733, P = 0475 The response to less probable deviant repetitions

733, P = 0.475. The response to less probable deviant repetitions (mean = −1.548 μV, SE = 0.333 μV) was similar to the first deviant tone response (mean = −1.885 μV, SE = 0.363 μV). Within anisochronous sequences, the repetition × repetition

probability interaction was not significant: F1,14 = 0.487, P = 0.497. The response to highly probable deviant repetitions (mean = −1.418 μV, SE = 0.430 μV) was similar to the first deviant tone response (mean = −1.896 μV, SE = 0.344 μV). Likewise, the response to less probable deviant repetitions (mean = −1.593 μV, SE = 0.250 μV) was similar to the first deviant tone response (mean = −2.294 μV, SE = 0.348 μV). The pattern of significant findings suggests that temporal information is required for the computation of higher-order predictions in audition based on deviant repetition probability (see Fig. 2). The four-way interaction of repetition, Epigenetic inhibitor repetition probability, laterality and side was not significant within either temporal regularity level (see the main experiment section of Table 2). However, within isochronous sequences a significant repetition × repetition probability × laterality

interaction was found: F1,14 = 4.605, P = 0.05, partial η2 = 0.248. Follow-up tests were conducted separately for central and lateral electrode positions. A significant repetition × repetition probability interaction emerged for centrally located electrodes: F1,14 = 5.071, P = 0.041, partial η2 = 0.266. A significant hypoxia-inducible factor cancer difference between first deviant tones and highly Sucrase probable deviant repetitions was shown using t-tests: t14 = −2.692, P = 0.018. Here too, the response to highly probable deviant repetitions (mean = −0.912 μV, SE = 0.362 μV) was largely attenuated compared with the first deviant tone response (mean = −1.878 μV, SE = 0.504 μV). And again, no difference was found between first deviant tones and less probable deviant repetitions: t14 = −0.893, P = 0.387. As for lateral electrodes, the repetition × repetition probability interaction was not significant: F1,14 = 2.274, P = 0.154. The error response attenuation

effect reflecting higher-order predictions is thus localized at frontocentral electrode locations, irrespective of side. Additionally, the omnibus anova yielded a significant repetition probability × side interaction: F1,14 = 4.614, P = 0.05, partial η2 = 0.248. However, follow-up t-tests failed to reach statistical significance (all P ≥ 0.12). Within anisochronous sequences, we further observed a significant repetition × laterality × side interaction: F1,14 = 6.355, P < 0.024, partial η2 = 0.312. Follow-up tests were conducted separately for central and lateral electrode positions. A main effect of repetition was found at central electrode locations: F1,14 = 4.620, P < 0.050, partial η2 = 0.248. First deviant tones (mean = −1.847 μV, SE = 0.274 μV) yielded a larger response than deviant tone repetitions (mean = −1.

In each case, 5-HT1A

receptors have been implicated in th

In each case, 5-HT1A

receptors have been implicated in the response. To determine whether there are different subgroups of 5-HT cells activated during nicotine administration and withdrawal, we mapped the appearance of Fos, a marker of neuronal activation, in 5-HT cells of the dorsal raphe nucleus (DR) and median raphe nucleus (MR). selleck products To understand the role of 5-HT1A receptor feedback inhibitory pathways in 5-HT cell activity during these conditions, we administered a selective 5-HT1A receptor antagonist and measured novel disinhibited Fos expression within 5-HT cells. Using these approaches, we found evidence that acute nicotine exposure activates 5-HT neurons rostrally and in the lateral wings of the DR, whereas there is 5-HT1A receptor-dependent inhibition of cells located ventrally at both the rostral level and mid-level. Previous chronic nicotine exposure did not modify the pattern of activation produced by acute nicotine exposure, but increased 5-HT1A receptor-dependent inhibition of 5-HT cells in the caudal DR. This pattern was nearly reversed during nicotine withdrawal, when there was evidence for caudal activation selleck screening library and mid-level and rostral 5-HT1A receptor-dependent inhibition. These results suggest that the distinct behavioral states produced by nicotine exposure and withdrawal correlate with reciprocal rostral–caudal patterns of activation and 5-HT1A receptor-mediated

inhibition of DR 5-HT neurons. The complementary patterns of activation and inhibition suggest that 5-HT1A receptors may help to shape distinct topographic patterns of activation within the

