although capsaicin can stimulate the apoptosis of cells, no increase in cleaved PARP (poly (ADP-ribose) polymerase), an apoptotic indicator, was observed at a working concentration. All of the drugs tested (capsazepine, a TRPV1 antagonist; 5CPPSS-50, an LXRα inhibitor; and T0070907, a PPARγ inhibitor) had no effect on chlamydial inhibition in the presence of capsaicin. In addition, we also confirmed that capsaicin inhibited Chlamydia pneumoniae growth, indicating a phenomena not specific to C. trachomatis. Thus, we conclude that capsaicin can block chlamydial growth without the requirement of host cell protein synthesis, but by another, yet to be defined, mechanism. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Transgene delivery to endothelial cultures derived from porcine carotid artery ex vivo.

PubMed

Andoh, J; Sawyer, B; Szewczyk, K; Nortley, M; Rossetti, T; Loftus, I M; Yáñez-Muñoz, R J; Hainsworth, A H

2013-10-01

Carotid artery disease is a widespread cause of morbidity and mortality. Porcine models of vascular disease are well established in vivo, but existing endothelial systems in vitro (e.g. human umbilical vein endothelial cells, rat aortic endothelial cultures) poorly reflect carotid endothelium. A reliable in vitro assay would improve design of in vivo experiments and allow reduction and refinement of animal use. This study aimed (1) to develop ex vivo endothelial cultures from porcine carotid and (2) to test whether these were suitable for lentivector-mediated transgene delivery. Surplus carotid arteries were harvested from young adult female Large White pigs within 10 min post-mortem. Small sectors of carotid artery wall (approximately 4 mm×4 mm squares) were immobilised in a stable gel matrix. Cultures were exposed to HIV-derived lentivector (LV) encoding a reporter transgene or the equivalent integration-deficient vector (IDLV). After 7-14 days in vitro, cultures were fixed and labelled histochemically. Thread-like multicellular outgrowths were observed that were positive for endothelial cell markers (CD31, VEGFR2, von Willebrand factor). A minority of cells co-labelled for smooth muscle markers. Sensitivity to cytotoxic agents (paclitaxel, cycloheximide, staurosporine) was comparable to that in cell cultures, indicating that the gel matrix permits diffusive access of small pharmacological molecules. Transgene-expressing cells were more abundant following exposure to LV than IDLV (4.7, 0.1% of cells, respectively). In conclusion, ex vivo adult porcine carotid artery produced endothelial cell outgrowths that were effectively transduced by LV. This system will facilitate translation of novel therapies to clinical trials, with reduction and refinement of in vivo experiments.

Changes in gene expression linked to methamphetamine-induced dopaminergic neurotoxicity.

PubMed

Xie, Tao; Tong, Liqiong; Barrett, Tanya; Yuan, Jie; Hatzidimitriou, George; McCann, Una D; Becker, Kevin G; Donovan, David M; Ricaurte, George A

2002-01-01

The purpose of these studies was to examine the role of gene expression in methamphetamine (METH)-induced dopamine (DA) neurotoxicity. First, the effects of the mRNA synthesis inhibitor, actinomycin-D, and the protein synthesis inhibitor, cycloheximide, were examined. Both agents afforded complete protection against METH-induced DA neurotoxicity and did so independently of effects on core temperature, DA transporter function, or METH brain levels, suggesting that gene transcription and mRNA translation play a role in METH neurotoxicity. Next, microarray technology, in combination with an experimental approach designed to facilitate recognition of relevant gene expression patterns, was used to identify gene products linked to METH-induced DA neurotoxicity. This led to the identification of several genes in the ventral midbrain associated with the neurotoxic process, including genes for energy metabolism [cytochrome c oxidase subunit 1 (COX1), reduced nicotinamide adenine dinucleotide ubiquinone oxidoreductase chain 2, and phosphoglycerate mutase B], ion regulation (members of sodium/hydrogen exchanger and sodium/bile acid cotransporter family), signal transduction (adenylyl cyclase III), and cell differentiation and degeneration (N-myc downstream-regulated gene 3 and tau protein). Of these differentially expressed genes, we elected to further examine the increase in COX1 expression, because of data implicating energy utilization in METH neurotoxicity and the known role of COX1 in energy metabolism. On the basis of time course studies, Northern blot analyses, in situ hybridization results, and temperature studies, we now report that increased COX1 expression in the ventral midbrain is linked to METH-induced DA neuronal injury. The precise role of COX1 and other genes in METH neurotoxicity remains to be elucidated.

Gibberellic Acid Regulates Chalcone Synthase Gene Transcription in the Corolla of Petunia hybrida 1

PubMed Central

Weiss, David; van Blokland, Rik; Kooter, Jan M.; Mol, Joseph N. M.; van Tunen, Arjen J.

1992-01-01

The pigmentation of Petunia hybrida corollas is regulated by gibberellic acid (GA3). It controls the increase of flavonoid enzyme levels and their corresponding mRNAs. We have used an in vitro culture system for corollas to study the regulatory role of GA3 in the expression of flavonoid genes. By determining steady-state mRNA levels, we show that the accumulation of chalcone synthase (chs) mRNA in young corollas is dependent on the presence of both sucrose and GA3 in the culture medium. Whereas sucrose had a general metabolic effect on gene expression, the stimulatory role of GA3 was specific. Analysis of nascent transcripts in isolated corolla nuclei showed that changes in steady-state chs mRNA levels correlated very well with changes in the transcription rate. We therefore conclude that GA3 controls the expression of chs at the transcriptional level. Preculturing the corollas in sucrose medium without GA3 resulted in a lower chs mRNA level. The expression could be reinduced by the addition of GA3. The hormone is thus required for the induction but also for the maintenance of chs transcription. The delayed reinduction of chs expression, the lag time in the kinetics of chs mRNA accumulation, and the inhibitory effect of cycloheximide on the action of GA3 suggest that GA3 controls chs transcription in an indirect manner. Our data support a model in which GA3 induces the production of a regulatory protein such as a receptor or a trans-acting factor that is directly involved in chs transcription. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6 PMID:16668613

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sun-Mi, E-mail: lala1647@hanmail.net; Lee, Chung Won, E-mail: vasculardoctorlee@gmail.com; Kim, Bo-Young, E-mail: kimboyoung@pusan.ac.kr

We attempted to determine the effects of a milieu rich in cholesterol molecules on expression of chemokine CXCL8. A high-cholesterol diet led to an increased transcription of the IL-8 gene in the arteries and elevated levels of CXCL8 in sera of ApoE{sup −/−} mice, compared with those of wild-type C57BL/6 mice. Treatment of THP-1 monocyte/macrophage cells with 27-hydroxycholesterol (27OHChol) resulted in transcription of the IL-8 gene and increased secretion of its corresponding gene product whereas cholesterol did not induce expression of CXCL8 in THP-1 cells. 27OHChol-induced transcription of the IL-8 gene was blocked by cycloheximide, but not by polymyxin B.more » Treatment of THP-1 cells with 27OHChol caused translocation of p65 NF-κB subunit into the nucleus and up-regulation of CD88. Inhibition of NF-κB and CD88 using SN50 and W-54011, respectively, resulted in reduced transcription of the IL-8 gene and attenuated secretion of CXCL8 induced by 27OHChol. We propose that oxidatively modified cholesterol like 27OHChol, rather than cholesterol, is responsible for sustained expression of CXCL8 in monocytes/macrophages in atherosclerotic arteries. - Highlights: • Consumption of a high-cholesterol diet leads to increased CXCL8 expression in ApoE{sup −/−} mice. • 27OHChol, but not cholesterol, up-regulates expression of CXCL8 in macrophages. • 27OHChol enhances nuclear translocation of NF-κB and expression of CD88 in macrophages. • Inhibition of NF-κB or CD88 results in decreased CXCL8 expression induced by 27OHChol. • 27OHChol up-regulates CXCL8 expression via NF-κB and CD88 in macrophages.« less

Toward the Standardization of Mycological Examination of Sputum Samples in Cystic Fibrosis: Results from a French Multicenter Prospective Study.

