In Experiment 1, we examined the

In Experiment 1, we examined the Selleckchem Torin 1 effects of unilateral lesions of CeA and/or VTA on rats’ acquisition of conditioned responses to visual cues paired with food. Contrary to the results of previous studies that examined interactions

of CeA with either SNc or DLS, rats with contralateral disconnection lesions of CeA and VTA were unimpaired in their acquisition of cue-directed responses. By contrast, rats with lesions of both structures in the same hemisphere failed to learn cue-directed responses, but were normal in their acquisition of conditioned responses directed to the food cup. In Experiment 2, we attempted to characterize the influence of VTA on CeA by examining FOS induction in CeA by a visual cue for food in rats with unilateral lesions of VTA. The results suggested an excitatory influence of VTA on CeA in the presence of food cues. Implications of these results for brain circuits involved in learned orienting and incentive motivation are discussed. “
“Synapsins are abundant synaptic vesicle (SV)-associated proteins thought

to mediate synaptic vesicle mobility and clustering at most synapses. We used synapsin triple knock-out (TKO) mice to examine the morphological and functional consequences Ganetespib of deleting all synapsin isoforms at the calyx of Held, a giant glutamatergic synapse located in the auditory brain stem. Quantitative three-dimensional (3D) immunohistochemistry of entire calyces showed lower amounts of the synaptic vesicle protein vGluT1 while the level of the active zone marker bassoon was unchanged in TKO terminals. Examination of brain lysates by ELISA revealed a strong reduction in abundance of several synaptic vesicle proteins, while proteins of the active zone cytomatrix or postsynaptic density were unaffected. Serial section scanning electron microscopy of large 3D-reconstructed segments confirmed a decrease in the number of SVs to approximately 50% in TKO calyces. Short-term depression tested at stimulus

frequencies ranging from 10 to 300 Hz was accelerated only at frequencies above 100 Hz and the time course of recovery from depression was slowed in calyces lacking synapsins. Histone demethylase These results reveal that in wild-type synapses, the synapsin-dependent reserve pool contributes to the replenishment of the readily releasable pool (RRP), although accounting only for a small fraction of the SVs that enter the RRP. In conclusion, our results suggest that synapsins may be required for normal synaptic vesicle biogenesis, trafficking and immobilization of synaptic vesicles, yet they are not essential for sustained high-frequency synaptic transmission at the calyx terminal. “
“Department of Neuroscience, Physiology and Pharmacology, University College, London, UK The K+-Cl− cotransporter type 2 is the major Cl− extrusion mechanism in most adult neurons.

, 2005, 2008; Nguyen et al, 2007) Whereas previous studies have

, 2005, 2008; Nguyen et al., 2007). Whereas previous studies have examined wag31-dependent functions by expressing the gene with an acetamide-inducible promoter (Kang et al., 2008), a tetracycline-inducible promoter (Hamasha et al., 2009), or a heat shock promoter (Kang et al., 2005), this current study is the first

to examine wag31Mtb expression using its native promoter. This promoter appears to be upregulated by the mycobacterial stringent response (Figs 1 and 2). The stringent response is necessary for persistent M. tuberculosis infections learn more in mammalian hosts (Dahl et al., 2003; Klinkenberg et al., 2010). Here, we report that the stringent response is needed for higher expression of wag31, suggesting a potential connection between Wag31 and virulence. Although Wag31 is involved in mycobacterial cell wall synthesis, Wag31 may be playing some alternative roles during the infection process. Cao et al. (2008) recently reported that Wag31Mtb stimulates XCL2 expression in macrophages. XCL2 is a chemokine in macrophages that serves as a chemoattractant for CD8+ and CD4+ T cells. Therefore, wag31Mtb expression may contribute to

the formation of granulomas that are extremely diminished in size selleck compound and in numbers in animals infected with M. tuberculosisΔrel strains (Dahl et al., 2003; Klinkenberg et al., 2010). Although traditionally thought to function as a host defense strategy, the role of the granuloma is being re-evaluated as providing a potential benefit to mycobacterial pathogens (Flynn, 2004).

