Cells treated

by Plu1962 alone displayed a dramatic decrease in density of green fluorescence (Fig. 4c). This implied that Plu1962 alone could depolymerize microtubules to a certain extent. We next investigated the possible mechanisms responsible for the rapid cell death caused by binary toxin using Apoptosis and Necrosis Assay Kit. The intact membrane of live cells excludes charged cationic dyes, such as trypan blue, propidium, or ethidium, and short incubation with these dyes results in selective AG-014699 cost labeling of dead cells, while live cells show minimal dye uptake. Loss of plasma membrane integrity leading to increased permeability to PI was found to be characteristic of necrosis. Most of the CF-203 cells treated with 0.6 μmol L−1 mixture of Plu1961/Plu1962 showed strong blue fluorescence and red fluorescence. Conversely, weak blue fluorescence and no red fluorescence were detected in control cells (Supporting Information, Fig. S1). Moreover, incubation of CF-203 cells with mixture of Plu1961/Plu1962 (0.6 nM) failed to induce DNA ladder fragmentation, a hallmark of apoptosis, even after incubation for 24 h (data not shown). Taken together, we therefore assumed that Plu1961/Plu1962 exhibited necrotic cytotoxicity in CF-203 cells. Five mammalian cell lines (B16, 4T1,

HeLa, Hep 3B, HCT116) were also used to examine the cytotoxicity of binary toxin. Neither Plu1961 nor Plu1962 alone could inhibit the growth of all tested mammalian cell lines. Unexpectedly, the mixture of Plu1961/Plu1962 (1.6 μmol L−1) exhibited no ERK inhibitor cytotoxic effect on all tested mammalian cell Molecular motor lines (data not shown). We then co-expressed Plu1961 and Plu1962 in BL21 (DE3). Lysate from BL (Bi) exhibited strong cytotoxicity against B16, 4T1, and HeLa cells (Fig. 5). Hep 3B and HCT116 cells were insensitive to BL (Bi) lysate (data not shown). In the present study, we identified a XaxAB-like binary toxin from P. luminescens, which exhibits cytotoxicity against insect midgut CF-203 cells and

some mammalian cell lines. Both Plu1961 and Plu1962 were necessary to restore full cytotoxicity against CF-203 cells. XaxAB and Plu1961/Plu1962 show no homology to any other protein with known function, indicating that they constitute a distinct family of binary toxins (Vigneux et al., 2007). Photorhabdus luminescens proliferates in the hemolymph before the insect dies and must therefore be able to escape the insect immune response. Cell-mediated immunity comes into play immediately after the insect hemocoel is penetrated by a foreign body (Ribeiro & Brehelin, 2006). It was reported that injection of wild-type E. coli into Manduca sexta resulted in rapid encapsulation of all of the bacteria by the insect hemocytes, completely clearing the infection from the hemocoel.

A decoding accuracy of 77.6% was obtained for the non-feedback condition, which is high considering that decoding was performed on a single TR without averaging multiple scans. We also tested if neurofeedback of scan-by-scan brain state classification

results can improve decoding performance by using a feedback condition learn more in which the relative mix of the face and place picture was adjusted depending on classification results. However, neurofeedback did not significantly improve decoding performance (see Supporting Information). When we analysed the results of TR-by-TR decoding performance, we did not observe an improvement in accuracy over time for feedback trials. This contradicted our expectation that neurofeedback of the attended stimulus in the form of its enhancement in the hybrid picture would result in higher decoding performance. From a purely perceptual point of view, enhancement of the target picture should make classification easier as an enhanced target picture would resemble more closely the neural patterns that the classifier was originally trained on. To examine why no improvement in decoding accuracy was observed in the feedback condition, we computed classifier prediction probability as a function of TR (see Supporting Information). We indeed

observed an increase in GDC-0449 ic50 the prediction probability of attended stimuli for successful feedback trials. However, we also observed a decrease in the prediction probability for unsuccessful feedback trials. This is because in the feedback condition, visibility of the attended picture increased as a trial progressed, irrespective of whether it was the target or distractor picture. As a result, when both successful and unsuccessful trials were combined and the TR-by-TR prediction

