Through pregnancy, it is routine to monitor LFT tests at each antenatal clinic appointment as

a marker for potential obstetric complications (HELLP, pre-eclampsia, acute fatty liver, etc.), particularly in the final trimester. Finally, in those diagnosed late and not receiving HBV treatment incorporated into HAART, LFT flares may be seen shortly after delivery, which in some relates to HBeAg seroconversion and reappearance or a marked increase in HBV DNA levels. Where acute HBV has been diagnosed, there are no data to support management and each case needs to be managed with specialist advice. Data suggest that lamivudine, as part of HAART, does not completely protect against the development of acute HBV infection, although it is unknown whether this is also the case with tenofovir with or without lamivudine/emtricitabine. Although there is a theoretical risk of high HBV Pictilisib cost DNA levels and the linked association with increased risk of

transmission combined with the potential for acute hepatitis and threat to maternal and fetal health, the presumption would be that this would be Selleck Galunisertib abrogated by the patient already being on HAART incorporating tenofovir and either emtricitabine or lamivudine. 6.1.4 Where pegylated interferon or adefovir is being used to treat HBV in a woman who does not yet require HIV treatment and who discovers she is pregnant, treatment should be switched to a tenofovir-based HAART regimen. Grading: 1C If a woman on pegylated interferon becomes Cetuximab price pregnant, it should be discontinued and changed to a tenofovir-based HAART regimen because of the antiproliferative effect of the drug. Few data are available on the risk of congenital malformation

with first trimester exposure to the newer therapies telbivudine (FDA category B) and entecavir (FDA Category C). The outcome of the pregnancy should be reported to the Interferon Pregnancy and Antiretroviral Pregnancy Registries. 6.1.5 As there is no evidence of any adverse effect on maternal or neonatal health if women become pregnant while taking ART active against HBV, treatment should be continued. Grading: 1C For tenofovir, emtricitabine and lamivudine, APR [1] and the Development of Antiretroviral Therapy Study (DART) have not identified any increased risk in prevalence or any specific pattern of anomaly, even when administered in the first trimester. Hence, when a patient becomes pregnant on an anti-HBV viral agent as part of their HAART (tenofovir, lamivudine or emtricitabine), as for HIV management, HAART should be continued. This is because the potential risk to the fetus from drug exposure is outweighed by that of a hepatitis flare or liver disease progression if the drug(s) were to be discontinued in addition to HIV virological rebound and risk of MTCT. Because entecavir has activity against HIV, it is not recommended unless given with active HAART in a coinfected patient.

We hypothesized that HIV-infected children with hyperlipidaemia have higher levels of selected biomarkers associated with vascular inflammation pathways BGJ398 compared with HIV-infected children without hyperlipidaemia and HEU children (with

and without hyperlipidaemia). Furthermore, we sought to determine whether metabolic, anthropometric and disease- or treatment-specific factors are associated with higher levels of these biomarkers. Participants were enrolled in the Adolescent Master Protocol (AMP), part of the Eunice Kennedy Shriver National Institute of Child Health and Human Development-supported Pediatric HIV/AIDS Cohort Study (PHACS). Eligible children were between 7 and 16 years old and born to HIV-infected mothers. The AMP study has enrolled 451 children with perinatal

HIV infection and 227 HEU children. Enrolment began in March 2007 at 15 sites in the USA and Puerto Rico, and recruitment was completed in December 2009. The overall aims of the prospective PHACS study are to determine the impact of HIV infection and ARV therapy on several clinical outcomes in pre-adolescents and adolescents, including growth, nutrition and cardio-metabolic risk. The PHACS protocol requires that all HIV-infected children have fasting lipids measured annually. The protocol also SCH727965 purchase requires that repository specimens be drawn at the entry visit for all participants. As these are paediatric patients, the amount of specimen varies across individuals. As of August 2009, there were 357 HIV-infected children enrolled in the study who had fasting lipids measured at the entry visit and 184 HEU children with an entry visit. We determined that 226 of 357

