We did observe an asymmetry in the increase in error rates on anti-saccade trials, with short-duration SEF stimulation causing a larger increase in contralateral (Fig. 2A) vs. ipsilateral anti-saccade errors (Fig. 2B). A three-way repeated-measures analysis of variance (anova) of error rate across

the factors of task (pro- or anti-saccades), direction (contra- or ipsilateral to stimulation) and time of stimulation (including control trials) revealed significant effects of task and time of stimulation (P < 10−5), and significant two-way and three-way interactions between all factors (task and direction: P = 0.02; task and stimulation time: P < 10−5; direction and stimulation time: P = 0.003; task, direction and MG-132 supplier stimulation time: P = 0.03). Subsequent two-way repeated-measures anovas of error rates on pro- or anti-saccade trials revealed a far greater influence of stimulation time on anti-saccade vs. pro-saccade trials, suggesting that the three-way interaction between task, direction and stimulation is primarily

driven by the anti-saccade error rate. The filled symbols in Fig. 2 show data that differed significantly from the respective Selleck RG 7204 control trials (paired t-tests, Bonferroni-corrected for multiple comparisons), and the frequency histograms in Fig. 2C and D represent the change in error rate vs. control trials for pro- or anti-saccades for each stimulation interval. The greater impact of ICMS-SEF on anti-saccade error rate across our sample can be appreciated by gauging the degree of shift of these histograms away from zero (rightward shifts convey increases in error rate). Note also that the histograms shifts tend to be greater for contralateral vs. ipsilateral anti-saccade errors for the later stimulation intervals, emphasizing some degree of laterality to the change others in anti-saccade error rate. The influence of short-duration ICMS-SEF on RTs is shown in Fig. 3 in a similar fashion. As with error rates, the influence of ICMS-SEF on correct

RTs is highly dependent on the task, and on the timing of stimulation relative to cue presentation (Fig. 3). Short-duration ICMS-SEF during the fixation interval exerted only a minor effect on RTs, but exerted a much greater effect when delivered after cue onset on anti-saccade trials, progressively prolonging the RTs of correctly performed anti-saccades in either direction. Interestingly, short-duration ICMS-SEF had little effect on the RTs of contralateral pro-saccades, although we did observe a modest increase in the RTs for pro-saccades to an ipsilateral cue for later stimulation times. Finally, the RTs of anti-saccade errors displayed a dependency with saccade direction, becoming shorter for errors made to contralateral cues, and longer for errors to ipsilateral cues.

These observations,

combined with the above-mentioned demonstrations of human resistin storage in neutrophil granules and resistin release in response to microbial stimuli, indicate that neutrophil granules were the source of the resistin released in our study. This conclusion is supported by the simultaneous release of resistin and granule-associated elastase (Fig. 4a and b). We have little information on how degranulation of neutrophils is stimulated by leukotoxin. Johansson et al. (2000) reported that leukotoxin induced degranulation of PMNs and that the polyclonal antibodies against LFA-1 subunits had no effect on degranulation. Moreover, signals involved in triggering degranulation by neutrophils stimulated by leukotoxin are poorly understood. Integrins, which are heterodimeric transmembrane adhesion receptors localized at cell–matrix Metabolism inhibitor contact sites, link extracellular matrix components to the actin cytoskeleton and interact with multiple structural and signaling molecules. LFA-1, a member of the β2-integin family, including CD11a and CD18, is a leukotoxin receptor located on the learn more surface of neutrophils (Lally et al., 1997). The significant decrease in leukotoxin-induced resistin release from

neutrophils pretreated with TS1/18 in the present study provides evidence for the involvement of CD18 in resistin release (Fig. 5a), as a recent study reported that CD18 is essential for the biological effect induced by leukotoxin (Dileepan et al., 2007). Our results differ from those reported by Johansson et al. (2000), and we cannot completely explain the discrepancy. It is possible the polyclonal antibodies used by Johansson et al. (2000) were less effective than the monoclonal antibodies that we used in the inhibition study. Furthermore, the inhibition