probabilities were computed again, we did not observe any difference between feedback and non-feedback conditions. Hence, no significant Dapagliflozin difference between feedback and non-feedback conditions was observed. A number of other design choices may have affected performance in the feedback condition. First, because feedback and non-feedback trials were conducted in interleaved mini-blocks, it might have weakened any learning effect as subjects would not have been able to discover a consistent strategy due to frequent switching between the feedback and non-feedback trials. In future studies, rather than using a within-subject design for feedback and non-feedback conditions, a between-subject design should be used. Second, the duration of feedback was chosen to be 12 TRs (24 s) as a compromise between the number of trials and the experiment duration. This might have been too short for any significant strategy learning. Previous real-time studies have used trial durations ranging from 15 to 60 s conducted over the course of multiple days (see Weiskopf et al., 2005 for a review).

Parents were encouraged to discuss their own and their child’s experiences of dental care. The interview data were systematically coded using key theme headings, and summary charts constructed to facilitate the analysis. Results.  A sense of ‘uneasiness’ pervaded the parents’ comments and perceptions of the dental care provided for their children. This was conceptualized as parents ‘remembering in words’ and

‘repeating through actions’ their own childhood dental experiences. They remembered and repeated their childhood experiences by delaying dental treatment for themselves and their children. Conclusions.  Acknowledging the influence of parental dental experience would help ensure that parents of young children access routine care for their children and themselves. “
“International Journal of Paediatric Dentistry LDK378 2010; 20:

144–150 Background.  The early mutans streptococci (MS) bacteria colonization is connected to early childhood caries. The aim PCI-32765 of this study is to examine associations between the MS-colonization and background factors in young children, in order to enhance the oral health program in a low caries prevalence community. Subjects and Design.  An age cohort of 512 children was screened for MS in the oral biofilm at the age of 18 months. The caretakers were, using a structured form, interviewed of demographical factors and habits connected to oral health: antibiotic treatments, child’s appetite, frequency of night feeding, use of sugary products or drinks, and maternal xylitol use. The associations were evaluated with logistic regression analysis. Results.  Mutans streptococci colonization was significantly associated with both the occupation of the caretaker and the non-Finnish background. Conclusion.  The early else MS-colonization, in preschool children, strongly associates with the socioeconomic status of the family. “
“International Journal of Paediatric Dentistry 2011;

21: 96–102 Background.  Oral mucosal lesions can result from irritation caused by orthodontic appliances or malocclusion, but their frequency is not known. Aim.  To examine the frequency of oral mucosal lesions in wearers of orthodontic appliances in comparison to children with malocclusion. Design.  This study comprised 111 subjects: 60 wearers of orthodontic appliances and 51 controls (aged between 6 and 18 years). Type and severity of mucosal lesions, their topography, gingival inflammation, and oral hygiene status were determined by using clinical indices. Results.  Mucosal lesions were more present in wearers of orthodontic appliances than in children with malocclusion. Gingival inflammation, erosion, ulceration, and contusion were the most common findings in orthodontic patients. The severity of gingival inflammation was in correlation with oral hygiene status; the poorer oral hygiene, the more severe gingival inflammation was.

Site-directed mutagenesis

was performed in all the conserved amino acids. Growth profile Epigenetics inhibitor of wild-type catalytic domain and its mutant variant was analysed by performing endogenous toxicity assay. Homogeneity of the purified recombinant wild-type catalytic domain and its mutants was confirmed by Western blot. Structural integrity of the purified recombinant proteins was analysed by intrinsic tryptophan fluorescence and circular dichroism. Escherichia coli strain DH5α (Bethesda Research Laboratories) was used as the host for cloning. The E. coli strains, TOP10 and BL 21(DE3) pLysS, were used in the expression studies. E. coli XL-Blue cells were used for the site-directed mutagenesis studies. The plasmid vector pGEM-T Easy from Promega (Madison, WI) was used for PCR cloning. LB medium was used for growing bacterial strains. Ampicillin, kanamycin and chloramphenicol were used at 100, 35 and 25 μg mL−1, respectively. Primer PColF with PstI site at 5′ and primer 2 with HindIII site at 3′ were used to amplify xcinA alone, from the 4.3-kb genomic DNA fragment. The amplified 1.7-kb product

was ligated in pGEM-T Easy vector producing pJS2 plasmid. Plasmid was digested with PstI and HindIII, and the released DNA fragment of 1.7 kb was ligated to pBAD vector resulting in plasmid pJSR2. For catalytic domain, forward primer PDomF with PstI and backward primer 2 with HindIII site were used for PCR amplification. Amplified 318-bp product was cloned in pGEM-T Easy vector producing pJS3. 318 bp was excised from pJS3 by digestion with PstI and HindIII and ligated to pBAD vector, resulting pJSR3 construct. pJSR2, pJSR3 and pBAD without insert were

