Potential adjustments of feeding style in a culturally sensitive manner may be addressed at a pre-travel visit. Breastfeeding women and their infants can travel safely, but need special attention to protect the infant. A critical goal is to maintain Selleck Y27632 adequate hydration. Geographic areas where clean water and sanitation are lacking pose particular hurdles to any traveler and are especially difficult for the breastfeeding woman. Careful planning and assessment of local resources are important to preserve the health of infant and mother. The authors thank

Drs I. Dale Carroll and Robert Steffen, and Brenda Phipps, BS, IBCLC, for their thoughtful review of the manuscript and helpful comments. The authors state that they have no conflicts of interest to declare. “
“We present a 31-year-old man who, after a Conus textile sting acquired in New Caledonia, developed a cutaneous abscess on a buttock. The abscess was accompanied by pain, paraesthesia, general malaise, and fever. Complete remission was achieved by sodium

hypochlorite packs and oral amoxicillin/clavulanic acid, metronidazole, and tramadol. A 31-year-old man was admitted because of Cilomilast an abscess located in the right buttock. The patient stated that the abscess had appeared 2 weeks earlier, during a trip to New Caledonia (South-West Pacific Ocean). The patient claimed that he was snorkeling, when he observed a beautiful shell: he picked it up and put it in the back pocket of the bathing suit. Some minutes later, the patient complained of a burning sensation in the right buttock. Three hours later, a painful swelling appeared in the same area. Two days later, fever (<38°C) and general malaise appeared. Before admission to our department, the patient was unsuccessfully treated at other DOK2 centers with topical antiseptics, clotrimazole and hydrocortisone butyrate, and oral paracetamol. Dermatological examination revealed an abscess: it was round, 3.5 cm in diameter, red in color, with two fistulas discharging pus. The lesion was surrounded by erythematous edema, hard in consistency (Figure 1). The patient complained of severe pain, local paraesthesia, and fever (37.8°C). General physical examination

revealed nothing pathological. Laboratory examinations showed leucocytosis (12.700 white blood cells/mm3, with 9.300 neutrophils/mm3), and increase in erythrocyte sedimentation rate (71 mm at the first hour) and C-reactive protein (7.9 mg/L). Bacteriological examinations of the abscess were positive for Escherichia coli, Staphylococcus aureus, and Peptostreptococcus sp. The shell was classified as a 10.3 cm long specimen of Conus textile Linnaeus 1758 (Figure 2). The patient was treated with sodium hypochlorite packs and, on the basis of antibiogram results, with oral amoxicillin/clavulanic acid [minimum inhibitory concentration (MIC): ≤0.03 µg/mL for both Escherichia coli and S aureus; 3 g/d for 10 d], oral metronidazole (MIC: ≤0.

Potential adjustments of feeding style in a culturally sensitive manner may be addressed at a pre-travel visit. Breastfeeding women and their infants can travel safely, but need special attention to protect the infant. A critical goal is to maintain selleck compound adequate hydration. Geographic areas where clean water and sanitation are lacking pose particular hurdles to any traveler and are especially difficult for the breastfeeding woman. Careful planning and assessment of local resources are important to preserve the health of infant and mother. The authors thank

Drs I. Dale Carroll and Robert Steffen, and Brenda Phipps, BS, IBCLC, for their thoughtful review of the manuscript and helpful comments. The authors state that they have no conflicts of interest to declare. “
“We present a 31-year-old man who, after a Conus textile sting acquired in New Caledonia, developed a cutaneous abscess on a buttock. The abscess was accompanied by pain, paraesthesia, general malaise, and fever. Complete remission was achieved by sodium