Also, elevated wag31 expression may enhance M. tuberculosis survival in macrophages by enhancing resistance to oxidative stress. Wag31 may do this by stabilizing penicillin-binding protein 3 (PBP3) against cleavage by the M. tuberculosis metalloprotease Rv2869c. This metalloprotease is essential for M. tuberculosis cells heptaminol to infect mice lungs, and it likely acts to regulate the bacterial lipid and membrane composition necessary for survival in the host (Madinoshima & Glickman, 2005). However, without protection by Wag31 binding, PBP3 is susceptible to deleterious cleavage by Rv2869c, leading to reduced survival of M. tuberculosis within macrophages (Mukherjee et al., 2009). We thank Christine Davitt for assistance with TEM analysis, Gerhard Munske for help with proteomic identification of Wag31, and Mike Konkel for assistance with antibody production. This research was supported by internal funds from the University of Minnesota Duluth. “
“Eradication of Helicobacter pylori with traditional therapy often fails in clinical treatment. As a result, a novel efficacious therapeutic agent is strongly needed. Allitridi, a proprietary garlic derivative, has been successfully used to treat both systemic fungal and bacterial infections in China. Our previous study has shown a dose-dependent inhibitory effect of allitridi on H. pylori growth. However, the antibacterial mode of action of allitridi is still unclear.

Three female BALB/c mice were injected intraperitoneally with the

Three female BALB/c mice were injected intraperitoneally with the bacterial suspension at a volume of 0.5 mL. Twenty-four hours later, the mice were sacrificed, injected intraperitoneally with 1 mL of sterile PBS, kept for 1 min with gentle massage over the abdomen and then extracted. After serial dilution, the samples were spread selleck on the LB plates and incubated at 37 °C overnight.

Of the colonies recovered from the same mice, 20 were randomly picked and identified by PCR with primers O1 and O2. To calculate the competitive indices, the ratio of yncD-deleted mutant to wild type recovered from the abdominal cavity was determined and then normalized by dividing by the ratio of yncD-deleted mutant to wild type in the initial inoculum. Female BALB/c mice aged 6–8 weeks (five groups with three mice per group) were immunized once intranasally with 109 CFU of YGC102 or PBS (as control). Thirty days later, the mice of the control group were challenged with 103 CFU of wild type, whereas the mice of the other four groups were challenged respectively with 104, 105, 106 and 107 CFU of the strain using the porcine gastric mucin model as described Roxadustat manufacturer above. The survival of the mice was monitored for 7 days. A promoterless egfp gene from pEGFP-N2 was isolated by digestion with EcoRI and HindIII

and was subcloned into the corresponding sites of the pBR322 plasmid, resulting in the pBGPL plasmid. The yncD promoter region was amplified by PCR using the primers EPR1 and EPR2 (Table 1). The promoter fragment was ligated directly with PMD18-T vector and subcloned as NcoI fragments into the corresponding sites of pBGPL resulting in the pBGP plasmid. The generated plasmid was electroporated into the YGC101 strain to generate YGC104 strain. The YGC104 strain cells were inoculated into the indicated media (for the heat-shock experiment, cells were incubated at

45 °C for 10 min) and grown at 37 °C for 5 h to allow expression of enhanced green fluorescent protein (EGFP). Then, the bacteria were diluted with PBS and analyzed in a flow Teicoplanin cytometer (BD FACSCanto II) with the gates set to forward and side scatters characteristic of the bacteria. The optical detector FL1-H was used for this measurement. For each condition assessed, 10 000 bacterial cells were analyzed and the mean fluorescent intensity of the bacteria was obtained. Each experiment was performed in triplicate. Comparisons of expression values among the groups were performed by t-test. Total RNA was isolated from bacterial cells of Ty2 wild type incubated under each condition using the SV Total RNA Isolation System (Promega). Additional treatments with RNase-Free DNase I (Takara) were performed to eliminate any genomic DNA. The quantity and quality of the total RNA was determined with an ND-1000 spectrophotometer (NanoDrop). The cDNAs were synthesized using the PrimeScript RT reagent kit (Takara).

Translocation of bacteria across both monolayers may also be occu

Translocation of bacteria across both monolayers may also be occurring partly by an active invasion mechanism, and although this requires further investigation, it explains the relatively high number of bacteria translocated by Caco-2. Compared to viable bacteria, a severe reduction in transport

of heat-killed Salmonella was previously observed (Martinez-Argudo & Jepson, 2008), suggesting a role for bacterial-directed invasion in the translocation process. Previous studies have shown that V. parahaemolyticus activates the intracellular MAPK signalling pathways to exert its effects on host cells. As a result, we investigated the role of MAPK LY294002 molecular weight activation in the bacterial translocation across M cell-like co-cultures. Immunoblotting experiments demonstrated that the MAPK was endogenously activated in uninfected co-cultures and therefore no increased activation was observed upon infection with V. parahaemolyticus (data not shown).