PubMed

Coron, Noémie; Pihet, Marc; Fréalle, Emilie; Lemeille, Yolande; Pinel, Claudine; Pelloux, Hervé; Gargala, Gilles; Favennec, Loic; Accoceberry, Isabelle; Durand-Joly, Isabelle; Dalle, Frédéric; Huet, Frédéric; Fanton, Annlyse; Boldron, Amale; Loeuille, Guy-André; Domblides, Philippe; Coltey, Bérengère; Pin, Isabelle; Llerena, Catherine; Troussier, Françoise; Person, Christine; Marguet, Christophe; Wizla, Nathalie; Thumerelle, Caroline; Turck, Dominique; Bui, Stéphanie; Fayon, Michael; Duhamel, Alain; Prévotat, Anne; Wallaert, Benoit; Leroy, Sylvie; Bouchara, Jean-Philippe; Delhaes, Laurence

2018-02-01

Fungal respiratory colonization of cystic fibrosis (CF) patients emerges as a new concern; however, the heterogeneity of mycological protocols limits investigations. We first aimed at setting up an efficient standardized protocol for mycological analysis of CF sputa that was assessed during a prospective, multicenter study: "MucoFong" program (PHRC-06/1902). Sputa from 243 CF patients from seven centers in France were collected over a 15-month period and submitted to a standardized protocol based on 6 semi-selective media. After mucolytic pretreatment, sputa were plated in parallel on cycloheximide-enriched (ACT37), erythritol-enriched (ERY37), benomyl dichloran-rose bengal (BENO37) and chromogenic (CAN37) media incubated at 37 °C and on Sabouraud-chloramphenicol (SAB27) and erythritol-enriched (ERY27) media incubated at 20-27 °C. Each plate was checked twice a week during 3 weeks. Fungi were conventionally identified; time for detection of fungal growth was noted for each species. Fungal prevalences and media performances were assessed; an optimal combination of media was determined using the Chi-squared automatic interaction detector method. At least one fungal species was isolated from 81% of sputa. Candida albicans was the most prevalent species (58.8%), followed by Aspergillus fumigatus (35.4%). Cultivation on CAN37, SAB27, ACT37 and ERY27 during 16 days provided an optimal combination, detecting C. albicans, A. fumigatus, Scedosporium apiospermum complex and Exophiala spp. with sensitivities of 96.5, 98.8, 100 and 100%. Combination of these four culture media is recommended to ensure the growth of key fungal pathogens in CF respiratory specimens. The use of such consensual protocol is of major interest for merging results from future epidemiological studies.

Preferential cytotoxicity of bortezomib toward highly malignant human liposarcoma cells via suppression of MDR1 expression and function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Yamei; Wang, Lingxian; Wang, Lu

2015-02-15

Liposarcoma is the most common soft tissue sarcoma with a high risk of relapse. Few therapeutic options are available for the aggressive local or metastatic disease. Here, we report that the clinically used proteasome inhibitor bortezomib exhibits significantly stronger cytotoxicity toward highly malignant human liposarcoma SW872-S cells compared with its parental SW872 cells, which is accompanied by enhanced activation of apoptotic signaling both in vitro and in vivo. Treatment of cells with Jun-N-terminal kinase (JNK) inhibitor SP60015 or the translation inhibitor cycloheximide ameliorated this enhanced apoptosis. Bortezomib inhibited MDR1 expression and function more effectively in SW872-S cells than in SW872more » cells, indicating that the increased cytotoxicity relies on the degree of proteasome inhibition. Furthermore, the pharmacological or genetic inhibition of sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2, which is highly expressed in SW872-S cells, resulted in partial reversal of cell growth inhibition and increase of MDR1 expression in bortezomib-treated SW872-S cells. These results show that bortezomib exhibits preferential cytotoxicity toward SW872-S cells possibly via highly expressed SERCA2-associated MDR1 suppression and suggest that bortezomib may serve as a potent agent for treating advanced liposarcoma. - Highlights: • We compare the cytotoxicity of different drugs between SW872-S and SW872 cells. • Highly malignant liposarcoma cells SW872-S show hypersensitivity to bortezomib. • Apoptotic signaling is robustly enhanced in bortezomib-treated SW872-S cells. • Bortezomib has strong suppression on MDR1 expression and function in SW872-S cells. • Inhibition of SERCA2 protects SW872-S cells from bortezomib.« less

Neutral endopeptidase up-regulation in isolated human umbilical artery: involvement in desensitization of bradykinin-induced vasoconstrictor effects.

PubMed

Pelorosso, Facundo Germán; Halperin, Ana Verónica; Palma, Alejandro Martín; Nowak, Wanda; Errasti, Andrea Emilse; Rothlin, Rodolfo Pedro

2007-02-01

Previous reports show that bradykinin B(2) receptors mediate contractile responses induced by bradykinin (BK) in human umbilical artery (HUA). However, although it has been reported that BK-induced responses can desensitize in several inflammatory models, the effects of prolonged in vitro incubation on BK-induced vasoconstriction in HUA have not been studied. In isolated HUA rings, BK-induced responses after a 5-h in vitro incubation showed a marked desensitization compared with responses at 2 h. Inhibition of either angiotensin-converting enzyme (ACE) or neutral endopeptidase (NEP), both BK-inactivating enzymes, failed to modify responses to BK at 2 h. After 5 h, ACE inhibition produced only a slight potentiation of BK-induced responses. In contrast, BK-induced vasoconstriction at 5 h was markedly potentiated by NEP inhibition. Moreover, NEP activity, measured by hydrolysis of its synthetic substrate (Z-Ala-Ala-Leu-p-nitroanilide), showed a 2.4-fold increase in 5-h incubated versus 2-h incubated tissues, which was completely reversed by cycloheximide (CHX) treatment. Furthermore, CHX significantly potentiated BK-induced responses, suggesting that NEP-mediated kininase activity increase at 5 h depends on de novo protein synthesis. In addition, under NEP inhibition, CHX treatment failed to produce an additional potentiation of BK-induced vasoconstriction. Still, NEP up-regulation was confirmed by Western blot, showing a 2.1-fold increase in immunoreactive NEP in 5-h incubated versus 2-h incubated HUA. In summary, the present study provides strong pharmacological evidence that NEP is up-regulated and plays a key role in desensitization of BK-induced vasoconstriction after prolonged in vitro incubation in HUA. Our results provide new insights into the possible mechanisms involved in BK-induced response desensitization during sustained inflammatory conditions.

Heat shock protein 70 in the rat nasal cavity: localisation and response to hyperthermia.

PubMed

Simpson, Sharon A; Alexander, David J; Reed, Celia J

2004-06-01

Heat shock proteins (HSPs) are a group of proteins that are rapidly induced in response to physiological stress, including hyperthermia and exposure to toxicants. Thus they may provide a useful index of toxicity in in vitro systems for screening for toxicity. We have recently developed a rat nasal explant system for investigating upper respiratory tract toxicity, and the aims of this study were to localise HSP70 within the rat nasal cavity and to characterise its response to hyperthermia. Constitutively, HSP70 was found to be predominantly localised to the sustentacular cells, basal cells and Bowman's glands of the olfactory epithelium (OE), with the most intense immunohistochemical staining at levels 3 and 4 of the posterior of the rat nasal cavity. Ethmoturbinates (ETs) and liver slices were exposed to heat shock (37 degrees and 43 degrees C, respectively) for 45 min and then returned to normal culture temperatures (31 degrees and 37 degrees C, respectively) for 24 h. In ETs, HSP72 was maximally induced 4-fold at 4 h after heat shock, and levels then returned to those of control tissue. ATP concentrations were markedly decreased up to 4 h after heat shock and then returned to control levels. In contrast, HSP72 levels in liver slices increased and ATP levels decreased steadily throughout the 24 h culture period. ETs were also able to withstand a 45-min heat shock at 43 degrees C, that is 12 degrees C above normal culture temperature. Incubation of ETs with cycloheximide prior to heat shock reduced the ability of the OE to recover from heat shock at 37 degrees C. Thus the OE of the rat nasal cavity expresses HSP72, and this protein appears to play an important role in the ability of the tissue to withstand hyperthermia.

