The majority of local and systemic reactions were mild or moderate, and there were no significant differences between the two vaccines.41 Additionally, in multiple clinical trials, there have been no cases of Guillain–Barré syndrome observed with ACWY-CRM. Studies are currently ongoing to assess immunogenicity and safety of ACWY-CRM in older adults aged 55 to 65. Vaccination with ACWY-CRM results in a protective immune response in adolescents (aged 11–18 y), which is comparable Staurosporine to that observed with MPSV4 and ACWY-D and is statistically significantly different for certain serogroups.40,45 A phase II multicenter US study in adolescents

(aged 11–17 y) reported significantly greater immunogenicity at 1 month postvaccination with ACWY-CRM compared with MPSV4. Significantly more subjects achieved hSBA titer ≥1 : 8 after 1 month with ACWY-CRM compared with MPSV4 for serogroups A, C, and Y (p < 0.001; Figure 2). By 12 months, significantly more adolescents http://www.selleckchem.com/products/ensartinib-x-396.html were protected against serogroups C, W-135, and Y with ACWY-CRM (p < 0.01). Levels of hSBA GMTs remained significantly higher with ACWY-CRM for serogroups W-135 and Y (p < 0.001) and were comparable between vaccines for A and C.45 In the subsequent phase III study in 2,170 adolescents (aged 11–18 y), the percentage of subjects with a postvaccination hSBA titer ≥1 : 8 with ACWY-CRM was superior compared with the response to ACWY-D for serogroups

A, W-135, and Y and was noninferior for serogroup C (lower limit of the two-sided 95% CI >0%) (Figure 3).40 The level of hSBA GMTs was significantly higher with ACWY-CRM versus ACWY-D for all four serogroups. The percentage of seroresponders was significantly higher for ACWY-CRM Palmatine (68%–75%) than for ACWY-D (41%–66%) for serogroups A, W-135, and Y, and comparable for serogroup C (75% vs 73%, respectively).40 Immune response was found to persist at 22 months, with a statistically significantly higher (p < 0.05) proportion of subjects achieving hSBA titer ≥1 : 8 in the ACWY-CRM

group compared with the ACWY-D group for serogroups A, W-135, and Y.46 Overall, tolerability was comparable among the vaccines.40,45 Pain at injection site was the most common local reaction in both studies, reported by 44% to 56% of subjects; with no difference between groups. The most common systemic reaction in both studies was headache.40,45 Significantly more adolescents reported nausea with ACWY-CRM compared with MPSV4 (p = 0.009); no other significant difference in adverse effects was noted.45 In children (aged 2–10 y), a single-center, phase II US study (N = 619) reported a superior protective immune response with ACWY-CRM compared with MPSV4 for all four serogroups at 1 and 12 months.47 One month after administration, 73% to 92% of children in the ACWY-CRM group had an hSBA titer ≥1 : 8 for all serogroups versus 37% to 65% for MPSV4.

[1,8,28] This formative role of simulated-patient methods seeks to improve quality of advice regarding non-prescription medicines.[16] Performance Selleckchem Depsipeptide feedback provided to pharmacists and their staff after a simulated-patient visit appears to be an important aspect of the simulated-patient method, as it allows for gradual and ongoing fine-tuning of practice behaviour over time.[8,18] However, little is known on how feedback has been delivered to pharmacists and their staff post simulated-patient visits. Although simulated-patient methods as an educational tool have been used in the pharmacy setting for over a decade, systematic reviews of simulated-patient studies

have not investigated feedback provision.[19,23] Furthermore, the review by Mesquita et al. highlighted that no studies found in their review had focused on children’s medicines, which often require unique counselling GDC-0973 purchase information.[19] Therefore there is a need for further knowledge on how feedback is being provided in the pharmacy setting and on how pharmacists and their staff perceive these methods in pharmacy education, as well as exploring how simulated patients can be used to improve the quality use of medicines in children. The aim of this bibliographic review was to explore the use of the

simulated-patient method in the community pharmacy setting involving non-prescription medicines. Previous reviews have mainly focussed on simulated-patient scenarios employed to assess communication Amrubicin skills of pharmacists and their staff and outcome measures. This review, however, focuses on the purpose of the simulated-patient method, the types of scenarios employed to assess practice behaviour (with particular interest in whether scenarios have involved children’s medicines), as well as whether and how performance feedback