HIV-infected selleck chemicals children and 140 of 184 HEU children had an adequate volume of repository specimen to assay for vascular biomarkers. Among the HIV-infected children, we defined hyperlipidaemia according to the modified National Cholesterol Education Program (NCEP) criteria [total cholesterol > 200 mg/dL, low-density lipoprotein (LDL) cholesterol > 130 mg/dL, triglycerides > 110 mg/dL (≤ 9 years) or > 150 mg/dL (≥ 10 years), or high-density lipoprotein (HDL) cholesterol < 35 mg/dL] [23]. Among the 357 HIV-infected children (all eligible children), 41% met the dyslipidaemia definition. In our subsample of 226 (those included in this analysis), 39% met the hyperlipidaemia definition. Our subsample was similar by age, sex, race, CD4 category, and body mass index (BMI) z-score when compared with children who did not have repository samples.

l. is also traditionally considered to be highly polymorphic (Jumpponen & Trappe, 1998; Gams, 2000). Likewise, there are still some disagreements between the morphological and the molecular identification of Phialophora spp. (Yan et al., 1995; de Hoog et al., 1999; Ulrich et al., 2000; Sieber, 2002). Species formerly classified in the genus are now known to belong to different orders of Ascomycetes. Gams (2000) began to sort out the taxonomy of Phialophora spp. and erected Harpophora for anamorphs of Gaeumannomyces and Magnaporthe within the Magnaporthaceae. Its morphological characteristics include fast-growing, thin colonies with ‘runner hyphae’ and more or less pigmented phialides coupled with cylindrical,

hyaline selleck chemicals and strongly curved conidia. Up to now, four species combinations have been described within Harpophora, i.e. Harpophora radicicola (type species, previously Phialophora radicicola) (McKeen, 1952; Walker, 1980), Harpophora maydis (Cephalosporium maydis) (Samra et al., 1963), Harpophora graminicola (Phialophora graminicola) (Hornby et al., 1977; Walker, 1980) and Harpophora zeicola (Phialophora zeicola) (Deacon & Scott, 1983). In addition, the anamorphs of Gaeumannomyces spp. belong here without being separately named as anamorph species. We have recently started an

examination of the endophytic fungal community in wild rice (Oryza granulata) roots in China, during which we found a new species, which is described here as Harpophora oryzae.

The site of study is located in Xishuangbanna, Yunnan http://www.selleckchem.com/products/VX-770.html province, southwest of China (22°04′–22°17′N; 100°32′–100°44′E). In September of 2007 and 2008, we collected samples from two sites in Xishuangbanna. Healthy and intact wild rice plants with bulk soil were packed in a box and carefully transported to the laboratory within 48 h. For isolation of endophytic fungi, healthy roots (free of detectable lesions) of the sample rice plants were gently rinsed with tap water, immersed in ethanol (75% v/v) for 30 s, then in sodium hypochlorite (1% w/v) for 10 min and finally rinsed three times in sterile-distilled water. Roots were cut into segments of 0.6 cm length and transferred to a malt extract agar (MEA) Fenbendazole plate containing 2% malt extract and 2% agar (w/v) supplemented with chloramphenicol (50 mg L−1) to prevent bacterial growth. Six root fragments were placed on one plate and incubated at 25 °C in permanent darkness. After the emergence of fungal hyphae, these were cut off and subcultured. Isolates were stored by covering a culture on potato dextrose agar (PDA) slants with sterile liquid paraffin at 25 °C and by preservation in aqueous 15% v/v glycerol additionally containing glucose (10 g L−1), yeast extract (1 g L−1) and casein hydrolysate (1 g L−1) at −70 °C. Light-microscopic analysis was performed using an Olympus BX51 microscope. Images were acquired using axiovision 3.1. For the determination of spore characteristics, specimens were mounted in water.

