No such enhancement was observed in the thi3Δ strain. However, Pdc2p expressed striking transactivation activity in a Thi3p-independent fashion when the C-terminal region containing the Thi3p-interacting domain was shortened (Nosaka et al., 2008). Based on these observations, we proposed a mechanism for the transcriptional activation of THI genes mediated by Pdc2p in response to thiamin starvation as follows. When intracellular check details TPP is abundant and occupies the TPP-binding sites of Thi3p, the C-terminal domain of Pdc2p masks the internal domain responsible for the transactivation activity. Upon thiamin deprivation,

the dissociation of TPP from Thi3p is followed by the interaction of Thi3p with the C-terminal domain

of Pdc2p, which in turn causes a conformational change in Pdc2p. As a result, the C-terminal domain is removed from the transactivation domain; thus, Pdc2p can exert full transactivation activity by recruiting general transcription factors efficiently. It is likely that Pdc2p binds the upstream region of THI genes, and Mojzita & Hohmann (2006) noted that Pdc2p actually binds DNA, although the experimental see more data were not published. In this paper, we demonstrated, using chromatin immunoprecipitation (ChIP) assays, that Pdc2p interacts with the upstream region of THI genes, the sequences of which are different from the target sequence of Thi2p. It was also found that Pdc2p interacts with PDC5. Interestingly, the association of Pdc2p or Thi2p with the target DNA sequences of THI genes was enhanced by thiamin starvation, whereas the association of Pdc2p with the PDC5 promoter was unaffected. Furthermore, we identified a DNA element in the upstream region of

PDC5, which can bind to Pdc2p and is required for the expression of PDC5. The TA-cloning vector pGEM® T-Easy (Promega) was used to clone PDC2 gene and the PDC5 promoter isolated from yeast genomic DNA by PCR using Ex Taq™ DNA polymerase (Takara Bio, Otsu, Japan) with specific primers. The expression vectors are listed in Table 1. In general, the target sequence was PCR-amplified from the vector pGEM-PDC2 or pGEM-PDC5-promoter OSBPL9 using specific primers into which restriction sites were designed, and the fragment obtained was digested with the restriction enzymes and subcloned into expression vectors. The PDC5 promoter-lacZ plasmids (B593ΔX series) carried an in-frame fusion between the inserted promoter-associated start codon and the lacZ coding sequence. All PCR primers are available on request. Escherichia coli strains DH5α and BL21(DE3)pLysS were used to amplify plasmids and express the recombinant proteins, respectively. Saccharomyces cerevisiae strains YPH500 (MATα ura3-52 his3-Δ200 leu2-Δ1 trp1-Δ63 ade2-101 lys2-801), NKC18 (thi3::HIS3 in YPH500), and NKC19 (thi2::HIS3 in YPH500) (Nosaka et al., 2005) were used in this study.

No such enhancement was observed in the thi3Δ strain. However, Pdc2p expressed striking transactivation activity in a Thi3p-independent fashion when the C-terminal region containing the Thi3p-interacting domain was shortened (Nosaka et al., 2008). Based on these observations, we proposed a mechanism for the transcriptional activation of THI genes mediated by Pdc2p in response to thiamin starvation as follows. When intracellular Target Selective Inhibitor Library high throughput TPP is abundant and occupies the TPP-binding sites of Thi3p, the C-terminal domain of Pdc2p masks the internal domain responsible for the transactivation activity. Upon thiamin deprivation,

the dissociation of TPP from Thi3p is followed by the interaction of Thi3p with the C-terminal domain

of Pdc2p, which in turn causes a conformational change in Pdc2p. As a result, the C-terminal domain is removed from the transactivation domain; thus, Pdc2p can exert full transactivation activity by recruiting general transcription factors efficiently. It is likely that Pdc2p binds the upstream region of THI genes, and Mojzita & Hohmann (2006) noted that Pdc2p actually binds DNA, although the experimental buy ABT-263 data were not published. In this paper, we demonstrated, using chromatin immunoprecipitation (ChIP) assays, that Pdc2p interacts with the upstream region of THI genes, the sequences of which are different from the target sequence of Thi2p. It was also found that Pdc2p interacts with PDC5. Interestingly, the association of Pdc2p or Thi2p with the target DNA sequences of THI genes was enhanced by thiamin starvation, whereas the association of Pdc2p with the PDC5 promoter was unaffected. Furthermore, we identified a DNA element in the upstream region of