DR. “
“The dorsolateral prefrontal and the posterior parietal cortex have both been implicated in the guidance of visual attention. Traditionally, posterior parietal cortex has been thought to guide visual bottom-up attention and prefrontal cortex to bias attention through top-down information. More recent Erastin in vivo studies suggest a parallel time course of activation of the two areas in bottom-up attention tasks, suggesting a common involvement, though these results do not necessarily imply identical roles. To address the specific roles of the two areas, we examined the influence of neuronal activity recorded from the prefrontal and parietal cortex of monkeys as they performed attention tasks based on choice probability and on correlation between reaction time and neuronal activity. The results revealed that posterior parietal but not dorsolateral prefrontal activity correlated with behavioral choice during the fixation period, prior to the appearance of the stimulus, resembling a bias factor. This preferential influence of posterior parietal activity on behavior was transient, so that dorsolateral prefrontal activity predicted choice after the appearance of the stimulus. Additionally, reaction time was better predicted by posterior parietal activity.

Reelin Western blots were performed as described by Krstic et al

Reelin Western blots were performed as described by Krstic et al. (2012b). RNA was extracted using a GenElute Mammalian Total RNA Miniprep Selleck ALK inhibitor Kit (Sigma, St Louis, MO, USA) according to the manufacturer’s instructions. Total RNA was quantified by absorbance spectroscopy and RNA integrity and quality was assessed by 1.0%

agarose gel electrophoresis. Total RNA (1 μg) was transcribed to cDNA with SuperScript II (Invitrogen, Carlsbad, CA, USA) using random hexamer primers according to the manufacturer’s instructions. For quantitative real-time PCR (qPCR), 20 ng of cDNA was used, and single transcript levels of genes were detected with the HOT FIREPol EvaGreen qPCR Mix (Solis BioDyne, Tartu, Estonia) and an AB7900 thermocycler. Primers used for detection of synaptic

transcripts were as follows: β-actin, AGTGTGACGTTG ACATCCGTA (sense), GCCAGAGCAGTAATCTCCTTCT (antisense); Gephn, GGCGACCGAGGGAATGAT (sense), CCACCCAACAAAGAAGGATCTT (antisense); Gabra1, GGTTGACCGTGAGAGCTGAA (sense), CTACAACCACTGAACGGGCT Selleck MLN0128 (antisense); Gabra2, CAGTGGCCCATAACATGACAAT (sense), GGACATTCGGCTTGGACTGT (antisense); CamKIIa, CCCCTTTCGCCTACATGTGA (sense), GGCTACAGTGGAGCGGCTTA (antisense). Data were analysed using the comparative CT method (Schmittgen & Livak, 2008). Images from immunoperoxidase staining were acquired with a color digital camera using either bright- or dark-field illumination (Zeiss Axioskop microscope, Jena, Germany) and assembled with Photoshop. A sharpening filter was applied to all images. Immunofluorescence images were captured by laser scanning confocal microscopy, using a 40 × lens, NA 1.4, 1024 × 1024 pixels (Zeiss LSM Nabilone 700). Final illustrations were prepared from the maximal intensity projection of stacks of images spaced at 0.5 μm. Images were