, 2006). Thus, we defined an extended consensus sequence (CG-N-TAT-N2-G-N6-CTA-N-ATA-N-CG) based on the three strongly repressed Mo-boxes upstream of the morA, mopA, and anfA genes (Fig. 1a; Consensus R). The major difference between MopA/MopB-repressed

Mo-boxes and the MopA-activated mop-Mo-box seems to lie in the right half-site. Therefore, we investigated whether this region of the mop-Mo-box either selectively facilitates binding of MopA and/or discriminates against binding Ferroptosis tumor of MopB. Several rationally designed single-base substitutions were introduced to convert the anfA-Mo-box into the mop-Mo-box and vice versa (Materials and methods). Specifically, mutations T3A, A7G, T17C, A18T, A23T, and C24T converted the anfA-Mo-box toward the mop-Mo-box (Fig. 1b), while mutations A3T, T16C, C17T, T18A, C19T, T23A, and T24C made the mop-Mo-box more similar to the anfA-Mo-box (Fig. 1c). Mutations A18G, T21C, and C24A probed for the principal importance of highly conserved nucleotides, which were exchanged for nucleotides not occurring in any of the Mo-boxes. To prove that the mop-Mo-box was essential for MopA-dependent mop

gene activation, the triple mutation T4A-A5T-G7C was constructed to destroy the conserved left half-site of the mop-Mo-box (Fig. 1c). In addition to these single-base substitutions, the anfA- and mop-Mo-boxes were SB203580 in vitro exchanged against each other (anfAmop and mopanfA). In anfAmop, the entire 25-bp anfA-Mo-box was replaced Staurosporine ic50 by the mop-Mo-box (Fig. 1b). In contrast, in mopanfA, only the first 22 nucleotides were replaced, because nucleotides 23–25 of the mop-Mo-box overlap with the −35 region of the mop promoter and are thus essential for mop gene expression (Fig. 1c). The effects of Mo-box mutations on anfA transcription were examined by lacZ reporter fusions. For this purpose, wild-type and mutant anfA promoter fragments were cloned into the low-copy broad-host-range vector pML5, thus creating transcriptional

fusions to the promoterless lacZ reporter gene (Fig. 1b; Table 1; Materials and methods). These reporter plasmids were transferred into R. capsulatus wild-type and mutant strains defective for mopA, mopB, or both. The resulting reporter strains were grown in minimal medium under Mo-limiting and Mo-replete conditions before determination of β-galactosidase activities (Fig. 2). In addition to these in vivo studies, the in vitro effects of selected anfA-Mo-box mutations on binding by MopA and MopB were analyzed by DNA mobility shift assays (Fig. 3). For this purpose, 209-bp anfA promoter fragments (PanfA; Fig. 1b) were PCR amplified and used for gel-shift assays with increasing amounts of the regulators (Materials and methods). The effects of Mo-box mutations on anfA gene expression and regulator binding may be summarized as follows: (1) In the R.

However, different conclusions were reached concerning the ratio of synchronous to asynchronous Silmitasertib clinical trial release (synchronicity ratio) and its dependence on the identity of the postsynaptic target cell. Whereas Daw et al. (2009) and Karson et al. (2009) suggested that the synchronicity ratio is independent of the identity of the postsynaptic target cell, Ali and Todorova report that this ratio is larger for synapses formed between

CCK-interneurons than for synapses between CCK-interneurons and pyramidal neurons. Accordingly, they suggest that factors governing asynchronous GABA release are synapse-specific and determined in part by the postsynaptic target. Alternatively, these divergent results may be explained by differences in experimental conditions (room versus physiological temperature, number of presynaptic action potentials, current-clamp versus voltage-clamp recording, and/or age of the animals) and the methods used to quantify asynchronous release. Despite these differences, all three papers unequivocally demonstrate asynchronous release at interneuron-interneuron synapses. Asynchronous transmitter release and modulation of synaptic transmission by presynaptic CB1 receptors are hallmarks of the function of synapses formed by CCK-interneurons. How are these two properties interrelated? Ali & Todorova (2010) found that the CB1 receptor inverse agonist AM-251 increased the synchronicity Selleckchem CHIR-99021 ratio, whereas the