Compound C finally electroplated Roflumilast in the E. coli TOP10 cells and gave rise to JSR2, JSR3 and JSR4 strains, respectively. All these strains were studied by endogenous toxicity assays. For the isolation of individual domain proteins, the Ni-NTA purified catalytic–immunity domain protein complex was dialysed against 20 mM glycine–HCl buffer, pH 3.0, overnight and purified by a Sepharose-SP column (HiTrap SP; Amersham Biosciences) as described earlier (Singh & Banerjee, 2008). The catalytic domain was eluted first with NaCl gradient (0–2 M, pH 3) followed by the immunity domain with 20 mM sodium phosphate buffer, pH 8.0. The individual domains were dialysed against 50 mM sodium phosphate buffer, pH 8.0, for further studies. The 64-kDa xenocin was purified as described earlier (Singh & Banerjee, 2008), and SDS-PAGE was performed by following the procedure described by Laemmli (1970). Antiserum against purified recombinant xenocin was raised in rabbit with standard protocol. Western blot was performed with 500 ng each purified sample with standard molecular protocol using anti-xenocin serum (1 : 2000 dilution). Site-directed mutagenesis in the catalytic domain of xenocin was performed by Quick Change Site-Directed kit (stratagene) as per recommended protocol by manufactures. Ligation and transformation of competent E.

Genomic DNA from N. punctiforme was used as a template for the hupSL promoter-hupS- and the hupSL intergenic region-hupL-containing DNA fragments. The gfp-modified hup-operon PCR product was cloned into the pBluescript II SK+ plasmid (Stratagene) before subcloning into pSUN119 (Argueta et al., 2004) using SmaI and SacI (Fermentas), generating plasmid pSHG. The complete sequence of the gfp-modified hup-operon is available (Supporting Information). Finally, pSHG was transferred into N. punctiforme by electroporation

and positive clones were selected as described previously (Holmqvist et al., 2009), using 10 μg mL−1 neomycin (creating Vorinostat manufacturer the SHG culture). The GFP and phycobilisome/photosystem II emission of WT, SHG, and GFP control [N. punctiforme containing the pPMQAK1-Ptrc1O-GFP plasmid (Huang et al., 2010)] cultures were examined as described previously (Cardona et al., 2009). Nonconfocal differential interference contrast (DIC) reference images were produced on a separate channel. GFP was excited using 488-nm laser light and emission was detected from 500 to 540 nm. Confocal microscopy settings, laser effects and PMT voltages were kept identical to enable comparison of GFP fluorescence signal strength for studying the BIBW2992 solubility dmso cellular localization of GFP, but not for studying

the subcellular localization. Overlay images were produced from confocal red autofluorescence, confocal GFP fluorescence, and nonconfocal DIC images using the las af software (Leica). Image processing was performed using Photoshop Depsipeptide molecular weight CS4 Extended (Adobe Systems). The red autofluorescence (in magenta) was enhanced for clarity.

The GFP fluorescence was not edited. Heterocyst isolations were performed as in our previous work on N. punctiforme (Cardona et al., 2009; Ow et al., 2009), using protocols originally established by (Almon & Böhme, 1980). Chlorophyll a measurements were carried out as reported previously (Holmqvist et al., 2009). Proteins from isolated heterocysts were extracted as described (Ow et al., 2009; Agervald et al., 2010) using denaturing buffer [50 mM Tris-HCl, pH 7.8, 14.2 mM β-mercaptoethanol, 2% sodium dodecyl sulphate (SDS)] or native buffer, (25 mM BisTris, pH 7, and 20% glycerol) supplemented with Complete Mini, EDTA-free protease inhibitor cocktail tablets (Roche). The protein concentrations were determined using colorimetric Bradford protein assay (Bio-Rad Laboratories) and 50 μg total proteins were separated on 12% SDS-PAGE gels run at 200 V. To examine whether HupS–GFP forms a complex with HupL, attempts were made to extract HupS–GFP under native conditions, with no success. To examine the solubility of HupS–GFP, proteins from equal amounts of SHG cultures were extracted as above, but using buffers containing no detergents, mild nonionic detergents (0–2% Triton X-100 or 0–5% dodecyl maltoside), or strongly denaturing additives (7 M urea and 2 M thiourea) (see Supporting Information, Fig. S1, for details).

