We do not deny the validity of most studies that use CB1 antibodies; however, we emphasize the need for additional controls and careful interpretation of immunolabeling results. The authors are grateful to Ruth Rappaport, PhD, for her editorial assistance in the preparation of the manuscript. This research was supported by US Public Health Service.

There were no conflicts JNK inhibitor ic50 of interest. Abbreviations BLAST basic local alignment search tool BSA bovine serum albumin CB1 cannabinoid type 1 receptor DAB-Ni Ni-intensified 3,3′-diaminobenzidine-4HCl DMSO dimethylsulfoxide DTT dithiothreitol E embryonic day KO knockout L15 last 15 amino acids L31 last 31 amino acids NH amino-terminus SLP-2 stomatin-like protein 2 WIN WIN 55,212-2 “
“In this study, we examined how risk and delay influence rats’ decision-making, and the role of the ventral hippocampus (VHC) and orbitofrontal cortex (OFC) in the valuation of these two factors.

We used a touchscreen testing method in which rats with VHC lesions, OFC lesions and sham control surgery made choices in two decision-making tasks. In the delay discounting task, rats chose between two visual stimuli, one of which indicated a small, immediate reward, and the other of which indicated a large, delayed reward. In the probability discounting task, two stimuli indicated, instead, a small, certain reward or a large, uncertain reward. The two lesion groups showed a double dissociation with respect to the two tasks. Rats with VHC lesions were intolerant Roflumilast Selleckchem Mitomycin C of delay, and were strongly biased towards the small, immediate reward. However, the same rats were indistinguishable from sham controls in the probability discounting task. The opposite pattern was observed for rats with OFC lesions; they performed normally in the delay discounting task, but showed a reduced tolerance for uncertainty as compared with sham-operated controls. These data support the conclusion that the VHC and OFC contribute differentially to decision-making that involves delayed or uncertain outcomes. This provides a means

for understanding the neural basis of a range of neurological and psychiatric patients who show impaired decision-making and executive dysfunction. “
“The prevention of cone loss during retinal degeneration is a major goal of most therapeutic strategies in retinal degenerative diseases. An intriguing issue in the current research in this field is to understand why a genetic mutation that affects rods eventually leads to cone death. The main objective of the present study was to investigate to what extent rescuing rods from degeneration affects the survival of cones and prevents functional impairment of the visual performance. To this purpose, we compared rod and cone viabilities by both ex vivo and in vivo determinations in the rd10 mutant mouse, a validated model of human retinitis pigmentosa.

Assessment of the risk of protocol-defined virological failure at 48 weeks favoured TDF-FTC (RR 0.76, 95% CI 0.53–1.07), although the effect was not statistically significant and heterogeneity in the analysis was relatively high (I2 46%). Assessment of protocol-defined virological failure at 96 weeks showed a significant difference favouring TDF-FTC (RR 0.73, 95%

CI 0.59–0.92). Data were only available from one study [4] for this analysis; U0126 research buy however, this was by far the largest of the three trials and the quality of evidence assessment for this outcome was rated as high. The difference in virological failure was assessed by the Writing Group to be large enough to be above the clinical threshold for decision-making. The difference equates to a number needed to treat to prevent one case

of virological failure of approximately 20 patients treated for 1 year. The results of ACTG 5202 [2-4] are complicated by early termination of those individuals with a baseline VL >100 000 copies/mL at the recommendation Erlotinib of the data and safety monitoring board due to significantly inferior performance in those subjects receiving ABC-3TC. No difference in virological efficacy between the TDF-FTC and ABC-3TC arms was seen in those in the lower VL stratum (baseline VL <100 000 copies/mL). The subsequent 96-week analysis, after discontinuation of those subjects in the higher VL stratum, may therefore underestimate the difference between the two backbones. HLA-B*57:01 screening was not routine in ACTG 5202 and this potentially may have influenced some of the safety endpoints, but