In-frame seven amino-acid duplication in AIP arose over the last 3000 years, disrupts protein interaction and stability and is associated with gigantism

PubMed Central

Salvatori, Roberto; Radian, Serban; Diekmann, Yoan; Iacovazzo, Donato; David, Alessia; Gabrovska, Plamena; Grassi, Giorgia; Bussell, Anna-Marie; Stals, Karen; Weber, Astrid; Quinton, Richard; Crowne, Elizabeth C; Corazzini, Valentina; Metherell, Lou; Kearney, Tara; Du Plessis, Daniel; Sinha, Ajay Kumar; Baborie, Atik; Lecoq, Anne-Lise; Chanson, Philippe; Ansorge, Olaf; Ellard, Sian; Trainer, Peter J; Balding, David; Thomas, Mark G

2017-01-01

Objective Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene are associated with pituitary adenoma, acromegaly and gigantism. Identical alleles in unrelated pedigrees could be inherited from a common ancestor or result from recurrent mutation events. Design and methods Observational, inferential and experimental study, including: AIP mutation testing; reconstruction of 14 AIP-region (8.3 Mbp) haplotypes; coalescent-based approximate Bayesian estimation of the time to most recent common ancestor (tMRCA) of the derived allele; forward population simulations to estimate current number of allele carriers; proposal of mutation mechanism; protein structure predictions; co-immunoprecipitation and cycloheximide chase experiments. Results Nine European-origin, unrelated c.805_825dup-positive pedigrees (four familial, five sporadic from the UK, USA and France) included 16 affected (nine gigantism/four acromegaly/two non-functioning pituitary adenoma patients and one prospectively diagnosed acromegaly patient) and nine unaffected carriers. All pedigrees shared a 2.79 Mbp haploblock around AIP with additional haploblocks privately shared between subsets of the pedigrees, indicating the existence of an evolutionarily recent common ancestor, the ‘English founder’, with an estimated median tMRCA of 47 generations (corresponding to 1175 years) with a confidence interval (9–113 generations, equivalent to 225–2825 years). The mutation occurred in a small tandem repeat region predisposed to slipped strand mispairing. The resulting seven amino-acid duplication disrupts interaction with HSP90 and leads to a marked reduction in protein stability. Conclusions The c.805_825dup allele, originating from a common ancestor, associates with a severe clinical phenotype and a high frequency of gigantism. The mutation is likely to be the result of slipped strand mispairing and affects protein–protein interactions and AIP protein stability. PMID:28634279

In-frame seven amino-acid duplication in AIP arose over the last 3000 years, disrupts protein interaction and stability and is associated with gigantism.

PubMed

Salvatori, Roberto; Radian, Serban; Diekmann, Yoan; Iacovazzo, Donato; David, Alessia; Gabrovska, Plamena; Grassi, Giorgia; Bussell, Anna-Marie; Stals, Karen; Weber, Astrid; Quinton, Richard; Crowne, Elizabeth C; Corazzini, Valentina; Metherell, Lou; Kearney, Tara; Du Plessis, Daniel; Sinha, Ajay Kumar; Baborie, Atik; Lecoq, Anne-Lise; Chanson, Philippe; Ansorge, Olaf; Ellard, Sian; Trainer, Peter J; Balding, David; Thomas, Mark G; Korbonits, Márta

2017-09-01

Mutations in the aryl hydrocarbon receptor-interacting protein ( AIP ) gene are associated with pituitary adenoma, acromegaly and gigantism. Identical alleles in unrelated pedigrees could be inherited from a common ancestor or result from recurrent mutation events. Observational, inferential and experimental study, including: AIP mutation testing; reconstruction of 14 AIP -region (8.3 Mbp) haplotypes; coalescent-based approximate Bayesian estimation of the time to most recent common ancestor (tMRCA) of the derived allele; forward population simulations to estimate current number of allele carriers; proposal of mutation mechanism; protein structure predictions; co-immunoprecipitation and cycloheximide chase experiments. Nine European-origin, unrelated c.805_825dup-positive pedigrees (four familial, five sporadic from the UK, USA and France) included 16 affected (nine gigantism/four acromegaly/two non-functioning pituitary adenoma patients and one prospectively diagnosed acromegaly patient) and nine unaffected carriers. All pedigrees shared a 2.79 Mbp haploblock around AIP with additional haploblocks privately shared between subsets of the pedigrees, indicating the existence of an evolutionarily recent common ancestor, the 'English founder', with an estimated median tMRCA of 47 generations (corresponding to 1175 years) with a confidence interval (9-113 generations, equivalent to 225-2825 years). The mutation occurred in a small tandem repeat region predisposed to slipped strand mispairing. The resulting seven amino-acid duplication disrupts interaction with HSP90 and leads to a marked reduction in protein stability. The c.805_825dup allele, originating from a common ancestor, associates with a severe clinical phenotype and a high frequency of gigantism. The mutation is likely to be the result of slipped strand mispairing and affects protein-protein interactions and AIP protein stability. © 2017 The authors.

1-Nitropyrene (1-NP) induces apoptosis and apparently a non-apoptotic programmed cell death (paraptosis) in Hepa1c1c7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Asare, Nana; Landvik, Nina E.; Lagadic-Gossmann, Dominique

2008-07-15

Mechanistic studies of nitro-PAHs (polycyclic aromatic hydrocarbons) of interest might help elucidate which chemical characteristics are most important in eliciting toxic effects. 1-Nitropyrene (1-NP) is the predominant nitrated PAH emitted in diesel exhaust. 1-NP-exposed Hepa1c1c7 cells exhibited marked changes in cellular morphology, decreased proliferation and different forms of cell death. A dramatic increase in cytoplasmic vacuolization was observed already after 6 h of exposure and the cells started to round up at 12 h. The rate of cell proliferation was markedly reduced at 24 h and apoptotic as well as propidium iodide (PI)-positive cells appeared. Electron microscopic examination revealed thatmore » the vacuolization was partly due to mitochondria swelling. The caspase inhibitor Z-VAD-FMK inhibited only the apoptotic cell death and Nec-1 (an inhibitor of necroptosis) exhibited no inhibitory effects on either cell death or vacuolization. In contrast, cycloheximide markedly reduced both the number of apoptotic and PI-positive cells as well as the cytoplasmic vacuolization, suggesting that 1-NP induced paraptotic cell death. All the MAPKs; ERK1/2, p38 and JNK, appear to be involved in the death process since marked activation was observed upon 1-NP exposure, and their inhibitors partly reduced the induced cell death. The ERK1/2 inhibitor PD 98057 completely blocked the induced vacuolization, whereas the other MAPKs inhibitors only had minor effects on this process. These findings suggest that 1-NP may cause apoptosis and paraptosis. In contrast, the corresponding amine (1-aminopyrene) elicited only minor apoptotic and necrotic cell death, and cells with characteristics typical of paraptosis were absent.« less

A role for polyamines in glucose-stimulated insulin-gene expression.

PubMed Central

Welsh, N

1990-01-01

The aim of the present study was to evaluate the possible role for polyamines in the glucose regulation of the metabolism of insulin mRNA of pancreatic islet cells. For this purpose islets were prepared from adult mice and cultured for 2 days in culture medium RPMI 1640 containing 3.3 mM- or 16.7 mM-glucose with or without the addition of the inhibitors of polyamine biosynthesis difluoromethylornithine (DFMO) and ethylglyoxal bis(guanylhydrazone) (EGBG). Culture at the high glucose concentration increased the islet contents of both insulin mRNA and polyamines. The synthesis of total RNA, total islet polyamines and polyamines associated with islet nuclei was also increased. When the combination of DFMO and EGBG was added in the presence of 16.7 mM-glucose, low contents of insulin mRNA, spermine and spermidine were observed. Total islet polyamine synthesis was also depressed by DFMO + EGBG, unlike islet biosynthesis of polyamines associated with nuclei, which was not equally decreased by the polyamine-synthesis inhibitors. Total RNA synthesis and turnover was not affected by DFMO + EGBG. Finally, actinomycin D attenuated the glucose-induced enhancement of insulin mRNA, and cycloheximide counteracted the insulin-mRNA attenuation induced by inhibition of polyamine synthesis. It is concluded that the glucose-induced increase in insulin mRNA is paralleled by increased contents and rates of polyamine biosynthesis and that an attenuation of the increase in polyamines prevents the increase in insulin mRNA. In addition, the results are compatible with the view that polyamines exert their effects on insulin mRNA mainly by increasing the stability of this messenger. PMID:2241922

Fungal and bacterial contributions to nitrogen cycling in cheatgrass-invaded and uninvaded native sagebrush soils of the western USA

USGS Publications Warehouse

DeCrappeo, Nicole; DeLorenze, Elizabeth J.; Giguere, Andrew T; Pyke, David A.; Bottomley, Peter J.

2017-01-01

AimThere is interest in determining how cheatgrass (Bromus tectorum L.) modifies N cycling in sagebrush (Artemisia tridentata Nutt.) soils of the western USA.MethodsTo gain insight into the roles of fungi and bacteria in N cycling of cheatgrass-invaded and uninvaded sagebrush soils, the fungal protein synthesis inhibitor, cycloheximide (CHX), and the bacteriocidal compound, bronopol (BRO) were combined with a 15NH4+ isotope pool dilution approach.ResultsCHX reduced gross N mineralization to the same rate in both sagebrush and cheatgrass soils indicating a role for fungi in N mineralization in both soil types. In cheatgrass soils BRO completely inhibited gross N mineralization, whereas, in sagebrush soils a BRO-resistant gross N mineralization rate was detected that was slower than CHX sensitive gross N mineralization, suggesting that the microbial drivers of gross N mineralization were different in sagebrush and cheatgrass soils. Net N mineralization was stimulated to a higher rate in sagebrush than in cheatgrass soils by CHX, implying that a CHX inhibited N sink was larger in the former than the latter soils. Initial gross NH4+ consumption rates were reduced significantly by both CHX and BRO in both soil types, yet, consumption rates recovered significantly between 24 and 48 h in CHX-treated sagebrush soils. The recovery of NH4+ consumption in sagebrush soils corresponded with an increase in the rate of net nitrification.ConclusionsThese results suggest that cheatgrass invasion of sagebrush soils of the northern Great Basin reduces the capacity of the fungal N consumption sink, enhances the capacity of a CHX resistant N sink and alters the contributions of bacteria and fungi to gross N mineralization.

