1 software package (Noldus Software, Wageningen, The Netherlands). The distance moved in a cage was calculated in 30-min time bins. Locomotor activity, the sleep–wake cycle and histamine release time series were

initially examined for the presence of statistically significant periods with lengths from 3 to 30 h by use of the Lomb–Scargle VE-822 manufacturer method (Ruf, 1999) implemented in lsp software (Refinetti et al., 2007). Identified periods were subjected to a multiple cosinor analysis (Bingham et al., 1982; Libre Office Calc, The Document Foundation) to obtain their mesor, orthophase and amplitude values. To verify the applicability of cosinor analysis, all of the time series were tested for zero amplitude and sinusoidality (whenever applicable). The parameters of periodicity in the population rhythm were separately estimated and tested for significance with the cosinor procedure. Cross-correlation Bafetinib analysis was performed with spss 15.0 (SPSS, Armonk, NY, USA). The correlation between histamine release and power spectrum frequencies was computed for individual mice with Spearman correlation coefficients. To obtain average correlation coefficients, the values were subjected to Fisher Z-transformation.

They were then averaged and reverse transformed. If no periodicity was detected, the data sets were compared by the use of two-way anova with time and strain as factor variables, and P ≤ 0.05 was considered to be significant. For the measurement of histamine and 1-methylhistamine concentrations and HDC and HNMT activities, samples were collected every 4 h for two consecutive days (as described above), and then approximated by use of a multiple cosinor procedure with a major period set to 24 h and a first harmonic of 12 h. When a period was considered to be non-significant, it was removed from the model, and the time series was further examined by use of a single cosinor model.

The significance levels SPTLC1 were set to P ≤ 0.05 in all experiments, unless otherwise stated. The temporal pattern of hdc transcript expression in C57BL/6J mice was assessed with quantitative radioactive in situ hybridization. It was measured in E2/E3 and E4/E5 subpopulations of histaminergic neurons in the TMN region of the hypothalamus at 4-h intervals over a period of 24 h. No significant periodicity in mRNA expression was found in either group [E2/E3, F2,27 = 2.15 (P = 0.137); E4/E5, F2,27 = 0.96 (P = 0.38); Fig. 1]. The average expression levels [mean ± standard deviation (SD)] were 0.168 ± 0.028 μCi/g/pixel for the E2/E3 group, and 0.117 ± 0.017 μCi/g/pixel for the E4/E5 group. The activity of both enzymes was measured in hypothalamic, striatal and cortical samples of C57BL/6J mice. The enzymatic activity of HDC showed no 24-h periodicity in any structures analysed (Table 1), as estimated by multiple cosinor analysis. It was approximately three-fold higher in hypothalamic samples than in the other regions.

4). AroS was readily phosphorylated, with the maximum incorporation of [γ-32P]ATP reached within 5 min as shown by the intensities of the bands in the auotoradiograph (Fig. 4a). Identification of the putative phosphoacceptor residue was carried out by site-directed Selleck Gefitinib mutagenesis of the only two histidine residues present in the phosphotransfer domain (DHp): His273 and His292. While the autophosphorylation activity of the AroS226–490H292N mutant was unaffected compared with the wild-type protein (Fig. 4c, lanes 2 and 3, respectively), the AroS226–490H273N mutant protein was defective in autophosphorylation (Fig. 4c, lane 1). Similar protein concentrations were used in these experiments

as can be seen in Fig. 4b and d. Thus, we demonstrated that AroS exhibits sensor histidine Dabrafenib datasheet kinase activity and that His273 is required for autophosphorylation most likely as the phosphoaccepting residue. 1D 1H NMR spectra of AroS226–490, AroS226–490H273N and AroS226–490H292N mutant proteins, recorded on a 1H frequency of 700 MHz on a Bruker Advance III spectrometer at 25 °C, were similar (see Supporting Information, Fig. S1), exhibiting characteristic features of a folded polypeptide, thus excluding