was delivered to pharmacists and their staff, and how these simulated-patient methods were perceived by participants. This review will inform the design of a simulated patient intervention to improve the management of common childhood ailments in community pharmacy. The databases IPA (International Pharmaceutical Abstracts), EMBASE and MEDLINE were searched using the following key words and search strategy: (‘pseudo patient’ OR ‘pseudo customer’ OR ‘standardised patient’ OR ‘standardized patient’ OR ‘shopper patient’ OR ‘mystery shopper’ OR ‘simulated patient’ OR ‘pseudo patron’ OR ‘covert participant’ OR ‘surrogate shopper’ OR ‘disguised shopper’) AND ((‘community’ AND ‘pharmacy’) OR ‘community pharmacy’) in all three databases The search strategy and review protocol were jointly developed by TX and RM. Data collection and extraction was carried out by TX. The search was limited to articles published in the English language, from 1990 to 2010 (Tables 1–3).

However, it is not precisely known which species of microorganisms play the principal part in these beneficial properties. Some major Dabrafenib cost health benefits of probiotics and their proposed mechanisms are illustrated in Table 1. Several probiotic bacteria have been introduced in the market, and the range of products in which probiotic bacteria are added is increasing (Table 2).

Some of the major health attributes of probiotics are discussed in the following sections. Resistance to enteric pathogens Antagonism activity Adjuvant effect increasing antibody production Systemic immune effect Colonization resistance Limiting access of enteric pathogens (pH, bacteriocins/defensins, antimicrobial peptides, lactic acid production, and toxic oxygen metabolites) Aid in lactose digestion Bacterial lactase acts on lactose in the small intestine Small bowel Ibrutinib mouse bacterial overgrowth Lactobacilli influence the activity of overgrowth flora, decreasing toxic metabolite production Normalization of a small bowel microbial community Antibacterial characteristics Immune system modulation Strengthening of nonspecific and antigen-specific

defense against infection and tumors Adjuvant effect in antigen-specific immune responses Regulating/influencing Th1/Th2 cells, production of anti-inflammatory cytokines Decreased release of toxic N-metabolites Anticolon cancer effect Antimutagenic activity Detoxification of carcinogenic metabolites Alteration in pro-cancerous enzymatic activity of colonic microorganisms Stimulation

of immune function Influence on bile salt concentration Decreased detoxification/excretion of toxic microbial metabolites Increased bifidobacterial cell counts and shift from a preferable protein- to carbohydrate-metabolizing microbial community, less toxic and for putrefactive metabolites, improvements of hepatic encephalopathy after the administration of bifidobacteria and lactulose Prevention of antigen translocation into blood stream Prevent excessive immunologic PRKD3 responses to increased amount of antigen stimulation of the gut Blood lipids, heart disease Assimilation of cholesterol by bacterial cell Alteration in the activity of BSH enzyme Antioxidative effect Bacterial peptidase action on milk protein results in antihypertensive tripeptides Cell wall components act as ACE inhibitors Adhesion to urinary and vaginal tract cells Competitive exclusion Inhibitor production (H2O2, biosurfactants) Infection caused by Helicobacter pylori Competitive colonization Inhibition of growth and adhesion to mucosal cells, decrease in gastric H.

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“Streptococcus pyogenes causes a broad spectrum of acute infections and is the bacterium most frequently Sirolimus isolated from patients with pharyngitis. A number of antibiotics including penicillin have been shown to be effective, although antibiotic treatment

failure in cases of streptococcal pharyngitis have been reported. Herein, we aimed to elucidate the features of recurrent strains using clinical isolates. Ninety-three S. pyogenes organisms were obtained from Japanese patients with recurrent pharyngitis. Following genetic characterization, M-type isolates from patients with recurrent pharyngitis differed from those obtained at initial onset in 11 of 49 episodes, and pulsed field gel electrophoresis analysis showed different patterns in those cases. Additionally, spe genotyping revealed Linsitinib in vitro that the Spe type of the strains obtained at secondary onset corresponded with those from the initial onset in 22 cases. Furthermore, antibiotic