endocannabinoid anandamide decreased it. This finding raises the interesting possibility that synchronous and asynchronous release are differentially affected during DSI. Whether other presynaptic receptors on the terminals of CCK-interneurons have similar effects needs to be determined. Furthermore, Phosphatidylinositol diacylglycerol-lyase the computational significance of asynchronous GABA release in principal neuron-interneuron networks remains to be elucidated. Ali & Todorova (2010)

suggest that asynchronous GABA release modulates the time windows of inhibition, thereby controlling spike timing among local circuit interneurons. “
“This revised Figure 2A corrects the time-points listed for the studies by Kippin et al . (2005), Tanaka et al. (2007) and Tropepe et al. (1997) in the published paper of Hamilton et al. (2013). The authors apologize for any inconvenience caused by this error. “
“Brain plasticity is a double-edged sword. It allows for individuals to learn and adapt to their environment, but peculiarities may also alter the brain and contribute to maladaptive outcomes. Here, in the very interesting study conducted by Frey and colleagues, the authors used measures derived from event-related potentials (ERPs) to assess visuo-spatial maps within the visual cortex in youths with autism spectrum disorders (ASD) and controls. Based on the observation that some individuals with ASD tend to not fixate on a target (i.e. they exhibit off-center fixations), Frey and colleagues hypothesized that this fixation pattern would impact the development of the visual cortex.

The clinical syndrome that results depends on a number of factors including the Leishmania species and immune response of the host. Here, we report successful treatment of lingual leishmaniasis complicating visceral disease in an immunocompetent patient. A 50-year-old National Guardsman with no significant medical problems presented with a 2-week history of a painful central tongue ulcer preceded by 2 weeks of tongue edema. He was deployed to Saudi Arabia during Operation Desert Storm in 1991 and to Iraq and Kuwait during Operation Iraqi Freedom (2002–2003). The lesion appeared 6 years after he returned from his last Rapamycin in vitro deployment. A 1.5-cm central cavitary lesion extending to the circumvallate

papillae with surrounding erythema, a smaller

0.5 cm lesion lateral to the midline, and oral candidiasis were noted on examination. Physical examination did not reveal any hepatosplenomegaly, lymphadenopathy, or abnormal skin findings. The platelet count was 115,000 µL but the white blood cell count and hemoglobin level were normal. Aspartate aminotransferase and alanine aminotransferase were 113 U/L and 132 U/L, respectively. The alkaline phosphatase level was 571 U/L, but the measurements of the total bilirubin, albumin, total protein, and renal function were normal. Incisional biopsy of the central tongue cavity done at presentation revealed squamous papilloma with candidiasis. The patient received nystatin suspension but no systemic antifungal therapy. During the subsequent 15 weeks, four additional lateral lesions developed and the central lesion enlarged. Laser excision biopsy of the central selleck screening library lesion was done to determine a definitive

etiology of the ulcers. Histopathologic evaluation showed marked non-caseating granulomas. The lesions continued to worsen, and 2 weeks later a partial glossectomy was done. Histopathologic examination revealed the presence of numerous intracellular amastigotes (Figure 1A and B). After the amastigotes were discovered, the previous biopsies were reexamined and were also noted to contain amastigotes. Review heptaminol of additional history revealed that the patient had experienced intermittent night sweats and an unintentional 40-pound weight loss over the last 5 years. While serving with the US military in Saudi Arabia in 1991, he had developed pruritic white and red macules on his arms, neck, and back. These lesions eventually waned and never became ulcerative. Punch biopsy of the back performed in 2004 revealed only a perivascular lymphocytic infiltrate. Since 2000, he had been noted to have thrombocytopenia. Liver function tests were noted to be abnormal in 2004 and liver biopsy demonstrated non-necrotizing granulomas, but no specific diagnosis was made. He recalled being bitten by various insects and had contact with various animals including dogs during both deployments. He lived in Tennessee and denied any additional travel history.