the possibility that the loss of autophosphorylation of AroS226–490H273N is due to protein missfolding. To address whether AroR is the cognate response regulator for AroS, an expression construct coding for the receiver domain of AroR (residues 1–125) was cloned and expressed in E. coli and recombinant protein AroR1–125 was purified. The transphosphorylation reaction was carried out such that AroS226–490 was first incubated with [γ-32P]ATP for 10 min to generate a population of phosphorylated AroS226–490 and then purified AroR1–125 was added to the reaction mixture. The transphosphorylation reaction of AroS226–490 with AroR1–125 was incubated at room temperature for 1 and 10 min. Figure 5 clearly shows the autophosphorylation of AroS226–490 and the subsequent transfer of the phosphate group to AroR1–125 (Fig.

5a, lanes 3 and 4). Phosphorylation of AroR1–125 is AroS-dependent as omission of AroS226–490 from the reaction mixture (Fig. 5a, lane 2 and c, lane 2) leads to no Aurora Kinase AroR phosphorylation – an expected observation, given that the receiver domains are unable to undergo ATP-dependent autophosphorylation. Direct phosphotransfer from AroS to AroR confirms that these two proteins are a cognate sensor response regulator pair. To determine which aspartate residue is involved in the phosphorelay mechanism, purified protein variants of AroR1–125 containing single mutations (D13N, D53N and D58N) were tested for their ability to undergo transphosphorylation. Figure 5b shows that both AroR1–125D13N and AroR1–125D53N mutants show a reduced phosphorylation level (Fig. 5b, lanes 3–6) compared with wild-type AroR1–125 (Fig.

4). AroS was readily phosphorylated, with the maximum incorporation of [γ-32P]ATP reached within 5 min as shown by the intensities of the bands in the auotoradiograph (Fig. 4a). Identification of the putative phosphoacceptor residue was carried out by site-directed RG7204 concentration mutagenesis of the only two histidine residues present in the phosphotransfer domain (DHp): His273 and His292. While the autophosphorylation activity of the AroS226–490H292N mutant was unaffected compared with the wild-type protein (Fig. 4c, lanes 2 and 3, respectively), the AroS226–490H273N mutant protein was defective in autophosphorylation (Fig. 4c, lane 1). Similar protein concentrations were used in these experiments

as can be seen in Fig. 4b and d. Thus, we demonstrated that AroS exhibits sensor histidine selleckchem kinase activity and that His273 is required for autophosphorylation most likely as the phosphoaccepting residue. 1D 1H NMR spectra of AroS226–490, AroS226–490H273N and AroS226–490H292N mutant proteins, recorded on a 1H frequency of 700 MHz on a Bruker Advance III spectrometer at 25 °C, were similar (see Supporting Information, Fig. S1), exhibiting characteristic features of a folded polypeptide, thus excluding

the possibility that the loss of autophosphorylation of AroS226–490H273N is due to protein missfolding. To address whether AroR is the cognate response regulator for AroS, an expression construct coding for the receiver domain of AroR (residues 1–125) was cloned and expressed in E. coli and recombinant protein AroR1–125 was purified. The transphosphorylation reaction was carried out such that AroS226–490 was first incubated with [γ-32P]ATP for 10 min to generate a population of phosphorylated AroS226–490 and then purified AroR1–125 was added to the reaction mixture. The transphosphorylation reaction of AroS226–490 with AroR1–125 was incubated at room temperature for 1 and 10 min. Figure 5 clearly shows the autophosphorylation of AroS226–490 and the subsequent transfer of the phosphate group to AroR1–125 (Fig.

5a, lanes 3 and 4). Phosphorylation of AroR1–125 is AroS-dependent as omission of AroS226–490 from the reaction mixture (Fig. 5a, lane 2 and c, lane 2) leads to no Dapagliflozin AroR phosphorylation – an expected observation, given that the receiver domains are unable to undergo ATP-dependent autophosphorylation. Direct phosphotransfer from AroS to AroR confirms that these two proteins are a cognate sensor response regulator pair. To determine which aspartate residue is involved in the phosphorelay mechanism, purified protein variants of AroR1–125 containing single mutations (D13N, D53N and D58N) were tested for their ability to undergo transphosphorylation. Figure 5b shows that both AroR1–125D13N and AroR1–125D53N mutants show a reduced phosphorylation level (Fig. 5b, lanes 3–6) compared with wild-type AroR1–125 (Fig.