To determine whether the MAPK pathways are involved in bacterial translocation across the co-culture model, cells were pretreated with MAPK inhibitors (15 μM SP600125, 40 μM PD98059 and 5 μM SB203580, which inhibit the JNK, p38 and ERK pathways, respectively) 2 h prior to infection and maintained throughout the experiment. Co-cultures treated with SP600125, PD98059 and SB203580 displayed 1.2-, 6.6- Obeticholic Acid and 2.0-fold decreases in translocation, respectively, 1 h postinfection (Fig. 2a). Two hour postinfection, co-cultures treated with SP600125 and PD98059 displayed a 1.3- and 1.7-fold decrease in translocation, respectively,

while cells treated with SB203580 displayed a 1.8-fold increase in bacterial translocation (Fig. 2b). Statistical analysis of the data concludes that only the differences observed between untreated wt-infected co-cultures and those-treated with the ERK pathway inhibitor at 1 h postinfection are significant. The ERK signalling pathway is one of the most important in eukaryotic cells with roles in cell proliferation, differentiation and survival. PD98059 specifically inhibits the phosphorylation of ERK by inhibiting the activity of upstream MEK1/2, with limited off-target effects Fossariinae (Davies et al., 2000). These data indicate that ERK activity plays a role in the translocation of V. parahaemolyticus across the co-culture model during the early stages of infection. Studies investigating enteropathogenic E. coli have demonstrated that the bacterial TTSS inhibit the translocation of the bacteria across co-cultures, therefore, the influence of V. parahaemolyticus TTSS on M cell-like co-culture translocation was investigated (Martinez-Argudo et al., 2007). Individual single TTSS mutants were employed as previous studies have indicated that each TTSS delivers unique effectors into the host cell and each mediates unique effects on the host cell and in vivo (Park et al., 2004a, b; Hiyoshi et al., 2010; Matlawska-Wasowska et al., 2010).

However, upon completion of their lytic cycle, they exit the cell

However, upon completion of their lytic cycle, they exit the cell using lysozymes (Moak & Molineux, 2000), which hydrolyze the same peptidoglycan bond as LTs do, but without the creation of anhydromuropeptides. ORFs encoding enzymes with LT active site-like domains (Blackburn & Clarke, 2001) have been identified within chromosomal or plasmid-borne operons associated with T3S and T4S systems (Koraimann, 2003). Koraimann (2003) termed these putative LTs ‘specialized LTs’ to indicate that they have a unique biological function Veliparib in vivo not associated with basic peptidoglycan metabolism.

The peptidoglycan-lytic activity of putative specialized LTs has often been demonstrated with zymograms on peptidoglycan-containing gels. However, proteins that bind but do not hydrolyze peptidoglycan can still produce zones of clearing on a zymogram by sequestering peptidoglycan away from the stain; for this reason, zymograms intended to demonstrate lytic activity should be interpreted with caution (Dijkstra & Keck, 1996b; Kohler et al., 2007). Work by Zahrl et al. (2005) and Kohler et al. (2007) demonstrated cleavage specificity against the MurNAc-GlcNAc linkage for a number of specialized LTs involved in T3S (IpgF, Shigella FK506 supplier flexneri; IagB, Salmonella enterica) and T4S (VirB1, Agrobacterium

tumefaciens, Brucella suis; TrbN, Pseudomonas sp.; P19, E. coli plasmid R1; HP0523, Helicobacter pylori; AtlA, Neisseria gonorrhoeae). AtlA, one of two N. gonorrhoeae LTs involved in T4S (Kohler et al., 2005, 2007), was also shown to produce 1,6-anhydromuropeptides, the definitive

sign of an LT-catalyzed reaction. Degradation by AtlA does not appear to contribute to the overall pools of peptidoglycan monomer that N. gonorrhoeae releases to the extracellular environment, suggesting that its activity is reserved for the creation of localized gaps to permit T4S system assembly (Kohler et al., 2007). Although specialized LTs degrade peptidoglycan, their activities are typically nonessential; loss of the putative LT in most cases decreases, but does not abrogate, secretion of effectors and thus virulence. The observed decreases are often due to a reduction in surface components including flagellin or needle filaments, pilin (Viollier & Shapiro, 4-Aminobutyrate aminotransferase 2003; Hoppner et al., 2004; Yu et al., 2010), and in some cases, structural components from the inner or outer membranes (Baron et al., 1997; Viollier & Shapiro, 2003). As most bacteria encode a number of different LTs, it is likely that assembly of T3S and T4S complexes can continue, albeit less efficiently, by taking advantage of temporary breaks in the sacculus that are created during normal peptidoglycan metabolism. While most studies have examined the involvement of specialized LTs in macromolecular complex assembly, other peptidoglycan-degrading activities may also be involved in this process. In fact, three different enzymatic mechanisms of peptidoglycan cleavage have been associated with flagellar assembly.

