After 10 min preincubation at 37 °C, the reaction was initiated b

After 10 min preincubation at 37 °C, the reaction was initiated by the addition of 1 or 6 mM Ala–Ala. When necessary, chloramphenicol (100 μg mL−1) was added 20 s before the addition of the dipeptide. Separation of intracellular and extracellular fractions was performed by the silicone oil method (Klingenberg & Pfaff, 1967), where cells (1 mL) were placed onto the upper layer (0.5 mL) of a 3 : 2 mixture of silicone oil AR20 and AR200 (Wacker Chemie, Germany) with the lower layer (0.15 mL) consisting of 20% (w/w) perchloric

acid. After centrifugation (20 000 g, 23 °C, 1 min), the upper layers were recovered as the extracellular fraction. The cell pellets were suspended Mitomycin C using a bath-type sonicator (15 s, 23 °C) followed by centrifugation (20 000 g, 23 °C, 5 min). The resulting supernatant was neutralized with 2 M Na2CO3 to obtain the intracellular fraction. Amino acids in each fraction were quantified by an HPLC system (LC-10A, Shimadzu, Japan). To calculate the intracellular click here amino acid concentration, the intracellular volume was assumed to be 2.03 μL mg−1 dry cell weight (Schneider et al., 2004). The MIC of Ala–Ala was determined by the agar dilution method, in which minimal agar plates were supplemented with 50 μg mL−1d-alanine, and twofold serial dilutions of the dipeptide. Cells were grown overnight in minimal medium containing 50 μg mL−1

of d- and l-alanine. Subsequently, the cells were diluted with minimal medium, and then spotted on peptide-containing plates (1 × 104–3 × 104 cells). The MICs were scored after 44 h incubation at 37 °C. The MICs of drugs were determined by the agar dilution method SPTLC1 using Luria agar containing 50 μg mL−1d-alanine and serial dilutions of the drugs. In order to investigate the presence of an export system for l-alanine

in E. coli, we isolated mutants lacking the system by exploiting the screening method with Ala–Ala, which had been applied to isolate amino acid exporter mutants of C. glutamicum. This would enable isolation of a mutant by selecting dipeptide-hypersensitive clones, in which the lack of the l-alanine export system might cause growth arrest due to the excessive accumulation of l-alanine inside the cell. However, such accumulation may not occur if internal l-alanine is degraded. Escherichia coli is indeed known to have the metabolic pathway by which l-alanine is metabolized via d-alanine to pyruvate (Wild et al., 1985). To test l-alanine degradation, we determined the level of l-alanine and Ala–Ala in the culture supernatant during growth of the wild-type E. coli strain, MG1655, in minimal medium supplemented with Ala–Ala. Consequently, l-alanine appeared transiently and then disappeared completely (Fig. 1a), indicating that MG1655 does degrade l-alanine and also has an l-alanine export function. In addition, we recently found that E. coli has three aminotransferases (AvtA, YfbQ and YfdZ) involved in l-alanine synthesis from pyruvate (unpublished data).

048; Fig 1B) Corresponding changes in hit rates

(ie t

048; Fig. 1B). Corresponding changes in hit rates

(i.e. the number of correctly recognized pictures) and in false alarms (i.e. falsely recognized pictures) did not reach significance (see Table 1 for a summary of results). As d′ is the most sensitive indicator of encoding, taking into account also the subject’s response bias, this pattern basically indicates an enhancing effect of tSOS on encoding of pictures, although this influence appears to be of moderate size, and is masked with measures (such as hit rate) that are confounded by response bias. There was also a tendency for there to be, overall, more correct responses (i.e. hit rate plus correct rejections) and fewer incorrect responses (i.e. false alarms plus misses) selleck after tSOS than after sham stimulation (total rate of BGB324 molecular weight correct responses, 0.84 ± 0.02 vs. 0.82 ± 0.02; total rate of incorrect responses, 0.16 ± 0.02 vs. 0.18 ± 0.02; F1,12 = 3.41, P = 0.09; Fig. 1B). In the word pair learning task, subjects overall learned significantly more word pairs after tSOS than after sham stimulation (mean number of learnt words: 52.40 ± 3.99 vs. 47.41 ± 4.28; F1,12 = 5.07, P = 0.044; Fig. 1C and Table 1). The analyses of word pair recall included an additional factor, ‘learning trial’ (L1–L5). Learning significantly improved over the five learning trials in both conditions (F4,48 = 316.98, P < 0.001), with the stimulation condition