Nonetheless, GPD activity was KU-57788 mouse detected almost exclusively in the membrane fraction. Extensive washing of the membrane preparations with increasing concentrations of NaCl (up to 1 M) in 10 mM Tris buffer (pH 7.5) with or without 10 mM EDTA did not affect the levels of GPD activity in the membranes (data not shown), suggesting that the GPD is not a loosely bound membrane protein adsorbed onto the membrane surface. The identification

of the M. hyorhinis GPD was further strengthened by showing its homology to the active GPD of M. pneumoniae and to the GPD of Thermoanaerobacter tengcongensis (GenBank accession no. 2PZ0_A) where strictly conserved residues involved in the activity were identified (Fig. 2, Shi et al., 2008). We suggest that GPD is an essential enzyme in the turnover of glycerophospholipids, the major building blocks of the lipid bilayer of M. hyorhinis membranes. First, the fatty acids are cleaved resulting in the formation of glycerophosphodiesters, which are then further cleaved by GPD to yield glycerol-3-phosphate (Schmidl et al., 2011). Upon incubation

of M. hyorhinis extracts with radiolabeled PG, a decrease in the radioactivity of the PG band with a concomitant increase in the radioactivity of the lysophospholipid and FFA fractions were noticed (data not shown), suggesting a phospholipase activity. The activity was almost exclusively associated with isolated membrane preparations (data not shown). When reaction mixtures containing M. hyorhinis membranes and the fluorescent substrate C12-NBD-PC were incubated for up to 4 h at 37 °C, two fluorescently labeled breakdown products were detected on the TLC plates, the Dapagliflozin in vivo major being C12-NBD-LPC with nonfluorescent fatty acid in position 1 hydrolyzed and the minor C12-NBD-FFA (Fig. 3), suggesting the activity of a PLA in M. hyorhinis membranes. In control experiments, using snake venom PLA2, the breakdown product of C12-NBD-PC was exclusively C12-NBD-FFA. The PLA activity of M. hyorhinis was neither stimulated by Ca2+ (0.1–10 mM) nor inhibited

by EGTA (5 mM) and had a broad pH spectrum (pH 7.0–8.5). Quantitative analysis of the fluorescence Astemizole products obtained by the hydrolysis of C12-NBD-PC by M. hyorhinis membranes is shown in Table 1. The ratio of C12-NBD-LPC to C12-NBD-FFA after treatment of C12-NBD-PC with M. hyorhinis membranes was 2.5 after a short incubation period (up to 1 h) and 0.8 after a prolonged incubation period (4 h), suggesting that M. hyorhinis possess a nonspecific PLA activity capable of hydrolyzing both position 1 and position 2 of the C12-NBD-PC, but with a somewhat higher affinity to position 1. The possibility that M. hyorhinis possess a PLA1 (Istivan & Coloe, 2006) or PLA2 (Rigaud & Leblanc, 1980) as well as a lysophospholipase (Gatt et al., 1982) was excluded as we were unable to demonstrate lysophospholipase activity using C12-NBD-LPC (data not shown). The in silico analysis of M.

