Anesthetic pretreatment confers thermotolerance on Saccharomyces cerevisiae yeast.

Saccharomyces cerevisiae yeast, when pretreated with elevated temperatures, undergo adaptive changes that promote survival after an otherwise lethal heat stress. The heat shock response, a cellular stress response variant, mediates these adaptive changes. Ethanol, a low-potency anesthetic, promotes thermotolerance possibly through heat shock response activation. Therefore, we hypothesized other anesthetic compounds, like ethanol, may invoke the heat shock response to promote thermotolerance. To test this hypothesis, we pretreated yeast with a series of non-volatile anesthetic and anesthetic-related compounds and quantified survival following lethal heat shock (52 °C for 5 min). Most compounds invoked thermoprotection and promoted survival with a potency proportional to hydrophobicity: tribromoethanol (5.6 mM, peak survival response), trichloroethanol (17.8 mM), dichloroethanol (100 mM), monochloroethanol (316 mM), trifluoroethanol (177.8 mM), ethanol (1 M), isopropanol (1 M), propofol (316 µM), and carbon tetrabromide (32 µM). Thermoprotection conferred by pretreatment with elevated temperatures was "left shifted" by anesthetic co-treatment from (in °C) 35.3 ± 0.1 to 32.2 ± 0.1 with trifluoroethanol (177.8 mM), to 31.2 ± 0.1 with trichloroethanol (17.8 mM), and to 29.1 ± 0.3 with tribromoethanol (5.6 mM). Yeast in postdiauxic shift growth phase, relative to mid-log, responded with greater heat shock survival; and media supplementation with tryptophan and leucine blocked thermoprotection, perhaps by reversing the amino acid starvation response. Our results suggest S. cerevisase may serve as a model organism for understanding anesthetic toxicity and anesthetic preconditioning, a process by which anesthetics promote tissue survival after hypoxic insult.

Effect of dietary supplementation of nitrocompounds on Salmonella colonization and ileal immune gene expression in laying hens challenged with Salmonella Enteritidis.

Foodborne disease caused by Salmonella Enteritidis (SE) is one of the important public health and economic concerns. A study was conducted to determine the effect of supplementation with 2-nitroethanol (NE) and 2-nitropropanol (NP) on Salmonella recovery of internal organs as well as on the immune gene expression in the ileum of laying hens. Thirty-six White Leghorns were orally gavaged with nalidixic acid resistant Salmonella Enteritidis (SENR). Hens were housed individually in wire-laying cages and randomly assigned to six dietary treatments: T1 = SENR unchallenged (negative control), T2 = SENR challenged (positive control), T3 = SENR challenged + 100 ppm NE, T4 = SENR challenged + 200 ppm NE, T5 = SENR challenged + 100 ppm NP, and T6 = SENR challenged + 200 ppm NP. Hens were sampled at 7 days post inoculation (dpi). Ceca, liver with gall bladder (L/GB), and ovary samples were collected for bacteriology, and ileum samples were collected for analysis of immune gene expression. T3 and T6 significantly reduced (P < 0.05) cecal SENR count, whereas T4 and T5 were not different from T2, the SENR challenged control. There was no significant difference in SENR reduction in the L/GB or ovary after supplementation of either nitrocompounds. Pro- and anti-inflammatory cytokines such as interferon (IFN)-γ, interleukin (IL)-1B, IL-6, toll-like receptors (TLR)-4, and IL-10 all were significantly upregulated (P < 0.05) after SENR challenge. Supplementation at both levels of NE and NP showed a significant immune gene expression response in the ileum with reduction of IFN-γ, IL-6, TLR-4, and IL-10 mRNA expression. Overall, nitrocompounds such as NE and NP can be used in the intervention strategy to reduce Salmonella infection in hens.

Differential Intraocular Pressure Measurements by Tonometry and Direct Cannulation After Treatment with Soluble Adenylyl Cyclase Inhibitors.