showing better learning performance from the second presentation onwards (L1, F1,12 = 0.38, P = 0.561; L2, F1,12 = 4.36, P = 0.059; L3, F1,12 = 6.15, P = 0.029; L4, F1,12 = 5.21, P = 0.041; L5, F1,12 = 3.42, P = 0.089;

Fig. 1C). tSOS also significantly improved cued recall in a delayed retrieval test performed ~90 min after learning (77.14 ± 4.13 vs. 69.93 ± 5.58 after sham stimulation; F1,12 = 6.03, P = 0.03; Fig. 1C). Analyses of word list recall in the Verbal Learning and Memory Test included an additional factor, ‘learning trial’. Mean encoding of the word many list across L1–L5 also tended to be enhanced after tSOS, as compared with sham stimulation (number of recalled words: 12.44 ± 0.33 vs. 11.83 ± 0.45; F1,14 = 3.78, P = 0.072; Fig. 1D; see Table 1 for performance during single learning trials). Interestingly, learning of the IL tended to be worse after tSOS than after sham stimulation (6.93 ± 0.79 vs. 8.80 ± 0.72; F1,14 = 4.26, P = 0.058), pointing to stronger proactive interference resulting from enhanced encoding of the list to be learnt first in the tSOS condition. This interpretation was confirmed by calculating the ratio between learning of the IL and the mean learning of the original list (i.e. IL divided by mean L1–L5), which indicated a significantly lower ratio of interference learning after tSOS than after sham stimulation (0.56 ± 0.06 vs. 0.74 ± 0.05; F1,14 = 8.27, P = 0.012).

The frequency of transient desaturations emphasises the importanc

The frequency of transient desaturations emphasises the importance of adequate monitoring during sedation. The study highlights the need for more consistent reporting of adverse effects.

The authors declare no conflict of http://www.selleckchem.com/products/BIBW2992.html interest. “
“International Journal of Paediatric Dentistry 2012; 22: 406–418 Background.  As a result of numerous rapid and exciting developments in tissue engineering technology, scientists are able to regenerate a fully functional tooth in animal models, from a bioengineered tooth germ. Advances in technology, together with our understanding of the mechanisms of tooth development and studies dealing with dentally derived stem cells, have led to significant progress in the field of tooth regeneration. Aim and design.  This review focuses on some of the recent advances in tooth bioengineering technology, the signalling pathways in tooth development, and in dental stem cell biology. These factors are highlighted in respect of Palbociclib manufacturer our current knowledge of tooth regeneration. Results and conclusion.  An understanding of these new approaches in tooth regeneration should help to prepare clinicians to use this new and somewhat revolutionary therapy while also enabling them to partake in future clinical trials. Tooth bioengineering promises to be at the forefront of the next generation of dental treatments.


“Oral health literacy is a newly emerging field with considerable research potential. To validate an original instrument, the Hong Kong Oral Health Casein kinase 1 Literacy Assessment Task (HKOHLAT-P) for paediatric dentistry. A convenient