, 1999). Membrane topology of Chr3N and Chr3C is antiparallel. The C-terminal end of Chr3N is located in the cytoplasm, whereas the C terminus of Chr3C lies in the periplasm (Fig. 1b and d). Jiménez-Mejía et al. (2006) reported a 13-TMS topology for P. aeruginosa ChrA protein, a member of the long-chain CHR family of the CHR superfamily. The two homologous halves of ChrA,

formed by six TMSs each, displayed antiparallel membrane topology between them. It was proposed that this structure arose from the duplication of an equally oriented six-TMS ancestral protein domain followed by insertion of a central TMS (TMS7); this insertion might have caused the repeated domains to adopt the opposite orientation in a native parallel structure (Jiménez-Mejía check details et al., 2006). Topologic inversion of halves of membrane proteins has been widely reported and is considered a common evolutionary process for these polypeptides (Ichihara et al., 2004;

Rapp et al., 2006). It was proposed that membrane proteins with two antiparallel domains arose from ancestral monodomain proteins with dual topology (Rapp et al., 2006), that is, proteins that may insert into the membrane in either orientation (a ‘flip-flopping’ protein; Bowie, 2006). This dual topology ancestor may form homodimers displaying opposite orientation in the membrane. Gene duplication followed by sequence divergence would result in heterodimeric proteins with subunits of fixed but opposite orientation. Experimental evidence supporting this evolutionary Selleck Gefitinib pathway has been obtained from the analysis of proteins of the small multidrug resistance (SMR) family (reviewed in Bay et al., 2008). Antiparallel

arrangement of E. coli homodimeric EmrE transporter has been widely reported (see Chen et al., 2007), although a parallel structure oxyclozanide has also been claimed (Steiner-Mordoch et al., 2008). Another SMR family member, the EbrAB protein pair, has also been assigned antiparallel membrane topology (Kikukawa et al., 2007). Closely homologous proteins RnfA and RnfE from E. coli (Saaf et al., 1999) and NqrD and NqrE from Vibrio cholerae (Duffy & Barquera, 2006), both pairs being NADH-oxidoreductases constituted by six-TMS monomers, showed a completely opposite membrane topology. Members of several 10-TMS transporter families are also constituted by 2 five-TMS repeat units arranged in opposite membrane orientations (Saier, 2003; Lolkema et al., 2005). Aquaporins (Murata et al., 2000), ClC chloride channels (Dutzler et al., 2002), AmtB ammonia transporters (Khademi et al., 2004), and members of the DUF606 family of bacterial transporters (Lolkema et al., 2008) are all additional examples of proteins composed of two repeated halves with opposite membrane orientations. Indeed, the antiparallel domain organization is observed more frequently in the 3D structures of membrane proteins than the parallel domain organization (Lolkema et al., 2008).

bovis/BCG narK2X promoter inactive. To confirm that no other trans-acting factor (e.g. repressor) contributed to the loss of promoter activity in M. bovis/BCG, the pnarK2 plasmid (harbouring the M. tb WT narK2 promoter) was introduced into M. bovis and BCG strains and the GFP reporter assay was performed under hypoxic conditions. The M. tb WT narK2X promoter was well induced and to the same level in M. bovis and BCG as in M. tb (Table 3), which suggests that the −6TC mutation, and not a trans-acting negative regulator, is responsible for the absence

of narK2X promoter activity in M. bovis and BCG. To further validate that the M. tb narK2X promoter behaves similarly in M. bovis/BCG and M. tb, two additional truncated narK2X promoter GSK1120212 cost selleck GFP reporter constructs pnarK2Δdown and pnarK2Δup (described in Chauhan & Tyagi,

2008a) were also assessed for GFP fluorescence in M. bovis and BCG under hypoxic conditions. In pnarK2Δdown, the so-called ‘downstream inhibitory region’ is removed (+14 to +57 with respect to M. tb narK2X TSP), whereas in pnarK2Δup, the so-called ‘upstream activating region’ (described by Hutter & Dick, 2000) is deleted (−122 to −220 relative to M. tb narK2X TSP). A similar level of hypoxic induction was observed for both promoter constructs in all three strains (Table 3), demonstrating that the M. tb narK2X promoter has similar activity in M. tb, M. bovis and BCG. These results suggest that all the trans-acting (including DevR) and cis elements that control the narK2X promoter are functionally conserved in M. tb, M. bovis and BCG, except for the −6T/C SNP. A putative −10 element was recognized upstream of the experimentally detected TSP of narK2 that was reported previously (Chauhan & Tyagi, 2008a). In the present study, it was functionally characterized by individually mutating additional nucleotides at −4, −5, −7 and −8 positions with respect to the TSP (Fig. 1).