background-subtracted and filtered with a Gaussian filter, but no change in brightness and contrast was applied. In this protocol, ACSF-perfused living tissue is fixed by immersion in aldehyde solution (4% paraformaldehyde dissolved in sodium phosphate buffer). We systematically tested the duration of fixation to determine the time required for entire blocks (hemi-brain cut sagitally along the midline or coronal block containing either the entire hippocampal formation or the entire cerebellum) to be fixed while preserving optimal antigenicity of proteins of interest. For such tissue blocks (up to 2–3 cm3), we saw no difference in staining quality/intensity at different tissue depths, suggesting that a homogeneous fixation was achieved even after a short fixation (60–90 min). However, fixation of entire brains was not appropriate, possibly because fixative did not penetrate through the ventricular system. No tissue block was fixed longer than 6 h, prior to washing and cryoprotection.

In the past, it has also been used with several haloarchaeal spec

In the past, it has also been used with several haloarchaeal species including H. volcanii and it was shown that it also induces oxidative stress at the high salt concentrations of the haloarchaeal cytoplasm (May & Dennis, 1989; May et al., 1989; Joshi & Dennis, 1993). Paraquat was added in concentrations from 1 μM to 4 mM to exponentially growing cultures of H. volcanii (Fig. 4). All cultures continued to grow for several hours and then exhibited a difference from the nonstressed

control. The growth curves after the addition of paraquat in concentrations from 1 to 100 μM were identical, the cultures entered the transition phase earlier than the wild type, but the final growth yields were the same as that of the wild type. The selleck inhibitor growth yields of cultures after the addition of 1 mM or higher concentrations of paraquat were lower than that of the wild type. The effect was relatively mild after the addition of 1 and 2 mM paraquat, in contrast to the addition of 4 mM, which led to a considerable reduction in growth yield (<50% after paraquat addition compared with the nonstressed control). The next application was the optimization of the supplementation of amino acid auxotrophic mutants. The tryptophan auxotrophic mutant H53 with

a deletion of the trpA gene was compared with the tryptophan prototrophic strain selleck H26 (Fig. 5a). Growth of the prototrophic strain is independent of the addition of tryptophan and the growth curves in the absence of tryptophan and in the presence of three different concentrations were absolutely identical. In contrast, mutant H53 was totally unable to grow without tryptophan addition. Growth after the addition of 2 and 10 μg mL−1 was strictly tryptophan limited, while the growth yield after the addition of 50 μg mL−1 was the same as that of the prototrophic strain and thus growth could fully be supplemented. Unexpectedly, the auxotrophic

mutant H53 grew faster than the prototrophic strain H26 after the addition of 10 and 50 μg mL−1 tryptophan. As already mentioned, the growth rate of the prototrophic strain was not influenced by tryptophan addition. PIK3C2G It seems that H. volcanii does not benefit from external tryptophan as long as the biosynthesis gene trpA is intact, in contrast to the expectation that saving of the energetically very costly tryptophan biosynthesis would be beneficial. Another unexpected result was obtained as the leucine auxotrophic mutant H66 with a deletion of the leuB gene was supplemented with leucine (Fig. 5b). Again, the growth curves of the prototrophic control strain H26 were independent of the addition of leucine. As expected, the auxotrophic mutant H66 was unable to grow in the absence of leucine.

In the past, it has also been used with several haloarchaeal spec

In the past, it has also been used with several haloarchaeal species including H. volcanii and it was shown that it also induces oxidative stress at the high salt concentrations of the haloarchaeal cytoplasm (May & Dennis, 1989; May et al., 1989; Joshi & Dennis, 1993). Paraquat was added in concentrations from 1 μM to 4 mM to exponentially growing cultures of H. volcanii (Fig. 4). All cultures continued to grow for several hours and then exhibited a difference from the nonstressed

control. The growth curves after the addition of paraquat in concentrations from 1 to 100 μM were identical, the cultures entered the transition phase earlier than the wild type, but the final growth yields were the same as that of the wild type. The Torin 1 growth yields of cultures after the addition of 1 mM or higher concentrations of paraquat were lower than that of the wild type. The effect was relatively mild after the addition of 1 and 2 mM paraquat, in contrast to the addition of 4 mM, which led to a considerable reduction in growth yield (<50% after paraquat addition compared with the nonstressed control). The next application was the optimization of the supplementation of amino acid auxotrophic mutants. The tryptophan auxotrophic mutant H53 with