sample of 200 child/parent dyads attending a dental hospital in Hong Kong was selected. Convergent validity was tested by examining the association of HKOHLAT-P scores with those derived from the Test of Functional Health Literacy in Dentistry (TOFHLiD) and Hong Kong Rapid Estimate of Adult Literacy in Dentistry (HKREALD-30). The predictive validity of HKOHLAT-P was determined by testing the association between HKOHLAT-P and children’s caries experience (dmft) and the Chinese Early Childhood Oral Health Impact Scale (ECOHIS). The test-retest reliability and internal consistency of HKOHLAT-P were also evaluated. HKOHLAT-P was positively correlated with TOFHLiD and HKREALD-30 (P < 0.01), and was negatively correlated with children's dmft and ECOHIS. In the regression model, HKOHLAT-P was associated with TOFHLiD, HKEALD-30, children's dmft, and ECOHIS (P < 0.05) after controlling for participants' demographic characteristics. The intra-class correlation coefficient of HKOHLAT-P was 0.63 and the Cronbach's α was 0.71. Initial testing of HKOHLAT-P suggested that it is a valid and reliable instrument. "
“International Journal of Paediatric Dentistry 2012; 22: 310–316 Background.  Generalized aggressive periodontitis (GAP) in primary teeth is a rare periodontal disease that occurs during or soon after eruption of the primary teeth. An association with systemic diseases is a possibility. Case Report.

Practitioners caring for immigrant patients should be made aware

Practitioners caring for immigrant patients should be made aware of the significant burden of travel-related illnesses in these communities, especially in children. They should remain up to date on recommended preventive measures for travelers, and know when to refer the more complex cases to specialized travel health clinics. For example, health professionals need to make sure that their patients’ routine vaccinations are complete, and those who prescribe malaria chemoprophylaxis should receive adequate training to avoid medication errors. The increasing Selleckchem AG 14699 resistance of Salmonella typhi to antibiotics points to the need of a more effective and targeted promotion of typhoid

fever vaccination among VFRs. It is worth mentioning that the benefits of prevention among VFRs extend beyond immigrant communities. For example, hepatitis A vaccination of young VFRs could help prevent outbreaks in day-care centers, which are attended by more than two-thirds of Quebec children between the ages

of 6 mo and 5 y.22 At the government level, a health information package on routine vaccinations and other recommended preventive measures before a person’s return trip to their country of origin should be provided to new immigrants. Moreover, recent initiatives in Quebec’s Provincial Selleckchem MLN0128 Health Insurance provisions, such as the reimbursement of anti-malarial drugs and the use of the combined hepatitis A and B vaccine in the school-based vaccination program, could

lower the burden of these diseases among young VFRs in coming years. The data sources can bias some of our estimates. Notifiable diseases are often under-diagnosed and under-reported.23–26 Epidemiological questionnaires are not uniform between public health departments, thus resulting in missing information, particularly on the trip purpose and pre-travel consultation. Statistics Canada data estimate the number of trips, and not the number of travelers. This can result in an overestimation in the number of travelers, second because one can make several trips a year. However, this may be less significant for VFRs, since they typically return to their country of origin with their children during summer holidays. Statistics show that VFRs travel back to Quebec mainly in the third quarter (33%),5 which is consistent with our results. Our study examined all reported cases of malaria, hepatitis A, and typhoid fever among Quebec travelers between 2004 and 2007, providing a complete picture of the epidemiologic situation. Since we used a similar methodology to the one used in 2000 to 2002, one can compare changes over time and identify the groups that require more preventive work.7 Typhoid fever data should be interpreted with caution because of their small numbers, although our results are consistent with other studies.7,8,27 It would be interesting to examine the pre-travel consultation determinants among VFRs in a future study.