of leukotoxin-induced resistin release from neutrophils incubated with PP1 indicates that an Src family tyrosine kinase participates in resistin release (Fig. 5a). Src family tyrosine kinases have been reported to be important mediators acting downstream of integrins to affect adhesion-dependent degranulation of neutrophils (Mocsai et al., 1999). Although PP1 inhibited adhesion-dependent degranulation, it had no effect on adhesion-independent Farnesyltransferase degranulation induced by phorbol 12-myristate 13-acetate. The results obtained from experiments with TS1/18 and PP1 suggest that leukotoxin binds to LFA-1 on the surface of neutrophils and then activates an Src family tyrosine kinase, leading to the release of resistin from neutrophils by degranulation, as well as adhesion-dependent degranulation. Release of resistin and elastase still occurred, but a lower level, when stimulated by the mutant strain (Fig. 4). Moreover, pretreatment with TS1/18 or PP1 inhibited release of resistin and elastase from neutrophils stimulated by the mutant strain (Fig. 5a and b). Another molecule of A. actinomycetemcomitans might interact with CD18.

(2006). The barley cultivar Rihane, which covers >70% of the barley area in Tunisia, was used as a control. Disease severity was assessed 17 days after inoculation according to the rating scale described by Ceoloni (1980). The differential cultivars were scored for resistance (R) and susceptibility (S), and a matrix showing reaction patterns was constructed for the 79 pathotype responses vs. the 19 differential cultivars. Cluster analysis was performed on the pathotype matrix using the unweighted pair group method with arithmetic

averaging of darwin software (http://darwin.cirad.fr/darwin) to determine patterns of pathogenicity of Tunisian R. secalis AZD2281 research buy isolated from local barley landraces and the cultivar Rihane. The susceptibility percentage of 19 differential barley cultivars with known resistance genes to 79 R. secalis isolates sampled from different hosts (Rihane cv. and local

barley landraces) was calculated by host and by differential cultivar to determine the possible resistance genes. To detect new sources of resistance, the reaction spectrum of the 79 R. secalis isolates was compared with pairs of differentials with the same resistance genes Jet and Steudel and Kitchin and Abyssinian for differences in pathogenic reaction. Fungal mycelial DNA was extracted according to the Von Korff et al. (2004) method. Seven microsatellite loci

developed for R. secalis (Linde et al., 2005) were used to fingerprint the 79 isolates. Loci were amplified by multiplex PCR with group I (GA-SSR7, GA-SSR3, GA-R2 and CA-SSR1) Cyclopamine and II (TAC-SSR6, GA-SSR4 and TAC-SSR1) primers on either a Biometra T-gradient or an AB-GeneAmp PCR System 9700 thermocycler, subjected to capillary electrophoresis, and per-locus allele assignments were carried out using an ABI PRISM 310 Genetic Analyzer as described by Linde et al. (2005). SSR data were used to assess the level of genetic polymorphism and clustering. For each locus, we determined the total number of alleles and unique alleles by host and by virulence group. Patterns of genetic variation were determined by host through cluster analysis using the unweighted pair RG7420 in vivo group method as above. The relationship between variation in pathogenicity and the haplotype of microsatellite markers was compared for isolates having the same haplotype, by examining their reaction spectra to 19 differential cultivars. The ratio of the number of differential cultivars showing a coincident reaction to isolates with the same haplotype relative to the differential cultivars was used to calculate the degree of coincidence as described by Takeuchi & Fukuyama (2009). A total of 79 pathotypes were sampled from either Rihane cultivar (43) or local barley landraces (36) from 17 localities.

All patients who have originated or spent significant time (more

than 1 month) in sub-Saharan Africa should have Schistosoma serology performed. Any patient with an eosinophilia (absolute eosinophil count greater than 0.4 × 109 cells/L) on FBC who has originated or spent significant time (more than 1 month) in the Tropics (areas excluding Europe/Russia, this website North America and Australasia) should be investigated further depending on geographical exposure [13, 14]: please liaise with a physician with a specialist interest or with an infectious diseases unit. Such tests will probably include (but not be limited to) stool examinations for ova, cysts and parasites, and serologies for helminths such as Strongyloides, filaria and Schistosoma (if not already performed). Patients who spend further time in the Tropics should have these tests repeated as required. It is preferable to perform all such investigations in asymptomatic patients at least 3 months after their last tropical exposure. Individuals with exposure ALK mutation longer than 1 month in sub-Saharan Africa should have screening with Schistosoma serology. Those with an

eosinophilia (absolute eosinophil count greater than 0.4 × 109 cells/L) who originate from or report significant time spent in tropical areas (more than 1 month) may have a helminthic infection