, 2008). An untreated control was included. Bacteria were collected after 20 min of treatment before significant growth differences were observed due to the antimicrobial effect of the drugs. It is noted that we observed a weak growth inhibition at the two highest concentrations of thioridazine. Total RNA was prepared by a hot acid–phenol procedure (Moazed et GSK-3 inhibitor review al., 1986). Total nucleic acid concentrations and purity were estimated using absorbance readings (260 nm/280 nm) on a NanoDrop (Saveen Werner). The genes were analyzed by either Northern blotting or primer extension. For genes larger than 1000 bp we performed primer extensions to obtain

a clear result. Primer extension analyses Metformin chemical structure were performed as described previously (Klitgaard et al., 2008) and Northern blot analyses were carried out as described elsewhere (Nielsen et al., 2010). All primers and DNA probes used for primer extension and Northern

blot analyses, respectively (Table 1), were labelled at the 5′ end with 32P γATP. The primer extension and Northern blot products were visualized by autoradiography and/or phosphor imaging using a Typhoon scanner (GE Healthcare). Spot intensities were quantified using imagequant 5.0 software (Molecular Dynamics) and gene expression ratios were calculated relative to the untreated control. Expression levels on Northern blots were normalized to the 16S rRNA gene levels on the reprobed membrane preliminary to the calculation of expression ratios. Treatments were compared with the untreated control and only changes of at least twofold up- or downregulation were considered. Expression of the mecA gene has previously been shown to be induced by oxacillin and to be reduced yet again when oxacillin was

combined with thioridazine (Klitgaard et al., 2008). Related to this, it was interesting to comprehend whether other PBPs and genes involved in β-lactam resistance were affected by the combinatorial treatment or if the effect was specific to the non-native PBP2a. pbpB is transcribed from three different promoters: P1 and P1′ are located upstream of the first gene in the operon (recU) and the VraSR-regulated P2 is located immediately upstream of pbpB; the latter will be described in coherence Amylase with the VraSR regulon below. The distal P1 and P1′ promoters of pbpB were unaffected by the drug addition (Fig. 2a and b) besides a slight induction of pbpB P1′ by oxacillin as observed previously (Utaida et al., 2003). In contrast, the level of pbpD transcript was reduced at the highest concentrations of thioridazine (Fig. 2c). The femAB gene products were induced by oxacillin. This induction was further increased by addition of low concentrations of thioridazine; however, at higher thioridazine concentrations the induction is diminished (Fig. 2d).

Its termini contain the inverted repeat sequence 5′-CCTGC … GCAGG-3′, and its 5′-ends are covalently capped with protein (Overhage et al., 2005; Parschat et al., 2007). Our previous sequence analysis of pAL1 and predictions of possible secondary structures formed by potential telomeric 3′-overhangs indicated significant differences of the ‘left’ and ‘right’ terminus of pAL1, raising the question of whether each terminus of pAL1 is recognized, or even capped, by a specific protein (Parschat et al., www.selleckchem.com/products/ABT-263.html 2007). Rhodococcal plasmids pHG201 and pHG205 are other examples of actinomycete

linear plasmids that do not show striking homology between their ‘left’ and ‘right’ telomere sequences (Kalkus et al., 1998), but their TPs have not been described. In contrast, the