PDC5, which can bind to Pdc2p and is required for the expression of PDC5. The TA-cloning vector pGEM® T-Easy (Promega) was used to clone PDC2 gene and the PDC5 promoter isolated from yeast genomic DNA by PCR using Ex Taq™ DNA polymerase (Takara Bio, Otsu, Japan) with specific primers. The expression vectors are listed in Table 1. In general, the target sequence was PCR-amplified from the vector pGEM-PDC2 or pGEM-PDC5-promoter Dolutegravir supplier using specific primers into which restriction sites were designed, and the fragment obtained was digested with the restriction enzymes and subcloned into expression vectors. The PDC5 promoter-lacZ plasmids (B593ΔX series) carried an in-frame fusion between the inserted promoter-associated start codon and the lacZ coding sequence. All PCR primers are available on request. Escherichia coli strains DH5α and BL21(DE3)pLysS were used to amplify plasmids and express the recombinant proteins, respectively. Saccharomyces cerevisiae strains YPH500 (MATα ura3-52 his3-Δ200 leu2-Δ1 trp1-Δ63 ade2-101 lys2-801), NKC18 (thi3::HIS3 in YPH500), and NKC19 (thi2::HIS3 in YPH500) (Nosaka et al., 2005) were used in this study.

The finding that reported levels of pain and functional disability increased markedly with time since diagnosis is to be expected, given the progressive nature of the disease. The fact that reports of negative emotions, depression and a negative outlook towards the future (with respect to their pain) also rose following diagnosis is particularly interesting, and may suggest a psychological element to the increased reports of pain. Indeed, a study conducted by Veale

et al.[31] found that patients Trametinib diagnosed with OA reported a significantly reduced quality of life relative to people who fulfilled the criteria for OA, but had not yet been informed of their ‘diagnosis’. This would appear to indicate that psychological factors

play a major role in the pain and disability associated with arthritis, Crizotinib order and highlights the need to address psychosocial health in any effective patient-centric management program. Current management protocols were generally regarded as being of only medium effectiveness, a result that is largely in line with previous studies, and indicative of the current lack of effective management programs and interventions.[31] However, the levels of supplement and over-the-counter (OTC) medication usage were significantly higher than those reported in other studies.[32, 33] It is unclear why this is the case, although it is possible that the higher prevalence of ‘self-diagnosed’ patients within the cohort may lead to an increased reliance on readily obtainable supplements and OTC pain medications. However, the estimates are in line with findings in the US that suggest Avelestat (AZD9668) that between 30% and 47% of older adults with OA use complementary or alternative medicine (CAM).[34,

35] US expenditures for CAM therapies averaged $1127 per year per patient, compared with $1148 for traditional therapies and musculoskeletal conditions account for 16% of CAM use.[36] Concerns about the side-effects associated with prescription medications such as non-steroidal anti-inflammatory drugs (NSAIDs) were also common, resulting in relatively high non-compliance rates, and potentially a move toward supplements and OTC medications that may be erroneously viewed as ‘safer’ within the community. The fact that more patients were using supplements than were undertaking other patient-centric interventions such as weight loss and exercise – that actually have been shown to improve pain and functional disability – highlights the need for increased patient education and information.[10] Two major limitations of this study are common to virtually all online questionnaires. The first is the issue of self-reporting, and the inherent difficulties involved in requiring patients to describe their own conditions, disease states and treatments.

The amygdala consists of many nuclei that are extensively interconnected. The basolateral amygdaloid complex (BLA), which includes the lateral (LA) and basal (BA) nuclei, is considered to be an important site where sensory inputs converge and associations between the CS and the US are formed (Maren, 1999; LeDoux, 2000). Surrounding the BLA are γ-aminobutyric acid containing (GABAergic) interneurons of the intercalated cell masses (ITCs), which are thought to gate Pembrolizumab information flow into and out of the BLA (Paréet al., 2004; Marowsky et al., 2005; Pape, 2005). These structures influence the central nucleus of the amygdala