hypochlorite packs and oral amoxicillin/clavulanic acid, metronidazole, and tramadol. A 31-year-old man was admitted because of selleck chemical an abscess located in the right buttock. The patient stated that the abscess had appeared 2 weeks earlier, during a trip to New Caledonia (South-West Pacific Ocean). The patient claimed that he was snorkeling, when he observed a beautiful shell: he picked it up and put it in the back pocket of the bathing suit. Some minutes later, the patient complained of a burning sensation in the right buttock. Three hours later, a painful swelling appeared in the same area. Two days later, fever (<38°C) and general malaise appeared. Before admission to our department, the patient was unsuccessfully treated at other SB-3CT centers with topical antiseptics, clotrimazole and hydrocortisone butyrate, and oral paracetamol. Dermatological examination revealed an abscess: it was round, 3.5 cm in diameter, red in color, with two fistulas discharging pus. The lesion was surrounded by erythematous edema, hard in consistency (Figure 1). The patient complained of severe pain, local paraesthesia, and fever (37.8°C). General physical examination

revealed nothing pathological. Laboratory examinations showed leucocytosis (12.700 white blood cells/mm3, with 9.300 neutrophils/mm3), and increase in erythrocyte sedimentation rate (71 mm at the first hour) and C-reactive protein (7.9 mg/L). Bacteriological examinations of the abscess were positive for Escherichia coli, Staphylococcus aureus, and Peptostreptococcus sp. The shell was classified as a 10.3 cm long specimen of Conus textile Linnaeus 1758 (Figure 2). The patient was treated with sodium hypochlorite packs and, on the basis of antibiogram results, with oral amoxicillin/clavulanic acid [minimum inhibitory concentration (MIC): ≤0.03 µg/mL for both Escherichia coli and S aureus; 3 g/d for 10 d], oral metronidazole (MIC: ≤0.

raciborskii capable of the CYN synthesis (Neilan et al., 2003; Haande et al., 2008; Antal et al., 2011). However, CYN was detected in Finland (Spoof et al., 2006), Germany (Fastner et al., 2007; Wiedner et al., 2008), the Czech Republic (Bláhová

et al., 2008, 2009), Poland (Kokociński et al., 2009), France (Brient et al., 2009) and Italy (Messineo et al., 2010). In these cases, microscopic analysis indicated that suggested species Selleck Adriamycin of cyanobacteria that could produce CYN included: Anabaena lapponica in Finland (Spoof et al., 2006); Aphanizomenon sp., Aphanizomenon gracile, Aphanizomenon flos-aque and/or Anabaena sp. in Germany (Fastner et al., 2007; Wiedner et al., 2008); Aphanizomenon sp. including Aph. klebahnii in the Czech Republic (Bláhová et al., 2008, 2009); Aph. gracile and/or C. raciborskii in Poland (Kokociński et al., 2009); Aph. flos-aque and Anabaena planctonica in France (Brient et al., 2009); Aphanizomenon ovalisporum and/or C. raciborskii in Italy (Messineo et al., 2010). In further research, the possibility of using molecular analysis has allowed to determine toxigenic strains of cyanobacteria responsible for CYN production (Haande et al., 2008; Stüken & Jakobsen, 2010). However, in Europe, this information is still

poor. Preußel et al. (2006) determined three single filaments of toxigenic Aph. flos-aque in two German lakes based on the presence of ps gene sequences. Description of the toxigenic strain of Oscillatoria from the Tarn River in France was based on the presence of cyrJ X-396 gene (Mazmouz et al., 2010). Additionally, that study indicated a high homology to cyr genes previously identified for C. raciborskii strains isolated from Australian water bodies (Mihali et al., 2008). The presence of cyr genes (cyrA/aoaA and cyrB/aoaB) was also confirmed for the strains of Aphanizomenon sp. in Germany (Stüken & Jakobsen, 2010). Recently, CYN synthetase gene (pks) was detected in one of the samples contained C. raciborskii