susceptibility testing revealed that more than half of the strains were resistant to macrolides and lincosamides, which was a much greater ratio as compared with the strains obtained from initial onsets in previous studies. Our results suggest that recurrence and reinfection are often confused during the diagnosis of repetitive and persistent streptococcal pharyngitis. Moreover, the present S. pyogenes

organisms were less susceptible to antibiotics, which raises caution about their appropriate use in clinical practice. Streptococcus pyogenes, also known as Group A Streptococcus, is a common human pathogen that causes a broad spectrum of acute infectious diseases ranging from noninvasive diseases, such as crotamiton pharyngitis, skin infections, and acute rheumatic fever, to more life-threatening invasive infections, including myositis, necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome (Cunningham, 2000). Streptococcal pharyngitis is frequently observed in infants and adolescents, and most bacterial pharyngitis cases are caused by S. pyogenes. A variety of antibiotics have been suggested to be effective for treating streptococcal pharyngitis, including penicillins, cephalosporins, macrolides, and lincosamides. Currently, penicillin remains the treatment of choice, because of its proven efficacy and safety, narrow spectrum, and low cost (Dajani et al., 1995; Bisno et al., 2002). However, antibiotic treatment failure has been reported in clinical cases of streptococcal pharyngitis (Macris et al., 1998; Kuhn et al., 2001). Several theories have been proposed to account for this phenomenon, including the coexistence of β-lactamase-producing bacteria (Brook, 1994) and internalization of S.

Our data on the stress response behavior of a V. choleraeΔphoB mutant suggests that PhoB modulates stress response but differs from the pattern reported for rpoS and ppk mutants (Yildiz & Schoolnik, 1998; Jahid et al., 2006). For instance, global gene expression profiling of an rpoS mutant revealed that RpoS positively affects the expression of the catalase peroxidase PerA (VC1560) and cytochrome c551 peroxidase RG7420 nmr VC0089 (Silva et al., 2008). In contrast, deletion of phoB in V. cholerae was found to affect the expression the alkylhydroperoxidase VC0731 (von Kruger et al., 2006) reported to protect against oxidative stress under

conditions of phosphate starvation (Moreau et al., 2001). These results are in agreement with our data, suggesting that RpoS and PhoB activate different stress response mechanisms in V. cholerae. Taken together, our results suggest that under conditions of phosphate limitation, elevated expression of HapR and expression of PhoB act to diminish biofilm formation by diminishing VpsT and VpsR, respectively. In parallel, induction of PhoB under conditions of phosphate limitation modulates stress response in an RpoS-independent manner to provide planktonic cells with resistance mechanisms that could be specifically tailored to the phosphate-deprived environment. The finding Z VAD FMK that phosphate limitation and expression

of PhoB appears to induce V. cholerae to switch to a planktonic life style poses an intriguing question. The planktonic life style could provide fitness when survival depends on interspecies competition for limiting amounts of soluble phosphate. A model for the integration of cell density and nutritional signals in the regulation of biofilm formation is shown in Fig. 6. According to this model, high cell density, carbon starvation and phosphate limitation promote a planktonic life style by enhancing the expression of the negative factor HapR and PhoB (in the case of phosphate limitation). Interestingly, the opposing effects

of CRP and PhoB on Mannose-binding protein-associated serine protease VpsR expression suggest that VpsR might function to finely adjust the transition between life styles in response to the carbon–phosphate ratio in the environment. Clearly, more research is required to clarify how the complex interplay between cell density and nutritional signals in the aquatic environment coordinately affect biofilm formation, stress response and the persistence of V. cholerae. The present study was supported by grant GM008248 from the National Institute of General Medical Sciences to A.J.S and PHS grant AI63187 from the National Institute of Allergy and Infectious Disease to J.A.B. Table S1. Strains, plasmids and primers Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