and should be further assessed (see text) (III). Thorough contact tracing and partner notification are essential; careful documentation of this, and Sulfite dehydrogenase eventual outcomes, should be performed. A patient may wish to delay disclosure to partners; some delay may be acceptable if there is no urgency (i.e. no ongoing risk behaviour). Attempts should be made to encourage and support disclosure, counselling should be provided and contacts should be tested; if the patient refuses to cooperate, then additional action may be required. Testing of children is a sensitive area and specialist input should be sought. Interventions shown to reduce transmission risk such as ART, pre- and post-exposure prophylaxis for seronegative partners, and diagnosis and treatment of STIs may all be relevant depending on specific circumstances. Asymptomatic individuals should be offered STI screening at least yearly with consideration of more frequent screening dependent on risk [1]. There is some evidence that adding syphilis serology to routine HIV monitoring reduces time with undiagnosed syphilis and therefore potentially contributes to a reduction in onward syphilis transmission [2]. Therefore, in individuals or groups at increased risk of syphilis (currently MSM), syphilis serology should be considered with routine HIV follow-up (2–4 times yearly).

In addition, SK activation GSK2126458 in the latDS prevented LTD induction. Greater SK function in the latDS than in the NAS was not secondary to differences in sodium or inwardly rectifying potassium channel function, and apamin enhancement of firing did not reflect indirect action through cholinergic interneurons.

Thus, these data demonstrate that SKs are potent modulators of action potential generation and LTD in the dorsal striatum, and could represent a fundamental cellular mechanism through which habits are regulated. “
“Recently, genome-wide association between schizophrenia and an intronic variant in AMBRA1 (rs11819869) was reported. Additionally, in a reverse genetic approach in adult healthy subjects, risk allele carriers showed a higher medial prefrontal cortex blood oxygen level-dependent (BOLD) response during a flanker task examining motor inhibition as an aspect of impulsivity. To test whether this finding can be expanded to further aspects of impulsivity, we analysed the effects of the rs11819869 genotype on impulsivity-related traits on a behavioral, temperament and neural level in a large sample of healthy adolescents. learn more We consider this reverse genetic approach specifically suited for use in a healthy adolescent sample, as these individuals

comprise those who will eventually develop mental disorders in which impulsivity is implicated. Healthy adolescents from the IMAGEN study were included in the neuropsychological analysis (n = 848) and a functional magnetic resonance imaging (fMRI) task (n = 512). Obatoclax Mesylate (GX15-070) Various aspects of impulsivity were assessed using the Temperament and Character Inventory-Revised, the Substance Use Risk Profile Scale, the Cambridge Cognition Neuropsychological Test Automated Battery, and the Stop Signal Task (SST) in the fMRI paradigm. On a behavioral level, increased delay aversion was observed in risk allele carriers. Furthermore, risk allele carriers showed a higher BOLD response in an orbito-frontal target region during the SST, which declined to

trend status after Family Wise Error correction. Our findings support the hypothesis that the schizophrenia-related risk variant of rs11819869 is involved in various aspects of impulsivity, and that this involvement occurs on a behavioral as well as an imaging genetics level. “
“The auditory N1 event-related potential has previously been observed to be attenuated for tones that are triggered by human actions. This attenuation is thought to be generated by motor prediction mechanisms and is considered to be important for agency attribution. The present study was designed to rigorously test the notion of action prediction-based sensory attenuation. Participants performed one of four voluntary actions on each trial, with each button associated with either predictable or unpredictable action effects. In addition, actions with each hand could result in action effects that were either congruent or incongruent with hand-specific prediction.

[10] In spite of avoidance behavior, a traveler may still be bitten by an animal in the developing world where there is a reasonable risk of exposure to rabies infection. If the traveler has a contingency plan to deal with such a scenario, she/he will know to go to the nearest center of safe medical care within a

few days or as soon as possible for appropriate rabies PEP. Unfortunately, many cases of travel-related rabies infection were associated with the exposed person grossly underestimating the significance of the incident and not seeking medical care until the onset of rabies symptoms.[17-20] Prior to the onset of symptoms, some travelers also died of rabies as a result of seeking but not receiving timely rabies PEP even at sites of medical selleck chemicals llc excellence.[21-24] In addition, recent studies document inadequate Belnacasan cost rabies PEP and animal bite aftercare provided to travelers following high-risk exposures in various developing countries.[25-27] Knowing