from the Vela Lake in Portugal (Moreira et al., 2011). However, the presence of CYN was not described. In Poland, as it has already been mentioned, the presence of CYN was described in two shallow eutrophic lakes: Bytyńskie cAMP (BY) and Bnińskie (BN) located in the western part of the country (Kokociński et al., 2009). Microscopic analysis indicated Aph. gracile and/or C. raciborskii as potential producers of CYN in the studied water samples. In the present study, in which the genetic analyses were used for the first time (to the best of our knowledge), the previous research has been followed up to confirm and develop this theory. The possibility of using cyrJ gene for early warning of CYN-producing cyanobacteria was also tested. Moreover, the objective of the study included an analysis of genetic identity of Polish cyanobacterial samples with known genomic sequences of CYN-producing cyanobacteria based on cyrJ gene product and characterization of the strain of C.

60 ± 0.04 × 106), with the anterior half of both segments being more densely innervated than the posterior half. Dorsoventral and mediolateral decreasing gradients of SERT varicosities occur in both pallidal segments, but are statistically significant only in the GPi. The neuronal density being significantly greater in the GPe (3.41 ± 0.23 × 103 neurons/mm3) than in the GPi (2.90 ± 0.11 × 103), the number of 5-HT axon varicosities per pallidal neuron was found to be superior in the GPi (201 ± 27) than in the GPe (156 ± 26). At the electron microscopic level, www.selleckchem.com/products/Vorinostat-saha.html SERT+ axon varicosities are comparable in size and vesicular

content in GPi and GPe, where they establish mainly asynaptic contacts with unlabeled profiles. Less than 25% of SERT+ varicosities display a synaptic specialization, which is of the symmetrical or asymmetrical type and occurs exclusively on pallidal dendrites. No SERT+ axo-axonic synapses are present, suggesting that 5-HT exerts its well-established modulatory Selleck LDK378 action upon various pallidal afferents mainly through diffuse transmission, whereas its direct control of pallidal neurons results from both volumic and synaptic release of the transmitter. “
“D-cycloserine (DCS) is currently under clinical trials for a number of neuropsychiatric conditions and has been found to augment fear extinction in rodents and exposure therapy

in humans. However, the molecular mechanism of DCS action

in these multiple modalities remains unclear. Here, we describe the effect of DCS administration, alone or in conjunction with extinction training, on neuronal activity (c-fos) and neuronal plasticity [phospho-extracellular signal-regulated kinase triclocarban (pERK)] markers using immunohistochemistry. We found that intraperitoneal administration of DCS in untrained young rats (24–28 days old) increased c-fos- and pERK-stained neurons in both the prelimbic and infralimbic division of the medial prefrontal cortex (mPFC) and reduced pERK levels in the lateral nucleus of the central amygdala. Moreover, DCS administration significantly increased GluA1, GluN1, GluN2A, and GluN2B expression in the mPFC. In a separate set of animals, we found that DCS facilitated fear extinction and increased pERK levels in the infralimbic prefrontal cortex, prelimbic prefrontal cortex intercalated cells and lateral nucleus of the central amygdala, compared with saline control. In the synaptoneurosomal preparation, we found that extinction training increased iGluR protein expression in the mPFC, compared with context animals. No significant difference in protein expression was observed between extinction-saline and extinction-DCS groups in the mPFC. In contrast, in the amygdala DCS, the conjunction with extinction training led to an increase in iGluR subunit expression, compared with the extinction-saline group.

60 ± 0.04 × 106), with the anterior half of both segments being more densely innervated than the posterior half. Dorsoventral and mediolateral decreasing gradients of SERT varicosities occur in both pallidal segments, but are statistically significant only in the GPi. The neuronal density being significantly greater in the GPe (3.41 ± 0.23 × 103 neurons/mm3) than in the GPi (2.90 ± 0.11 × 103), the number of 5-HT axon varicosities per pallidal neuron was found to be superior in the GPi (201 ± 27) than in the GPe (156 ± 26). At the electron microscopic level, see more SERT+ axon varicosities are comparable in size and vesicular