, 2005; Green et al, 2007; Marcos & DuPont, 2007), has come to l

, 2005; Green et al., 2007; Marcos & DuPont, 2007), has come to light. The strain carries 17-AAG in vitro the binary toxin gene CdtB, and has an 18-base-pair deletion in the toxin repressor gene, tcdC, which means that it generates approximately 16–23 times more toxin than other strains (Warny et al., 2005). Infection is associated with a high risk of acute clinical deterioration and a poor response to metronidazole

therapy (Spigaglia & Mastrantonio, 2002; Pepin et al., 2005), making it a major concern for healthcare worldwide. Clostridium difficile ribotype 027 was initially rare in the United Kingdom; however, when outbreaks at Stoke Mandeville and the Royal Devon and Exeter Hospitals were investigated in 2004–2005, type 027 was found to predominate in their cases (Anon, 2006), check details and this ribotype has now been detected in the majority of countries around the world (Kuijper et al., 2007). It is clear, then, that C. difficile is a significant burden on the healthcare profession and patients. With the ever-increasing availability of genomic information, however, greater insight into the evolution and variation of C.

difficile genomes is now possible (Stabler et al., 2006, 2009; He et al., 2010). The Clostridb database (http://xbase.bham.ac.uk/clostridb/) (Chaudhuri & Pallen, 2006), an excellent publicly accessible resource for those interested in comparative genomics of the genus Clostridium, currently contains genome sequences of 18 strains of clostridia, including two genomes of C. difficile, namely C. difficile 630 and C. difficile qcd32_g58, a representative of the predominant

NAP1/BI/027 strain in Quebec (Loo et al., 2005). The 4.29 Mb genome of C. difficile strain 630 and its Galactosylceramidase 7.8 kb plasmid encode a remarkable number of genes associated with resistance to antimicrobial agents, as well as virulence factors, host adherents and surface structures (Sebaihia et al., 2007). Genome sequences have been generated recently for a further six strains, including CD196, an early, nonepidemic, ribotype 027 strain (Stabler et al., 2009), the R20291 isolate responsible for the UK Stoke Mandeville outbreak, and 21 other hypervirulent ribotype 027 strains isolated over the past two decades (He et al., 2010). A further six hypervirulent isolates associated with the Quebec outbreak and a reference ATCC43255 strain are at the draft genome sequence stage (McGill University and Génome Québec Innovation Centre), while the human microbiome project at Baylor College of Medicine has draft genome sequences for two strains (NAP07, NAP08) at the time of writing. These genomic data, along with recently developed tools for Clostridial functional genomics (Heap et al., 2009), make it possible for researchers to adopt a systems approach to the dissection of the physiology and biochemistry of this pathogen.

In this review article, we describe some of the latest advances i

In this review article, we describe some of the latest advances in our knowledge on the role of the endocannabinoid system, in its most recent and wider conception, in pain pathways, by focusing on: (1) neuron–glia interactions; and (2) emerging data on endocannabinoid cross-talk with neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor. “
“Chronic N-methyl-d-aspartate

receptor (NMDAR) hypofunction has been proposed as a contributing factor to symptoms of schizophrenia. FK506 mouse However, it is unclear how sustained NMDAR hypofunction throughout development affects other neurotransmitter systems that have been implicated in the disease. Dopamine neuron biochemistry and activity were examined to determine whether sustained NMDAR hypofunction causes

a state of hyperdopaminergia. We report that a global, genetic reduction in NMDARs led to a remodeling of dopamine neurons, substantially affecting two key regulators of dopamine homeostasis, i.e. UK-371804 cell line tyrosine hydroxylase and the dopamine transporter. In NR1 knockdown mice, dopamine synthesis and release were attenuated, and dopamine clearance was increased. Although these changes would have the effect of reducing dopamine transmission, we demonstrated that a state of hyperdopaminergia existed in these mice because dopamine D2 autoreceptors were desensitized. In support of this conclusion, NR1 knockdown dopamine neurons have higher tonic firing rates. Although the tonic firing rates are higher, phasic signaling is impaired, and dopamine overflow cannot be achieved with exogenous high-frequency stimulation that models phasic firing. Through the examination of several parameters of dopamine neurotransmission, we provide evidence that chronic NMDAR hypofunction leads to a state of elevated synaptic dopamine. Compensatory mechanisms to attenuate hyperdopaminergia also impact the ability to generate dopamine surges through phasic firing. “
“Elimination of granule cells (GCs) in the olfactory bulb (OB) is not a continual event but is promoted during a short time window in the postprandial period, typically