Insights into the working mechanism of water filtered infrared A (wIRA) irradiation on Chlamydia trachomatis serovar E

NASA Astrophysics Data System (ADS)

Kuratli, Jasmin; Pesch, Theresa; Marti, Hanna; Blenn, Christian; Borel, Nicole

2018-02-01

Infections with Chlamydia trachomatis are the major cause for infectious blindness and still represent the most common bacterial sexually transmitted disease worldwide. Considering the possible side effects of antibiotic therapy and the increasing threat of antibiotic resistance, alternative therapeutic strategies are needed. Previous studies showed a reduction of C. trachomatis infectivity after irradiation with water filtered infrared A alone (wIRA) or in combination with visible light (wIRA/VIS). In this study, we aimed to gain further insight into the working mechanism of wIRA/VIS by analyzing cytokine and chemokine levels of infected and non-infected HeLa cells following triple dose irradiation at 24, 36 and 40 hours post infection. Subsequently, we examined the influence of cytokines on irradiation and chlamydial infection using a cytokine/chemokine inhibitor (Azelastine) and by IL-6 and IL-8 gene silencing. A triple dose irradiation significantly reduced chlamydial infectivity in HeLa cells without inducing the chlamydial stress response. The reducing effect was present regardless of the addition of cycloheximide (CHX), a host protein synthesis inhibitor. Chlamydial infection, wIRA/VIS treatment and the combination of both revealed a similar release pattern of a subset of pro-inflammatory cytokines (IL-6, IL-8, RANTES, Serpin E1). The addition of Azelastine induced the chlamydial stress response in non-irradiated samples. This effect was even more pronounced in wIRA/VIS-treated conditions. Silencing of IL-6 and IL-8 resulted in a lower chlamydial infectivity. However, wIRA/VIS treatment of infected and silenced cells reduced the chlamydial infectivity similar to wIRA/VIS treated control cells. Further studies are needed to elucidate the working mechanism of wIRA/VIS.

Parathyroid hormone induces transcription of collagenase in rat osteoblastic cells by a mechanism using cyclic adenosine 3',5'-monophosphate and requiring protein synthesis

NASA Technical Reports Server (NTRS)

Scott, D. K.; Brakenhoff, K. D.; Clohisy, J. C.; Quinn, C. O.; Partridge, N. C.

1992-01-01

Collagenase is synthesized and secreted by rat osteoblastic cells in response to PTH. We have previously demonstrated that this effect involves a substantial increase in collagenase mRNA via transcription. Northern blots and nuclear run-on assays were performed to further investigate the induction of collagenase by PTH in the rat osteoblastic cell line UMR 106-01. Detectable amounts of collagenase mRNA were not apparent until 2 h of PTH treatment, showed the greatest abundance at 4 h, and declined to approximately 30% of maximum by 8 h. The changes in the rate of transcription of the collagenase gene in response to PTH paralleled and preceded the changes in the steady state mRNA levels. After an initial lag period of about 1 h, collagenase transcription rates increased from very low levels to a maximal response at 2 h, returning to about 50% of maximum by 10 h. The increased transcriptional rate of the collagenase gene was found to be dependent on the concentration of PTH, with a half-maximal response at approximately 7 x 10(-10) M rat PTH-(1-34) and a maximal effect with a dose of 10(-8) M. The PTH-mediated induction of collagenase transcriptional activity was completely abolished by cycloheximide, while transcription of the beta-actin gene was unaffected by the translation inhibitor. These data suggest that a protein factor(s) is required for PTH-mediated transcriptional induction of collagenase. Since PTH increases intracellular levels of several potential second messengers, agents that mimic these substances were employed to determine which signal transduction pathway is predominant in the PTH-mediated stimulation of collagenase transcription.(ABSTRACT TRUNCATED AT 250 WORDS).

Dynamic Proteomics Emphasizes the Importance of Selective mRNA Translation and Protein Turnover during Arabidopsis Seed Germination*

PubMed Central

Galland, Marc; Huguet, Romain; Arc, Erwann; Cueff, Gwendal; Job, Dominique; Rajjou, Loïc

2014-01-01

During seed germination, the transition from a quiescent metabolic state in a dry mature seed to a proliferative metabolic state in a vigorous seedling is crucial for plant propagation as well as for optimizing crop yield. This work provides a detailed description of the dynamics of protein synthesis during the time course of germination, demonstrating that mRNA translation is both sequential and selective during this process. The complete inhibition of the germination process in the presence of the translation inhibitor cycloheximide established that mRNA translation is critical for Arabidopsis seed germination. However, the dynamics of protein turnover and the selectivity of protein synthesis (mRNA translation) during Arabidopsis seed germination have not been addressed yet. Based on our detailed knowledge of the Arabidopsis seed proteome, we have deepened our understanding of seed mRNA translation during germination by combining two-dimensional gel-based proteomics with dynamic radiolabeled proteomics using a radiolabeled amino acid precursor, namely [35S]-methionine, in order to highlight de novo protein synthesis, stability, and turnover. Our data confirm that during early imbibition, the Arabidopsis translatome keeps reflecting an embryonic maturation program until a certain developmental checkpoint. Furthermore, by dividing the seed germination time lapse into discrete time windows, we highlight precise and specific patterns of protein synthesis. These data refine and deepen our knowledge of the three classical phases of seed germination based on seed water uptake during imbibition and reveal that selective mRNA translation is a key feature of seed germination. Beyond the quantitative control of translational activity, both the selectivity of mRNA translation and protein turnover appear as specific regulatory systems, critical for timing the molecular events leading to successful germination and seedling establishment. PMID:24198433

c-myc, c-fos, and c-jun regulation in the regenerating livers of normal and H-2K/c-myc transgenic mice.

PubMed Central

Morello, D; Fitzgerald, M J; Babinet, C; Fausto, N

1990-01-01

We investigated the mechanisms of regulation of c-myc, c-fos, and c-jun at the early stages of liver regeneration in mice. We show that the transient increase in steady-state levels of c-myc mRNA at the start of liver regeneration is most probably regulated by posttranscriptional mechanisms. Although there was a marked increase in c-myc transcriptional initiation shortly after partial hepatectomy, a block in elongation prevented the completion of most transcripts. To gain further information on the mechanism of regulation of c-myc expression during liver regeneration, we used transgenic mice harboring the human c-myc gene driven by the H-2K promoter. In these animals, the murine c-myc responded to the growth stimulus generated by partial hepatectomy, whereas the expression of the transgene was constitutive and did not change in the regenerating liver. However, the mRNA from both genes increased markedly after cycloheximide injection, suggesting that the regulation of c-myc mRNA abundance in the regenerating liver differs from that occurring after protein synthesis inhibition. Furthermore, we show that in normal mice c-fos and c-jun mRNA levels and transcriptional rates increase within 30 min after partial hepatectomy. c-fos transcriptional elongation was restricted in nongrowing liver, but the block was partially relieved in the regenerating liver. Nevertheless, for both c-fos and c-jun, changes in steady-state mRNA detected after partial hepatectomy were much greater than the transcriptional increase. In the regenerating liver of H-2K/c-myc mice, c-fos and c-jun expression was diminished, whereas mouse c-myc expression was enhanced in comparison with that in nontransgenic animals. Images PMID:2111449

Subcellular localization and characterization of G protein-coupled receptor homolog from lymphocystis disease virus isolated in China.