content in GPi and GPe, where they establish mainly asynaptic contacts with unlabeled profiles. Less than 25% of SERT+ varicosities display a synaptic specialization, which is of the symmetrical or asymmetrical type and occurs exclusively on pallidal dendrites. No SERT+ axo-axonic synapses are present, suggesting that 5-HT exerts its well-established modulatory Regorafenib price action upon various pallidal afferents mainly through diffuse transmission, whereas its direct control of pallidal neurons results from both volumic and synaptic release of the transmitter. “
“D-cycloserine (DCS) is currently under clinical trials for a number of neuropsychiatric conditions and has been found to augment fear extinction in rodents and exposure therapy

in humans. However, the molecular mechanism of DCS action

in these multiple modalities remains unclear. Here, we describe the effect of DCS administration, alone or in conjunction with extinction training, on neuronal activity (c-fos) and neuronal plasticity [phospho-extracellular signal-regulated kinase selleck kinase inhibitor (pERK)] markers using immunohistochemistry. We found that intraperitoneal administration of DCS in untrained young rats (24–28 days old) increased c-fos- and pERK-stained neurons in both the prelimbic and infralimbic division of the medial prefrontal cortex (mPFC) and reduced pERK levels in the lateral nucleus of the central amygdala. Moreover, DCS administration significantly increased GluA1, GluN1, GluN2A, and GluN2B expression in the mPFC. In a separate set of animals, we found that DCS facilitated fear extinction and increased pERK levels in the infralimbic prefrontal cortex, prelimbic prefrontal cortex intercalated cells and lateral nucleus of the central amygdala, compared with saline control. In the synaptoneurosomal preparation, we found that extinction training increased iGluR protein expression in the mPFC, compared with context animals. No significant difference in protein expression was observed between extinction-saline and extinction-DCS groups in the mPFC. In contrast, in the amygdala DCS, the conjunction with extinction training led to an increase in iGluR subunit expression, compared with the extinction-saline group.

60 ± 0.04 × 106), with the anterior half of both segments being more densely innervated than the posterior half. Dorsoventral and mediolateral decreasing gradients of SERT varicosities occur in both pallidal segments, but are statistically significant only in the GPi. The neuronal density being significantly greater in the GPe (3.41 ± 0.23 × 103 neurons/mm3) than in the GPi (2.90 ± 0.11 × 103), the number of 5-HT axon varicosities per pallidal neuron was found to be superior in the GPi (201 ± 27) than in the GPe (156 ± 26). At the electron microscopic level, Olaparib SERT+ axon varicosities are comparable in size and vesicular

content in GPi and GPe, where they establish mainly asynaptic contacts with unlabeled profiles. Less than 25% of SERT+ varicosities display a synaptic specialization, which is of the symmetrical or asymmetrical type and occurs exclusively on pallidal dendrites. No SERT+ axo-axonic synapses are present, suggesting that 5-HT exerts its well-established modulatory see more action upon various pallidal afferents mainly through diffuse transmission, whereas its direct control of pallidal neurons results from both volumic and synaptic release of the transmitter. “
“D-cycloserine (DCS) is currently under clinical trials for a number of neuropsychiatric conditions and has been found to augment fear extinction in rodents and exposure therapy

in humans. However, the molecular mechanism of DCS action

in these multiple modalities remains unclear. Here, we describe the effect of DCS administration, alone or in conjunction with extinction training, on neuronal activity (c-fos) and neuronal plasticity [phospho-extracellular signal-regulated kinase Immune system (pERK)] markers using immunohistochemistry. We found that intraperitoneal administration of DCS in untrained young rats (24–28 days old) increased c-fos- and pERK-stained neurons in both the prelimbic and infralimbic division of the medial prefrontal cortex (mPFC) and reduced pERK levels in the lateral nucleus of the central amygdala. Moreover, DCS administration significantly increased GluA1, GluN1, GluN2A, and GluN2B expression in the mPFC. In a separate set of animals, we found that DCS facilitated fear extinction and increased pERK levels in the infralimbic prefrontal cortex, prelimbic prefrontal cortex intercalated cells and lateral nucleus of the central amygdala, compared with saline control. In the synaptoneurosomal preparation, we found that extinction training increased iGluR protein expression in the mPFC, compared with context animals. No significant difference in protein expression was observed between extinction-saline and extinction-DCS groups in the mPFC. In contrast, in the amygdala DCS, the conjunction with extinction training led to an increase in iGluR subunit expression, compared with the extinction-saline group.