appears not to have influenced the primary virological of outcome. In the higher VL strata the number of patients with suspected hypersensitivity reactions was equal between both arms and virological failure in these patients was infrequent. With regard to the assessment of the other critical and important outcomes, including drug resistance, discontinuation for adverse events and lipodystrophy, no difference was shown between TDF-FTC and ABC-3TC. No data were available to assess quality of life outcomes. For grade 3/4, adverse events (all) and grade 3/4 alanine transaminase/aspartate transaminase elevation there were trends that favoured TDF-FTC (see Appendix 3.1). Although the rate of drug resistance was not different between the NRTI backbones, the number developing drug resistance was higher numerically in those receiving ABC-3TC, given the higher rate of virological failure. The only outcome that significantly favoured ABC-3TC was bone mineral density but no difference in bone fractures was identified.

Like HPr, Crh becomes (de)phosphorylated in vitro at residue Ser46 by the metabolite-controlled HPr kinase/phosphorylase HPrK/P. Depending on its phosphorylation state, Crh exerts regulatory functions in connection with carbohydrate metabolism. So far, knowledge on phosphorylation of Crh in vivo has been limited and derived from indirect evidence. Here, we studied the dynamics of Crh phosphorylation directly by non-denaturing gel electrophoresis followed by Western analysis. The results confirm that HPrK/P is the single kinase catalyzing phosphorylation of Crh in vivo. Accordingly, phosphorylation of Crh is triggered by the carbon source as observed

previously for HPr, but with some differences. Phosphorylation of both proteins occurred during HDAC inhibitor exponential growth and disappeared upon exhaustion of the carbon source. During exponential growth, ~ 80% of the Crh molecules were phosphorylated when cells utilized a preferred carbon source. The reverse distribution, i.e. around 20% of Crh molecules phosphorylated, was obtained upon utilization of less favorable substrates. This clear-cut classification of the substrates into two groups has not previously been observed for HPr(Ser)~P formation. The likely reason for this difference is the additional

PTS-dependent phosphorylation of HPr at His15, which limits accumulation of HPr(Ser)~P. The histidine protein (HPr) of the carbohydrate : phosphotransferase system (PTS) has a dual role in Firmicutes bacteria. OSBPL9 In its transport function HPr delivers phosphoryl-groups from Enzyme STAT inhibitor I (EI) to the Enzyme II (EII) transport proteins, which phosphorylate their sugar substrates during uptake. During this phosphoryl-group transfer, HPr becomes transiently phosphorylated at residue His15. In addition, HPr also exerts important regulatory functions (Deutscher et al., 2006). It is the key player in carbon catabolite repression (CCR), which allows the bacteria to repress functions for the utilization

of secondary carbon sources when a preferred substrate is simultaneously present (Deutscher, 2008; Görke & Stülke, 2008). To be active in CCR, HPr must be phosphorylated at a different site, Ser46. HPr(Ser)~P binds the global transcriptional regulatory protein CcpA, which thereby gains DNA-binding activity (Fujita, 2009). Phosphorylation as well as de-phosphorylation of HPr at Ser46 is catalyzed by a single enzyme, the HPr kinase/phosphorylase (HPrK/P). The decision as to whether kinase or phosphorylase activity will prevail is controlled by the quality of the available carbon source. Preferred carbon sources such as glucose or fructose, which allow the fastest growth rates, activate the kinase function of HPrK/P and thereby trigger the formation of HPr(Ser)~P.

