The following six-step protocol discriminated nine of the 11 species Aspergillus species of the section Flavi– five of the economically important and widespread species and four recently described species. The primer set targeting the aflT gene designed by Tominaga et al. (2006) successfully amplified 11 type strains of Aspergillus section Flavi, but none of the other species and genera were tested. Afaflt-F and Afaflt-R separated the 11 species into two groups. Species of the first group (A. flavus/A. oryzae/A. minisclerotigenes/A. parvisclerotigenus) presented the amplified Epigenetic Reader Domain inhibitor target fragment, whereas no amplification was observed for species of the second group (A.

parasiticus/A. sojae/A. nomius/A. tamarii/A. arachidicola/A. bombycis/A. pseudotamarii). Within the second group, the AflR-F and AflR-R primers amplified the target products only for A. parasiticus, A. sojae and A. arachidicola, and not for A. nomius, A. tamarii, A. bombycis and A. pseudotamarii, confirming the data of Chang et al. (1995). For the nonamplified species during the third step, the Anits-F

and Anits-R primers amplified only A. nomius, as expected. For the species group obtained in the second step (A. flavus/A. oryzae/A. minisclerotigenes/A. parvisclerotigenus), the presence of a 3.8-kb band in the A. flavus SmaI restriction pattern only and of 2.7-kb and 1-kb bands Inhibitor Library high throughput in the A. oryzae restriction pattern differentiated A. flavus from A. oryzae (Fig. 2a), as previously demonstrated by Klich & Mullaney (1987). Furthermore, the SmaI pattern of A. minisclerotigenes did not present a 3.8-kb band (Fig. 2b). Unfortunately, A. parvisclerotigenus could not be differentiated from A. flavus after the SmaI digest (Fig. 2b). This step consists in analyzing RAPD profiles of the unresolved groups A. parasiticus/A. ADP ribosylation factor sojae/A. arachidicola and A.

tamarii/A. bombycis/A. pseudotamarii. The presence of a major 2.0-kb band in the A. parasiticus amplification pattern obtained with OPB-10 allowed us to distinguish A. parasiticus from A. sojae (Fig. 3a), as demonstrated previously by Yuan et al. (1995). Furthermore, using the OPA-04 primer, a major band of 1.7 kb was observed in the pattern of A. arachidicola and not in the two other patterns (Fig. 3a). The two RAPD amplifications thus allowed the discrimination of the three species. RAPD patterns of A. bombycis obtained with OPA-04, OPB-10 and OPR-01 were clearly different from those of A. tamarii and A. pseudotamarii (Fig. 3b). The A. pseudotamarii amplification pattern obtained with OPR-01 produces a 3000-bp and a 500-bp major band, allowing its discrimination from A. tamarii. The PCR profiles (+ or −) obtained for the four primer sets are summarized in Table 1, as well as the RAPD and SmaI digestion results. Finally, a decision-making tree (Fig. 4) was set up and will serve as the molecular key tool for Aspergillus section Flavi strain identification.

During 2005–2007, a third of women delivered vaginally, half by elective CS and the remainder by emergency CS. In contrast, at the start of the HAART era, two-thirds of women delivered by elective CS. We document geographical variation in mode of delivery in the HAART era, with an increasing proportion of vaginal deliveries, mainly in the United Kingdom, Belgium and the Netherlands. In multivariable analysis of MTCT risk among MCPs with maternal HIV