4f and h). A polyclonal antibody to flagella from B. bacilliformis (Scherer et al., 1993), which is closely related to A. felis, did not recognize Afipia flagellae, indicating substantial diversity in their flagella amino acid sequences. DNA was isolated and sequenced outward using primer Kan-2 FP01 for the transposon-flanking regions. We have obtained two sequences that prove an integration of the transposon in genes involved in flagella biosynthesis (Fig. 5). The sequence of mutant G4 (Fig. 4f) showed high similarities to FlhA, a part of the flagella export apparatus in different click here bacteria species (Ghelardi et al., 2002; McMurry et al., 2004). This mutant was detected using nonpermeabilized cells

and CSD11, because the CSD11 target antigen (flagellin) did not appear on the bacterial cell surface due to an export defect. When the same screen was performed in the presence of sodium dodecyl sulphate (SDS) to permeabilize the bacterial cells, this mutant showed weak signals due to the accessibility of the flagellar antigen under these conditions and was therefore not detected (data not shown). In an SDS-polyacrylamide gel electrophoresis (PAGE) with bacterial lysate material, the signal was not intense enough to be detected (Fig. 4i). One possible reason could be the degradation of the protein when export is disturbed as shown for a flhA mutant of Bacillus thuringiensis (Ghelardi et

al., 2002). Such fragments might be too small for detection in standard Western-blotting experiments with SDS-PAGE, while they are detected using colony blots. Similar reasons probably caused the identification of a total of seven flagella-deficient Tigecycline cost mutants in a screen without SDS permeabilization, while in its presence none of these clones was identified (data not shown). The mutant D5 (Fig. 4g) likely codes for a defective flagellin gene, probably in the region corresponding to the protein’s C-terminus. This would explain why the flagellin is still detected by CSD11 antibody but the protein had a reduced molecular weight. Because of the ∼420 remaining amino acids before insertion of the transposon (Fig. 5a), the expected mass of the truncated flagellin would be about 40–45 kDa, which

is the mass of the observed truncated flagellin in Western blot (Fig. 4i). Because the C-terminus of flagellin is these well conserved and important for assembly of the flagellar filament (Beatson et al., 2006), a defect in this area can result in unstable shortened flagella, which were observed using immunofluorescence (Fig. 4g) as well as scanning electron microscopy (Fig. 4b). In summary, we provide evidence that we have developed a genetic system to mutagenize Afipia spp. and a suitable vector system for gene cloning in this genus. We further present the first mutant A. felis, in this case with defects in flagella biogenesis. The tools presented here will help to analyse the unusual phagosome biogenesis of A. felis in macrophages (Lührmann et al.

succinogenes S85. The 16S rRNA gene copy numbers for these strains at 96 h of incubation were significantly higher (P < 0.05) in triculture than in monocultures and two-member coculture (Fig. 2a). Scanning electron microscopy (SEM) observations showed that all three strains attached to rice straw in monoculture (Fig. 2b, i–iii). In the triculture, the

three strains were shown to ABT199 be closely located on the rice straw (Fig. 2b, iv). Although the positive interaction between rumen bacteria has been reported in the previous studies, the present result is the first demonstration of synergism between the newly cultured group U2 bacterium R-25 and F. succinogenes. The extent of increase in DM digestion by coculture of strain R-25 and F. succinogenes S85 was comparable with the previous coculture studies using the combinations of F. succinogenes and several nonfibrolytic species, where DM digestion was enhanced in coculture at 1.05–1.18-fold (Dehority & Scott, 1967; Kudo et al., 1987; Osborne & Dehority, 1989; Fondevila & Dehority,