a deletion of the trpA gene was compared with the tryptophan prototrophic strain selleck inhibitor H26 (Fig. 5a). Growth of the prototrophic strain is independent of the addition of tryptophan and the growth curves in the absence of tryptophan and in the presence of three different concentrations were absolutely identical. In contrast, mutant H53 was totally unable to grow without tryptophan addition. Growth after the addition of 2 and 10 μg mL−1 was strictly tryptophan limited, while the growth yield after the addition of 50 μg mL−1 was the same as that of the prototrophic strain and thus growth could fully be supplemented. Unexpectedly, the auxotrophic

mutant H53 grew faster than the prototrophic strain H26 after the addition of 10 and 50 μg mL−1 tryptophan. As already mentioned, the growth rate of the prototrophic strain was not influenced by tryptophan addition. Prostatic acid phosphatase It seems that H. volcanii does not benefit from external tryptophan as long as the biosynthesis gene trpA is intact, in contrast to the expectation that saving of the energetically very costly tryptophan biosynthesis would be beneficial. Another unexpected result was obtained as the leucine auxotrophic mutant H66 with a deletion of the leuB gene was supplemented with leucine (Fig. 5b). Again, the growth curves of the prototrophic control strain H26 were independent of the addition of leucine. As expected, the auxotrophic mutant H66 was unable to grow in the absence of leucine.

8% and 587 ± 49% of ExPEC strain PCN033 were killed with hyperi

8% and 58.7 ± 4.9% of ExPEC strain PCN033 were killed with hyperimmune mouse sera against OmpC and OmpF, respectively. The results indicated that sera from both OmpC- and OmpF-immunized mice could mediate a significantly higher level of opsonophagocytic killing of ExPEC than sera from mice that received adjuvant

alone. The evidence for recombination signals was found in the E. coli ompC alignment by two programs, SBP and GARD, used for testing recombination. Staurosporine cell line Three potential recombination breakpoints with significant phylogenetic incongruence were identified at the nucleotide positions 492, 744 and 981 in the alignment of ompC. Four non-recombinant alignments together with the relevant trees of topological congruity were generated for the subsequent selection analysis. Based on the FEL inference, the selection profile of the ompC coding region is illustrated in Fig. 5a. The porin showed significant evidence for positive selection, with seven codon sites (47, 189, 223, 237, 322, 324 and 325) under positively selected force. Structural mapping of these sites is shown in Fig. 5b. According to the predicted topology of OmpC by pred-tmbb, all positively selected sites were located in the extracellular space and outer membrane surface. In addition, 48 negatively selected sites were detected at the 0.1 significance level. For ompF, two recombination

breakpoints were identified at the nucleotide positions 234 and 809 in the gene alignment. Notably, none of the positively selected sites was detected in the ompF gene through FEL inference. The E. coli genes ompC and ompF each encode an outer AZD0530 ic50 membrane protein. OmpC has a narrower pore and is preferentially expressed under higher osmolar pressure compared with OmpF (Nikaido, 2003). OmpC and OmpF have both been functionally confirmed to be beta barrel porins (Basle et al., 2006), which are important HAS1 for