this, it may be more prudent in some high-risk travel environments (eg, India or Africa) to offer rabies PrEP to any concerned traveler.[10] Where cost is a barrier, the intradermal method of administration is a cost-effective alternative to intramuscular injections.[28] Unlike animal avoidance, rabies immunization is a passive act and does not require active participation of the traveler. In general, passive interventions tend to be more successful than active ones that require the client’s adherence throughout the trip. If the properly primed traveler [eg, with post-series rabies virus neutralizing antibodies (RVNA) titer ≥0.5 IU/mL] is potentially exposed to rabies, then the management becomes an urgent and not an emergent matter.[14] Rabies PrEP may be seen as addressing a manageable risk, because it simplifies post-exposure aftercare. Rabies immune globulin (RIG) is not required for PEP in an adequately “PrEPed” traveler; and RIG is often unavailable in many developing countries.[10,

12-14, 25-27] However, rabies PrEP may also be seen as addressing rabies exposure as a preventable risk rather than simply a manageable one. Veterinarians and other animal handlers receive rabies PrEP for occupational reasons, because they may experience inapparent BCKDHB rabies exposure during the course of their careers.[12, 14] As a precaution, these individuals are tested at regular intervals to assure having adequate RNVA (>0.5 IU/mL) as a surrogate for protection against rabies infection, because inapparent exposures would never result in post-exposure rabies vaccination. This has been an accepted occupational health practice for several decades.[13] To our knowledge, there have been no reported cases of rabies among animal handlers who have received a proper rabies PrEP series using a World Health Organization (WHO)-recommended vaccine of cell-culture origin.

[73] Moreover, it should be noted that adding anti-TNF-α to RA synovial cell cultures did not increase IL-23 cell-associated levels, check details whereas a reduction (non-significant) in p19 mRNA levels was observed.[10, 22, 73] In mice, systemic IL-23 exposure induced chronic arthritis, severe bone loss, and expanded myeloid lineage osteoclast precursors in the bone marrow, which resulted in

increased osteoclast differentiation and systemic bone loss as observed in RA and other types of autoimmune arthritis.[60, 74] Moreover, in conflict with its effects, IL-23 also dose-dependently inhibited osteoclastogenesis in a CD4+ T lymphocyte-dependent manner. Like IL-12, IL-23 acts synergistically with IL-18 to block osteoclastogenesis but, unlike IL-12, IL-23 action depends on T cell granulocyte-macrophage AZD0530 colony-stimulating factor (GM-CSF) production. Thus, IL-23 is able to inhibit osteoclast formation indirectly via T cells.[75] In RA, expression of IL-22 was found to be up-regulated in synovium with ability to induce synovial fibroblast proliferation

and chemokine production.[76, 77] The high levels of IL-22 were expressed both in the lining and the sublining layers of RA synovial tissues.[77, 78] The paucity of IL-22-producing CD4 T cells in synovial fluid (SF) lends support to the notion that the primary source of IL-22 in the joint is synovial fibroblasts and/or macrophages but not T cells, based on the report of Ikeuchi et al.[76, 77] In RA, IL-21 can regulate the function of T, B, NK and DC cells, and pro-inflammatory cytokine secretion in immune responses. IL-21 expression shows a correlation with the presence of Th17 cells in the synovium, SF and peripheral blood in RA patients. It has been reported that human CCR6+ CD4+ T cells can produce high levels of both IL-21 and IL-17. Similar to mouse T cells, IL-21 auto-regulates its own production in human CD4+ T cells.[79] In addition, IL-21 forms a positive-feedback autocrine loop involving homeostatically activated CD4+ cells, which is essential in the progression of autoimmune

arthritis by mechanisms dependent on follicular Th cell development, autoreactive B cell maturation, and RANKL induction, but is independent from Th17 cell function.[80] Here, we have focused Pyruvate dehydrogenase on the role of Th17 cells in inducing and perpetuating chronic inflammation, cartilage damage and bone erosion which are hallmark phases of joint destruction (Fig. 1). The aim of current and emerging therapies is to seek a way for disrupting the inflammatory Th17 network and shifting the immune system back toward homeostasis.[81] In this section, the potential dynamic of Th17 cell populations and their interplay with other inflammatory cells in inducing tissue inflammation in organ-specific autoimmunity are reviewed.[82] Various animal models have demonstrated key roles of IL-17A (henceforth called IL-17) and Th17 cells in immunopathology and joint damage of arthritis.