PURPOSE: To validate the increase in intraocular pressure (IOP) caused by soluble adenylyl cyclase (sAC) inhibitors and determine reasons behind variation in IOP measurements performed by tonometry. METHODS: C57BL/6J mice were administered DMSO solubilized sAC inhibitors (KH7 or LRE-1) by intraperitoneal injection. Two hours post-treatment, mice were anesthetized with avertin or ketamine/xylazine/acepromazine (KXA). IOP was measured by a rebound tonometer or direct cannulation of the anterior chamber. Spectral-domain optical coherence tomography was used to measure anterior chamber depth and corneal thickness in live mice. Outflow facility was measured in perfused, enucleated mouse eyes. RESULTS: Compared with DMSO controls, KH7 treatment caused an increased IOP in avertin- and KXA-anesthetized mice when measured by direct cannulation [avertin: 14.4 ± 2.1 mmHg vs. 11.1 ± 1.0 mmHg (P = 0.003); KXA: 14.4 ± 1.0 mmHg vs. 11.3 ± 0.8 mmHg (P < 0.001)] and tonometry [avertin: 10.8 ± 1.4 mmHg vs. 7.4 ± 0.6 mmHg (P < 0.001); KXA: 11.9 ± 0.9 mmHg vs. 10.3 ± 1.7 mmHg (P = 0.283)]. However, treatment with KH7 in nonanesthetized mice showed a significant decrease in IOP measured by tonometry and compared with DMSO-treated animals [13.1 ± 2.6 mmHg vs. 15.6 ± 0.5 mmHg (P = 0.003)]. Both KH7- and DMSO-treated groups anesthetized with avertin showed increased corneal thickness, whereas KH7-treated mice anesthetized with KXA exhibited a shallower anterior chamber compared with untreated mice. KH7 decreased outflow facility by 85.1% in nonanesthetized, enucleated eyes (P < 0.003). CONCLUSIONS: Systemically administered DMSO and anesthesia have significant effects on anterior chamber characteristics, resulting in altered IOP readings measured by tonometry. In the presence of DMSO and anesthesia, tonometry IOP readings should be confirmed with direct cannulation.

Evolution of matrinic ethanol derivatives as anti-HCV agents from matrine skeleton.

Twenty-two novel 12N-substituted matrinic ethanol derivatives were synthesized and evaluated for their antiviral activities against HCV taking compound 1 as the lead. The SAR study indicated that the shortening of the 11-butyl chain to ethyl chain did not affect the activity significantly. Out of the target compounds, matrinic ethanol 6a demonstrated a potential anti-HCV effect with an EC50 value of 3.2µM and a SI value of 96.6. The free hydroxyl arm in 6a made it possible as a parent structure to prepare pro-drug for the potential application in HCV treatment. This study provided powerful information on further strategic optimization and development of this kind of compounds into a novel family of anti-HCV agents.

Antimicrobial Metabolites from a Marine-Derived Actinomycete in Vietnam's East Sea.

Two new compounds, a quinoline alkaloid (1) and a 1,4-dioxane derivative (2), were isolated from culture broth of the marine-derived actinomycete Micromonospora sp. (strain G019) by bioassay-guided fractionation. This actinomycete strain was isolated from sediment, collected at Cát Bà Peninsula, Vietnam. The taxonomic identification was achieved by analysis of 16S rRNA gene sequences. On the basis of morphological and phylogenetic evidence, strain G019 was assigned to the genus Micromonospora. The structures of 1 and 2 were established by spectroscopic data analysis, including one- and two-dimensional NMR, and MS. Compound 1 was found to have antibacterial activity against Escherichia coli (MIC: 48 µg/mL), Salmonella enterica (MIC: 96 µg/mL) and Enterococcus faecalis (MIC: 128 µg/mL), while compound 2 showed inhibitory activity against Enterococcusfaecalis (MIC: 32 µg/mL) and Candida albicans (MIC: 64 µg/mL).

Induction of murine TNBS colitis is strictly controlled by a modified method using continuous inhalation anesthesia with sevoflurane.