with postprandial sleep. However, the neuronal mechanisms for the enhanced GC elimination during the postprandial period are not understood. Here, we addressed the question of whether top-down inputs of 4-Aminobutyrate aminotransferase centrifugal axons from the olfactory cortex (OC) during the postprandial period are involved in the enhanced GC elimination in the OB. Electrical stimulation of centrifugal axons from the OC of anesthetized mice increased GC apoptosis. Furthermore, pharmacological suppression of top-down inputs from the OC to the OB during the postprandial period of freely behaving mice by γ-aminobutyric acid (GABA)A receptor agonist injection in the OC significantly decreased GC apoptosis. Remarkable apoptotic GC elimination in the sensory-deprived OB was also suppressed by pharmacological blockade of top-down inputs.

In this review article, we describe some of the latest advances i

In this review article, we describe some of the latest advances in our knowledge on the role of the endocannabinoid system, in its most recent and wider conception, in pain pathways, by focusing on: (1) neuron–glia interactions; and (2) emerging data on endocannabinoid cross-talk with neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor. “
“Chronic N-methyl-d-aspartate

receptor (NMDAR) hypofunction has been proposed as a contributing factor to symptoms of schizophrenia. www.selleckchem.com/products/dabrafenib-gsk2118436.html However, it is unclear how sustained NMDAR hypofunction throughout development affects other neurotransmitter systems that have been implicated in the disease. Dopamine neuron biochemistry and activity were examined to determine whether sustained NMDAR hypofunction causes

a state of hyperdopaminergia. We report that a global, genetic reduction in NMDARs led to a remodeling of dopamine neurons, substantially affecting two key regulators of dopamine homeostasis, i.e. BMS-354825 price tyrosine hydroxylase and the dopamine transporter. In NR1 knockdown mice, dopamine synthesis and release were attenuated, and dopamine clearance was increased. Although these changes would have the effect of reducing dopamine transmission, we demonstrated that a state of hyperdopaminergia existed in these mice because dopamine D2 autoreceptors were desensitized. In support of this conclusion, NR1 knockdown dopamine neurons have higher tonic firing rates. Although the tonic firing rates are higher, phasic signaling is impaired, and dopamine overflow cannot be achieved with exogenous high-frequency stimulation that models phasic firing. Through the examination of several parameters of dopamine neurotransmission, we provide evidence that chronic NMDAR hypofunction leads to a state of elevated synaptic dopamine. Compensatory mechanisms to attenuate hyperdopaminergia also impact the ability to generate dopamine surges through phasic firing. “
“Elimination of granule cells (GCs) in the olfactory bulb (OB) is not a continual event but is promoted during a short time window in the postprandial period, typically

with postprandial sleep. However, the neuronal mechanisms for the enhanced GC elimination during the postprandial period are not understood. Here, we addressed the question of whether top-down inputs of 3-oxoacyl-(acyl-carrier-protein) reductase centrifugal axons from the olfactory cortex (OC) during the postprandial period are involved in the enhanced GC elimination in the OB. Electrical stimulation of centrifugal axons from the OC of anesthetized mice increased GC apoptosis. Furthermore, pharmacological suppression of top-down inputs from the OC to the OB during the postprandial period of freely behaving mice by γ-aminobutyric acid (GABA)A receptor agonist injection in the OC significantly decreased GC apoptosis. Remarkable apoptotic GC elimination in the sensory-deprived OB was also suppressed by pharmacological blockade of top-down inputs.