1996; Sawanon et al., 2011). Growth and fermentation patterns of F. succinogenes S85 were altered www.selleckchem.com/products/MDV3100.html in coculture with strain R-25. Higher level of 16S rRNA gene copy number (at 96 h) and succinate production (at 48 h) of F. succinogenes S85 suggest that strain R-25 had a positive effect on fermentation activity of F. succinogenes S85. Enzyme activity in coculture of strain R-25 with F. succinogenes S85 partly supports this suggestion. Although extracellular activity of CMCase and xylanase was significantly higher in coculture of strains R-25 and F. succinogenes S85, activity of extracellular CMCase and xylanase from strain R-25

alone was almost negligible. Therefore, elevated extracellular activity of fibrolytic enzyme in the coculture is likely to be solely attributable to F. succinogenes S85. Possible explanations Ribonucleotide reductase of this positive alteration of F. succinogenes S85 activity by strain R-25 include the consumption of oligosaccharides and hydrogen, which can accumulate in the monoculture. Previous research has shown that endoglucanase activity of F. succinogenes S85 is repressed by cellobiose (McGavin et al., 1990). Furthermore, the consumption of hydrogen by methanogenic archaea leading to increased ATP production and/or organic acid concentration of fibrolytic strains has been reported as interspecies hydrogen transfer (Latham & Wolin, 1977; Williams et al., 1994; Rychlik & May, 2000). Consumption of oligosaccharides and hydrogen to produce lactate by strain R-25 could lead to the maintenance of the fibrolytic activity of F. succinogenes S85, resulting in enhanced DM digestion in coculture.

larvae. The three indigenous strains were screened by PCR amplification for the presence of binary toxin genes. Among the strains, only ISPC-8

showed the presence of bin genes, whereas these genes were absent in ISPC-5 and ISPC-6 strains. The results agree with the medium larvicidal activity of ISPC-5 and ISPC-6 strains, as also noted earlier (de Barjac et al., 1985; Charles et al., 1996). Most of the highly toxic strains (1593, 2362) of B. sphaericus showed the presence of these genes (Yousten, 1984; Baumann et al., 1987). The binA (1.1 kb) and binB (1.3 kb) genes from ISPC-8 were PCR amplified (Fig. 1). The sequences of binA (GenBank accession no. EU3753086) and binB (GenBank accession no. EU3753089) from ISPC-8 were compared with other highly toxic strains 1593/2362. The BinA protein differed by one amino acid (R197M), whereas BinB differs by two amino acids (H99P,

P174S) as compared with standard 1593/2362 strains. selleck compound The insecticidal activity of this organism is mainly due to the presence of Bin (41.9 and 51.4 kDa) proteins (Broadwell & Baumann, 1987). The Bin proteins from ISPC-8 were purified using ion-exchange and gel-filtration chromatography. Selleckchem Palbociclib These proteins coeluted as a single peak on a gel filtration column with an elution volume that corresponded to an ∼65-kDa protein. The eluted peak showed two distinct bands of BinA and BinB when resolved on 12% SDS-PAGE (Fig. 2). The apparent molecular mass of ∼65 kDa is much lower than the complex of the BinA/BinB monomer, which essentially should show an elution volume corresponding to ∼93 kDa. These results indicate that the BinA and BinB proteins did not interact under these elution conditions, but coeluted, Bcl-w most likely due to the resolution in the gel