RNA was pelleted at 16 000 g for 20 min at 4 °C, washed once with 1 mL of 70% ethanol, repelleted and briefly air-dried before being resuspended in 100 μL of RNase-free water. The

resuspended RNA was then further purified using the Qiagen RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The pure RNA was stored at −80 °C. RNA was DNase treated using the Ambion turbo-free DNA kit according to the manufacturer’s instructions. cDNA was synthesized using the high-capacity cDNA reverse transcription kit (Applied Biosystems). A total of ∼1.2−1.5 μg of RNA was used in a 20-μL reaction in all cases. cDNA was synthesized using a PCR cycle of 25 °C for 10 min, JNK inhibitors library 37 °C for 120 min and 85 °C for 5 s. qRT-PCR was performed using the custom-made Taqman gene expression assays (Applied Biosystems). A total of 60 ng of cDNA was used in each 20 μL reaction. Reactions were performed in 20 μL containing 10 μL 2 × Taqman gene expression Mastermix (Applied Biosystems),

1 μL Taqman gene expression assay (Applied Biosystems) and 9 μL cDNA (60 ng). The real-time PCR cycle was carried out in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) (50 °C for 2 min, 95 °C for 10 min and then 40 cycles of 95 °C for 15 s, followed by 60 °C for 1 min). The fold change in the expression levels of each of the genes was calculated using the ΔΔCt method (Livak & Schmittgen, 2001). RNA was extracted from mid-log cultures of M. smegmatis as described above, and the 5′RACE system for the rapid amplification of cDNA ends SD-208 (Version 2.0, Invitrogen) was used according to the manufacturer’s instructions, using the primers cpn60.1 gsp1, cpn60.1 gsp2 and cpn10 gsp2. cDNA was tailed at the 5′ ends using poly-cytosine and transcriptional start sites were identified by detection

of the junction of this poly-C tail in the sequenced cDNA. The promoterless lacZ E. coli–Mycobacterium shuttle vector pSD5B was used to analyse promoter activity (Jain et al., Rutecarpine 1997). Fragments of varying lengths upstream of the cpn60 or cpn10 genes were amplified with primers containing XbaI and SphI sites, or XbaI sites alone. The products were digested as appropriate and ligated into plasmid pSD5B. The resultant recombinant plasmids contained the various promoter regions just upstream of the lacZ gene (Table 1 and Fig. 1). Each of the pSD5B constructs containing a promoter region was electroporated into M. smegmatis mc2155 cells. The strains were grown in liquid media at 37 °C for 2 days, after which their absorbance at OD600 nm was measured. Each culture (100 μL) was added to 900 μL Z buffer (30 °C). A drop each of 0.1% sodium dodecyl sulphate and chloroform was then added to the tubes, which were vortexed to lyse the cells. The reaction was started by adding 200 μL ONPG (4 mg mL−1) and mixing well. When a significant yellow colour developed, the reaction was stopped by addition of 500 μL 0.

Microarray data

have been submitted to ArrayExpress under accession number A-MEXP-1990. Total RNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Cells were disrupted in RLT buffer provided with the Kit in Fast Protein tubes (Qbiogene, Carlsbad, CA) using the Ribolyser (Hybaid, Heidelberg, Germany) (30 s, level 6.5) before spin column purification according to the RNeasy Mini Kit RNA purification protocol. Fluorescent-labeled amplified RNA was prepared using the MessageAmp II-Bacteria RNA Amplification Kit (Applied Biosystems, Darmstadt, Galunisertib Germany). Starting from 500 ng total RNA, cDNA carrying a terminal T7 promoter was synthesized. Subsequent in vitro transcription resulted in aminoallyl-modified RNA that was labeled ABT-737 manufacturer with Cy3- or Cy5-N-hydroxysuccinimidyl ester dyes (GE Healthcare, Little Chalfont, UK). Uncoupled dye was removed applying the RNeasy MinElute Kit (Qiagen). Processing of microarrays before hybridization included the following washes: once in 0.1% Triton-X100 (5 min, 20 °C); twice in 0.032% (w/v) HCl (2 min, 20 °C); once in 0.1 M KCl (10 min, 20 °C); once in H2O (1 min, 20 °C); once in 0.064% (w/v) HCl,