5 billion. Conclusions Unnecessary spending on pharmacy charges has the potential to outstrip the estimated cost of medicines wastage in the UK. The cost-effectiveness of restricted prescription lengths for the cheaper, mostly generic medications merits an urgent re-examination. “
“Objective  Problem drinking is an increasing concern Rapamycin cost to many governments worldwide including those of England and New Zealand. Screening and brief

intervention (SBI) is effective at reducing alcohol consumption and preventing escalation of hazardous drinking patterns into harmful drinking or dependence. Community pharmacy has been suggested as a potential site from which to provide readily accessible SBI services. This paper explores the views of 40 pharmacists on the prospect of providing SBI for alcohol health promotion purposes, focusing particularly upon potential barriers and incentives to provision of these services. The aim was to explore the views of community pharmacists toward the development of SBI for risky drinkers through semi-structured interviews. Methods  Qualitative, tape-recorded interviews conducted with 22 English pharmacists and 18 New Zealand pharmacists. Data collection continued until theme FG-4592 datasheet saturation

occurred. Transcribed interviews were thematically analysed. Key findings  Pharmacists considered there was a place for alcohol health promotion in community pharmacy. However, not all participants were positive about this potential new role and some expressed apprehension about implementing SBI services due to concerns about offending or alienating customers. Other barriers included lack of experience and confidence, problems faced with other health promotion initiatives, time, privacy and remuneration.

Other pharmacists were more positive, seeing potential in terms of remaining competitive. Selleckchem Decitabine Facilitators included a public health campaign to raise awareness of problem drinking, having appropriate screening tools available and training for pharmacists. Conclusion  There appears to be potential for alcohol SBI services in community pharmacy, and interventions designed to reduce barriers and enhance incentivisation need to be implemented and evaluated. “
“The objective of this article was to assess if Australian pharmacy staff prevent potential adverse reactions in warfarin patients requesting over-the-counter (OTC) analgesia. Mystery shoppers entered 170 pharmacies across Australia to request OTC analgesia for a hypothetical patient with a wrist injury who currently takes warfarin following a heart valve replacement. The request was made to the first pharmacist or non-pharmacist staff member to approach the mystery shopper. The interaction was audio-taped and assessed by a pharmacist. The OTC analgesic recommended was assessed for the potential to cause an adverse bleeding event.

5 billion. Conclusions Unnecessary spending on pharmacy charges has the potential to outstrip the estimated cost of medicines wastage in the UK. The cost-effectiveness of restricted prescription lengths for the cheaper, mostly generic medications merits an urgent re-examination. “
“Objective  Problem drinking is an increasing concern this website to many governments worldwide including those of England and New Zealand. Screening and brief

intervention (SBI) is effective at reducing alcohol consumption and preventing escalation of hazardous drinking patterns into harmful drinking or dependence. Community pharmacy has been suggested as a potential site from which to provide readily accessible SBI services. This paper explores the views of 40 pharmacists on the prospect of providing SBI for alcohol health promotion purposes, focusing particularly upon potential barriers and incentives to provision of these services. The aim was to explore the views of community pharmacists toward the development of SBI for risky drinkers through semi-structured interviews. Methods  Qualitative, tape-recorded interviews conducted with 22 English pharmacists and 18 New Zealand pharmacists. Data collection continued until theme EPZ-6438 supplier saturation

occurred. Transcribed interviews were thematically analysed. Key findings  Pharmacists considered there was a place for alcohol health promotion in community pharmacy. However, not all participants were positive about this potential new role and some expressed apprehension about implementing SBI services due to concerns about offending or alienating customers. Other barriers included lack of experience and confidence, problems faced with other health promotion initiatives, time, privacy and remuneration.

Other pharmacists were more positive, seeing potential in terms of remaining competitive. Sclareol Facilitators included a public health campaign to raise awareness of problem drinking, having appropriate screening tools available and training for pharmacists. Conclusion  There appears to be potential for alcohol SBI services in community pharmacy, and interventions designed to reduce barriers and enhance incentivisation need to be implemented and evaluated. “
“The objective of this article was to assess if Australian pharmacy staff prevent potential adverse reactions in warfarin patients requesting over-the-counter (OTC) analgesia. Mystery shoppers entered 170 pharmacies across Australia to request OTC analgesia for a hypothetical patient with a wrist injury who currently takes warfarin following a heart valve replacement. The request was made to the first pharmacist or non-pharmacist staff member to approach the mystery shopper. The interaction was audio-taped and assessed by a pharmacist. The OTC analgesic recommended was assessed for the potential to cause an adverse bleeding event.