Both −4AC Tacrolimus (FK506) and −5TC mutations significantly or completely reduced the promoter activity and demonstrated the importance of these nucleotides in promoter function. Taken together, the results of mutation analysis indicate that ‘ATT’ nucleotides present at −4, −5 and −6 positions are essential for promoter activity and likely to be recognized by the transcriptional machinery. Note that the −5TC mutation was reported to be present in BCG by Hutter and Dick (2000), and the introduction of this mutation significantly reduced inducible promoter activity by ∼7-fold, but did not abolish it (Fig. 1). The −7GC or −8GT mutations, which also altered the overlapping putative SigC −10 sequence, did not adversely affect narK2X promoter activity. Although the −10 element of the narK2X promoter showed only a modest resemblance (2/6) to the SigA consensus sequence (Unniraman et al.

It is reasonable to expect that geographically/ecologically distinct populations of streptococci might be responsible for the absence of some sk gene alleles or detection of novel ones. Carfilzomib mw The sk5 allele was the most commonly found variant detected in 13 (17%) of all 76 strains. The most prevalent gene alleles among GAS isolates were sk1, sk5, sk16 and sk18. GCS and GGS strains were distributed among sk5, sk6, sk10, sk11, sk16 and sk17 gene alleles (Fig. 2). Although six variants including sk5, sk10, sk11, sk12, sk13 and sk14 were previously

reported as skcg gene-specific alleles (Tewodros et al., 1996), we could identify several GAS strains among our isolates that belonged to sk5 and sk11 variants. This finding is in accordance with prior proposition on horizontal gene transfer of either the entire sk or fragments of sk between GAS and GCS/GGS strains (Kalia & Bessen, 2004). Therefore, the presence of particular gene alleles might not be restricted to GCS/GGS or GAS strains, and their detection might be solely dependent on the population of the streptococci under study and the geographical regions from where they were isolated. While

the majority of GCS/GGS isolates in the present study were classified in previously identified sk gene alleles (sk5, sk6, sk10 and sk11), most of GAS isolates belonged to the new allelic variants (sk15-sk28). This finding is consistent with the prior hypothesis for high intragenic recombination levels of ska, which accounted for the high variation rate of ska among GAS (Kapur Y-27632 research buy et al., 1995; Kalia & Bessen, 2004). Although a number of sk gene alleles such as sk1, sk2 and sk6 were previously

proposed as SKN (Malke, 1993), in accordance with several other reports (Tewodros et al., 1993, 1996; Haase et al., 1994), identification of these alleles in our study among strains that were isolated from uncomplicated clinical diseases (Fig. 2) implies that Histone demethylase there is no association between sk allelic variants and disease manifestation. As shown in Fig. 2, a wide range of Plg activation levels displayed by different SK variants ranged from 9 to 182 IU mL−1. These results are consistent with previous observations for a wide variation of SK activity levels in a PCR/RFLP pattern (Tewodros et al., 1995) or even in a specific SK cluster (McArthur et al., 2008). In fact, beside SK variations in their primary structure, other upstream regulatory regions of SK gene were also proposed for differences in SK activities of the streptococci (Malke et al., 2000). SDS-PAGE analysis of the mid log phase proteins of the culture supernatants (as expected) did not show the presence of either the zymogene (40 kDa) or the active form (28 kDa) of the SpeB protease (Fig. S1). It indicated the reliability of SK activity data (i.e.

europaea, we extended our study to test whether psRNA11, like RyhB, is also an iron-dependent sRNA. The transcript levels of psRNA11 under iron-replete and iron-depleted conditions were examined by real-time PCR and Northern analysis (not shown) in wild type and fur:kanP mutant N. europaea strains. Compared with wild-type

cells grown under iron-replete conditions, transcript levels for psRNA11 in wild-type cells slightly increased when iron was limited. In the fur:kanP mutant, the psRNA11 transcript levels were about 50% higher in both iron-replete and iron-depleted conditions, relative to that in the control wild type grown in iron-replete conditions. The sdhC transcript levels decreased significantly in wild-type N. europaea in iron-depleted conditions, and in mutant N. europaea, regardless this website of iron availability.