BACKGROUND: Trinitrobenzenesulfonic acid (TNBS)-induced colitis is one of the most widely used experimental colitis models. However, there is no standard procedure for inducing colitis by TNBS because it is difficult to achieve a uniform distribution of colitis. We have developed a modified method of murine TNBS-induced colitis that involves inhalation anesthesia with sevoflurane combined with both single and repeated TNBS administrations. AIMS: To compare the usefulness of our newly developed method for inducing murine TNBS-induced colitis with that of conventional intraperitoneal anesthesia. METHODS: TNBS in ethanol was administered to C57BL/6J mice held in an inverted vertical position either under continuous inhalation anesthesia with sevoflurane, in accordance with our newly developed method, or by intraperitoneal injection with 2.5 % avertin, in accordance with the conventional procedure. Body weight change, cytokine profile, and histological findings were examined during the course of colitis. RESULTS: The dispersion of anesthesia time, TNBS retention time, and nadir weight during the course of colitis was decreased using the newly developed method compared with the conventional procedure. Optimization of the modified TNBS-induced colitis, as evidenced by the predominant expression of Th1 and Th17 cytokines on day 7, was attained by the injection of 2.25 mg TNBS in 55 % ethanol. Regulation of the TNBS retention time using inhalation anesthesia with sevoflurane allowed strict control of the disease severity of TNBS-induced colitis. Using the modified method we were also able to develop a chronic TNBS-induced colitis model by repeated TNBS administration without excessive mortality of the mice. CONCLUSIONS: Our modified method for murine TNBS-induced colitis using continuous inhalation anesthesia with sevoflurane provides a better experimental colitis model following both single and repeated TNBS administrations.

Chemical synthesis and characterization of palm oil-based difatty acyl thiourea.

Difatty acyl thiourea (DFAT), which has biological activities as antibiotics and antifungal, has been synthesized from palm oil and thiourea using sodium ethoxide as catalyst. Ethyl fatty ester (EFE) and glycerol were produced as by-products. The synthesis was carried out by reflux palm oil with thiourea in ethanol. In this process, palm oil converted to about 81% pure DFAT after 11 hour and molar ratio of thiourea to palm oil was 6.0: 1 at 78 degrees C. Elemental analysis, Fourier transform iInfrared (FTIR) spectroscopy and (1)H nuclear magnetic resonance (NMR) technique were used to characterize both DFAT and EFE.

Antinociceptive effect of stimulating the occipital or retrosplenial cortex in rats.

UNLABELLED: A role for the occipital or retrosplenial cortex in nociceptive processing has not been demonstrated yet, but connections from these cortices to brain structures involved in descending pain-inhibitory mechanisms were already demonstrated. This study demonstrated that the electrical stimulation of the occipital or retrosplenial cortex produces antinociception in the rat tail-flick and formalin tests. Bilateral lesions of the dorsolateral funiculus abolished the effect of cortical stimulation in the tail-flick test. Injection of glutamate into the same targets was also antinociceptive in the tail-flick test. No rats stimulated in the occipital or retrosplenial cortex showed any change in motor performance on the Rota-rod test, or had epileptiform changes in the EEG recording during or up to 3 hours after stimulation. The antinociception induced by occipital cortex stimulation persisted after neural block of the retrosplenial cortex. The effect of retrosplenial cortex stimulation also persisted after neural block of the occipital cortex. We conclude that stimulation of the occipital or retrosplenial cortex in rats leads to antinociception activating distinct descending pain-inhibitory mechanisms, and this is unlikely to result from a reduced motor performance or a postictal phenomenon. PERSPECTIVE: This study presents evidence that stimulation of the retrosplenial or occipital cortex produces antinociception in rat models of acute pain. These findings enhance our understanding of the role of the cerebral cortex in control of pain.

Synthesis and characterization of N,N'-carbonyl difatty amides from palm oil.

N,N'-Carbonyl difatty amides (CDFAs) have been synthesized from palm oil using sodium ethoxide as catalyst. Ethyl fatty esters (EFEs) were produced as a by-product as well as glycerol. The synthesis was carried out by reflux palm oil and urea in presence of ethanol. In this process, palm oil gave 79% pure CDFAs after 8 hours and molar ratio of urea to palm oil was 6.2: 1 at 78 degrees C. Both CDFAs and EFEs have been characterized using elemental analysis, Fourier transform infrared (FTIR) spectroscopy and (1)H nuclear magnetic resonance (NMR) technique.