dynamic interactions with the host immune system (Massari et al., 2003). Additionally, OmpC and OmpF are involved in antibiotic resistance and bacterial virulence (Negm & Pistole, 1999; De et al., 2001; Kumar et al., 2010). In this study, the immunogenic properties of porcine ExPEC OmpC and OmpF were investigated using a mouse model. Both porins OmpC and OmpF of ExPEC can provide high protection against lethal infection with the highly virulent strain PCN033. In addition, OmpC and OmpF both could induce high titers of IgG antibodies, indicating that these two proteins have good immunogenic properties. The type of immune responses was reflected by the two IgG subclasses produced through immunization, IgG1 and IgG2a. In mice, serum IgG1 is associated with a Th2-type response, whereas serum IgG2a is associated with a Th1-type response, which is particularly effective at mediating bacterial opsonophagocytosis (Unkeless et al., 1988). Our study showed that OmpC and OmpF elicited high titers of IgG2a, although less than IgG1, which indicated that OmpC and OmpF could induce significant Th1/Th2 immune responses.

2b) On the other hand, although the motA and motB mutants produc

2b). On the other hand, although the motA and motB mutants produced flagella, they were still unable to move because MotA and MotB formed a proton channel that transferred proton-motive force to drive the flagella (Asai et al., 2003); either motA or motB gene mutations resulted in the production of nonfunctional flagella (Figs 2b and 3c). These data demonstrate that the swarming of C. freundii is dependent on functional flagella, as in other swarming bacteria (Kearns, 2010). The largest gene cluster identified in our study is involved in the synthesis of lipopolysaccharide. Altogether, 13 mutants were isolated,

of which six mutated genes –wzx, rfaL, rfbX, rfaJ/CKO_05084, rfaJ/CKO_05086, and rfaG– were identified. The swarming ability of these mutants was dramatically decreased (two of them are shown in Fig. 3g and h as examples). As observed directly buy PLX4032 under inverted microscope, only a few bacterial cells were actively motile in the swarming colonies of these mutants and these were mainly distributed at the edges. In the central region, most cells formed aggregates that scarcely moved (Videos S2 and S3). In contrast, selleck chemical in wild-type colonies, all swarming cells were actively motile (cells in the edge of colonies were less active) and no aggregation was observed (Video S1). The hydrophilicity

of these mutants was decreased compared with the wild type (Fig. S2), which could have led to the aggregation. In a previous study, many transposon swarming mutants isolated in Salmonella enterica serovar Typhimurium have been shown to have mutations in the lipopolysaccharide biosynthetic pathway (Toguchi et al., 2000). The authors suggested that

the O antigen directly or indirectly improved the surface wettability required for swarm colony expansion. Our observation showed that the polysaccharide structure on the cell surface had important role not only in overcoming Parvulin friction between bacterial cells and media surface, but also in reducing intercellular interaction. The poorly motile aggregates formed with bacteria on the agar surface because of the O antigen defects could account for the defective swarming in addition to the decreased wettability of the agar surface. rcsC and rcsD mutants were identified in this study, and both mutants displayed defective swarming behavior (Fig. 3a and b). The products of rcsC and rcsD, together with RcsB, constitute the regulator of the capsule synthesis (Rcs) phosphorelay system. The regulator RcsB is activated by the transfer of a phosphate group from its cognate sensor, RcsC, through a histidine-containing phosphotransmitter (Hpt) domain intermediate called RcsD (previously called YojN; Takeda et al., 2001). The Rcs system has been implicated in the regulation of bacterial responses to osmotic and other kinds of membrane stress, growth at low temperatures in the presence of glucose and zinc, and growth on solid surfaces (Carballes et al.

The Writing Group drew up a list of questions reflecting day-to-d

The Writing Group drew up a list of questions reflecting day-to-day practice and queries. It was acknowledged that the level of evidence for many of these topics was poor but recognized that there was a need to provide guidance. These guidelines have expanded on all areas relevant to the clinical care of HIV-positive pregnant women. The guidelines are intended to inform and aid healthcare workers in the management of pregnant women with HIV. They are not intended to be prescriptive or restrictive and it is recognized that situations will arise where the optimum management may deviate from these recommendations and new data will emerge to better inform practice. A particular focus has been

obstetric management. An increasing number of women are aiming for and achieving a vaginal delivery but the rate of emergency Caesarean sections has increased. It is hoped that the