Another putative target of pRNA11, the FecI-like ECF σ factor encoded by NE1071 was upregulated ABT-888 order in iron-limited conditions in wild-type cells, and in the fur:kanP mutant, the transcript levels increased almost four times in both iron-replete and iron-depleted conditions, suggesting the involvement of Fur in the regulation of psRNA11 (Fig. 2a). Compared with untreated cells, the transcript levels for sdhC and sdhA were significantly lower in chloromethane- and chloroform-treated cells (Gvakharia et al., 2007). The transcript levels of psRNA11, sdhC, and sdhA were also analyzed in chloroform- and chloromethane-treated wild-type cells. In chloromethane-treated cells, psRNA11 was at significantly higher levels after 30 min of treatment (Fig. 2b). In chloroform-treated cells, psRNA11 was slightly at higher levels after 30 min (Fig. 2b). The results of real-time PCR Northern analysis, and microarrays experiments support the notion that psRNA11 influences the transcription of the of sdhCDAB operon. Recent systematic searches of bacterial genomes have considerably

increased the number of known small RNAs (Sittka et al., 2008). Direct cloning and parallel sequencing applied to the bacterial genome of V. cholerae demonstrated the complexity of the sRNA component of a bacterial transcriptome (Liu et al., 2009). Although the number of identified sRNAs in bacteria is Epothilone B (EPO906, Patupilone) increasing, the biological role of the vast majority of these noncoding genes is still unclear. The present study was motivated by extensive analysis of N. europaea transcriptome in response to various stimuli, in which some changes in gene transcriptional profiles were explained by documented regulatory mechanisms in N. europaea, while others were not (Gvakharia et al., 2007). We hypothesized that sRNAs are part of a regulatory network that regulates bacterial adaptation to environmental changes and stress conditions and may be responsible for some of the unexplained changes in gene transcriptional profiles observed in N. europaea.

pneumophila (Newton et al., 2007; D’Auria et al., 2008) and would therefore also be an important aspect in host–pathogen interaction. The VNTR

analysis performed at our lab (Coil et al., 2008) identified a gene with a VNTR region that displayed a high homology with eukaryotic collagen. Here, we describe the initial characterization of this L. pneumophila gene, lpg 2644, with a VNTR region, encoding an outer membrane motif and containing a collagen-like repeat region. The gene was therefore annotated lcl (Legionella collagen-like). The origin of strains and the selection based on sequence-based type (SBT) and repeat pattern are described in detail Selleck JNK inhibitor elsewhere (Coil et al., 2008). Legionella strains were grown at 37 °C on buffered charcoal yeast extract (BCYE) agar plates or in buffered yeast extract broth supplemented with α-ketoglutarate, l-cysteine and ferric pyrophosphate (Edelstein, 1981), Escherichia coli was grown in Luria–Bertani medium (Miller, 1972), and if necessary, supplemented with ampicillin (50 μg mL−1) or chloramphenicol (25 μg mL−1). Strains were grown overnight Ribociclib in 5 mL of BCYE. Genomic DNA was isolated from 1 mL of this culture using a Wizard® Genomic DNA Purification Kit (Promega) according to the manufacturer’s recommendations.