(CEA), a major source of output neurons projecting to downstream targets (LeDoux, 2000). Conditioned fear responses can be inhibited by repeated non-reinforced presentations of the CS – a process termed extinction (Myers & Davis, 2007). Both fear conditioning and extinction are NMDAR-dependent (LeDoux, 2000; Myers & Davis, 2007). NMDAR-dependent synaptic plasticity has been described in various nuclei of the amygdala, including the LA (Blair et al., 2001), BA (Maren & Fanselow, 1995; Chapman et al., 2003), ITCs (Royer & Paré, 2002) and CEA (Fu & Shinnick-Gallagher, 2005; Samson & Paré, 2005). As PN-1 can regulate NMDAR function and synaptic plasticity, we compared the acquisition and extinction

of auditory fear conditioning in PN-1 KO mice and wild-type (WT) littermates. Then, in order to determine if the pattern of fear conditioning- http://www.selleckchem.com/B-Raf.html and extinction-induced biochemical responses distributed over the different nuclei of the amygdala was altered in these mice, we immunohistologically analysed Fos protein expression and, using immunoblot analysis of extracts of laser-microdissected subregions, measured phosphorylated alpha-calcium/calmodulin protein kinase II (pαCamKII) levels. PN-1 heterozygote mice (Lüthi et al., 1997) and PN-1HAPN−1-lacZ/HAPN−1-lacZ (PN-1 reporter mice; Kvajo et al., 2004) were derived and backcrossed into the C57BL/6J (RCC, Füllinsdorf, Switzerland) background in our animal facility. Heterozygous mating generated PN-1−/− (PN-1 KO) and PN-1+/+ (WT) littermates. All Anacetrapib experimental animals

were male, except females were used for PN-1 immunohistology, 4–8 months old, housed on a 12-h day/night cycle with ad libitum access to food and water. Mice were singly housed for at least 2 weeks for all experiments. A total of 101 mice were used in these experiments. All animal experiments were approved by the Swiss Veterinary Authorities and carried out in accordance with the European Communities Council directive (86/609/EEC). All studies took place during the light portion of the cycle. Mice were handled gently for 2–5 min/day for 5 days. Fear conditioning and extinction sessions took place in two different contexts, basically as described (Herry et al., 2006). Briefly, mice were submitted to fear conditioning protocols in which a 30-s tone CS (7.

Short-interval intracortical inhibition assesses the excitability of intrinsic GABAA circuits in the motor cortex (Di Lazzaro et al., 1998). In our experiments, attention to one area of the skin had no effect on SICI evoked in a nearby hand muscle; in contrast, SICI was reduced (i.e.

less effective inhibition) in a distant muscle. At first sight, the lack of effect in nearby muscles differs from that reported by Thomson et al. (2008) who found that SICI was reduced in the FDI muscle when participants selleck chemicals attended to cutaneous input from the index finger. However, Thomson et al. (2008) required participants to react to the cutaneous input by abducting the index finger, whereas there was no motor requirement in the present task. In addition, they did not compare Panobinostat cell line the amount of SICI with that seen at rest (as in the present task), but with the amount of SICI that

was measured when participants received inputs to the opposite hand. The reduction in SICI that we observed in a muscle distant from the locus of attention was unexpected and has not been reported previously by others. Indeed, the combined results from experiments 1 and 2 suggest that there may even be a spatial gradient in this effect as attention to the skin in the mid-dorsum had no effect on SICI in experiment 1, whereas attention to the skin overlying the ADM muscle reduced SICI in experiment 2. This contrasts with the findings of Conte et al. (2008) who found that attention to the hand in

general had no effect on SICI in a hand muscle. In addition, Ridding & Rothwell (1999) noted that electrical stimulation of cutaneous afferents had much no effect on SICI in distant muscles. A likely explanation is that our task differed from previous work in terms of the specificity of the locus of attention, task difficulty as well as different methodological approaches, such as the definition of the baseline resting state [listening to music or reading (Rosenkranz & Rothwell, 2006), closing eyes (Conte et al., 2007), resting with eyes open (Thomson et al., 2008) or the combination of attention paradigms with motor tasks or with simultaneous vibration input to the hand]. It could be, for example, that individuals in the experiments of Conte et al. (2007) paid attention to varying regions of the hand at different times throughout the experiment, so that no overall effects on SICI were seen. The decreased SICI observed in muscles distant from the focus (internal focus) is similar to the decreased SICI during the visual discrimination task (external focus). In both cases, the muscle studied is distant from the locus of attention, and could, as in the visual task, be affected by a general increase in arousal during task performance.