The only significant result of this analysis was that neck EMG responses evoked during the post-cue interval tended to be greater with the head unrestrained. Subsequent analyses of data restricted to that obtained with the head restrained revealed the same pattern of results emphasized below, and hence our results are not simply due to the inclusion of head-unrestrained

data. We therefore pooled data across head-restrained and head-unrestrained sessions. We also pooled data across stimulation of the right and left SEF, and refer to cue locations, saccades and muscles as being contra- or ipsilateral to the side of SEF stimulation. Our convention is to refer to saccade direction, and hence a correct contralateral learn more anti-saccade requires the monkey to look away from an ipsilateral cue. A contralateral anti-saccade error is one where the monkey saccades incorrectly to a contralateral this website cue. We first analysed whether short-duration ICMS-SEF directly evoked saccadic eye movements. During the fixation interval, saccades following stimulation but preceding cue

onset occurred on fewer than 1% of all appropriate stimulation trials. We also found no consistent difference in the change of eye position during the fixation interval between control trials and trials with stimulation during this interval (a t-test of the eye position changes reached significance in only three of the 52 sessions, and only one of these sessions showed the contralateral change in eye position that would be expected from stimulation). These analyses show that the animals maintained fixation during short-duration ICMS-SEF. We also found that the proportion of express saccades, which we leniently defined as RTs between 60 and 120 ms, was 2.5 ± 5.8% on control trials, and never exceeded 3% for trials with stimulation delivered at any interval. FAD These analyses emphasize the inability of short-duration ICMS-SEF to directly evoke saccades, even when

delivered during the post-cue interval. On control trials, both monkeys generated higher error rates (Fig. 2) and longer RTs (Fig. 3) on anti- vs. pro-saccade trials. Furthermore, the RTs of anti-saccade errors approached the RTs of pro-saccades (Fig. 3), and are not in the range of express saccades. These patterns replicate those reported in previous studies in monkeys generating intermixed pro- and anti-saccades (Amador et al., 1998; Bell et al., 2000). The influence of short-duration ICMS-SEF on error rates is shown in Fig. 2, collapsed across all experimental sessions. Short-duration ICMS-SEF exerted a negligible influence on either pro- or anti-saccades when delivered during the fixation interval (i.e. to the left of the vertical dashed line), but progressively impacted error rates the later it was delivered during the post-cue interval.

The only significant result of this analysis was that neck EMG responses evoked during the post-cue interval tended to be greater with the head unrestrained. Subsequent analyses of data restricted to that obtained with the head restrained revealed the same pattern of results emphasized below, and hence our results are not simply due to the inclusion of head-unrestrained

data. We therefore pooled data across head-restrained and head-unrestrained sessions. We also pooled data across stimulation of the right and left SEF, and refer to cue locations, saccades and muscles as being contra- or ipsilateral to the side of SEF stimulation. Our convention is to refer to saccade direction, and hence a correct contralateral FK228 price anti-saccade requires the monkey to look away from an ipsilateral cue. A contralateral anti-saccade error is one where the monkey saccades incorrectly to a contralateral Ruxolitinib ic50 cue. We first analysed whether short-duration ICMS-SEF directly evoked saccadic eye movements. During the fixation interval, saccades following stimulation but preceding cue

onset occurred on fewer than 1% of all appropriate stimulation trials. We also found no consistent difference in the change of eye position during the fixation interval between control trials and trials with stimulation during this interval (a t-test of the eye position changes reached significance in only three of the 52 sessions, and only one of these sessions showed the contralateral change in eye position that would be expected from stimulation). These analyses show that the animals maintained fixation during short-duration ICMS-SEF. We also found that the proportion of express saccades, which we leniently defined as RTs between 60 and 120 ms, was 2.5 ± 5.8% on control trials, and never exceeded 3% for trials with stimulation delivered at any interval. Mannose-binding protein-associated serine protease These analyses emphasize the inability of short-duration ICMS-SEF to directly evoke saccades, even when