RNA <400 copies/mL, elective CS was associated with an 80% decreased MTCT risk. However, among women with viral loads <50 copies/mL there were only two transmissions overall. Although clinical trials are the gold standard for clinical care, observational studies often provide initial evidence for trial inception and design. Use of elective CS find more BIBW2992 clinical trial as a PMTCT intervention is a case in point: the ECS first published results showing an association between reduced MTCT risk and elective CS in 1994 [5],

with subsequent confirmation from a large meta-analysis [9]. Our finding here that the peak elective CS rate occurred in 1999, when the mode of delivery trial was published [8], is probably largely explained by participating clinicians changing their practices before the trial results were released based on the observational evidence they helped to provide; furthermore, some women were concomitantly enrolled in both the trial and the ECS. The somewhat paradoxical finding of a declining elective CS rate in the years immediately following the trial publication may be partly explained by the concurrent implementation of antenatal HAART

instead of mono- or dual therapy for PMTCT, when the first studies suggesting the benefit of HAART for decreasing MTCT risk were published [24–27] and guidelines started to change. In the Netherlands, for instance, the national guideline in 2000 only mentioned an elective CS as a rescue therapy in case of HAART failure or refusal [28]. Other European studies have also documented declining elective CS rates in the HAART era. In an analysis from the French Niclosamide Perinatal Study involving over 5000 pregnant women receiving antenatal ART and delivering between 1997 and 2004, the elective CS rate declined from 56% in 2000 to 41% in 2004 [4]. In the United Kingdom and Ireland National Study of HIV in Pregnancy and Childhood (NSHPC), the elective CS rate peaked in 1999 at 66%, declining to around 50% in 2006. The emergency CS rate we report here was relatively stable but high and ranged from 15% to 17% in the HAART era; the French Perinatal Study also reported stable emergency CS rates between 1997 and 2004, but higher at around 29% [4].

niger N402 after 24 h of growth click here on MM or MM without Iron (iron omitted from trace elements) followed by the addition of selected compounds (Table 1). Cultures were harvested 30 min after addition of the compound, and RNA was extracted using TRIzol reagent (Invitrogen). Expression levels of hemA, hemB, hemF, hemH and met1 (Table 2) were examined, and actin was used as loading control. Recently, all potential A. niger haem and sirohaem biosynthesis genes were identified (Franken et al., 2011). Northern analysis on several haem and sirohaem genes was carried out on mRNA samples isolated from cultures grown under different conditions, in response to supplementation

with haem sources, various haem intermediates and iron as metal-ligand of haem (Fig. 1). Under standard iron conditions, only the expression of hemA was found to be responsive to the addition of iron-containing supplements. With the exception of ALA, all conditions appear to result in a small upregulation of hemA under standard iron conditions and would suggest a positive regulation by iron and possibly haem. However, the changes in expression are very limited compared to the levels obtained for solvent control conditions (MQ and DMSO).When precultured under iron-limited conditions, a modest repression of hemA, hemF and hemH was observed. However, hemA and hemH are directly iron-responsive upon (high)iron addition. buy BMS-354825 Increased expression of hemA and hemH was also observed upon the addition of

hemin and haemoglobin, whereas the final haem intermediate protoporphyrin IX did not alter the expression of any of the selected genes. ALA supplementation reduced the expression of all examined haem biosynthetic genes. This reduced expression was not observed for the sirohaem synthesis gene met1. Haemoglobin addition resulted in reduced met1 expression. The haemoglobin-induced expression

of the haem biosynthetic pathway under both standard and iron-limited conditions might not be specific as addition of another haem-free protein BSA had a similar effect. A deletion strain of hemA (An17g01480) was constructed in A. niger. 50 μM ALA was supplemented during transformation of pΔhemA to AB4.1, as the deletion was expected to be conditionally lethal. Transformants were prescreened on MM and DOCK10 MM containing 50 μM ALA. ALA-requiring mutant strains were analysed by Southern analysis. One of the strains showing to be a correct deletion strain was designated ΔhemA (results not shown). Growth of ΔhemA could be restored to wild type by supplementing 100 μM ALA in MM or 500 μM ALA in CM. Decreasing ALA concentrations led to a strong, dose-dependent growth reduction. Complementation of ΔhemA on DNA level, by inserting a functional hemA fragment restored all phenotypic defects, indicating that the observed phenotype is specific for ΔhemA (results not shown). To test whether ΔhemA is able to utilize exogenous haem sources, fresh conidia were spotted on MM or CM containing hemin as haem source (Fig.