This work was funded by the Università Cattolica

del Sacro Cuore, progetti di ricerca d’interesse d’Ateneo – D.3.2 – Anno 2006 to R.C., Lattobacilli contro l’influenza aviare. “
“Persisters are suggested to be the products of a phenotypic variability that are quasi-dormant forms of regular bacterial cells highly tolerant to antibiotics. Our previous investigations revealed that a decrease in antibiotic tolerance of Escherichia coli cells could be reached through the inhibition of key enzymes of polyamine synthesis (putrescine, spermidine). We therefore assumed that polyamines could be involved in persister cell formation. Data obtained in our experiments with the polyamine-deficient E. coli strain demonstrate that the formation of persisters tolerant to netilmicin is highly BMN 673 nmr upregulated by putrescine in a concentration-dependent manner when cells enter the stationary phase. This period is also accompanied by dissociation selleck chemical of initially homogenous subpopulation of persister cells to some fractions differing in their levels of tolerance to netilmicin. With three independent experimental approaches, we demonstrate that putrescine-dependent upregulation of persister cell formation is mediated by stimulation of rpoS expression. Complementary

activity of putrescine and RpoS results in ~ 1000-fold positive effect on persister cell formation. “
“The ataxic sticky (sti/sti) mouse is a spontaneous autosomal recessive mutant resulting from a disruption in the editing domain of the alanyl-tRNA synthetase (Aars) gene. The sticky phenotype is characterized by a small Phosphatidylinositol diacylglycerol-lyase body size, a characteristic unkempt coat and neurological manifestations including marked tremor and ataxia starting at 6 weeks of age. The present study was undertaken to examine the spatiotemporal features of Purkinje cell degeneration in the sticky mouse. Purkinje cell loss was found to be both progressive and patterned, with vermal lobules VI, IX and X, crus 1 of the hemisphere, and the flocculus

and paraflocculus being differentially resistant to degeneration. The pattern of Purkinje cell degeneration in sticky is not random – in general, the sphingosine kinase 1a-immunonegative Purkinje cell subset is preferentially susceptible to early cell death. In addition, zebrin II/aldolase C expression in the sticky cerebellum is profoundly downregulated, whereas the heat-shock protein 25 is both ectopically expressed in some scattered Purkinje cells and downregulated in other Purkinje cells in which it is normally expressed constitutively. Compared with many mouse mutants with patterned Purkinje cell death, in which successive stripes of cell loss are very clear, Purkinje cell loss in sticky shows a less clear-cut pattern between different Purkinje cell subtypes, with the result that preferential survival is less dramatic. This may represent a secondary consequence of the downregulation of zebrin II expression.

Recent literature has described the emergence of a multidrug-resistant USA-300 strain

that has accumulated genes conferring resistance not only to beta-lactams, but also to fluoroquinolones, tetracyclines, macrolides, clindamycin and mupirocin [17]. These strains appear to be common among MSM, with an overall prevalence of 26 cases per 100 000 persons in MSM in one study where MSM status was identified as a risk factor for multidrug-resistant USA-300 strain, independent of HIV status [18]. Selleck Dasatinib Of all MRSA infections among our MSM population, only one (5.3%) was a multidrug-resistant isolate, and it was not a USA-300. According to our definition of multidrug resistance (resistance Dinaciclib manufacturer to more than two classes of antimicrobials other than beta-lactams) we had a total of eight multidrug-resistant isolates,

five of which were USA-300. Our study had several limitations. Not all patients seen in our ID clinic undergo MRSA surveillance cultures, not all isolates were available for PFGE, and complete antibiotic susceptibilities were not reported for each isolate. Additionally, although ART use within the previous year was documented, patient compliance was not assessed in our study. Unlike previous findings, our multivariate analysis did not show an increased risk of MRSA colonization or infection among injecting drug users, MSM or patients with prior incarceration. This may be explained

by our patient population, our sample size, or underreporting of homosexual activity and IDU. In summary, MRSA infection, and specifically USA-300 CA-MRSA infection, occurs in HIV-infected patients. As previously demonstrated, prior antibiotic exposure and a CD4 count <200 cells/μL proved to be independent risks for colonization or infection with MRSA in our study population. Most interesting was our novel finding that HIV-infected patients who received ART in the past year had a significantly decreased risk of MRSA colonization or infection. Further studies are warranted to determine whether HIV-infected patients with recurrent MRSA infections should be considered for earlier Mirabegron initiation of ART or offered decolonization. “
“The aim of the study was to determine the prevalence and risk factors for HIV-associated fatigue in the era of highly active antiretroviral therapy (HAART). A cross-sectional survey of 100 stable HIV-infected out-patients was carried out. Severity of fatigue was measured using the Fatigue Impact Scale (FIS). Symptoms of orthostatic intolerance (dysautonomia) were evaluated using the Orthostatic Grading Scale (OGS). Data for HIV-infected patients were compared with those for 166 uninfected controls and 74 patients with chronic fatigue syndrome (CFS)/myalgic encephalomyelitis (encephalopathy) (ME).