filtration. The purified proteins were tested against third-instar larvae of C. quinquefasciatus. The results showed higher toxicity of purified proteins with an LC50 value 6.32 ng mL−1 (Fig. 3). These results are particularly significant as there are very few reports on the toxicity of purified binary proteins. Baumann et al. (1991) have shown that the purified crystal from strain 2362 showed an LC50 value of 7 ng protein mL−1, whereas N NaOH-solubilized crystal yielded an LC50 dose of 2700 ng mL−1. The purified 51- and 42-kDa proteins from strain 2362 showed an LC50 value of 12 ng mL−1 (Baumann et al., 1991). When these Bin protein genes were expressed in Bacillus subtilis, the purified inclusion bodies showed an LC50 dose of 16 ng mL−1 (Baumann et al., 1991). Thus, a large variation in the LC50 doses and in the preparation methods has been observed. Perhaps more accurate estimates of toxicity profiles can be obtained using in situ folded BinA and BinB proteins in the assay, which may reveal the effect of amino acid substitutions observed in BinA and BinB proteins in our indigenous high-activity strain, ISPC-8.

2. The cultures were then incubated at 30 °C for 12 h. For qRT-PCR analysis of SYK-6 and ferC mutant, the cells prepared, as described earlier, were used as induced cells, while the cells incubated in LB medium for 12 h, were employed

as uninduced cells. For identification of an inducer, cells of SYK-6, FAK, and FBK were incubated in Wx-SEMP medium at 30 °C for 4 h. After the addition of 5 mM ferulate, 5 mM vanillin, or 10 mM vanillate, the cells were further incubated for 6 h. Total RNAs were isolated as described previously (Kamimura et al., 2010) and then treated with RNase-free DNase I (Takara Bio Inc.) to remove contaminating DNA. RT-PCR and qRT-PCR analyses were carried out according to the previous reports (Kamimura et al., 2010; Kasai et al., 2010). A Beckman H 89 mouse dye D4 (D4)-labeled primer, PEferB, complementary to the ferB mRNA from 91 to 111 nucleotides downstream from the

ferB start codon, was used to INK-128 detect the start site of the ferB mRNA (Table S3). Primer extension reactions were performed as described previously (Kasai et al., 2010). The reporter plasmids carrying the ferB promoter-lacZ fusion with or without ferC were introduced into SME043 cells. The resulting transformants were grown in Wx medium containing 5 mM ferulate or grown in LB medium at 30 °C for 12 h. Preparation of cell extracts and β-galactosidase assays were performed as described previously (Kasai et al., 2010). The coding region of ferC was amplified by PCR using Ex Taq DNA polymerase (Takara Bio Inc.) together with NdeferC-F and R primer pair (Table S3). The 0.6-kb NdeI-XhoI fragment of the PCR product was inserted into pET-16b to generate pETRR1. E. coli BL21(DE3) cells harboring pETRR1 were grown in 100 mL of LB medium at 30 °C. When A600 nm of the culture reached 0.5, expression of ferC with an N-terminal His tag was induced for 6 h by adding 1 mM isopropyl-β-d-thiogalactopyranoside. After the incubation, cells were resuspended in 50 mM Tris–HCl buffer (pH 7.5) and broken by an ultrasonic disintegrator (UD-201; Tomy Seiko Co.). The supernatant obtained by centrifugation (19 000 g, 20 min) was applied to a His Spin Trap (GE Healthcare) previously equilibrated

with buffer A, consisting of 50 mM Tris–HCl (pH 7.5), 500 mM NaCl, and 100 mM imidazole. After the centrifugation at 100 g for 30 s, samples were washed twice with 500 μL of buffer Fossariinae A. His-tagged FerC (ht-FerC) was eluted with 400 μL of buffer B, consisting of 50 mM Tris–HCl (pH 7.5), 500 mM NaCl, and 500 mM imidazole, and resultant fractions were subjected to desalting and concentration by centrifugal filtration with an Amicon Ultra-0.5 10k filter unit (Millipore). The purity of the enzyme preparation was examined by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-PAGE). In vitro cross-linking of ht-FerC was performed as descried in a previous study (Kamimura et al., 2012). EMSAs for ht-FerC were performed with a DIG gel shift kit 2nd generation (Roche).