Membranes were prepared from E. coli murG(Ts);pAZI8952 grown at 42 °C in 0.2% arabinose to assay Mtu MurG. Unfortunately, Rapamycin chemical structure no MurG activity was detected in these membranes (see data below and Table 2). Activity was undetectable even in the membranes of transformants grown in 2% arabinose to obtain higher levels of Mtu MurG. The lack of MurG activity was surprising given that the Mtu murG complemented the E. coli (Ts) homologue and must have been functional. Activity was checked in the peptidoglycan synthesis assay in case the specific activity of the Mtu MurG protein was very low, because this assay is more sensitive than the MurG

assay (Chandrakala et al., 2001; Ravishankar et al., 2005). No cross-linked peptidoglycan synthesis was detected in these membranes (Table 2), whereas the expected level of activity was observed in the membranes of wild-type E. coli grown at 37 °C. The assay time, temperature and quantity

of protein were varied in an attempt to improve the sensitivity but peptidoglycan synthesis remained undetectable. Both MurG and peptidoglycan synthesis assays are dependent on having a functional MraY (Fig. 1a). However, the MraY enzyme was active in the membranes of the transformant, and the activity was similar to that in membranes from wild-type E. coli (strain AMA1004) grown at 37 °C (Table 1). This indicated that the block in peptidoglycan synthesis was downstream of the MraY and was probably due to the

lack of MurG activity in these membranes. Either the HDAC inhibitors in clinical trials Mtu MurG protein was unstable under Etomidate the conditions of membrane preparation and storage, or the specific activity of the Mtu MurG protein was below the limit of detection or the assay conditions were not appropriate for Mtu MurG. It is not obvious how membranes devoid of MurG can be made under normal circumstances, as murG is an essential enzyme. This result, while unexpected, offered an opportunity. Because the membranes contained the lipid carrier and all enzymes involved in peptidoglycan synthesis other than MurG, they provided a powerful assay system for MurG, provided that the addition of the pure enzyme could reconstitute the system. For ease of description, these membranes are referred to as E. coli(Ts) ΔMurG membranes. Membranes from wild-type E. coli or the Ts mutant cannot be used to assay exogenous MurG because the endogenous MurG activity would mask the activity. Solubilized, purified E. coli MurG (2 μg) was added to the membranes of E. coli(Ts) ΔMurG incubated with the two UDP-linked sugar precursors under conditions for peptidoglycan synthesis. Considerable cross-linked peptidoglycan was synthesized (Table 2). This indicates that the exogenous E. coli MurG protein was not only able to access the lipid carrier in the membrane, but also able to interact with other membrane and enzyme components to reconstitute peptidoglycan synthesis in these membranes.

Surface seawater samples were collected at Aburatsubo Inlet by using 50-mL Corning tubes (Sigma-Aldrich Japan, Tokyo, Japan) (Table 1). The method used for isolating luminous colonies was as described previously (Yoshizawa et al., 2009b). A Bio-Rad AquaPure Genomic DNA Kit (Bio-Rad Laboratories, Hercules, CA) was used to extract genomic DNA from 1 mL overnight cultures of strains grown in ZoBell broth. The

16S rRNA gene was amplified with bacterial universal primers (Lane, 1991). Other primers designed and used for amplification of the luxA gene, which encodes the alpha subunit of luciferase, were Vch LuxA-F (5′-GATCAAATGTCAAAAGGACG-3′) and Vch LuxA-R (5′-CCGTTTGCTTCAAAACCACA-3′). Genes encoding GPCR Compound Library clinical trial uridylate kinase (pyrH), a cell division protein (ftsZ), and a rod-shaped protein (mreB) were used for MLSA (Thompson et al., 2007). PCR primers for the three genetic loci and reaction conditions were used in accordance with the method of Sawabe et al.

(2007). TaKaRa EX Taq polymerase (TaKaRa Bio, Shiga, Japan) was used to amplify the genes. An ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) was used for sequencing. Multiple alignments of the sequences were performed with clustal w (version 1.6) (Thompson et al., 1994). Distances were calculated by using the Kimura 2-parameter model (Kimura, 1980). Clustering based on the neighbor-joining method (Saitou & Nei, 1987) was determined using bootstrap values based on 1000 replications (Felsenstein, 1985). Sequence data used for other Vibrio