niger N402 after 24 h of growth C646 on MM or MM without Iron (iron omitted from trace elements) followed by the addition of selected compounds (Table 1). Cultures were harvested 30 min after addition of the compound, and RNA was extracted using TRIzol reagent (Invitrogen). Expression levels of hemA, hemB, hemF, hemH and met1 (Table 2) were examined, and actin was used as loading control. Recently, all potential A. niger haem and sirohaem biosynthesis genes were identified (Franken et al., 2011). Northern analysis on several haem and sirohaem genes was carried out on mRNA samples isolated from cultures grown under different conditions, in response to supplementation

with haem sources, various haem intermediates and iron as metal-ligand of haem (Fig. 1). Under standard iron conditions, only the expression of hemA was found to be responsive to the addition of iron-containing supplements. With the exception of ALA, all conditions appear to result in a small upregulation of hemA under standard iron conditions and would suggest a positive regulation by iron and possibly haem. However, the changes in expression are very limited compared to the levels obtained for solvent control conditions (MQ and DMSO).When precultured under iron-limited conditions, a modest repression of hemA, hemF and hemH was observed. However, hemA and hemH are directly iron-responsive upon (high)iron addition. selleck screening library Increased expression of hemA and hemH was also observed upon the addition of

hemin and haemoglobin, whereas the final haem intermediate protoporphyrin IX did not alter the expression of any of the selected genes. ALA supplementation reduced the expression of all examined haem biosynthetic genes. This reduced expression was not observed for the sirohaem synthesis gene met1. Haemoglobin addition resulted in reduced met1 expression. The haemoglobin-induced expression

of the haem biosynthetic pathway under both standard and iron-limited conditions might not be specific as addition of another haem-free protein BSA had a similar effect. A deletion strain of hemA (An17g01480) was constructed in A. niger. 50 μM ALA was supplemented during transformation of pΔhemA to AB4.1, as the deletion was expected to be conditionally lethal. Transformants were prescreened on MM and cAMP MM containing 50 μM ALA. ALA-requiring mutant strains were analysed by Southern analysis. One of the strains showing to be a correct deletion strain was designated ΔhemA (results not shown). Growth of ΔhemA could be restored to wild type by supplementing 100 μM ALA in MM or 500 μM ALA in CM. Decreasing ALA concentrations led to a strong, dose-dependent growth reduction. Complementation of ΔhemA on DNA level, by inserting a functional hemA fragment restored all phenotypic defects, indicating that the observed phenotype is specific for ΔhemA (results not shown). To test whether ΔhemA is able to utilize exogenous haem sources, fresh conidia were spotted on MM or CM containing hemin as haem source (Fig.

Once the records had been reviewed, all unique identifiers associated with outbreaks and case reports were removed, including names, dates CT99021 cell line of birth, gender, countries of origin, job titles and duties on the vessel, vessel names, and cruise line names. Analysis was limited to varicella reports among crew members on cruise ships. Reports from cargo ships were not included since they do not carry trained medical personnel and follow different CDC recommendations

than those given to cruise ships. Passenger varicella cases and contacts were not included, since secondary cases associated with the index case would not be readily identified and only contact management information up to the time of disembarkation would be available. Categorical variables were described using frequencies and percentages, and continuous variables were described using ranges, means, and medians. This investigation was approved as non-research by the CDC Institutional Review Board. During 2005 to 2009, varicella reports comprised 357 (15%) of the total 2,305 maritime illness reports submitted to CDC during that period. Of the varicella reports, 278 (78%) were among cruise ship crew members. They were CP-690550 purchase predominantly male (80%), their median age was 29 years (range 20–66), and three-quarters

Thiamine-diphosphate kinase of the ill crew members were residents of Caribbean countries, Indonesia, the Philippines, or India. Excluding 2005, a partial reporting year, varicella was reported more commonly in the spring (28%) and winter (27%) months. During 2009, 94 cases of varicella among cruise ship crew members were reported to CDC Quarantine Stations. By manual review of each case report, 22 varicella clusters were identified. Four of the clusters were excluded because the cases were not considered epidemiologically linked. Therefore, after exclusion, 66/94 (70%) cases among crew members were associated with 18 outbreaks. The remaining 28 cases were considered isolated case reports.