1 × Nexterion blocking solution (Schott AG) (15 min, 50 °C); and once in H2O (1 min, 20 °C). Microarrays were dried by centrifugation (3 min, 185 g, 20 °C). Hybridization was performed in an EasyHyb hybridization solution (Roche, Mannheim, Germany) supplemented with sonicated salmon sperm DNA at 50 μg mL−1 in a final volume of 100 μL for 90 min at 45 °C much using the HS 4800 hybridization station (Tecan Trading AG, Switzerland). Before application to the microarrays, labeled samples were denatured

for 5 min at 65 °C. After hybridization microarrays were washed once in 2 × SSC, 0.2% sodium dodecyl sulfate (SDS) (w/v) (5 min, 42 °C), twice in 0.2 × SSC, 0.1% SDS (w/v) (1 min, 21 °C), twice in 0.2 × SSC (1 min, 21 °C), and once in 0.05 × SSC (1 min, 21 °C). Following the washes, slides were dried by centrifugation (3 min, 185 g, 20 °C) and scanned with a pixel size of 10 μm using the LS Reloaded microarray scanner (Tecan Trading AG). Four independent biological replicates including a dye swap were processed for each comparison. The mean signal and the mean background intensities were obtained for each spot of the microarray images using the imagene software 6.0 software (Biodiscovery Inc., Los Angeles) for spot detection, image segmentation, and signal quantification. Spots were flagged as ‘empty’ if R≤0.5 in both channels, where R=(signal mean−background mean)/background SD. The remaining spots were considered for further analysis. After subtractions of the local background intensities from the signal intensities and the introduction of a floor value of 20, the log2 value of the ratio of intensities was calculated for each spot using the formula Mi=log2(Ri/Gi).

“
“Chronic variable stress (CVS) exposure modifies the paraventricular nucleus of the hypothalamus (PVN) in a manner consistent with enhanced central drive of the hypothalamo-pituitary-adrenocortical (HPA) axis. As previous reports suggest that post-stress enhancement of norepinephrine (NE) action contributes

to chronic stress regulation at the level of the PVN, we hypothesised that PVN-projecting NE neurons were necessary for the stress facilitatory effects of CVS. Following intra-PVN injection of saporin toxin conjugated to a dopamine beta-hydroxylase (DBH) antibody (DSAP), in rats PVN DBH immunoreactivity was almost completely eliminated, but immunoreactive afferents www.selleckchem.com/products/ve-821.html to other key regions involved in stress integration were spared (e.g. DBH fiber densities were unaffected in the central nucleus of the amygdala). Reductions in DBH-positive fiber density were associated with reduced numbers of DBH-immunoreactive neurons in the nucleus of the solitary tract and locus coeruleus. Following 2 weeks of CVS, DSAP injection did not alter stress-induced adrenal hypertrophy or attenuation of body weight gain, see more indicating that PVN-projecting NE [and epinephrine (E)] neurons are not essential for these physiological effects of chronic stress. In response to acute restraint stress, PVN-targeted DSAP injection attenuated peak adrenocorticotrophic

hormone (ACTH) and corticosterone in controls, but only attenuated peak ACTH in CVS animals, suggesting that enhanced adrenal sensitivity compensated Rho for reduced excitatory drive of the PVN. Our data suggest that PVN-projecting NE/E neurons contribute to the generation of acute stress responses, and are required for HPA axis drive (ACTH release) during chronic stress. However, loss of NE/E drive at the PVN appears to be buffered by compensation at the level of the adrenal. “
“The relative contribution to brain cholinergic signaling by synaptic- and diffusion-based mechanisms

remains to be elucidated. In this study, we examined the prevalence of fast nicotinic signaling in the hippocampus. We describe a mouse model where cholinergic axons are labeled with the tauGFP fusion protein driven by the choline acetyltransferase promoter. The model provides for the visualization of individual cholinergic axons at greater resolution than other available models and techniques, even in thick, live, slices. Combining calcium imaging and electrophysiology, we demonstrate that local stimulation of visualized cholinergic fibers results in rapid excitatory postsynaptic currents mediated by the activation of α7-subunit-containing nicotinic acetylcholine receptors (α7-nAChRs) on CA3 pyramidal neurons. These responses were blocked by the α7-nAChR antagonist methyllycaconitine and potentiated by the receptor-specific allosteric modulator 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596).