Quantification of AI-2 in ZFF using the DPD standard curve (AI-2=

Quantification of AI-2 in ZFF using the DPD standard curve (AI-2=0.376 × IOD−28.93) indicated that the amount of AI-2 in ZFF varied with species. ZFFaph contained the highest concentration of AI-2 (1.66±0.25 μM) among the three species tested, followed by ZFFsoj (1.40±0.18 μM), both from zoospores at 104 mL−1 levels, while ZFFnic from zoospores at 5 × 105 mL−1 contained the least AI-2 (0.66±0.13 μM). Negative values were obtained for the SDW and CV8 controls, indicating

that zoospores produced AI-2, and the AI-2 activity was not from residual CV8 broth. To confirm Selleckchem Ku-0059436 the results from the luminescence assay, ZFFnic and ZFFsoj were analyzed using chemical methods. The formation of quinoxaline derived from DPD, specifically 2-(1,2-dihydroxyethyl)-3-methylquinoxaline, was detected by LC-MS. First, a peak identical to quinoxaline from synthetic DPD (Fig. 2d) was identified in the EIC at m/z 205 from ZFFsoj (Fig. 2a)

and ZFFnic (Fig. 3a, middle). Second, quinoxaline products from ZFF coeluted with the quinoxaline standard (Figs 2c and 3, top). Lastly, the fragmentation patterns of the quinoxaline-derived buy Erastin from ZFF samples were identical to those from synthetic DPD (Figs 3b, c and 4a, b). Furthermore, the quinoxaline peak was not detected in the negative control, a 103× dilution of the CV8 broth equivalent to 106 times what was left in ZFFs (Figs 2b and 3a, bottom). These cumulative results indicate that AI-2 originated from zoospores. Quantification of DPD-derived quinoxaline in ZFF samples also showed a variation in AI-2 concentrations with species. Phytophthora sojae produced a higher amount of AI-2 than P. nicotianae. Based on the standard curve generated from synthetic DPD, the AI-2 concentration in ZFFnic from a suspension of 106 zoospores mL−1 was 1.1±0.1 μM, while in ZFFsoj from a suspension of 5 × 104 zoospores mL−1, it was 10.1±2.0 μM (Fig. 3), similar to what was observed Carbohydrate in the

bioluminescence assay. AI-2 represents an interspecies signaling molecule and is involved in the regulation of luminescence, virulence factor secretion, and biofilm formation in bacteria (Vendeville et al., 2005; Xavier & Bassler, 2005). Here, we demonstrate for the first time the production of AI-2 by P. nicotianae, P. sojae, and P. aphanidermatum, members of Pythiaceae in the eukaryotic Stramenopila kingdom, which will provide an insight into the physiology and ecology of zoosporic pathogens. Detection of AI-2 in ZFF (Figs 1–4) raises a question regarding the AI-2 production pathway in oomycete species. Currently, there are three known pathways for AI-2 (DPD) production (Winzer et al., 2002; Hauck et al., 2003; Nichols et al., 2009). Within these pathways, the pentose-phosphate pathway used by some plant and bacterial species (Hauck et al., 2003; Tavender et al., 2008) is most likely to be adopted by oomycetes. The reasons are threefold. First, the LuxS-dependent pathway is only available in bacterial species (Sun et al., 2004).

It was believed that pharmacists recognised this and consequently

It was believed that pharmacists recognised this and consequently their demands for development had increased. In terms of ‘responding to changes in the profession’ pharmacist development was seen as an investment choice with business benefits balanced against costs. In-house training was considered to facilitate greater control over pharmacist development than more costly externally provided courses, for which changes in practice were not always evident. Support Bortezomib mouse for external courses such as postgraduate diplomas tended to be offered to pharmacists that were performing well and had demonstrated commitment to the company. Completion of these courses was

thought to result in some pharmacists leaving the company to pursue other roles and the unclear career pathway in community pharmacy was believed to contribute to this. DZNeP Results are based on the opinions of four individuals and whilst they may be representative of SLDMs at other LMCPs they cannot be generalised further. Participants believed that training and development