recommendations contained within ABT-888 in vitro these guidelines will enable a further increase in the proportion of vaginal deliveries and a reduction in the number of emergency Caesarean sections. Linked to this is the proposed starting gestation for women temporarily taking combination antiretroviral therapy (cART) in pregnancy, which has been brought forward depending PI3K activation on baseline viral load. It is anticipated that this will result in a larger proportion of women achieving a viral load of < 50 HIV RNA copies/mL by 36 weeks' gestation, thereby allowing them to plan for a vaginal delivery. Additional

guidance has been provided with regard to conception on cART, the choice of specific drugs or drug classes and the management of women with hepatitis B virus or hepatitis C virus co-infection. For the first time these guidelines have addressed the issue of continuation of cART post delivery in women with a baseline CD4 cell count of more than 350 cells/μL. The paediatric section provides further guidance on infant post-exposure prophylaxis (PEP), drug dosing and safety. It is clear that there exists an urgent need for paediatric syrup preparations for a wider variety of antiretroviral drugs because the current options, particularly in the case of maternal viral resistance, are limited. In key areas, the National Florfenicol Study of HIV in Pregnancy and Childhood (NSHPC) informs the management of HIV in pregnancy through comprehensive data collection, collation and analysis, and the need to interrogate the data continues as practice changes. Prevalence of HIV infection amongst women giving birth in the UK was monitored through an unlinked anonymous survey based on residual neonatal dried blood spots until 2013, when it was discontinued. This survey was in place in London from 1988, other selected English regions from 1990, and Scotland between 1990 and 2008. It provided an estimate of overall HIV prevalence in women giving birth regardless of whether or not they had been diagnosed.

While it seems clear that drug-resistant microorganisms often hav

While it seems clear that drug-resistant microorganisms often have point mutation(s) in drug-target molecule genes (Walsh, 2000), no reports have yet described how and why such mutations occur in clinically important drug-resistant bacteria. We have reported that oxygen can Cyclopamine mw induce DNA damage, causing mutations in rpoB and Rif resistance, in a strict anaerobe (Takumi et al., 2008). In the environment, there are numerous mutagens capable of damaging DNA and inducing mutation. Clinical drugs, such as some used in cancer therapy, may also be mutagenic (Kunz & Mis, 1989). Cigarette smoke also

contains many mutagenic chemicals (Fujita & Kamataki, 2001; Yim & Hee, 2001). Environmental microorganisms, especially indigenous microorganisms, may frequently be exposed to mutagens. Pseudomonas aeruginosa is an indigenous bacterium and emerging drug resistance in this bacterium is a growing concern (Jalal & Wretlind, check details 1998; Mouneimne et al., 1999; Akasaka et al., 2001; Wydmuch et al., 2005). In this study, we exposed P. aeruginosa to mutagens that are known to induce point mutation. The environmental concentrations of mutagens are similar or even higher than those we have used in the present experiments. The concentration of EMS in the Viracept case was 2.3 mg mL-1. (Gerber & Toelle, 2009), that of 1,6-DNP in soot was 0.41–0.71 μg g−1 (Schauer et al., 2004), and that of BCNU was 4 μg mL−1 in human plasma and

3.3 mg mL−1 in injection fluid (Petros et al., 2002). We have set the exposure time at 24 h because indigenous bacteria may be exposed 3-oxoacyl-(acyl-carrier-protein) reductase to these mutagens continuously in