species were from the online electronic taxonomic LBH589 order scheme for Vibrios (http://www.taxvibrio.lncc.br) and the GenBank database. In vivo light emission spectra of luminous strains were measured after incubation at 20 °C for 24–48 h on ZoBell 2216E agar medium. Fluorescence (emission Ureohydrolase and excitation) and light emission spectra (in vivo and in vitro) were measured with a Shimadzu Model RF-5300PC spectrofluorophotometer (Shimadzu, Kyoto, Japan). Light emission spectra were measured more than twice with the excitation lamp off. For all measurements, the wavelength scan rate was 50 nm s−1. Cells of V. azureus strain NBRC 104587T were grown in ZoBell broth at 27 °C. The cells were harvested in the second half of the exponential phase. Subsequent procedures were carried out at 4 °C. The cells of NBRC 104587T were osmotically lysed in 10 mM Na/K phosphate lysis buffer containing 10 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM dithiothreitol (DTT) (pH 7.0). The lysate was centrifuged for 60 min at 10 000 g, and the supernatant was collected. Proteins were fractionated by the addition of solid ammonium sulfate to the cell lysate. The proteins that precipitated at between 40% and 80% (NH4)2SO4 saturation were collected by centrifugation for 60 min at 10 000 g. The protein precipitates were then dissolved in 10 mM Na/K phosphate buffer (containing 0.1 mM EDTA, 1 mM DTT [pH 7.

Surface seawater samples were collected at Aburatsubo Inlet by using 50-mL Corning tubes (Sigma-Aldrich Japan, Tokyo, Japan) (Table 1). The method used for isolating luminous colonies was as described previously (Yoshizawa et al., 2009b). A Bio-Rad AquaPure Genomic DNA Kit (Bio-Rad Laboratories, Hercules, CA) was used to extract genomic DNA from 1 mL overnight cultures of strains grown in ZoBell broth. The

16S rRNA gene was amplified with bacterial universal primers (Lane, 1991). Other primers designed and used for amplification of the luxA gene, which encodes the alpha subunit of luciferase, were Vch LuxA-F (5′-GATCAAATGTCAAAAGGACG-3′) and Vch LuxA-R (5′-CCGTTTGCTTCAAAACCACA-3′). Genes encoding INCB024360 datasheet uridylate kinase (pyrH), a cell division protein (ftsZ), and a rod-shaped protein (mreB) were used for MLSA (Thompson et al., 2007). PCR primers for the three genetic loci and reaction conditions were used in accordance with the method of Sawabe et al.

(2007). TaKaRa EX Taq polymerase (TaKaRa Bio, Shiga, Japan) was used to amplify the genes. An ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) was used for sequencing. Multiple alignments of the sequences were performed with clustal w (version 1.6) (Thompson et al., 1994). Distances were calculated by using the Kimura 2-parameter model (Kimura, 1980). Clustering based on the neighbor-joining method (Saitou & Nei, 1987) was determined using bootstrap values based on 1000 replications (Felsenstein, 1985). Sequence data used for other Vibrio

species were from the online electronic taxonomic Romidepsin in vivo scheme for Vibrios (http://www.taxvibrio.lncc.br) and the GenBank database. In vivo light emission spectra of luminous strains were measured after incubation at 20 °C for 24–48 h on ZoBell 2216E agar medium. Fluorescence (emission Thiamet G and excitation) and light emission spectra (in vivo and in vitro) were measured with a Shimadzu Model RF-5300PC spectrofluorophotometer (Shimadzu, Kyoto, Japan). Light emission spectra were measured more than twice with the excitation lamp off. For all measurements, the wavelength scan rate was 50 nm s−1. Cells of V. azureus strain NBRC 104587T were grown in ZoBell broth at 27 °C. The cells were harvested in the second half of the exponential phase. Subsequent procedures were carried out at 4 °C. The cells of NBRC 104587T were osmotically lysed in 10 mM Na/K phosphate lysis buffer containing 10 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM dithiothreitol (DTT) (pH 7.0). The lysate was centrifuged for 60 min at 10 000 g, and the supernatant was collected. Proteins were fractionated by the addition of solid ammonium sulfate to the cell lysate. The proteins that precipitated at between 40% and 80% (NH4)2SO4 saturation were collected by centrifugation for 60 min at 10 000 g. The protein precipitates were then dissolved in 10 mM Na/K phosphate buffer (containing 0.1 mM EDTA, 1 mM DTT [pH 7.