PubMed

Huang, Youhua; Huang, Xiaohong; Zhang, Jing; Gui, Jianfang; Zhang, Qiya

2007-01-01

G protein-coupled receptors (GPCRs) constitute a large superfamily involved in various types of signal transduction pathways, and play an important role in coordinating the activation and migration of leukocytes to sites of infection and inflammation. Viral GPCRs, on the other hand, can help the virus to escape from host immune surveillance and contribute to viral pathogenesis. Lymphocystis disease virus isolated in China (LCDV-C) contains a putative homolog of cellular GPCRs, LCDV-C GPCR. In this paper, LCDV-C GPCR was cloned, and the subcellular localization and characterization of GPCR protein were investigated in fish cells. LCDV-C GPCR encoded a 325 amino acid peptide, containing a typical seven-transmembrane domain characteristic of the chemokine receptors and a conserved DRY motif that is usually essential for receptor activation. Transient transfection of GPCR-EGFP in fathead minnow (FHM) cells and epithelioma papulosum cyprini (EPC) cells indicated that LCDV-C GPCR was expressed abundantly in both the cytoplasm and nucleoplasm. Transient overexpression of GPCR in these two cells cannot induce obvious apoptosis. FHM cells stably expressing GPCR showed enhanced cell proliferation and significant anchorage-independent growth. The effects of GPCR protein on external apoptotic stimuli were examined. Few apoptotic bodies were observed in cells expressing GPCR treated with actinomycin D (ActD). Quantitative analysis of apoptotic cells indicated that a considerable decrease in the apoptotic fraction of cells expressing GPCR, compared with the control cells, was detected after exposure to ActD and cycloheximide. These data suggest that LCDV-C GPCR may inhibit apoptosis as part of its potential mechanism in mediating cellular transformation.

Kaempferol Modulates DNA Methylation and Downregulates DNMT3B in Bladder Cancer.

PubMed

Qiu, Wei; Lin, Jun; Zhu, Yichen; Zhang, Jian; Zeng, Liping; Su, Ming; Tian, Ye

2017-01-01

Genomic DNA methylation plays an important role in both the occurrence and development of bladder cancer. Kaempferol (Kae), a natural flavonoid that is present in many fruits and vegetables, exhibits potent anti-cancer effects in bladder cancer. Similar to other flavonoids, Kae possesses a flavan nucleus in its structure. This structure was reported to inhibit DNA methylation by suppressing DNA methyltransferases (DNMTs). However, whether Kae can inhibit DNA methylation remains unclear. Nude mice bearing bladder cancer were treated with Kae for 31 days. The genomic DNA was extracted from xenografts and the methylation changes was determined using an Illumina Infinium HumanMethylation 450 BeadChip Array. The ubiquitination was detected using immuno-precipitation assay. Our data indicated that Kae modulated DNA methylation in bladder cancer, inducing 103 differential DNA methylation positions (dDMPs) associated with genes (50 hyper-methylated and 53 hypo-methylated). DNA methylation is mostly relied on the levels of DNMTs. We observed that Kae specifically inhibited the protein levels of DNMT3B without altering the expression of DNMT1 or DNMT3A. However, Kae did not downregulate the transcription of DNMT3B. Interestingly, we observed that Kae induced a premature degradation of DNMT3B by inhibiting protein synthesis with cycloheximide (CHX). By blocking proteasome with MG132, we observed that Kae induced an increased ubiquitination of DNMT3B. These results suggested that Kae could induce the degradation of DNMT3B through ubiquitin-proteasome pathway. Our data indicated that Kae is a novel DNMT3B inhibitor, which may promote the degradation of DNMT3B in bladder cancer. © 2017 The Author(s)Published by S. Karger AG, Basel.

Characterization of RAD9 of Saccharomyces cerevisiae and evidence that its function acts posttranslationally in cell cycle arrest after DNA damage.

PubMed

Weinert, T A; Hartwell, L H

1990-12-01

In eucaryotic cells, incompletely replicated or damaged chromosomes induce cell cycle arrest in G2 before mitosis, and in the yeast Saccharomyces cerevisiae the RAD9 gene is essential for the cell cycle arrest (T.A. Weinert and L. H. Hartwell, Science 241:317-322, 1988). In this report, we extend the analysis of RAD9-dependent cell cycle control. We found that both induction of RAD9-dependent arrest in G2 and recovery from arrest could occur in the presence of the protein synthesis inhibitor cycloheximide, showing that the mechanism of RAD9-dependent control involves a posttranslational mechanism(s). We have isolated and determined the DNA sequence of the RAD9 gene, confirming the DNA sequence reported previously (R. H. Schiestl, P. Reynolds, S. Prakash, and L. Prakash, Mol. Cell. Biol. 9:1882-1886, 1989). The predicted protein sequence for the Rad9 protein bears no similarity to sequences of known proteins. We also found that synthesis of the RAD9 transcript in the cell cycle was constitutive and not induced by X-irradiation. We constructed yeast cells containing a complete deletion of the RAD9 gene; the rad9 null mutants were viable, sensitive to X- and UV irradiation, and defective for cell cycle arrest after DNA damage. Although Rad+ and rad9 delta cells had similar growth rates and cell cycle kinetics in unirradiated cells, the spontaneous rate of chromosome loss (in unirradiated cells) was elevated 7- to 21-fold in rad9 delta cells. These studies show that in the presence of induced or endogenous DNA damage, RAD9 is a negative regulator that inhibits progression from G2 in order to preserve cell viability and to maintain the fidelity of chromosome transmission.

Characterization of the selenite uptake mechanism in the coccolithophore Emiliania huxleyi (Haptophyta).

PubMed

Araie, Hiroya; Sakamoto, Kou; Suzuki, Iwane; Shiraiwa, Yoshihiro

2011-07-01

The marine coccolithophore Emiliania huxleyi (Haptophyta) requires selenium as an essential element for growth, and the active species absorbed is selenite, not selenate. This study characterized the selenite uptake mechanism using ⁷⁵Se as a tracer. Kinetic analysis of selenite uptake showed the involvement of both active and passive transport processes. The active transport was suppressed by 0.5 mM vanadate, a membrane-permeable inhibitor of H⁺-ATPase, at pH 8.3. When the pH was lowered from 8.3 to 5.3, the selenite uptake activity greatly increased, even in the presence of vanadate, suggesting that the H⁺ concentration gradient may be a motive force for selenite transport. [⁷⁵Se]Selenite uptake at selenite-limiting concentrations was hardly affected by selenate, sulfate and sulfite, even at 100 μM. In contrast, 3 μM orthophosphate increased the K(m) 5-fold. These data showed that HSeO₃⁻, a dominant selenite species at acidic pH, is the active species for transport through the plasma membrane and transport is driven by ΔpH energized by H⁺-ATPase. Kinetic analysis showed that the selenite uptake activity was competitively inhibited by orthophosphate. Furthermore, the active selenite transport mechanism was shown to be induced de novo under Se-deficient conditions and induction was suppressed by the addition of either sufficient selenite or cycloheximide, an inhibitor of de novo protein synthesis. These results indicate that E. huxleyi cells developed an active selenite uptake mechanism to overcome the disadvantages of Se limitation in ecosystems, maintaining selenium metabolism and selenoproteins for high viability.

Amyloid beta peptide as a physiological modulator of neuronal 'A'-type K+ current.

PubMed

Plant, Leigh D; Webster, Nicola J; Boyle, John P; Ramsden, Martin; Freir, Darragh B; Peers, Chris; Pearson, Hugh A

2006-11-01

Control of neuronal spiking patterns resides, in part, in the type and degree of expression of voltage-gated K(+) channel subunits. Previous studies have revealed that soluble forms of the Alzheimer's disease associated amyloid beta protein (Abeta) can increase the 'A'-type current in neurones. In this study, we define the molecular basis for this increase and show that endogenous production of Abeta is important in the modulation of Kv4.2 and Kv4.3 subunit expression in central neurones. A-type K(+) currents, and Kv4.2 and Kv4.3 subunit expression, were transiently increased in cerebellar granule neurones by the 1-40 and 1-42 forms of Abeta (100nM, 2-24h). Currents through recombinant Kv4.2 channels expressed in HEK293 cells were increased in a similar fashion to those through the native channels. Increases in 'A'-type current could be prevented by the use of cycloheximide and brefeldin A, indicating that protein expression and trafficking processes were altered by Abeta, rather than protein degredation. Endogenous Abeta production in cerebellar granule neurones was blocked using inhibitors of either gamma- or beta-secretase and resulted in decreased K(+) current. Crucially this could be prevented by co-application of exogenous Abeta (1nM), however, no change in Kv4.2 or Kv4.3 subunit expression occurred. These data show that Abeta is a modulator of Kv4 subunit expression in neurones at both the functional and the molecular level. Thus Abeta is not only involved in Alzheimer pathology, but is also an important physiological regulator of ion channel expression and hence neuronal excitability.

Prostanoid-induced expression of matrix metalloproteinase-1 messenger ribonucleic acid in rat osteosarcoma cells

NASA Technical Reports Server (NTRS)

Clohisy, J. C.; Connolly, T. J.; Bergman, K. D.; Quinn, C. O.; Partridge, N. C.