The various substrates form a hierarchy in their ability to trigger phosphorylation of HPr at Ser46 (Singh et al., 2008). Fructose-1,6-bisphosphate (FBP) has been identified as the main factor that allosterically activates kinase activity of HPrK/P, but other metabolites may also play a role (Jault et al.,

2000; Ramström et al., 2003). Bacillus subtilis and other Bacilli possess carbon-flux-regulating histidine protein (Crh), which shares over 40% sequence identity with HPr (Galinier et al., 1997). Crh lacks His15, but contains Ser46 and accordingly it becomes (de)phosphorylated by HPrK/P in vitro (Lavergne et al., 2002). Crh~P can likewise form a complex with CcpA and contributes to CCR, but to a weaker extent than HPr(Ser)~P. Hence, Crh~P can only partially replace HPr(Ser)~P in CCR (Galinier et al., 1997; Singh et al., Regorafenib research buy 2008). This weaker contribution of Crh~P to CCR can be ascribed to its much lower levels in the cell and its lower binding affinity for CcpA as compared with HPr(Ser~P) (Görke Selleckchem ABT888 et al., 2004; Seidel et al., 2005). Therefore, Crh was regarded for a long time as back-up factor for CCR. However, recently a distinct role for Crh has been identified. It was found that non-phosphorylated Crh binds to and inhibits activity of the metabolic enzyme methylglyoxal synthase, MgsA,

in B. subtilis (Landmann et al., 2011). MgsA catalyzes the formation of methylglyoxal from dihydroxyacetone-phosphate, initiating a glycolytic bypass. This pathway may relieve cells from Epothilone B (EPO906, Patupilone) sugar-phosphate stress, when carbohydrate uptake rates exceed the capacity of the lower branch of the Embden–Meyerhof–Parnas (EMP) pathway (Weber et al., 2005). To understand the physiological conditions under which Crh exerts its regulatory functions, it is crucial to know its phosphorylation state in vivo. Indirect evidence from studies on CCR suggested that the phosphorylation of Crh and HPr at their Ser46 sites has similar dynamics (Galinier et al., 1997; Singh et al., 2008). Direct proof of this hypothesis has so far been hindered technically by the low cellular abundance of Crh (Görke et al., 2004). This might explain why Crh~P was detected in only one of several phosphoproteome

studies (Eymann et al., 2007). In the present work, we overcame these limitations and analyzed phosphorylation of Crh in vivo in response to different nutritional conditions. A direct method was used that involves separation of Crh and Crh~P in cell extracts by non-denaturing gel electrophoresis. Crh was detected using a tailor-made sensitive antiserum specifically directed against a C-terminal peptide of Crh. Bacillus subtilis strains were grown at 37 °C in CSE minimal medium (C-medium supplemented with sodium succinate and potassium glutamate; Martin-Verstraete et al., 1995) supplemented with 50 mg L−1 tryptophan and 0.5% of the indicated carbon source. The strains used were B. subtilis 168 (trpC2; wild-type), QB7097 (trpC2 Δcrh::spec; Singh et al.

During laparotomy, two electrodes were implanted into the stomach and high-frequency low-energy GES (14 Hz, 5 mA) was applied.

The effects of 1 h GES were compared with sham stimulation. After GES, c-Fos expression was increased in the mucosal and submucosal layers of the stimulated area (174%). In the stomach, GES increased ghrelin mRNA (178%) and doubled the number of ghrelin-positive cells, resulting in elevated plasma levels of ghrelin (2.3 ± 0.2 vs. 1.6 ± 0.2 ng/mL). In the arcuate nucleus of the hypothalamus, GES increased c-Fos (277%) and agouti-related protein (AgRP) mRNA expression (135%). GES reduced BIBF 1120 purchase the number of c-Fos-positive cells throughout the nucleus of the solitary tract (between 93 and 75% from rostral to caudal levels) including catecholaminergic