Outbreak response by cruise ships reporting the 18 varicella outbreaks during 2009 included isolation of 66 (100%) of 66 cases, restriction of 66 (26%) of 255 close crew-contacts, and administration of post-exposure vaccine to 522 close contacts and other susceptible crew members (Table 2). No contacts received VZIG. The number of cases per outbreak ranged from 2 to 9. There were a total of 45 first-generation cases (range 1–6 per outbreak), 16 second-generation cases (range 0–4), and five additional-generation cases (range 0–2) (Figure 1). There was a slight but nonsignificant positive correlation between time to reporting and number of second- and additional-generation cases.

However, major limitations in the techniques used for the acquisition and analysis of functional magnetic resonance imaging (fMRI) data have hitherto precluded segregation of function with the amygdala in humans. Here, we used high-resolution fMRI in combination with a region-of-interest-based normalization method to differentiate functionally the contributions of distinct subregions within the human amygdala during two different types of instrumental conditioning: reward and avoidance learning. Through the application of a computational-model-based analysis, we found evidence for a dissociation

between the contributions of the basolateral and centromedial complexes in the representation of specific computational signals during click here learning, with the basolateral complex contributing more to reward learning, and the centromedial complex more to avoidance learning. These results provide unique insights into the computations being implemented within fine-grained amygdala circuits in the human brain. “
“Cerebellar function is regulated by cholinergic mossy fiber inputs that are primarily derived from the medial vestibular nucleus (MVN) and prepositus hypoglossi this website nucleus (PHN). In contrast to the growing

evidence surrounding cholinergic transmission and its functional significance in the cerebellum, the intrinsic and synaptic properties of cholinergic projection neurons (ChPNs) have not been clarified. In this study, we generated choline acetyltransferase (ChAT)-tdTomato transgenic rats, which specifically express the fluorescent protein tdTomato in cholinergic neurons, and used them to investigate the response properties of ChPNs identified via almost retrograde labeling using whole-cell recordings in brainstem slices. In response to current pulses, ChPNs exhibited two afterhyperpolarisation (AHP) profiles and three firing patterns; the predominant AHP and firing properties differed between the MVN and PHN. Morphologically, the ChPNs were separated into two types based on their soma size and dendritic extensions. Analyses of the firing responses to time-varying sinusoidal

current stimuli revealed that ChPNs exhibited different firing modes depending on the input frequencies. The maximum frequencies in which each firing mode was observed were different between the neurons that exhibited distinct firing patterns. Analyses of the current responses to the application of neurotransmitter receptor agonists revealed that the ChPNs expressed (i) AMPA- and NMDA-type glutamate receptors, (ii) GABAA and glycine receptors, and (iii) muscarinic and nicotinic acetylcholine receptors. The current responses mediated by these receptors of MVN ChPNs were not different from those of PHN ChPNs. These findings suggest that ChPNs receive various synaptic inputs and encode those inputs appropriately across different frequencies.