A recombinant plasmid pET-tbinB carrying a truncated binB gene (encoding amino acids 33–408), which was cloned from Bs 2297 into the this website NheI and XhoI sites of the pET-17b vector, was used as a template for mutagenesis. This truncated BinB still retained the full activity of the full-length BinB; thus, it was used as an active form of the toxin without in vitro proteolytic processing. Mutagenic oligonucleotide primers were purchased from Sigma Proligo

(Singapore). Each primer was designed to introduce or abolish a restriction endonuclease recognition site in order to differentiate between the wild-type and mutant plasmids (Table 1). The recombinant plasmid pET-tbinB encoding the 43-kDa truncated BinB was used as a template for mutagenesis, and the Y150A mutant plasmid was used as a template for generating the Y150F mutant. All mutant

plasmids were generated by PCR using a high-fidelity Pfu DNA polymerase following the procedure of the QuikChange™ site-directed mutagenesis method (Stratagene). PCR products were treated with DpnI to eliminate the AC220 cost DNA templates and then transformed into E. coli JM109. The recombinant plasmids were extracted and the desired mutations were selected by restriction endonuclease digestion. DNA sequences of mutant plasmids were verified by automated DNA sequencing at Macrogen Inc. (Korea). Each recombinant plasmid, extracted from E. coli JM109, was retransformed into E. coli BL21 (DE3) pLysS for protein expression and grown at 37 °C in a Luria–Bertani medium containing 100 μg mL−1 ampicillin and 34 μg mL−1 chloramphenicol until the OD600 nm of the culture reached 0.4. Then 0.1 mM of isopropyl-β-d-thiogalactopyranoside Bupivacaine (IPTG) was added to induce protein expression. The culture was further grown for 5 h and then collected by centrifugation at 6000 g. Cells were resuspended in phosphate buffer (100 mM KH2PO4, pH 6.5) and

then disrupted using a French press at 10 000 p.s.i. Cell lysates were centrifuged at 6000 g, 4 °C for 10 min to separate the pellet-containing inclusions and the supernatant. The inclusions were resuspended in phosphate buffer containing 0.1% Triton X-100, 0.83% NaCl and incubated on ice for 30 min. After centrifugation, inclusions were washed once with phosphate buffer and two times with distilled water. Partially purified inclusions were resuspended in distilled water and kept at −20 °C. The protein concentration of the partially purified inclusions was determined using the method of Bradford using Bio-Rad protein assay reagent with bovine serum albumin as a standard. The partially purified inclusions and supernatant were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The expression of mutant BinB inclusions was further detected by Western blot analysis.

A recombinant plasmid pET-tbinB carrying a truncated binB gene (encoding amino acids 33–408), which was cloned from Bs 2297 into the SP600125 mw NheI and XhoI sites of the pET-17b vector, was used as a template for mutagenesis. This truncated BinB still retained the full activity of the full-length BinB; thus, it was used as an active form of the toxin without in vitro proteolytic processing. Mutagenic oligonucleotide primers were purchased from Sigma Proligo