Cyclohexyl-ethanol derivatives from the roots of Incarvillea mairei.

Two new cyclohexyl-ethanol derivatives, incarvmareins A (1) and B (2), together with two known derivatives, 3 and 4, were isolated from the ethanolic extract of the roots of Incarvillea mairei. The structures of the new compounds were elucidated primarily on the basis of analysis of spectroscopic data.

Surface roughness analysis of fiber post conditioning processes.

The chemo-mechanical surface treatment of fiber posts increases their bonding properties. The combined use of atomic force and confocal microscopy allows for the assessment and quantification of the changes on surface roughness that justify this behavior. Quartz fiber posts were conditioned with different chemicals, as well as by sandblasting, and by an industrial silicate/silane coating. We analyzed post surfaces by atomic force microscopy, recording average roughness (R(a)) measurements of fibers and resin matrix. A confocal image profiler allowed for the quantitative assessment of the average superficial roughness (R(a)). Hydrofluoric acid, potassium permanganate, sodium ethoxide, and sandblasting increased post surface roughness. Modifications of the epoxy resin matrix occurred after the surface pre-treatments. Hydrofluoric acid affected the superficial texture of quartz fibers. Surface-conditioning procedures that selectively react with the epoxy-resin matrix of the fiber post enhance roughness and improve the surface area available for adhesion by creating micro-retentive spaces without affecting the post's inner structure.

The phytoestrogen coumestrol is a naturally occurring antagonist of the human pregnane X receptor.

Antagonizing the action of the human nuclear xenobiotic receptor pregnane X receptor (PXR) may have important clinical implications in preventing drug-drug interactions and improving therapeutic efficacy. We provide evidence that a naturally occurring phytoestrogen, coumestrol, is an antagonist of the nuclear receptor PXR (NR1I2). In transient transfection assays, coumestrol was able to suppress the agonist effects of SR12813 on human PXR activity. PXR activity was assessed and correlated with effects on the metabolism of the anesthetic tribromoethanol and on gene expression in primary human hepatocytes. We found that coumestrol was able to suppress the effects of PXR agonists on the expression of the known PXR target genes, CYP3A4 and CYP2B6, in primary human hepatocytes as well as inhibit metabolism of tribromoethanol in humanized PXR mice. Coumestrol at concentrations above 1.0 microm competed in scintillation proximity assays with a labeled PXR agonist for binding to the ligand-binding cavity. However, mammalian two-hybrid assays and transient transcription data using ligand-binding-cavity mutant forms of PXR show that coumestrol also antagonizes coregulator recruitment. This effect is likely by binding to a surface outside the ligand-binding pocket. Taken together, these data imply that there are antagonist binding site(s) for coumestrol on the surface of PXR. These studies provide the basis for development of novel small molecule inhibitors of PXR with the ultimate goal of clinical applications toward preventing drug-drug interactions.

Synthesis of 11C-labelled (R)-OHDMI and CFMME and their evaluation as candidate radioligands for imaging central norepinephrine transporters with PET.