He felt well and had no subjective fever. Physical examination revealed no other

petechial lesions in the conjunctivae or skin. There was no new heart murmur. Neurological screening examination was normal. No treatment was given. Occasional new splinter hemorrhages continued to appear in the ensuing 90 days. The patient was one of a group of eight adults (aged 42–81 y) who traveled together. All were in generally excellent health, and all took acetazolamide 500 mg twice daily beginning 2–3 days before arrival. For 1–2 days they toured in and around Cuzco, either walking without backpacks or taking vans. They then took a leisurely 3-hour train ride to Machu Picchu where they hiked the ruins, either with no backpack or with a light pack (weight <10 pounds). PD98059 order Examination of the other seven subjects 1–3 days after descent from altitude revealed that four had splinter hemorrhages. Thus, in total, five of eight persons who hiked ruins at Maccu Picchu had splinter hemorrhages (range 1–8 hemorrhages per hiker, median 1). Of the five who had splinter hemorrhages, three were taking 60 mg aspirin daily or three times weekly compared to one of the three who did not have hemorrhages. Only one of the subjects had symptoms

(headaches) that she attributed to altitude sickness. Selleckchem LDK378 Rennie,[5] a physician and mountain climber, described an association between ascent to altitude and splinter hemorrhages. While hiking in the Himalayas, he noted that hemorrhages appeared in his nail beds at 19,300 feet, after he carried a 60-pound backpack through the snow for 4 h. In his expedition, 7 of 15 fellow climbers had 1–19 subungual hemorrhages. Several of his proposed causes—trauma, extreme exertion, cold exposure, and/or impeded venous return by rucksack straps—have

been generally accepted,[4, 6, 7] but they clearly do not apply to the situation described herein. Decreased barometric pressure and hypoxemia appear to be the likely common features contributing to the appearance of these hemorrhages. Although Rennie dismissed capillary fragility as a possible explanation, Hunter et al.[8] used petechiometry to show that capillary fragility increases in proportion to altitude. Since these investigators Methane monooxygenase did not provide supplementary oxygen to any of their subjects, their method could not distinguish between low barometric pressure and low oxygen content of air. Low barometric pressure is likely, however, to be the principal cause, since the examination of hypoxemic patients in medical intensive care units does not regularly reveal splinter hemorrhages. Interestingly, retinal hemorrhages (Roth spots) have also been documented in mountain climbers at very high altitudes,[9] supporting the hypothesized role for capillary fragility. The present report describes the appearance of splinter hemorrhages in five of eight healthy adults who spent 2–3 leisurely days touring at an altitude of 8,000–11,000 feet.

In addition, utilization of 4-ABS as sole nitrogen source was examined by growing mutants in PB medium with 3 mM of 4-ABS and gluconate. After 5 days of incubation with shaking at 150 r.p.m., growth was quantified by measuring A600 nm. Cells were grown in PBN medium supplemented with 5 mM of gluconate and 4-ABS. Samples were withdrawn every 48 h, filter sterilized and stored at

−20 °C selleck for subsequent analysis. For thin layer chromatography (TLC) analysis, 7.5 μL of sample was spotted onto a C18 RP TLC plate (Merck). The plate was allowed to dry and developed in mobile phase of butanol–propanol–acetic acid–water at 8 : 4 : 1 : 1 (Feigel & Knackmuss, 1988). HPLC analysis was performed using Waters 600 equipped with a 4.6 × 250 mm Zorbax SB-Aq column (Agilent, Santa Clara, CA). The mobile phase consisted of 98% water, 1% methanol and 1% phosphoric acid (85%) at a flow rate of 1.0 mL min−1. Detection was carried out at 230 nm. 4-Sulfocatechol standard was synthesized according to published method (Saito & Kawabata, 2006). Chromogenic detection of diphenolic intermediate in catabolism of 4-ABS was done by growing cells on nutrient agar

supplemented with 50 μg mL−1p-toluidine and 0.5 mM FeCl3 (Parke, 1992). To complement RK40, the DNA region spanning phthalate dioxygenase-like gene and its putative promoter was amplified from wild-type PBC with Benzatropine primers PDOF 5′-TACTTGCCGGTCTCGTTCG-3′ and PDOR 5′-GTTCGGGGGTGTGCAGTC-3′, cloned into pGEM-T Easy vector (Promega) and Autophagy inhibitor order subcloned as an EcoRI fragment into pBBR1MCS-5 (Kovach et al., 1995) to give pHG5. A similar approach was applied to RK32 complementation using primers DEHF 5′-GTTGAGACGCTCGTTGACC-3′ and DEHR 5′-TTTGCCTGAGAAATGTGTCG-3′ to amplify the ORFs of transposase and putative dehydrogenase to give pHG6. Plasmids were transformed into mutants via electroporation. Oxygen uptake was measured using a Clark-type oxygen electrode (YSI 5905, Yellow Springs Instruments). Cells