Recent literature has described the emergence of a multidrug-resistant USA-300 strain

that has accumulated genes conferring resistance not only to beta-lactams, but also to fluoroquinolones, tetracyclines, macrolides, clindamycin and mupirocin [17]. These strains appear to be common among MSM, with an overall prevalence of 26 cases per 100 000 persons in MSM in one study where MSM status was identified as a risk factor for multidrug-resistant USA-300 strain, independent of HIV status [18]. EPZ015666 mw Of all MRSA infections among our MSM population, only one (5.3%) was a multidrug-resistant isolate, and it was not a USA-300. According to our definition of multidrug resistance (resistance INK 128 solubility dmso to more than two classes of antimicrobials other than beta-lactams) we had a total of eight multidrug-resistant isolates,

five of which were USA-300. Our study had several limitations. Not all patients seen in our ID clinic undergo MRSA surveillance cultures, not all isolates were available for PFGE, and complete antibiotic susceptibilities were not reported for each isolate. Additionally, although ART use within the previous year was documented, patient compliance was not assessed in our study. Unlike previous findings, our multivariate analysis did not show an increased risk of MRSA colonization or infection among injecting drug users, MSM or patients with prior incarceration. This may be explained

by our patient population, our sample size, or underreporting of homosexual activity and IDU. In summary, MRSA infection, and specifically USA-300 CA-MRSA infection, occurs in HIV-infected patients. As previously demonstrated, prior antibiotic exposure and a CD4 count <200 cells/μL proved to be independent risks for colonization or infection with MRSA in our study population. Most interesting was our novel finding that HIV-infected patients who received ART in the past year had a significantly decreased risk of MRSA colonization or infection. Further studies are warranted to determine whether HIV-infected patients with recurrent MRSA infections should be considered for earlier Montelukast Sodium initiation of ART or offered decolonization. “
“The aim of the study was to determine the prevalence and risk factors for HIV-associated fatigue in the era of highly active antiretroviral therapy (HAART). A cross-sectional survey of 100 stable HIV-infected out-patients was carried out. Severity of fatigue was measured using the Fatigue Impact Scale (FIS). Symptoms of orthostatic intolerance (dysautonomia) were evaluated using the Orthostatic Grading Scale (OGS). Data for HIV-infected patients were compared with those for 166 uninfected controls and 74 patients with chronic fatigue syndrome (CFS)/myalgic encephalomyelitis (encephalopathy) (ME).

The membranes were counterstained using corresponding donkey anti-guinea pig (1 : 5000; Jackson Immunoresearch, West Grove, PA, USA), goat anti-rabbit or anti-mouse (both 1 : 3000; Bio-Rad Laboratories, Hercules, CA, USA) horseradish peroxidase conjugates. For stripping between the immunoblot procedures, membranes were rinsed and incubated in Restore Western Blot Stripping Buffer (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. For visualization of the proteins, the membranes were exposed to the enhanced chemiluminescence detection system Lumigen PS-3 (1 : 40; GE Healthcare, Buckinghamshire, UK). No immunopositive bands were observed

when immunoblotting was performed with anti-CB1 antibodies pre-absorbed with the antigene peptide (5 μg/mL; Frontier Science, Japan). For immunoprecipitation, ~2.0 mg of total protein from mouse embryo (E16.5) brain mitochondrial fractions