(R)-1-(10,11-Dihydro-dibenzo[b,f]azepin-5-yl)-3-methylamino-propan-2-ol ((R)-OHDMI) and (S,S)-1-cyclopentyl-2-(5-fluoro-2-methoxy-phenyl)-1-morpholin-2-yl-ethanol (CFMME) were synthesized and found to be potent inhibitors of norepinephrine reuptake. Each was labelled efficiently in its methyl group with carbon-11 (t(1/2)=20.4 min) as a prospective radioligand for imaging brain norepinephrine transporters (NET) with positron emission tomography (PET). The uptake and distribution of radioactivity in brain following intravenous injection of each radioligand into cynomolgus monkey was examined in vivo with PET. After injection of (R)-[(11)C]OHDMI, the maximal whole brain uptake of radioactivity was very low (1.1% of injected dose; I.D.). For occipital cortex, thalamus, lower brainstem, mesencephalon and cerebellum, radioactivity ratios to striatum at 93 min after radioligand injection were 1.35, 1.35, 1.2, 1.2 and 1.0, respectively. After injection of [(11)C]CFMME, radioactivity readily entered brain (3.5% I.D.). Ratios of radioactivity to cerebellum at 93 min for thalamus, occipital cortex, region of locus coeruleus, mesencephalon and striatum were 1.35, 1.3, 1.3, 1.2 and 1.2, respectively. Radioactive metabolites in plasma were measured by radio-HPLC. (R)-[(11)C]OHDMI represented 75% of plasma radioactivity at 4 min after injection and 6% at 30 min. After injection of [(11)C]CFMME, 84% of the radioactivity in plasma represented parent at 4 min and 20% at 30 min. Since the two new hydroxylated radioligands provide only modest regional differentiation in brain uptake and form potentially troublesome lipophilic radioactive metabolites, they are concluded to be inferior to existing radioligands, such as (S,S)-[(11)C]MeNER, (S,S)-[(18)F]FMeNER-D(2) and (S,S)-[(18)F]FRB-D(4), for the study of brain NETs with PET in vivo.

Phototoxic activity of a thiophene polyacetylene from Leuzea carthamoides.

The thiophene polyacetylene (E)-1-[5-(hept-5-en-1,3-diynyl)-2-thienyl]ethan-1,2-diol, isolated from the roots of Leuzea carthamoides, showed phototoxic activity in the assay systems of histidine photo-oxidation, Artemia and Tubifex assays. The effects were compared with the standard photosensitizer xanthotoxin.

Resolution and synthesis of optically active alcohols with immobilized water-soluble proteins from green pea, soybean and buckwheat as new bio-catalysts.

Kinetic resolution of racemic alcohols, (+/-)-1-(4-substituted phenyl)ethanol and (+/-)-1-(2-naphthyl)ethanol, was done with immobilized green pea, soybean, or buckwheat proteins. The resolution was done stereoselectively by oxidizing only one enantiomer of a racemic alcohol to leave an optically active alcohol with a high purity. In addition, each protein could be reused consecutively at least three times without any decrease of yield or optical purity.

Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells.

In previous parts of this study we developed procedures for the high-efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two-stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion-body stability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HEDS), also known as oxidized beta-mercaptoethanol, to the permeabilization buffer (6 M urea + 3 mM ethylenediaminetetraacetate [EDTA]). 2-HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion-body fraction by centrifugation. In the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two-stage process yielded an 81% (w/w) recovery of the recombinant protein Long-R3-IGF-I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells.

Modulation of the cell kinetics of pig skin by the topical application of evening primrose oil or Lioxasol.

The daily topical application of two compounds, a cream containing 10% evening primrose oil (EPO) and Lioxasol (a compound used clinically to treat radiation burns), resulted in increased cell proliferative activity in the skin of female Large White pigs. The effect was most pronounced in the case of the EPO based cream, and was comparable in magnitude with that observed in a previous study on pig skin using orally administered EPO. There was an increase in the size of the rete pegs in the epidermis by 6 weeks after the start of application of the EPO cream. However, this did not translate into an increase in the total thickness of the viable epidermis (excluding the stratum corneum) due to a reduction in the density of rete pegs, from 2 weeks after treatment. Lioxasol had no overall effect on the size of the rete pegs. The labelling index (LI) of cells in the basal layer of the epidermis of pigs receiving a daily topical application of EPO increased progressively with time from the start of application. The LI was maximal (17.9 +/- 2.4%) at the end of the observation period (8 weeks) at which time it was a factor of approximately 2 higher than in the basal layer prior to treatment. A considerably less marked increase in the LI of the basal layer was seen after the application of Lioxasol. The overall increase was approximately 20%, relative to the LI in the untreated epidermis. Labelled cell nuclei were also counted in the papillary dermis. After the application of the EPO cream, no significant increase in the number of labelled cells was observed until week 8, at which time values were approximately twice those in untreated skin. In Lioxasol treated skin the effect on the numbers of labelled cells in the papillary dermis was more immediate, with a approximately 60% increase at 2 weeks. This enhanced level of labelling was maintained until the end of the observation period of 10 weeks. Studies on the cell kinetics of the skin using the alcohol component of the Lioxasol preparation suggested that alcohol rather than Lioxasol was the most significant ingredient. It was concluded that the EPO cream merited further evaluation as a potential modulator of skin response to ionizing radiation.