delivered during the post-cue interval. On control trials, both monkeys generated higher error rates (Fig. 2) and longer RTs (Fig. 3) on anti- vs. pro-saccade trials. Furthermore, the RTs of anti-saccade errors approached the RTs of pro-saccades (Fig. 3), and are not in the range of express saccades. These patterns replicate those reported in previous studies in monkeys generating intermixed pro- and anti-saccades (Amador et al., 1998; Bell et al., 2000). The influence of short-duration ICMS-SEF on error rates is shown in Fig. 2, collapsed across all experimental sessions. Short-duration ICMS-SEF exerted a negligible influence on either pro- or anti-saccades when delivered during the fixation interval (i.e. to the left of the vertical dashed line), but progressively impacted error rates the later it was delivered during the post-cue interval.

, 1991). A recent TMS study in animals shows that intermittent TBS increased the gamma power of the EEG, while cTBS had no significant effect in any of BMS-354825 manufacturer the principal EEG bands (Benali et al., 2011). McAllister et al. (2011) also found an absence of cTBS-modulation of the power spectrum recorded over the stimulated M1 during eyes-opened

resting, in humans. However, this study only recorded resting EEG up to 10 min, whereas we found significant modulation of resting EEG after 20 min. By contrast, Noh et al. (2012) observed that cTBS increased the power in theta and low beta bands over the stimulated M1 during eyes-opened resting, these effects lasting longer than the modulation of MEPs. In addition, they found an increase in high beta band at rest over the frontal electrodes. It has to be noted than in our study, recordings were performed with eyes closed whereas the studies above were performed with eyes opened. Moreover, Noh et al. (2012) used a shorter version of cTBS (300 pulses) whereas we used 600 pulses as in the

original protocol introduced by Huang et al. (2005). The shorter version of cTBS has been shown to induce facilitation of MEPs instead of inhibition (Gentner et al., 2008). However, Noh et al. (2012) reported an inhibition of MEPs, probably ABT199 related to the muscular activation performed during the measurement of AMT (see Gentner et al., 2008). These methodological

discrepancies might account for the different results observed across studies. Again, two mechanisms could explain our results. An increase (respectively a decrease) in power after cTBS could be related to an NADPH-cytochrome-c2 reductase increase (respectively a decrease) of the number of active oscillators, while the synchronization between these oscillators remained constant. Alternatively, our findings could be related to an increase (respectively a decrease) in phase alignment between these oscillators, while the number of active oscillators remained constant. Combined with our results on cTBS-induced modulation of TMS-induced oscillations, our results favor the second explanation. We propose that cTBS acts primarily on already active oscillators, aligning the phase of low-frequency oscillators while desynchronizing active high-frequency oscillators. This effect results in an increase of resting theta oscillations combined with a decrease in TMS-induced theta oscillations. Similarly, it leads to a decrease of resting beta oscillations combined with an increase in TMS-induced beta oscillations (see Fig. 7). Thus, this slowing of frequencies could constitute a marker of cortical inhibition after cTBS. The plasticity induced by TBS shares properties with LTP and LTD mechanisms of synaptic efficacy (Huang et al., 2005), but the exact mechanisms in humans remain largely unknown.

0, 1 mM dithiothreitol and 1 mM phenylmethanesulfonyl fluoride) selleck compound containing 5 mg mL−1 lysozyme. Lysate was centrifuged

at 20 000 g for 15 min, clear supernatant was passed through a HiTrap Heparin (GE Healthcare) column and proteins were eluted with 500 mM NaCl. The purine nucleotides were extracted and quantified using the modified protocols described in studies of cerebellar granule cells (Giannattasio et al., 2003). In brief, the cells were treated with lysozyme (2 mg mL−1) for 1 h on ice and nucleotides then extracted with ice-cold 0.5 M perchloric acid (PCA). The pH of the PCA extract was adjusted to pH 7.5 with 0.5 M KOH and incubated for 30 min on ice. The potassium perchlorate precipitate was removed by centrifugation at 20 000 g for 15 min and the supernatant was used for HPLC analysis. The individual nucleotides were identified on the basis of their retention Ribociclib manufacturer time in C-18 column and by spiking the complex spectra with corresponding standards. The peak area of each nucleotide was obtained as arbitrary units from the spectra recorded with unirradiated control and PIR samples