ends of Streptomyces check details linear replicons usually contain well-conserved terminal palindromic sequences (Zhang et al., 2006). The gene product of pAL1.102 is the only protein exhibiting a weak similarity to known (Streptomyces) TPs; however, due to the low sequence similarity, its annotation as a ‘putative terminal protein’ was tentative (Parschat et al., 2007). As a first step toward characterizing the telomere complex of pAL1, we identified the protein attached to both termini of pAL1 and demonstrated its specific deoxynucleotidylation in vitro. The strains and plasmids used in this study are listed in Table 1. For isolation of total DNA, A. nitroguajacolicus Rü61a [pAL1] was grown in a mineral salts medium (Parschat et al., 2003) on 8 mM sodium benzoate at 30 °C. Arthrobacter nitroguajacolicus Rü61a [pAL1, pART2malE-ORF102 or pART2malE-ORF103] was cultivated in a mineral salts medium supplemented with 4 mM 4-hydroxyquinaldine CHIR-99021 molecular weight and 140 μg mL−1 kanamycin. Cells were harvested by centrifugation at an OD600 nm of approximately 2.5. Escherichia coli DH5α clones containing derivatives of pMal-c2x or pART2 were grown in lysogeny broth (LB) (Sambrook & Russell, 2001) at 37 °C in the presence of 100 μg mL−1 ampicillin or 50 μg mL−1 kanamycin, respectively. For the synthesis of fusion

proteins of maltose-binding protein (MBP) and the protein encoded by pAL1.102 (termed pORF102), E. coli K12 ER2508 [pLysSRARE] harboring pMal-c2x-ORF102 was grown in LB with ampicillin (100 μg mL−1), chloramphenicol (34 μg mL−1), and auto induction solutions ‘5052’ and ‘M’ (Studier, 2005) at 30 °C. Cells were harvested by centrifugation at an OD600 nm of ∼5 and stored at −80 °C before use. Total DNA of A. nitroguajacolicus Rü61a [pAL1] was isolated according to Rainey et al. (1996). Plasmid DNA was isolated using the EZNA Plasmid Miniprep kit (Peqlab, Erlangen, Germany). Gel extraction of DNA fragments from agarose gels was performed with the Perfectprep gel cleanup kit (Eppendorf, Hamburg, Germany). For cloning purposes, DNA fragments were purified using the High Pure PCR Product Purification kit (Roche Diagnostics GmbH, Mannheim, Germany).

These aerial structures are decorated with a hydrophobic coating of rodlets consisting of chaplins and rodlins. Here, we show that rodlins and the surface-active peptide SapB are essential for development during growth in a medium with high osmolarity. To this end, both vegetative and aerial hyphae secrete SapB, whereas rodlins are only secreted by the spore-forming aerial hyphae. Streptomycetes are filamentous bacteria with a complex life cycle. Spore germination and subsequent growth results in the formation of a substrate mycelium, which consists of a network of interconnected hyphae. Following a period of vegetative growth, aerial hyphae are formed that eventually septate into

chains of spores (Claessen et al., 2006). The chaplins (Claessen et al., 2003; Elliot et al., 2003) MLN0128 molecular weight and SapB (Willey et al., 1991; Tillotson et al., 1998;

Kodani et al., 2004; Capstick et al., 2007) Napabucasin in vitro have been shown to fulfill a role in spore formation. Two of eight of the chaplins, ChpE and ChpH, are secreted into the environment before aerial growth has started (Claessen et al., 2003). They lower the surface tension of the medium thereby enabling hyphae to grow into the air (Claessen et al., 2003; Sawyer et al., 2011). Aerial hyphae secrete all chaplins, ChpA-H, which assemble on the hyphal surface into an amphipathic protein film that consists of amyloid-like fibrils (Claessen et al., 2003, 2004; Capstick et al., 2011; Sawyer et al., 2011). The rodlin proteins organize these chaplin fibrils into so-called rodlets (Claessen et al., 2004). Yet, under the conditions tested, rodlins were not essential for development (Claessen et al., 2002). SapB is a lantibiotic-like peptide of 2027 Da (Willey et al., 1991; Kodani et al., 2004). Like ChpE and ChpH, SapB lowers the surface tension and thus allows hyphae to grow