“The public health response to the spread of HIV relies on

“The public health response to the spread of HIV relies on behavioural changes, especially reductions

Rapamycin cell line in sexual and drug-use-related transmission risk behaviours (TRBs). While understanding the factors that dispose people towards risky behaviours is important scientifically, it can be difficult to distil the many predictors of sexual risk behaviours into a useful clinical tool for focused prevention efforts. Our goal was to evaluate the extent to which known predictors of sexual TRBs (self-efficacy, treatment optimism, engagement with medical care, awareness of risky behaviours, substance use, and relevant behavioural and socio-demographic characteristics) combined with additional attitude-related assessments to identify those who had engaged in recent sexual TRBs and may therefore be at risk of additional TRBs. In this study, we analysed data on beliefs and behaviours related to sex, substance use, HIV prevention and other relevant factors for 280 patients at a publicly funded HIV/AIDS clinic in Seattle. All participants completed a baseline audio computer-assisted self interview (ACASI)

as part of a larger trial focused on reducing TRBs. Our multivariate model yielded three screening questions that could prove effective in identifying HIV-positive patients in need of focused prevention resources. The MAPK Inhibitor Library supplier resulting screener holds promise as a brief and easily deployed tool that can selleck products be used by providers regardless of access to ACASI technology. Additional validation is needed and longitudinal evaluation is currently in progress. Approximately 1.3 million individuals in the USA are infected with HIV, which continues to spread at a rate of 40 000 new cases each year [1]. The development of combination antiretroviral therapies has shifted HIV infection into the realm of manageable chronic illness, with the life

expectancies of infected individuals increasing significantly over the past 20 years [2]. However, combination therapies are not yet cures, and, given the absence of an HIV vaccine, the onus for containing the spread of HIV continues to rest in the hands of those already infected (in combination with others at risk for infection). This has, in fact, been the case since the initial discovery and description of HIV, with advocates and activists from the gay community and the substance abuse treatment community promoting and helping to sustain behavioural changes to reduce the spread of HIV. At its heart, the effectiveness of the public health response to the spread of HIV relies on individual behavioural changes. There are, of course, many people who become infected with HIV without having engaged in any high-risk behaviours, but such behaviours [especially related to unprotected sex and injecting drug use (IDU)] are the clearest targets for public health interventions.

, 1994; Mullin et al, 1994;

Wingrove & Gober, 1994; Dutt

, 1994; Mullin et al., 1994;

Wingrove & Gober, 1994; Dutton et al., 2005). Direct evidence of FlbD binding to flagellar promoters in vivo has not been shown. FlbD activity is modulated by the trans-acting factor FliX that links class II flagellar assembly to class III/IV flagellar gene transcription in two ways (Wingrove & Gober, 1994; Muir et al., 2001; Muir & Gober, 2004). First, FliX stimulates the activation of class III genes by FlbD during the assembly of the basal body. Second, when flagellar assembly is blocked, FliX prevents the activation of the class III gene pathway by FlbD (Muir & Gober, 2002, 2004). Genetic and biochemical studies provide evidence for FliX binding directly to FlbD (Muir & Gober, Mitomycin C in vivo 2002, 2004) to prevent binding to ftr (Dutton et al., 2005); yet, whether FliX associates with FlbD-dependent promoters in vivo remains to be determined. TipF, a predicted 50-kDa protein with two N-terminal transmembrane domains, a coiled-coil region, and a C-terminal EAL domain, is required for flagellum biogenesis (Huitema et al., 2006). TipN, a membrane-embedded landmark protein,

dictates the proper localization of TipF and the flagellar structure (Huitema et al., 2006; Lam et al., 2006). Little is known about how TipF and TipN affect flagellar gene expression. Here, we use β-galactosidase promoter probe assays and quantitative chromatin immunoprecipitation (qChIP) analyses to explore how a ΔtipF mutation Ku-0059436 affects the activity of flagellar promoters when compared with WT, a flagellar assembly (ΔfliG) mutant, positioning