1994-01-01

Individual prostanoids have distinct potencies in activating intracellular signaling pathways and regulating gene expression in osteoblastic cells. The E-series prostaglandins (PGs) are known to stimulate matrix metalloproteinase-1 (MMP-1) synthesis and secretion in certain rodent and human osteoblastic cells, yet the intracellular events involved remain unclear. To further characterize this response and its signal transduction pathway(s), we examined prostanoid-induced expression of the MMP-1 gene in the rat osteoblastic osteosarcoma cell line UMR 106-01. Northern blot analysis demonstrated that prostaglandin E2 (PGE2) and PGE1 were very potent stimulators (40-fold) of MMP-1 transcript abundance, PGF2 alpha and prostacyclin were weak stimulators (4-fold), and thromboxane-B2 had no effect. The marked increase in MMP-1 transcript abundance after PGE2 treatment was first detected at 2 h, became maximal at 4 h, and persisted beyond 24 h. This response was dose dependent and elicited maximal and half-maximal effects with concentrations of 10(-6) and 0.6 x 10(-7) M, respectively. Cycloheximide, a protein synthesis inhibitor, completely blocked this effect of PGE2, suggesting that the expression of other genes is required. Nuclear run-on experiments demonstrated that PGE2 rapidly activates MMP-1 gene transcription, with a maximal increase at 2-4 h. The second messenger analog, 8-bromo-cAMP, mimicked the effects of PGE2 by stimulating a dose-dependent increase in MMP-1 messenger RNA (mRNA) levels, with a maximal effect quantitatively similar to that observed with PGE2. Thus, in UMR 106-01 cells, different prostanoids have distinct potencies in stimulating MMP-1 mRNA abundance. Our data suggest that PGE2 stimulation of MMP-1 synthesis is due to activation of MMP-1 gene transcription and a subsequent marked increase in MMP-1 mRNA abundance. This effect is dependent on de novo protein synthesis and is mimicked by protein kinase-A activation.

On the nature and origin of the calcium asymmetry arising during gravitropic response in etiolated pea epicotyls

NASA Technical Reports Server (NTRS)

Migliaccio, F.; Galston, A. W.

1987-01-01

Seven day old etiolated pea epicotyls were loaded symmetrically with 3H-indole 3-acetic acid (IAA) or 45Ca2+, then subjected to 1.5 hours of 1g gravistimulation. Epidermal peels taken from top and bottom surfaces after 90 minutes showed an increase in IAA on the lower side and of Ca2+ on the upper side. Inhibitors of IAA movement (TIBA, 9-hydroxyfluorene carboxylic acid) block the development of both IAA and Ca2+ asymmetries, but substances known to interfere with normal Ca2+ transport (nitrendipine, nisoldipine, Bay K 8644, A 23187) do not significantly alter either IAA or Ca2+ asymmetries. These substances, however, are active in modifying both Ca2+ uptake and efflux through oat and pea leaf protoplast membranes. We conclude that the 45Ca2+ fed to pea epicotyls occurs largely in the cell wall, and that auxin movement is primary and Ca2+ movement secondary in gravitropism. We hypothesize that apoplastic Ca2+ changes during graviresponse because it is displaced by H+ secreted through auxin-induced proton release. This proposed mechanism is supported by localized pH experiments, in which filter paper soaked in various buffers was applied to one side of a carborundum-abraded epicotyls. Buffer at pH 3 increases calcium loss from the side to which it is applied, whereas pH 7 buffer decreases it. Moreover, 10 micromolar IAA and 1 micromolar fusicoccin, which promote H+ efflux, increase Ca2+ release from pea epicotyl segments, whereas cycloheximide, which inhibits H+ efflux, has the reverse effect. We suggest that Ca2+ does not redistribute actively during gravitropism: the asymmetry arises because of its release from the wall adjacent to the region of high IAA concentration, proton secretion, and growth. Thus, the asymmetric distribution of Ca2+ appears to be a consequence of growth stimulation, not a critical step in the early phase of the graviresponse.

Inhibition of Protein Ubiquitination by Paraquat and 1-Methyl-4-Phenylpyridinium Impairs Ubiquitin-Dependent Protein Degradation Pathways.

PubMed

Navarro-Yepes, Juliana; Anandhan, Annadurai; Bradley, Erin; Bohovych, Iryna; Yarabe, Bo; de Jong, Annemieke; Ovaa, Huib; Zhou, You; Khalimonchuk, Oleh; Quintanilla-Vega, Betzabet; Franco, Rodrigo

2016-10-01

Intracytoplasmic inclusions of protein aggregates in dopaminergic cells (Lewy bodies) are the pathological hallmark of Parkinson's disease (PD). Ubiquitin (Ub), alpha (α)-synuclein, p62/sequestosome 1, and oxidized proteins are the major components of Lewy bodies. However, the mechanisms involved in the impairment of misfolded/oxidized protein degradation pathways in PD are still unclear. PD is linked to mitochondrial dysfunction and environmental pesticide exposure. In this work, we evaluated the effects of the pesticide paraquat (PQ) and the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP(+)) on Ub-dependent protein degradation pathways. No increase in the accumulation of Ub-bound proteins or aggregates was observed in dopaminergic cells (SK-N-SH) treated with PQ or MPP(+), or in mice chronically exposed to PQ. PQ decreased Ub protein content, but not its mRNA transcription. Protein synthesis inhibition with cycloheximide depleted Ub levels and potentiated PQ-induced cell death. The inhibition of proteasomal activity by PQ was found to be a late event in cell death progression and had neither effect on the toxicity of either MPP(+) or PQ, nor on the accumulation of oxidized sulfenylated, sulfonylated (DJ-1/PARK7 and peroxiredoxins), and carbonylated proteins induced by PQ. PQ- and MPP(+)-induced Ub protein depletion prompted the dimerization/inactivation of the Ub-binding protein p62 that regulates the clearance of ubiquitinated proteins by autophagy. We confirmed that PQ and MPP(+) impaired autophagy flux and that the blockage of autophagy by the overexpression of a dominant-negative form of the autophagy protein 5 (dnAtg5) stimulated their toxicity, but there was no additional effect upon inhibition of the proteasome. PQ induced an increase in the accumulation of α-synuclein in dopaminergic cells and membrane-associated foci in yeast cells. Our results demonstrate that the inhibition of protein ubiquitination by PQ and MPP(+) is involved in the dysfunction of Ub-dependent protein degradation pathways.

Down-regulation of adipose tissue lipoprotein lipase during fasting requires that a gene, separate from the lipase gene, is switched on.

PubMed

Bergö, Martin; Wu, Gengshu; Ruge, Toralph; Olivecrona, Thomas

2002-04-05

During short term fasting, lipoprotein lipase (LPL) activity in rat adipose tissue is rapidly down-regulated. This down-regulation occurs on a posttranslational level; it is not accompanied by changes in LPL mRNA or protein levels. The LPL activity can be restored within 4 h by refeeding. Previously, we showed that during fasting there is a shift in the distribution of lipase protein toward an inactive form with low heparin affinity. To study the nature of the regulatory mechanism, we determined the in vivo turnover of LPL activity, protein mass, and mRNA in rat adipose tissue. When protein synthesis was inhibited with cycloheximide, LPL activity and protein mass decreased rapidly and in parallel with half-lives of around 2 h, and the effect of refeeding was blocked. This indicates that maintaining high levels of LPL activity requires continuous synthesis of new enzyme protein. When transcription was inhibited by actinomycin, LPL mRNA decreased with half-lives of 13.3 and 16.8 h in the fed and fasted states, respectively, demonstrating slow turnover of the LPL transcript. Surprisingly, when actinomycin was given to fed rats, LPL activity was not down-regulated during fasting, indicating that actinomycin interferes with the transcription of a gene that blocks the activation of newly synthesized LPL protein. When actinomycin was given to fasted rats, LPL activity increased 4-fold within 6 h, even in the absence of refeeding. The same effect was seen with alpha-amanitin, another inhibitor of transcription. The response to actinomycin was much less pronounced in aging rats, which are obese and insulin-resistant. These data suggest a default state where LPL protein is synthesized on a relatively stable mRNA and is processed into its active form. During fasting, a gene is switched on whose product prevents the enzyme from becoming active even though synthesis of LPL protein continues unabated.

Regulation of expression of collagenase-3 in normal, differentiating rat osteoblasts

NASA Technical Reports Server (NTRS)

Winchester, S. K.; Bloch, S. R.; Fiacco, G. J.; Partridge, N. C.