into the air (Tillotson et al., 1998; Capstick et al., 2007). Production of SapB is encoded and controlled by the ramCSABR gene cluster. SapB is derived from the 42 amino acid prepeptide encoded by ramS, which is probably post-translationally modified by the action of RamC (O’Connor et al., 2002; Kodani et al., 2004; Willey et al., 2006). The ABC-transporter encoded by 3-mercaptopyruvate sulfurtransferase ramAB is generally believed to transport SapB outside of the cell (Kodani et al., 2004; Willey et al., 2006), while RamR is the transcriptional regulator that controls expression of ramCSAB (Keijser et al., 2002; O’Connor et al., 2002). Interestingly, SapB was shown to be required for differentiation on certain complex media, but not on minimal media with mannitol as the carbon source (Willey et al., 1991). Here, we show that this difference is because of the osmolarity of the medium. We furthermore demonstrate that in addition to SapB, the rodlet layer contributes to efficient aerial growth when hyphae encounter osmotic stress conditions.

intermedia ATCC 25611. In E. coli, the transcribed leader region of tnaA contains a 72-basepair (bp) region, tnaC, which encodes a 24-residue leader peptide that is necessary for tnaA operon expression. No such sequence corresponding to the leader peptide region was identified in P. intermedia ATCC 25611. The genes upstream (nhaD) and downstream (orfY) of tnaA in P. intermedia 25611 were homologues of the genes for Na+/H+ antiporter and inner membrane

protein, respectively. There was no significant level of identity between these sequences and any of the flanking genes of P. gingivalis W83, E. coli K-12, or F. nucleatum ATCC 25586 (Fig. 1a). The transcriptional regulation of the tnaA region in P. intermedia ATCC 25611 was characterized by RT-PCR. Transcripts corresponding to the regions spanning the borders buy AUY-922 of nhaD/tnaA and tnaA/orfY were undetectable, which indicated that tnaA of P. intermedia is not cotranscribed with any flanking genes (Fig. 1b). Thus, gene organization within the tnaA region of P. intermedia ATCC 25611 was more like that of P. gingivalis W83

than F. nucleatum ATCC 25586 and E. coli K-12. Given the high degree of amino acid similarity between TnaA of P. intermedia and P. gingivalis, these results suggested that the genetic origin of the tnaA region in these two bacteria may be similar. As to why tnaB was not identified at the tnaA locus in P. intermedia, it is possible that it may be located EPZ-6438 chemical structure at another locus, or may be unnecessary in these species of bacteria. Recombinant P. intermedia ATCC 25611 TnaA was expressed as a glutathione S-transferase fusion protein and then purified by cleavage of the protein bound to glutathione-sepharose 4B. Recombinant TnaA was sufficiently pure for

enzymatic characterization based on SDS-PAGE analysis. The molecular mass of the denatured polypeptide was in good agreement with the predicted molecular mass of the protein (51 kDa) (Fig. 2). To evaluate the quaternary structure of TnaA, the IMP dehydrogenase protein was examined by gel-filtration chromatography. The enzyme eluted at approximately 107.8 kDa, as estimated using a standard curve generated using commercially available protein molecular weight standards (data not shown), which corresponded to dimers of P. intermedia TnaA. This was different from the quaternary structure of P. gingivalis TnaA, which is 70% identical to P. intermedia TnaA at the amino acid level, but is stable as a tetramer (Yoshida et al., 2009). By contrast, incubation of the tetrameric form of E. coli TnaA in potassium phosphate buffer at 5 °C led to the conversion of approximately 24% of the protein to a dimeric form (Erez et al., 1998). The kinetic activity of recombinant TnaA from P. intermedia ATCC 25611 was evaluated by spectroscopy, and the results are summarized in Table 2. The Km of P. intermedia TnaA (0.23 ± 0.01 mM) was similar to that of other bacteria, including E. coli (0.32 mM), Bacillus alvei (0.27 mM), P. gingivalis (0.20 mM), and F. nucleatum (0.