neurons (81% at caudal level). Gastric emptying, CX-4945 order plasma glucose and heart rate variability were not affected by GES. This study shows that GES may improve appetite via stimulation of main orexigenic pathways, including ghrelin production in the stomach and AgRP in the hypothalamus, as well as by reducing the activity of catecholaminergic brainstem neurons. “
“Despite considerable progress, the mechanisms that control neural progenitor differentiation and behavior, as well as their functional integration into adult neural circuitry, are far from being understood. Given the complexity of the mammalian brain, non-mammalian models provide an excellent model to study neurogenesis, including both the cellular composition of the neurogenic microenvironment, and the factors required for precursor growth and maintenance. In particular, we chose to address the question of the control of progenitor proliferation by Sonic hedgehog (Shh) using the zebrafish dorsal mesencephalon, known as the optic tectum (OT), as a model system. Here we show that either inhibiting pharmacologically

or eliminating hedgehog (Hh) signaling by using mutants that lack essential components of the Hh pathway reduces neural progenitor Palbociclib cell proliferation affecting neurogenesis in the OT. On the contrary, pharmacological gain-of-function experiments result in significant increase in proliferation. Importantly, Shh-dependent function controls neural progenitor cell behavior as sox2-positive cell populations were lost in the OT in the absence of Hh signaling, as evidenced in slow-muscle-omitted (smu) mutants and with timed cyclopamine inhibition. Expressions of essential components of the Hh pathway reveal for the first time a late dorsal expression in the embryonic OT. Our observations argue strongly for a role of Shh in neural progenitor biology in the OT and provide comparative data to our current understanding of progenitor/stem cell mechanisms that place Shh as a key niche factor in the dorsal brain.

pIJ702-rshAbldG was constructed by inserting a bldG cassette prepared by PCR (primers

are shown in Supporting Information, Table MS-275 purchase S1) between the PstI and KpnI sites of pIJ702-rshA. The bldG cassette contained its own promoter region, which directed the expression of the coding sequence. For genetic complementation, the 957-bp DNA fragment containing the bldG coding sequence (cds) and its promoter region was amplified by PCR using the primers GCF/GCR and cloned between the EcoRI and HindIII sites of pKU463 to form pBG. pBG was then introduced into the bldG mutant by transformation to generate a kanamycin-resistant transformant carrying the plasmid integrated at the K38-1 site (nucleotide sequence accession no. AB251919). Disruption of bldG was carried out by a homologous recombination technique based on REDIRECT technology (Gust et al., 2003). The cosmid clone SGR1G10 used for bldG knockout in S. griseus containing the region corresponding to nucleotides 3874894–3914177 in the SB203580 supplier genome sequence database was obtained in our study using SuperCos1 (Stratagene, Japan,

Tokyo) as a vector. An apramycin resistance gene cassette was prepared by PCR using the primers DisGF/DisGR and used for the replacement for the bldG cds by in vivo recombination using λ RED. The resulting apramycin-resistant cosmids purified from Escherichia coli GM2163 were introduced into the wild-type strain of S. griseus. Apramycin-resistant recombinants were then screened and checked for true recombination by PCR using appropriate primer sets. All mutants obtained exhibited the identical bald phenotype; hence, one of them was designated as the bldG mutant. The transcriptional activities of the promoters preceding the rshA-sigH operon (PH1, the σH-dependent promoter), sigN (PN1), and rpp operon (Prpp) were studied by S1 protection analysis. Methods and conditions for RNA preparation and S1 nuclease mapping were previously

described (Kieser et al., 2000; Kelemen et al., 2001; Takano et al., 2007). The probes for PH1, PN1, and Prpp were prepared by PCR using primers HS1-F/HS1-R* for PH1, NS1-F/NS1-R* for PN1, and RS1-F/RS1-R* for Prpp, respectively (primers indicated with an asterisk were labelled at its 5′-end mafosfamide with [γ-32P] ATP using T4-polynucleotide kinase). Preparation of a C-terminally histidine-tagged RshA (RshA-6xHis) was described previously (Takano et al., 2003). For preparation of RshA carrying an N-terminal glutathione-S-transferase (GST-RshA), an rshA cassette was amplified using the PCR primers Rex-F/Rex-R and cloned between the BamHI/EcoRI sites of pGEX-4T-2 (GE Healthcare, Tokyo, Japan). For BldG-6xHis, a bldG cassette was amplified using primers Gex-F/Gex-R and cloned between the NdeI/HindIII sites of pET26-b+ (Takara Shuzo). The E.