(prepared PFT�� ic50 as above) was incubated overnight at +4 °C with 3 μL of made-in-guinea pig anti-CB1 sera (Frontier Science, Japan). Thirty microliters of a 1 : 1 slurry of protein A-sepharose (GE Healthcare, Buckinghamshire, UK) in phosphate-buffered saline was then added and antibody-bound protein was collected during a 2-h incubation at +4 °C. selleckchem The Sepharose beads were washed four times in 500 μL phosphate-buffered saline containing protease inhibitor cocktail (1 : 500; Calbiochem, La Jolla, CA, USA). The beads and bound protein were loaded in mini gel and separated using electrophoresis as above. The gel was then stained with SimplyBlue colloidal Coomassie (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The ~40-kDa band was cut from the gel and destained

in three washes of acetic acid : methanol : H2O (10 : 50 : 40) solution. The sample was submitted for in-gel tryptic digestion, followed by liquid chromatography, quadrupole/time-of-flight tandem mass spectrometry and peptide mass database searching (Keck Facility, Yale University, New Haven, CT, USA). Mouse neuroblastoma 2A cells were cultured in Dulbecco’s D-MEM/F12 medium containing 9% fetal bovine serum (all from Sigma-Aldrich, St Louis, MO, USA). For transfections, we cloned full-length SLP-2 from E14.5 embryo brain cDNA into pIRES2-EGFP (Clontech, Mountain View, CA, USA); transfections with pEGFP Urocanase (Clontech, Mountain View, CA, USA) were used as negative controls. Newly passaged cells at about 70–80% confluency were starved of serum overnight and transfected with 5 μg SLP-2 DNA using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s guidelines. After 24 h, cells were washed in phosphate-buffered saline, and immediately scraped and lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing protease (Roche, Indianapolis, IN, USA) and phosphatase (Sigma-Aldrich, St Louis, MO, USA) inhibitor cocktails.

0). Although this method was selleck compound applied to the consolidated sediment, prokaryotic DNA was not successfully extracted.

To modify the method established for opal-A from radiolarians, we raised the 1-h incubation temperature from 65 to 94 °C to dissolve the crystalline opal-CT that formed during burial diagenesis. When we conducted the modified DNA extraction, the congealed silica after the neutralization step. As 0.1 g wet sediment sample contained more silica than a single radiolarian cell. To avoid the congealed silica that hindered the subsequent purification step, aliquot was diluted with TE buffer in a range from 0- to fivefold volume before neutralization with 1 M Tris–HCl (pH 6.5). It was found that congealed silica was not visible after neutralization LGK-974 solubility dmso when the aliquot was diluted with a fivefold volume of TE buffer. Purified DNA extracts after neutralization

were subjected to qPCR analysis (Table 1). A fluorescent peak with a Tm of 86.4 °C corresponding to those of 16S rRNA gene sequences from mesophilic bacteria (85–87 °C; Kimura et al., 2006) was obtained during qPCR when the aliquot was diluted with 750 μL of TE buffer (Table 1). As the Tm from positive control cells of P. stutzeri (86.3–88.3 °C; Supporting Information, Table S1) was also similar to that of the sediment sample (86.4 °C), consistent with the extraction of bacteria DNA with fivefold dilution. However, dilution with volumes up to 600 μL resulted in fluorescent peaks with Tm not corresponding to those of 16S rRNA gene sequences from mesophilic bacteria (Table 1). Although gel formation was not evident when diluted with 300–600 μL, it is concluded that the recovery

DNA from the sediment sample was hampered by gel formation. Incubation time was optimized selleck monoclonal humanized antibody under constant NaOH concentration (0.33 N), dilution volume of TE buffer (fivefold volume) and incubation temperature (94 °C). Aliquot was incubated for 30–90 min, and the recovery of prokaryotic DNA was quantified by qPCR analysis (Fig. 1a and Table S1). Although prokaryotic DNA was not detected after heating for 30, 40 and 90 min, qPCR products with appropriate Tm (86.4–88.5 °C) were obtained by incubation for 50, 60, 70 and 80 min. We sequenced 22, 20, 32 and 20 clones for the samples incubated for 50, 60, 70 and 80 min, respectively (Table 2). Regardless of incubation time, dominant phylotypes were related to Cupriavidus metallidurans, Pseudomonas brenneri, Pseudomonas migulae or Acinetobacter sp. Phylotypes related to Mesorhizobium loti, Pelomonas aquatica or Pseudomonas putida were also detected from the samples at some incubation times. Cupriavidus metallidurans is capable of detoxifying a number of heavy metals and is known to thrive in environments enriched with metals. Close relatives of many phylotypes utilize nitrate or molecular oxygen for respiration, which is consistent with nitrate and/or nitrite-bearing pore water and high denitrification activities in the sediment samples (Suzuki et al., 2009).