(ΔtipN), and regulatory (fliX∷Tn5 and flbD∷Tn5) mutants. These experiments reveal, for the first time, the direct quantification of the occupancy of flagellar promoters by their cognate transcriptional regulators in vivo. Caulobacter crescentus NA1000, a synchronizable derivative of the CB15 wild-type strain (Evinger & Agabian, 1977), and derivatives were grown at 30 °C in peptone yeast extract (PYE) [2 g peptone, 1 g yeast extract, 0.2 g MgSO4, and 1 mL CaCl2 (0.5 M) per liter] (Poindexter, 1964; Johnson & Ely, 1977). β-Galactosidase activity (Miller, 1972) was measured at 30 °C with log-phase cultures grown in PYE–tetracycline (0.5 μg mL−1). Assays were performed in triplicate, with a minimum of two independent cultures for each promoter construct. For the generation of anti-FlbD antibodies, FlbD was overexpressed Sitaxentan in Escherichia coli Rosetta (DE3)/pLysS using pET28a (Novagen) as an N-terminal His6-tagged variant and purified using Ni-NTA agarose (Qiagen). Purified proteins were cut out from a 12.5% sodium dodecyl sulfate (SDS) polyacrylamide gel and used to immunize rabbits (Josman LLC). Cells (20 mL) were grown to the mid-log phase and cross-linked in 10 mM sodium phosphate (pH 7.6) and 1% formaldehyde for 10 min at room temperature and on ice for 30 min thereafter. Cells were then washed three times in phosphate-buffered saline (pH 7.4), resuspended in 500 μL of TES buffer [10 mM Tris-HCl (pH 7.

However, it did not affect HIV prevalence estimates in women In

However, it did not affect HIV prevalence estimates in women. In addition, the use of mortality rates to adjust survey HIV prevalence estimates in rural South Africa increased the overall prevalence by around 7% [21]. In situations of high nonparticipation rates in surveys 3-Methyladenine chemical structure conducted in low-income settings, it has also been suggested that the data collected should be carefully verified and the interviewers should be closely monitored to ensure validity of the results [25]. The current survey did not capture all subjects

who were absent from the household at the time of the invitation and at the time of the mobile team visit. Consequently, although the actual rate of refusal to participate in the study was relatively low, the number of absences could have introduced a bias. For instance, it could be hypothesized that sick individuals tend more frequently to stay at home than healthy individuals, and thus the HIV prevalence estimates may be biased towards a higher proportion Venetoclax nmr of infected people. As reported in most sub-Saharan countries [1, 6, 22, 26, 27], a gender disparity in the prevalence of HIV infection was also found in this study in all age groups,

although the only statistically significant difference in HIV prevalence between women (30.8%) and men (17.1%) was observed in the youngest age group (aged 18–27 years). This difference may be attributable to the previously demonstrated increased vulnerability of women to HIV infection [28-30]. Biological, social and behavioural risk factors (such as age differences between sexual partners)

have been suggested to contribute to the difference in HIV prevalence between the sexes in other African countries [30, 31]. In particular, in this area male partners are on average 5 years older than their female counterparts [32]. In addition, the observed gender difference in the youngest age group may be linked to the high migration rate of men in the Manhiça area (on average 100 per 1000 person- years) which peaks in 25-year-old men [11]. This migration pattern may indeed have contributed to a reduction in the number of young men present in Manhiça at the time of the survey. In addition, as previously mentioned, nonparticipation find more of men could also lead to a lower apparent HIV prevalence in men than in women [24]. At the end of 2010, the Mozambican Ministry of Health published the final results of the first population-based national survey on HIV infection prevalence, carried out in 2009 [4]. This national survey found an overall HIV prevalence of 11.5% in individuals aged 15–49 years, and stratification by regions showed a prevalence of 19.8% for Maputo Province. The difference between the results of the current survey in Manhiça (overall prevalence of about 40%) and those of the national survey in the same province may be explained by various factors.