1999-01-01

We investigated the regulation of collagenase-3 expression in normal, differentiating rat osteoblasts. Fetal rat calvarial cell cultures showed an increase in alkaline phosphatase activity reaching maximal levels between 7-14 days post-confluence, then declining with the onset of mineralization. Collagenase-3 mRNA was just detectable after proliferation ceased at day 7, increased up to day 21, and declined at later ages. Postconfluent cells maintained in non-mineralizing medium expressed collagenase-3 but did not show the developmental increase exhibited by cells switched to mineralization medium. Cells maintained in non-mineralizing medium continued to proliferate; cells in mineralization medium ceased proliferation. In addition, collagenase-3 mRNA was not detected in subcultured cells allowed to remineralize. These results suggest that enhanced accumulation of collagenase-3 mRNA is triggered by cessation of proliferation or acquisition of a mineralized extracellular matrix and that other factors may also be required. After initiation of basal expression, parathyroid hormone (PTH) caused a dose-dependent increase in collagenase-3 mRNA. Both the cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (8-Br-cAMP), and the protein kinase C (PKC) activator, phorbol myristate acetate, increased collagenase-3 expression, while the calcium ionophore, ionomycin, did not, suggesting that PTH was acting through the protein kinase A (PKA) and PKC pathways. Inhibition of protein synthesis with cycloheximide caused an increase in basal collagenase-3 expression but blocked the effect of PTH, suggesting that an inhibitory factor prevents basal expression while an inductive factor is involved with PTH action. In summary, collagenase-3 is expressed in mineralized osteoblasts and cessation of proliferation and initiation of mineralization are triggers for collagenase-3 expression. PTH also stimulates expression of the enzyme through both PKA and PKC pathways in the mineralizing osteoblast. Copyright 1999 Wiley-Liss, Inc.

Peptide-induced prostaglandin biosynthesis in the renal-vein-constricted kidney

PubMed Central

Myers, Stuart I.; Zipser, Robert; Needleman, Philip

1981-01-01

The ipsilateral kidney was removed from a rabbit 48h after unilateral partial renal-vein-constriction and was perfused with Krebs–Henseleit media at 37°C. Hourly administration of a fixed dose of bradykinin to the renal-vein-constricted kidney demonstrated a marked time-dependent increase in the release of bioassayable prostaglandin E2 and thromboxane A2 into the venous effluent as compared with the response of the contralateral control kidney. The renal-vein-constricted kidney produced up to 60 times more prostaglandin E2 in response to bradykinin after 6h of perfusion as compared with the contralateral kidney; thromboxane A2 was not demonstratable in the contralateral kidney. Inhibition of protein synthesis de novo in the perfused renal-vein-constricted kidney with cycloheximide lessened the hormone-stimulated increase in prostaglandin E2 by 94% and in thromboxane A2 by 90% at 6h of perfusion. Covalent acetylation of the renal cyclo-oxygenase by prior oral administration of aspirin to the rabbit inhibited initial bradykinin-stimulated prostaglandin E2 biosynthesis 71% at 1h of perfusion. However, there was total recovery from aspirin in the renal-vein-constricted kidney by 2h of perfusion after bradykinin stimulation. Total cyclo-oxygenase activity as measured by [14C]arachidonate metabolism to labelled prostaglandins by renal cortical and renal medullary microsomal fractions prepared from 6h-perfused kidneys demonstrated that renal-vein-constricted kidney-cortical cyclo-oxygenase activity was significantly greater than the contralateral-kidney-cortical conversion, whereas medullary arachidonate metabolism was comparable in both the renal-vein-constricted kidney and contralateral kidney. These data suggest that perfusion of a renal-vein-constricted kidney initiates a time-dependent induction of synthesis of prostaglandin-producing enzymes, which appear to be primarily localized in the renal cortex. The presence of the synthetic capacity to generate very potent vasodilator and vasoconstrictor prostaglandins in the renal cortex suggests that these substances could mediate or modulate changes in renal vascular resistance in pathological states. PMID:6798974

The multilevel antibiotic-induced perturbations to biological systems: Early-life exposure induces long-lasting damages to muscle structure and mitochondrial metabolism in flies.

PubMed

Renault, David; Yousef, Hesham; Mohamed, Amr A

2018-06-07

Antibiotics have been increasingly used over the past decades for human medicine, food-animal agriculture, aquaculture, and plant production. A significant part of the active molecules of antibiotics can be released into the environment, in turn affecting ecosystem functioning and biogeochemical processes. At lower organizational scales, these substances affect bacterial symbionts of insects, with negative consequences on growth and development of juveniles, and population dynamics. Yet, the multiple alterations of cellular physiology and metabolic processes have remained insufficiently explored in insects. We evaluated the effects of five antibiotics with different mode of action, i.e. ampicillin, cefradine, chloramphenicol, cycloheximide, and tetracycline, on the survival and ultrastructural organization of the flight muscles of newly emerged blow flies Chrysomya albiceps. Then, we examined the effects of different concentrations of antibiotics on mitochondrial protein content, efficiency of oxidative phosphorylation, and activity of transaminases (Glutamate oxaloacetate transaminase and glutamate pyruvate transaminase) and described the cellular metabolic perturbations of flies treated with antibiotics. All antibiotics affected the survival of the insects and decreased the total mitochondrial protein content in a dose-dependent manner. Ultrastructural organization of flight muscles in treated flies differs dramatically compared to the control groups and severe pathological damages/structures disorganization of mitochondria appeared. The activities of mitochondrial transaminases significantly increased with increased antibiotic concentrations. The oxidation rate of pyruvate + proline from isolated mitochondria of the flight muscles of 1-day-old flies was significantly reduced at high doses of antibiotics. In parallel, the level of several metabolites, including TCA cycle intermediates, was reduced in antibiotics-treated flies. Overall, antibiotics provoked a system-wide alteration of the structure and physiology of flight muscles of the blow fly Ch. albiceps, and may have fitness consequences at the organism level. Environmental antibiotic pollution is likely to have unwanted cascading ecological effects of insect population dynamics and community structure. Copyright © 2018 Elsevier Ltd. All rights reserved.

Differential expression of E-cadherin at the surface of rat beta-cells as a marker of functional heterogeneity.

PubMed

Bosco, Domenico; Rouiller, Dominique G; Halban, Philippe A

2007-07-01

The aim of this study was to assess whether the expression of E-cadherin at the surface of rat beta-cells is regulated by insulin secretagogues and correlates with insulin secretion. When cultured under standard conditions, virtually all beta-cells expressed E-cadherin observed by immunofluorescence, but heterogeneous staining was observed. Using fluorescence-activated cell sorting (FACS), two beta-cell sub-populations were sorted: one that was poorly labeled ('ECad-low') and another that was highly labeled ('ECad-high'). After 1-h stimulation with 16.7 mM glucose, insulin secretion (reverse hemolytic plaque assay) from individual ECad-high beta-cells was higher than that from ECad-low beta-cells. Ca2+-dependent beta-cell aggregation was increased at 16.7 mM glucose when compared with 2.8 mM glucose. E-cadherin at the surface of beta-cells was increased after 18 h at 11.1 and 22.2 mM glucose when compared with 2.8 mM glucose, with the greatest increase at 22.2 mM glucose + 0.5 mM isobutylmethylxanthine (IBMX). While no labeling was detected on freshly trypsinized cells, the proportion of stained cells increased in a time-dependent manner during culture for 1, 3, and 24 h. This recovery was faster when cells were incubated at 16.7 vs 2.8 mM glucose. Cycloheximide inhibited expression of E-cadherin at 2.8 mM glucose, but not at 16.7 mM, while depolymerization of actin by either cytochalasin B or latrunculin B increased surface E-cadherin at low glucose. In conclusion, these results show that expression of E-cadherin at the surface of islet beta-cells is controlled by secretagogues including glucose, correlates with insulin secretion, and can serve as a surface marker of beta-cell function.