The quality of the DNA was assessed by agarose gel electrophoresis. Standard PCRs were carried out using SuperTaq (HT Biotechnology). PCR amplification of the VNTR region of the lpg 2644 gene was accomplished with the primers 5′-TCACATCACAGATAGC-3′ and 5′-TTCCCAGCTCATTACG-3′, designed on the chromosome of L. pneumophila Philadelphia-1. Chromosomal

DNA from the different Legionella isolates was used as a template. The VNTR DNA fragments of lpg 2644 of all 108 strains were cloned into pGEM-T Rutecarpine Easy (Promega), introduced into TG1 competent cells and the constructs were purified using the Wizard Plus SV Minipreps DNA purification system (Promega). The size of the insert was checked through electrophoresis, using the initial PCR product as a reference for size. One clone that contained an insert of the exact size was selected for sequencing. Sequencing reactions were performed on this template DNA at the VIB Genetic Service Facility (Antwerp, Belgium). Acanthamoeba castellanii ATCC30234 was cultured in Acanthamoeba medium (PYG712) at room temperature. The THP-1 or U937 cell line was differentiated into macrophage-like cells by treatment with phorbol 12-myristate 13-acetate for 72 h in RPMI medium, containing 10% heat-inactivated fetal calf serum and 2 mM l-glutamine, at 37 °C and 5% carbon dioxide (CO2). The A549 cell line, a lung epithelial cell carcinoma, was maintained in DMEM medium, supplemented with 10% heat-inactivated fetal calf serum and 2 mM pyruvate, at 37 °C and 5% CO2. The lcl gene was amplified from L.

9), 1 mM dithiothreitol and 10 μg mL−1 leupeptin), resuspended in 833 μL of buffer A at a density of 6 × 108cells mL−1 and incubated on ice for 5 min. Epimastigotes were then permeabilized with 250 μg mL−1l-α-lysophosphatidylcholine palmitoyl for 1 min at 4 °C, washed twice with buffer A and brought to a final volume of 50 μL in buffer A. An equal amount of transcription cocktail buffer (75 mM sucrose, 20 mM potassium chloride, 3 mM magnesium chloride,

1 mM dithiothreitol, 10 μg mL−1 leupeptin, 25 mM creatinine phosphate, 0.6 mg mL−1 creatinine kinase, 2 mM ATP, 1 mM CTP and 1 mM GTP) containing 50 μCi of [α-32P]-UTP was added, followed by incubation at 28 °C. The time course was then monitored

by removing 5-μL aliquots at the indicated times (Fig. 1a). Macromolecules were precipitated Epacadostat nmr with cold (4 °C) trichloroacetic acid (TCA) containing 10 μg mL−1 of carrier tRNA and immobilized on a GF/C filter (Whatman). After these filters were washed with cold 10% TCA and dried, radioactivity was quantified by liquid scintillation. Additionally, a suspension MK0683 mw of isolated nuclei was used for the transcription assays. The nuclei were prepared essentially according to published methods for a related trypanosomatid (Martínez-Calvillo et al., 2001). Axenic cultures of T. cruzi epimastigotes undergo an exponential growth phase followed by a logarithmic transition phase before entering the stationary phase, in which the cells stop dividing. To compare the transcription rate 2-hydroxyphytanoyl-CoA lyase (RNA biosynthetic activity) of exponentially growing and stationary epimastigotes under our culture conditions, [α-32P]-UTP incorporation was measured in cells permeabilized with lysolecithin (Fig. 1a) and in nuclear suspensions (Fig. 1b). In both cases, epimastigotes in the exponential growth phase exhibited higher transcription

activity than cells derived from the stationary phase. Relative figures from the initial linear phase of the graphs indicate an approximately sixfold difference in permeabilized cells and 10-fold difference in the nuclear preparations. The higher estimate of activity in the nuclear suspension may be due to faster distribution of reactants in the assay. Based on published data, the vast majority of cellular transcription in T. cruzi corresponds to rRNA (Elias et al., 2001), which is synthesized in the nucleolus of eukaryotic organisms. Additionally, it is generally accepted that nucleolar organization correlates with cellular proliferation activity. To explore potential size differences in the nucleoli of epimastigotes growing in the exponential and stationary growth phases, nuclei from cultured cells were analysed by standard transmission electron microscopy.