While the functional genomic approaches allow the parallel charac

While the functional genomic approaches allow the parallel characterization of hundreds or thousands of transcripts, proteins or metabolites, the parallel generation and characterization

of many deletion mutants was long impossible or extremely tedious. In recent years, the methods for mutant construction have been improved for several bacterial model species to a level that allowed the generation of single deletion mutants of all genes of the respective genomes, i.e. for Escherichia coli, Bacillus subtilis and Acinetobacter baylyi (Kobayashi et al., 2003; Baba & Mori, 2008; de Berardinis et FK506 nmr al., 2008). In contrast to bacteria, such an approach has not been performed with any archaeal species. Haloferax volcanii is an archaeal model species that might be the first choice for the large-scale construction and characterization of deletion mutants. Its genome is available and transcriptomics,

proteomics and metabolomics have been established (for reviews, see J. Soppa, submitted; Soppa, 2006; Soppa et al., 2008). It was one of the first archaeal species that could be transformed (Charlebois CP-673451 research buy et al., 1987) and many molecular genetic tools have been established since then. A method for the construction of markerless in-frame deletion mutants has been established (Bitan-Banin et al., 2003) and several strains and plasmids have been developed to enhance its versatility (Allers et al., 2004). Recently, the generation of vectors for mutant construction has been optimized (Hammelmann & Soppa, 2008) and the optimized method has been successfully transferred to the microtiter plate format (K. Jantzer & J. Soppa, unpublished data). Recently, an alternative optimization of vector generation has been described that has also been described to be transferrable to the microtiter plate format (Blaby et al., 2010). Therefore, the generation of markerless in-frame deletion mutants of H. volcanii

in a middle- or high-throughput fashion has become feasible. A bottleneck for such a project would be the phenotypic characterization of mutants. It would be desirable that many conditions could be analyzed in parallel and a bona fide phenotyping approach could be performed. Recently, it has been described that the growth of H. volcanii in microtiter Chloroambucil plates is in fact possible and was applied for a phenotypic comparison of two sRNA gene deletion mutants with the wild type (Straub et al., 2009). However, several problems remained, for example evaporation of water and a suboptimal variance or replicates. Therefore, here, we describe an optimized method to cultivate H. volcanii in microtiter plates. First applications are reported, for example the optimization of growth parameters and the analysis of osmotolerance and the response to oxidative stress. Furthermore, the supplementation of amino acid auxotrophic mutants is described and the bona fide phenotyping of sRNA gene deletion mutants is exemplified.

3) Furthermore, the EHNA inhibition was long lasting, because no

3). Furthermore, the EHNA inhibition was long lasting, because no activity could be detected after passage in culture medium 1 and 6 h after the EHNA treatment (Table 1). The low ADA activity detected after 24 h (0.27 ± 0.05 nmol NH3 min−1 mg−1 protein) was probably due to new trophozoites grown after the incubation in the culture medium. We have evaluated the interaction of EHNA-treated T. vaginalis on NO production by human neutrophils stimulated with T. vaginalis. Figure 4 shows that neutrophils alone produced low levels of NO (1.98 ± 0.35 μM); however, when stimulated

with lipopolysaccharide (positive control), the concentration increased 35 times (70.26 ± 14.69 μM). When the trichomonad-culture supernatants from EHNA-treated Saracatinib nmr trichomonads and the T. vaginalis lysate were incubated with neutrophils, both conditions inhibited the NO production. On the other hand, and expectedly, the co-culture with intact T. vaginalis trophozoites produced a high CP 690550 amount of NO. However, when incubated in the presence of 1 h EHNA-treated parasites, the NO production effect was reverted. The same effect was observed with adenosine and inosine. In order to identify the ADA-related sequences on T. vaginalis genome, we performed a phylogenetic analysis. NCBI blast searches

of GenBank yielded two complete T. vaginalis ADA-related sequences (XP_001317231 and XP_001325125). Semi-quantitative RT-PCR experiments were performed and the relative abundance of ADA-related genes ada(125) and ada(231) mRNA vs. α-tubulin was determined by densitometry. As shown in Fig. 5a and b, both genes were expressed, although ada(231) in higher quantity when compared with the ada(125) : α-tubulin ratio. The phylogenetic tree was constructed using the neighbor-joining method and proportional (p) distance

(Fig. 5c). Four well-resolved terminal clades supported by high bootstrap values were identified, confirming the presence of two ADA orthologues for T. vaginalis. The first clade grouped consistently ADA1 vertebrate sequences and ADA-related sequence from T. spiralis. The second clade was formed BCKDHA by E. histolytica, D. discoideum and T. vaginalis sequences. The third clade grouped the ADAL sequences, whereas the fourth clade was formed by ADA2 sequences. Plasmodium falciparum and L. major ADA-related sequences were placed independently between the four clades mentioned. Trypanosoma brucei and E. coli were the most divergent sequences. The tree topology strongly suggests homologous functions on the T. vaginalis genome. In order to screen freshly isolated clinical isolates besides TV-VP60, we have determined ADA activity in five other T. vaginalis isolates.