were pregrown in 20 mL NB medium, harvested by centrifugation and grown in 50 mL 0.5 × NB medium with 5 mM 4-ABS for 36 h to induce 4-aminobenzenesulfonate 3,4-dioxygenase activity. Cells were then harvested, washed twice with 25 mM potassium phosphate buffer, pH 7.0, and resuspended in the same buffer containing 1 mM 4-ABS (OD600 nm of 0.15–0.2). Oxygen uptake was measured polarographically at 30 °C for 2 h. DNA sequences of insertion site in RK1, RK23, RK32 and RK40 were deposited in EMBL Nucleotide Sequence Database and assigned accession numbers FR720595, FR720597, FR720598 and FR720599, respectively. From three different electroporation experiments, approximately 10 000 kanamycin-resistant colonies were obtained, representing an average transformation efficiency of 1.7 × 105 CFU μg−1 transposon.

In addition, utilization of 4-ABS as sole nitrogen source was examined by growing mutants in PB medium with 3 mM of 4-ABS and gluconate. After 5 days of incubation with shaking at 150 r.p.m., growth was quantified by measuring A600 nm. Cells were grown in PBN medium supplemented with 5 mM of gluconate and 4-ABS. Samples were withdrawn every 48 h, filter sterilized and stored at

−20 °C Navitoclax research buy for subsequent analysis. For thin layer chromatography (TLC) analysis, 7.5 μL of sample was spotted onto a C18 RP TLC plate (Merck). The plate was allowed to dry and developed in mobile phase of butanol–propanol–acetic acid–water at 8 : 4 : 1 : 1 (Feigel & Knackmuss, 1988). HPLC analysis was performed using Waters 600 equipped with a 4.6 × 250 mm Zorbax SB-Aq column (Agilent, Santa Clara, CA). The mobile phase consisted of 98% water, 1% methanol and 1% phosphoric acid (85%) at a flow rate of 1.0 mL min−1. Detection was carried out at 230 nm. 4-Sulfocatechol standard was synthesized according to published method (Saito & Kawabata, 2006). Chromogenic detection of diphenolic intermediate in catabolism of 4-ABS was done by growing cells on nutrient agar

supplemented with 50 μg mL−1p-toluidine and 0.5 mM FeCl3 (Parke, 1992). To complement RK40, the DNA region spanning phthalate dioxygenase-like gene and its putative promoter was amplified from wild-type PBC with Guanylate cyclase 2C primers PDOF 5′-TACTTGCCGGTCTCGTTCG-3′ and PDOR 5′-GTTCGGGGGTGTGCAGTC-3′, cloned into pGEM-T Easy vector (Promega) and TGF-beta inhibitor subcloned as an EcoRI fragment into pBBR1MCS-5 (Kovach et al., 1995) to give pHG5. A similar approach was applied to RK32 complementation using primers DEHF 5′-GTTGAGACGCTCGTTGACC-3′ and DEHR 5′-TTTGCCTGAGAAATGTGTCG-3′ to amplify the ORFs of transposase and putative dehydrogenase to give pHG6. Plasmids were transformed into mutants via electroporation. Oxygen uptake was measured using a Clark-type oxygen electrode (YSI 5905, Yellow Springs Instruments). Cells