the environment. We selected Rif and CPFX, because the emergence of microorganisms resistant to Rif and to CPFX is a growing concern (Jalal & Wretlind, 1998; Wydmuch et al., 2005). In addition, both antibacterial agents have obvious target molecules and mutations related to these target molecules are known to confer drug resistance (Campbell et al., 2001; Mariam et al., 2004). Pseudomonas aeruginosa is inherently relatively resistant to Rif (Yee et al., 1996), but has been susceptible to high concentrations of Rif. At the same time, the emergence of Rif-resistant M. tuberculosis is also a growing concern (Yee et al., 1996; Murphy et al., 2006). CPFX has been highly effective in treating P. aeruginosa infections, but recently, CPFX-resistant P. aeruginosa has become a growing problem (Jalal & Wretlind, 1998). Rif- and CPFX-resistant P. aeruginosa emerged after exposure to EMS and MNU. Meanwhile, BCNU induced Rif resistance, and 1,6-DNP induced CPFX resistance. NNN did not increase Rif- or CPFX-resistant P. aeruginosa. While BP induced mutation in S. Typhimurium TA100, Rif- or CPFX-resistant P. aeruginosa did not result. Susceptibility to BP differs considerably among strains (Jemnitz et al., 2004). We supposed that the P. aeruginosa was not susceptible to the mutagenic action of BP metabolites.

We assigned these enzymes to group 2 Further analysis revealed s

We assigned these enzymes to group 2. Further analysis revealed several microorganisms (Agrobacterium vitis S4, Bordetella petrii DSM 12804, Vibrio vulnificus YJ016, Sideroxydans lithotrophicus ES-1) whose IDO homologues are expressed only in combination with a specific efflux pump (RhtA/RhtB exporter family) without AR in the same operon regulated by a LysR-type repressor. These dioxygenases were assigned to the third group [Fig. 1 (5, 6, 7)]. By way of

analogy to B. thuringiensis, we proposed that the operons from the Enzalutamide manufacturer first, second and third groups could be involved in the synthesis and excretion of special derivatives of the hydroxylated free l-amino acids produced by their corresponding IDO homologues. In several microbes that we assigned to the fourth group (e.g. Gluconacetobacter diazotrophicus PAl 5 and GSI-IX mouse Pseudomonas fluorescens Pf0-1), the IDO homologue genes belong to the operons assumed to be involved in the synthetic

process, one stage of which is hydroxylation of an unknown substrate [Fig. 1 (8, 9)]. In some bacteria (e.g. Burkholderia oklahomensis EO147, Burkholderia pseudomallei 668, Photorhabdus luminescens ssp. laumondii TTO1 and Photorhabdus asymbiotica ATCC 43949), the IDO is thought to be co-expressed with polyketide/nonribosomal peptide synthetase-like protein. We proposed that these dioxygenases can be involved in the synthesis of peptide antibiotics containing hydroxylated l-amino acid residues and may also hydroxylate free l-amino acids [Fig. 1 (10)]. We assigned these dioxygenases to the fifth group. Many bacteria encode IDO homologues that are not part of an operon structure and can hydroxylate unpredictable substrates, including free l-amino acids; we included these enzymes in the sixth group. Based on the data obtained thus far, we assumed that the free amino acid dioxygenases were likely to belong to any group except group number four. Eight members of the PF10014 family – IDO (group

1, as a control enzyme); PAA (group 2); AVI, BPE (group 3); PLU (group 5); MFL, GOX and GVI (group 6) – were arbitrarily chosen for cloning and expression in E. coli to examine their substrate specificities with regard to canonical l-amino acids (Table 1). Using standard methods, we expressed selected enzymes as his6-tag proteins and purified them to near homogeneity using conventional aminophylline IMAC. Because our goal was to identify enzymes possessing high hydroxylase activities with potential for biotechnology applications, we first performed a high-throughput analysis for dioxygenase substrate specificity with 20 canonical l-amino acids using TLC analysis of the reaction mixture products (Fig. 2a,b). We found that new amino group-containing substances are formed by hydroxylation reactions with l-isoleucine (IDO, PAA), l-leucine (all enzymes with exception of GVI and PLU), l-methionine (all enzymes, but the activity of PLU was rather low) and l-threonine (BPE, AVI) (Fig. 2c).