Different actions of endothelin-1 on chemokine production in rat cultured astrocytes: reduction of CX3CL1/fractalkine and an increase in CCL2/MCP-1 and CXCL1/CINC-1

PubMed Central

2013-01-01

Background Chemokines are involved in many pathological responses of the brain. Astrocytes produce various chemokines in brain disorders, but little is known about the factors that regulate astrocytic chemokine production. Endothelins (ETs) have been shown to regulate astrocytic functions through ETB receptors. In this study, the effects of ETs on chemokine production were examined in rat cerebral cultured astrocytes. Methods Astrocytes were prepared from the cerebra of one- to two-day-old Wistar rats and cultured in serum-containing medium. After serum-starvation for 48 hours, astrocytes were treated with ETs. Total RNA was extracted using an acid-phenol method and expression of chemokine mRNAs was determined by quantitative RT-PCR. The release of chemokines was measured by ELISA. Results Treatment of cultured astrocytes with ET-1 and Ala1,3,11,15-ET-1, an ETB agonist, increased mRNA levels of CCL2/MCP1 and CXCL1/CINC-1. In contrast, CX3CL1/fractalkine mRNA expression decreased in the presence of ET-1 and Ala1,3,11,15-ET-1. The effect of ET-1 on chemokine mRNA expression was inhibited by BQ788, an ETB antagonist. ET-1 increased CCL2 and CXCL1 release from cultured astrocytes, but decreased that of CX3CL1. The increase in CCL2 and CXCL1 expression by ET-1 was inhibited by actinomycin D, pyrrolidine dithiocarbamate, SN50, mithramycin, SB203580 and SP600125. The decrease in CX3CL1 expression by ET-1 was inhibited by cycloheximide, Ca2+ chelation and staurosporine. Conclusion These findings suggest that ETs are one of the factors regulating astrocytic chemokine production. Astrocyte-derived chemokines are involved in pathophysiological responses of neurons and microglia. Therefore, the ET-induced alterations of astrocytic chemokine production are of pathophysiological significance in damaged brains. PMID:23627909

Increased expression of the interleukin 1 receptor on blood neutrophils of humans with the sepsis syndrome.

PubMed Central

Fasano, M B; Cousart, S; Neal, S; McCall, C E

1991-01-01

Because of the potential importance of interleukin 1 (IL-1) in modulating inflammation and the observations that human blood neutrophils (PMN) express IL-1 receptors (IL-1R) and synthesize IL-1 alpha and IL-1 beta, we studied the IL-1R on blood PMN from a group of patients with the sepsis syndrome. We report a marked enhancement in the sites per cell of IL-1R expressed on sepsis-PMN of 25 consecutively studied patients compared to 20 controls (patient mean = 9,329 +/- 2,212 SE; control mean = 716 +/- 42 SE, respectively). There was no demonstrable difference in the Kd of IL-1R on sepsis-PMN (approximately 1 nM) as determined by saturation curves of 125I-IL-1 alpha binding and the IL-1R on sepsis-PMN had an apparent Mr approximately 68,000, a value like that of normal PMN. Cytofluorographic analysis indicated that the sepsis-PMN phenotype is a single homogeneous population with respect to IL-1R expression. In contrast, expression of the membrane complement receptor CR3 is not increased on sepsis-PMN. Similar increases in expression of IL-1R were not observed in various other inflammatory processes, including acute disseminated inflammation and organ failure not caused by infection, acute infection without organ failure, and immunopathologies such as active systemic lupus erythematosus and rheumatoid arthritis. Enhanced expression of IL-1R was not related simply to the state of myeloid stimulation. Increased expression of IL-1R on normal PMN was induced in vitro by incubating cells with recombinant human granulocyte-macrophage/colony-stimulating factor for 18 h and this response was inhibited by cycloheximide, suggesting the possibility that de novo synthesis of IL-1R might occur in PMN during the sepsis syndrome. Images PMID:1834697

1,25-Dihydroxyvitamin D3 inhibition of col1a1 promoter expression in calvariae from neonatal transgenic mice

NASA Technical Reports Server (NTRS)

Bedalov, A.; Salvatori, R.; Dodig, M.; Kapural, B.; Pavlin, D.; Kream, B. E.; Clark, S. H.; Woody, C. O.; Rowe, D. W.; Lichtler, A. C.

1998-01-01

We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on organ cultures of transgenic mouse calvariae containing segments of the Col1a1 promoter extending to -3518, -2297, -1997, -1794, -1763, and -1719 bp upstream of the transcription start site fused to the chloramphenicol acetyltransferase (CAT) reporter gene. 1,25(OH)2D3 had a dose-dependent inhibitory effect on the expression of the -3518 bp promoter construct (ColCAT3.6), with maximal inhibition of about 50% at 10 nM. This level of inhibition was consistent with the previously observed effect on the endogenous Col1a1 gene in bone cell models. All of the shorter constructs were also inhibited by 10 nM 1,25(OH)2D3, suggesting that the sequences required for 1, 25(OH)2D3 inhibition are downstream of -1719 bp. The inhibitory effect of 1,25(OH)2D3 on transgene mRNA was maintained in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the inhibitory effect on Col1a1 gene transcription does not require de novo protein synthesis. We also examined the in vivo effect of 1,25(OH)2D3 treatment of transgenic mice on ColCAT activity, and found that 48 h treatment caused a dose-dependent inhibition of CAT activity in calvariae comparable to that observed in organ cultures. In conclusion, we demonstrated that 1,25(OH)2D3 inhibits Col1A1 promoter activity in transgenic mouse calvariae, both in vivo and in vitro. The results indicate that there is a 1, 25(OH)2D3 responsive element downstream of -1719 bp. The inhibitory effect does not require new protein synthesis.

Changes in barrier function of a model intestinal epithelium by intraepithelial lymphocytes require new protein synthesis by epithelial cells.

PubMed Central

Taylor, C T; Murphy, A; Kelleher, D; Baird, A W

1997-01-01

BACKGROUND: Elements of the mucosal immune system may play an important part in regulating epithelial barrier function in the intestinal tract. Intraepithelial lymphocytes (IELs) represent a subtype of immunocyte which is strategically placed to regulate epithelial function at most mucosal sites. AIMS AND METHODS: An IEL derived cell line (SC1) was used to examine its effects on the model epithelium T84--a tumour derived cell line which retains the phenotype of colonic crypt cells. Transepithelial electrical resistance (TER) was used as a marker of epithelial integrity. RESULTS: Coculture of T84 cells with SC1 produced a significant fall in TER as did exposure of T84 monolayers to IEL derived supernatant. Recombinant interferon-gamma (rIFN gamma) also reduced TER in T84 monolayers. Cycloheximide prevented the effects of IEL supernatant and of rIFN gamma on TER. The fall in TER in response to rIFN gamma was attenuated by blocking antibodies, which did not alter the fall in resistance induced by IEL supernatant. Fractions of IEL supernatant, separated on the basis of size, evoked temporally distinct changes in TER. Ultrastructural studies support the hypothesis that the slow onset but severe fall in TER indicates catastrophic effects on the monolayer. The more rapid onset fall in TER was not associated with gross changes in monolayer morphology. Reduction of TER by IEL supernatant was not influenced by inhibitors of tyrosine phosphatase or of protein kinase C. Although herbimycin did reduce the rapid onset change in TER, the tyrosine kinase inhibitor genistein did not alter responses to IEL supernatant. CONCLUSIONS: Mucosal T cells may influence barrier function by a process involving new protein synthesis by epithelial cells. This model may have relevance in some inflammatory conditions of the gastrointestinal tract. Images PMID:9203943

Down-regulation of angiotensin II receptor subtypes and desensitization of cyclic GMP production in neuroblastoma N1E-115 cells.

PubMed

Reagan, L P; Ye, X; Maretzski, C H; Fluharty, S J

1993-01-01

Murine neuroblastoma N1E-115 cells possess membranous receptors for the octapeptide angiotensin II (AngII) whose density is substantially increased by in vitro differentiation. Incubation of differentiated N1E-115 cells with AngII produced a rapid decrease in receptor density, but did not alter the affinity of these receptors for either 125I-AngII or the high-affinity antagonist 125I-[Sarc1,Ile8]-AngII. This apparent down-regulation was dose related with an ED50 of 1 nM, and maximal decreases of approximately 90% were obtained with 100 nM AngII. Receptor loss from differentiated cell membranes was unaffected by incubations of membranes obtained from agonist-exposed cells with non-hydrolyzable analogues of GTP for 60 min at 37 degrees C to ensure dissociation of the ligand. Partial loss of AngII receptors was apparent within 5 min of agonist exposure, whereas maximal declines were not observed until 30 min. This temporal pattern resulted from a preferential decrease in the AT1 receptor subtype during the first 5 min, followed by a decline in both AT1 and AT2 receptors with longer periods of agonist exposure. The loss of membranous receptors was reversible with partial recovery observed after 4 h, and with nearly full recovery observed 18 h after exposure of the cells to AngII. However, the long-term recovery of receptor density was blocked by the protein synthesis inhibitor, cycloheximide. The heptapeptide angiotensin III produced a similar down-regulation of receptors, and the high-affinity antagonist [Sarc1,Thr8]-AngII blocked agonist-induced down-regulation. Finally, the apparent loss of cell surface AngII receptors decreased the ability of AngII to stimulate cyclic GMP production within intact N1E-115 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

PubMed Central

Martins, Rute; Proença, Daniela; Silva, Bruno; Barbosa, Cristina; Silva, Ana Luísa; Faustino, Paula; Romão, Luísa

2012-01-01

Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3′ untranslated region (UTR), along with exon seven. Therefore, this 3′ UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3′-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements. PMID:22530027