was required beyond that delivered up to registration to enable community pharmacists to perform effectively, thus supporting the current drive to change undergraduate and early career development. Externally provided postgraduate education has not been widely supported as a means of facilitating this because of concerns about costs, with little evidence available to demonstrate a positive return on investment. Instead the focus has been on in-house training which allows closer control of pharmacists’ development and the costs involved. Whilst completion of a postgraduate qualification was thought to result in people changing their career companies are using them as a reward when there may be a greater gain made by investing in those underperforming and less committed. If external postgraduate

education is to be more widely supported providers Methamphetamine should ensure courses are designed to deliver outcomes which justify the costs involved. 1. Howe H, Wilson K. Review of post-registration career development: Next steps. Report to Medical Education England Board 2012. 2. Seston L, Hassell K. Pharmacy Workforce Census 2008: Main findings. London, 2009. Bridget Coleman1, Apirati Yangphaibul2, Maja Begovic1 1Whittington Health, London, UK, 2UCL, School of Pharmacy, London, UK A pilot study assessed the contribution made by a non-medical prescribing pharmacist to a musculoskeletal (MSK) chronic pain clinic in primary care The clinic pharmacist performed a mean of 2.5 actions per patient (n = 32) to optimise therapy, reduce adverse effects and enhance adherence to medicines Members of the chronic pain team indicated that the pharmacist added value to patient care This new pharmacist role is being continued, developed and further evaluated.

5, lane 12) An extensive mutagenesis of the E coli ArgR was per

5, lane 12). An extensive mutagenesis of the E. coli ArgR was performed in order to determine its precise role in cer site-specific recombination. The ArgR protein binds DNA at ARG boxes localized in promoter regions of several genes of the arginine regulon and at the cer site, which

contains half an ARG box. It is quite likely that the DNA-binding activity of this protein is an important contributor to its role as an accessory factor in cer recombination by bringing two cer sites together. However, ArgR by itself cannot support cer recombination in the presence of the XerCD recombinases; PepA is also required for this reaction. Alén et al. (1997) have demonstrated that PepA and ArgR interact directly with cer, forming a complex in which the accessory sequences of two cer sites are interwrapped approximately three times in a right-handed fashion. Using pentapeptide Selleckchem ABT888 scanning mutagenesis, we isolated a series of ArgR mutants that showed an approximate 90% reduction in cer recombination, but were still able to repress an argA∷lacZ fusion effectively in vivo (Figs 1 and 2). The mutant proteins also displayed

sequence-specific DNA-binding activity (Fig. 3). All of Obeticholic Acid mouse the insertions mapped to the same amino acid, between residues 149 and 150 of ArgR (ArgR5aa). This region corresponds to the C-terminal region of ArgR, at the end of the α6-helix (Fig. 4). In order to show that the observed phenotype was due to the disruption of ArgR and was not caused by the additional five amino acids residues, we constructed an ArgR mutant that was truncated at this region. This protein lacks residues 150–156 (ArgR149), Anidulafungin (LY303366) but displays the same properties as ArgR5aa, namely a significant reduction

in cer site-specific recombination in vivo (Fig. 1b), and the ability to bind to DNA at near wild-type levels in vivo (Fig. 2) and in vitro (Fig. 3). Moreover, we were able to detect the same level of DNA retardation as the wild-type protein, which suggests that both ArgR149 and ArgR5aa bind to DNA as hexamers (Fig. 3). In addition, crosslinking studies have shown that wild-type and mutant proteins are capable of forming higher-order structures in solution, although ArgR149 does not appear to form hexamers as efficiently as either wild-type ArgR or ArgR5aa under the crosslinking conditions used (Fig. 5). It is possible that the small C-terminal deletion in ArgR149 prevents this protein from forming a stable hexameric structure. Similar results have been observed with the α A-crystallin protein, where deletions of the terminal 11 amino acids from the C-terminus significantly decreased the oligomeric size of the protein (Thampi & Abraham, 2003). Despite this, ArgR149 can still bind DNA effectively both in vivo and in vitro; the addition of DNA and l-arginine may allow this mutant to form more stable hexamers under these conditions.