and was then converted into yield mg-1 protein. The nuclease activity was measured as described earlier (Kota & Misra, 2008). The 500-ng heparin-purified proteins were incubated with 200 ng of 1-kb PCR product from D. radiodurans genome, in a buffer (10 mM Tris-HCl, pH 7.5, 3 mM MgCl2, 15 mM KCl and 2% glycerol) for 20 min at 37 °C. For ATP and calf intestinal

alkaline phosphatase (AP) treatment, the proteins were preincubated with these agents for 30 min at 37 °C. For phosphatase and protein kinase inhibitor treatment, the samples were treated with 10 mM sodium fluoride and different concentrations of protein kinase inhibitors, respectively, for 20 min. The treated samples were incubated with double-stranded DNA (dsDNA) substrate for 20 min at 37 °C and reaction products were analyzed on 1% agarose gel. Protein kinase activity was measured as described earlier (Rajpurohit et al., 2008). In brief, the cell-free extract was Pembrolizumab prepared from cells treated with γ radiation and equal amounts of protein (2 μg of each sample) were incubated with 50 μCi [32P] γ ATP (2500 Ci mmol−1) for 1 h at 37 °C. DNAse (50 μg mL−1) and RNAse (50 μg mL−1) were added and further incubated for 1 h. The mixture was passed through G-25 microspin columns (GE Healthcare) to remove the unincorporated radionucleotides and smaller nonproteinaceous phospho-contaminants. Incorporation of [32P] was measured by scintillation counting and the counts mg-1 protein were presented. The acid and alkaline phosphatases were assayed in 100 mM acetate buffer (pH 5.0) and 50 mM Tris-HCl buffer (pH 9.0), respectively, using disodium salt of p-nitrophenyl phosphate (pNPP) as described earlier (Bolton & Dean, 1972). In brief, 2 μg of total protein was incubated with the respective substrate in a corresponding buffer for 20 min.

Although these features were all seen in Δsahh

strain, a seemingly contradictory observation is that sahh transcript level is elevated in the hypovirus-infected strain. In a plant system, it has been reported that methylation pathway is targeted by geminivirus to HKI-272 cell line inhibit host antiviral defense of transcriptional gene silencing (Buchmann et al., 2009). Very recently SAHH was shown to be targeted by a geminivirus batasatellite-encoded protein to inhibit the SAHH activity and methylation-mediated transcriptional gene silencing (Yang et al., 2011). Should a hypovirus also regulate sahh at posttranslational level to inhibit its enzymatic function, one can hypothesize that elevated

sahh transcription and inhibition of SAHH activity can be two separate events incited by hypovirus infection. In this regard, actually measuring the in vivo SAHH activity by quantification of the SAH/SAM in hypovirus-infected strain will be vitally necessary. As SAHH regulation of the expression Selleckchem GSK2126458 of genes involved in key process of the cell is likely through its effects on intracellular methylation, further analysis of the genome methylation and global gene expression in Δsahh strain and testing the direct interaction of SAHH and hypovirus-encoded protein(s) will help to reveal the precise mechanisms by which SAHH regulates traits of chestnut blight fungus. This work was supported in part by the National Natural Science Foundation of China grants 31170137, 30130020, and 39925003 and the International Collaboration Key Project 2001CB711104. S.L. and R.L. contributed equally to this work. Please note: Wiley-Blackwell is not responsible for the content Florfenicol or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“The xnp1 remnant P2-type prophage of Xenorhabdus nematophila produces

xenorhabdicin that is active against closely related species. Xenorhabdicin had not been characterized previously in other Xenorhabdus species. Here, we show xenorhabdicin production in six different strains of Xenorhabdus bovienii. The sequenced genome of X. bovienii SS-2004 was found to possess a highly conserved remnant P2-type cluster (xbp1). Inactivation of the xbpS1 sheath gene resulted in loss of bacteriocin activity, indicating that the xbp1 locus was required for xenorhabdicin production. xbp1 and xnp1 contain a CI-type repressor, a dinI gene involved in stabilization of ssDNA-RecA complexes and are inducible with mitomycin C, suggesting that both loci are regulated by cleavage of the CI repressor.