Rapid in vivo hydrolysis of fatty acid ethyl esters, toxic nonoxidative ethanol metabolites.

Fatty acid ethyl esters (FAEE), esterification products of fatty acids and ethanol, are in use as fatty acid supplements, but they also have been implicated as toxic mediators of ethanol ingestion. We hypothesized that hydrolysis of orally ingested FAEE occurs in the gastrointestinal (GI) tract and in the blood to explain their apparent lack of toxicity. To study the in vivo inactivation of FAEE by hydrolysis to free fatty acids and ethanol, we assessed the hydrolysis of FAEE administered as an oil directly into the rat stomach and when injected within the core of low-density lipoprotein particles into the circulation of rats. Our studies demonstrate that FAEE are rapidly degraded to free fatty acids and ethanol in the GI tract at the level of the duodenum with limited hydrolysis in the stomach. In addition, FAEE are rapidly degraded in the circulation, with a half-life of only 58 s. Thus the degradation of FAEE in the GI tract and in the blood provides an explanation for the apparent lack of toxicity of orally ingested FAEE.

Discriminable excitotoxic effects of ibotenic acid, AMPA, NMDA and quinolinic acid in the rat laterodorsal tegmental nucleus.

Excitotoxins are valuable tools in neuroscience research as they can help us to discover the extent to which certain neurones are necessary for different types of behaviour. They have distinctive neurotoxic effects depending on where they are infused, and this study was conducted to delineate the neurotoxic profiles of excitotoxins in the laterodorsal tegmental nucleus (LDTg). Two 0.1 microl infusions of 0.1 M ibotenate, 0.1 M quinolinate, 0.04-0.1 M NMDA, or 0.05-0.015 M AMPA, were made unilaterally into the LDTg under either pentobarbitone or Avertin anaesthesia. The injection needle was oriented at an angle of 24 degrees from vertical in the mediolateral plane. After 23-27 days, sections through the mesopontine tegmentum were processed using standard histological procedures for NADPH-diaphorase histochemistry, tyrosine hydroxylase or 5-hydroxytryptamine immunohistochemistry, and Cresyl violet. Lesions were assessed in terms of the size of the damaged area (identified by reactive gliosis), the extent of cholinergic cell loss in the mesopontine tegmentum (by counting NADPH-diaphorase-positive neurones), and neuronal loss induced in the locus coeruleus and dorsal raphe nucleus. Ibotenate induced compact lesions in the LDTg (more than 80% cholinergic loss) and did little damage to the locus coeruleus and dorsal raphe nucleus. Quinolinate and low doses of AMPA and NMDA made very small lesions with less than 35% cholinergic loss, while at higher doses, AMPA and NMDA induced large areas of reactive gliosis but killed only a proportion of the cholinergic neurones. AMPA appeared to have a particular affinity for noradrenergic neurones in the locus coeruleus, with the 0.015 M dose injected into the LDTg typically destroying the majority of these neurones. The results are discussed in the context of what is known about the mechanisms of excitotoxins and the glutamate receptor profile of mesopontine neurones.

Selenotrisulfide inhibits initiation by RNA polymerase II but not elongation.

We previously reported that RNA polymerase II (purified from wheat germ) is inhibited by selenotrisulfides, the products of the reaction of selenite with sulfhydryl compounds [Frenkel, Walcott, and Middleton, Molecular Pharmacology 31, 112 (1987)]. We have now found that the initiation stage of the reaction is inhibited by selenotrisulfide but the elongation stage of the reaction is not. The actual start of the RNA chain is not inhibited by the selenotrisulfide, but rather the formation of the enzyme-DNA binary complex. Selenotrisulfide has a similar differential effect on initiation and elongation by RNA polymerase II from HeLa cells; in contrast, with E. coli RNA polymerase, it inhibits elongation as well.