were pregrown in 20 mL NB medium, harvested by centrifugation and grown in 50 mL 0.5 × NB medium with 5 mM 4-ABS for 36 h to induce 4-aminobenzenesulfonate 3,4-dioxygenase activity. Cells were then harvested, washed twice with 25 mM potassium phosphate buffer, pH 7.0, and resuspended in the same buffer containing 1 mM 4-ABS (OD600 nm of 0.15–0.2). Oxygen uptake was measured polarographically at 30 °C for 2 h. DNA sequences of insertion site in RK1, RK23, RK32 and RK40 were deposited in EMBL Nucleotide Sequence Database and assigned accession numbers FR720595, FR720597, FR720598 and FR720599, respectively. From three different electroporation experiments, approximately 10 000 kanamycin-resistant colonies were obtained, representing an average transformation efficiency of 1.7 × 105 CFU μg−1 transposon.

, 2011). Our results

show that: (1) ApSHMT catalyzes THF-dependent and THF-independent reactions; (2) ApSHMT is a salt-inducible gene; and (3) overexpression of the ApSHMT gene increases the levels of not only glycine and serine, but also choline and glycine betaine, and conferred tolerance to salinity stress. Escherichia coli strains DH5α, BL21, BL21 (DE3), and BL21 (DE3) pLys were grown in the Luria–Bertani (LB) medium or in M9 minimal medium. Chloramphenicol, kanamycin, and ampicillin antibiotics were used at a concentration of 25, 25, and 50 mg L−1, screening assay respectively, as required. A. halophytica cells were grown photoautotrophically (70 μE m−2 s−1) in BG11 liquid medium containing 18 mM NaNO3 and Turk Island salt solution at 30 °C, as previously described (Waditee et al., 2003). The growth of E. coli and cyanobacterial cells was monitored by measuring absorbance at 620 and 730 nm, respectively, with a Shimadzu UV-160A spectrophotometer. The ApSHMT coding sequence was Natural Product Library datasheet amplified from the genomic DNA of A. halophytica using the primer pair

ApSHMT-Nde 5′-CAACATATGGTGACGCAAACAAAC-3′ and ApSHMT-BamHI: 5′-AGGGATCCTTAT GCCATTGCGGG-3′, and thereby incorporating the 5′-NdeI and 3′-BamHI restriction sites. The PCR product was cloned into pCR2.1 vector and sequenced to exclude PCR errors. The full-length ApSHMT fragment was prepared by double digestion with NdeI and BamHI and ligated into the corresponding sites of pCold I vector (Takara, Tokyo, Japan). The expression construct was transformed first into E. coli strain DH5α and then strains BL21, BL21 (DE3), and

BL21 (DE3) pLys. Expression of recombinant ApSHMT was induced with 0.1 mM isopropyl β-d-thiogalactopyranoside at 16 °C. After 16 h, cells were harvested by centrifugation at 2300 g for 15 min. The bacterial pellets were resuspended Casein kinase 1 in buffer A (100 mM Tris-Cl, pH 8.0), sonicated, and centrifuged. Clear supernatant thus obtained was loaded onto the HisTrap FF column (GE Healthcare, Little Chalfont, UK). The column was washed extensively with buffer A containing 20 mM imidazole, followed by elution of the recombinant ApSHMT protein with buffer A containing 250 mM imidazole. Purified recombinant ApSHMT was used for biochemical characterization. For assay of THF-independent cleavage, the standard reaction mixture contained 10 μmol of dl-threo-3-phenylserine, 10 nmol of pyridoxal 5-phosphate (PLP), 100 μmol of Tris–HCl buffer (pH 8.5–9.0), and enzyme in a final volume of 0.5 mL. Incubation was performed at 30 °C for 10 min. The reaction was stopped by adding 0.5 mL of 1 M HCl. The benzaldehyde formed was determined by the 2,4-dinitrophenylhydrazine method (Misono et al., 2005). One unit of the enzyme was defined as the amount that catalyzed the formation of 1 μmol of benzaldehyde per minute in the reaction. Specific activity was expressed as units per milligram of protein. The THF-dependent cleavage was measured according to Simic et al.