(Singapore). Each primer was designed to introduce or abolish a restriction endonuclease recognition site in order to differentiate between the wild-type and mutant plasmids (Table 1). The recombinant plasmid pET-tbinB encoding the 43-kDa truncated BinB was used as a template for mutagenesis, and the Y150A mutant plasmid was used as a template for generating the Y150F mutant. All mutant

plasmids were generated by PCR using a high-fidelity Pfu DNA polymerase following the procedure of the QuikChange™ site-directed mutagenesis method (Stratagene). PCR products were treated with DpnI to eliminate the AZD2281 cell line DNA templates and then transformed into E. coli JM109. The recombinant plasmids were extracted and the desired mutations were selected by restriction endonuclease digestion. DNA sequences of mutant plasmids were verified by automated DNA sequencing at Macrogen Inc. (Korea). Each recombinant plasmid, extracted from E. coli JM109, was retransformed into E. coli BL21 (DE3) pLysS for protein expression and grown at 37 °C in a Luria–Bertani medium containing 100 μg mL−1 ampicillin and 34 μg mL−1 chloramphenicol until the OD600 nm of the culture reached 0.4. Then 0.1 mM of isopropyl-β-d-thiogalactopyranoside before (IPTG) was added to induce protein expression. The culture was further grown for 5 h and then collected by centrifugation at 6000 g. Cells were resuspended in phosphate buffer (100 mM KH2PO4, pH 6.5) and

then disrupted using a French press at 10 000 p.s.i. Cell lysates were centrifuged at 6000 g, 4 °C for 10 min to separate the pellet-containing inclusions and the supernatant. The inclusions were resuspended in phosphate buffer containing 0.1% Triton X-100, 0.83% NaCl and incubated on ice for 30 min. After centrifugation, inclusions were washed once with phosphate buffer and two times with distilled water. Partially purified inclusions were resuspended in distilled water and kept at −20 °C. The protein concentration of the partially purified inclusions was determined using the method of Bradford using Bio-Rad protein assay reagent with bovine serum albumin as a standard. The partially purified inclusions and supernatant were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The expression of mutant BinB inclusions was further detected by Western blot analysis.

However, users responded they were uncertain as to whether the new chart made it safer to prescribe, dispense and administer medicines. Users provided additional constructive feedback and identified ways in which the new chart design could be enhanced to further improve usability and safety aspects. A collaborative approach with involvement of relevant specialists and stakeholders resulted Metformin in the successful design and trial of a standard inpatient chart in five organisations. The pilot phase evaluation demonstrated some safety improvements, for example in the quality and visibility of

allergy status documentation, but also highlighted areas for further enhancement. Weight documentation which was low to begin with, decreased with the new design and this needed to be addressed through minor changes to the chart prior to implementation. Users reported an overall positive view of the new charts. 1. GMC. GMC Calls for a National Prescription Chart to Reduce Errors [press release]. 2009. See http://www.gmc-uk.org/news/5156.asp (last checked 26 April 2013). 2. Coombes ID, Stowasser DA, Reid C, Mitchell CA. Impact of a standard medication chart on prescribing errors: a before-and-after audit. Qual Saf Health Care 2009; 18: 478–485. Peter Rivers, Shoaib Haji, Hafizah Lorgat, Mohammed Mawji, Georgina Ridgway De Montfort University,

Leicester, UK The aim of the study was to observe the activities of care staff whilst administering medicines in care homes and Rutecarpine to understand the attitudes of staff towards medicines safety in the context of social care Interruptions constituted an selleckchem accepted part of the task of administering medicines Potential for harm caused by medication error should be balanced against priority for social care The CHUMS report 1 highlighted considerable risk of

making medication errors when administering medicines to elderly people in care homes although found no direct evidence of ‘severe harm’ to residents. In order to gain insight into the cause of such errors, the aim of this research was to describe activities that take place during medicine rounds. An aim was also to gain an understanding of the experience and attitudes of care staff when administering medicines in a social care setting. Non-participant observation of medicine rounds was conducted at breakfast and tea-time in four social services care homes. Staff were aware of being observed but this is unlikely to have substantially influenced routine medication-round activity or unplanned interruptions. Measures of activities and distractions were noted such as: a) time taken to complete medicine round, b) selecting doses, c) talking to residents, d) dealing with interruptions, e) documentation. In-depth interviews designed to seek carers’ views of the risks associated with administering medicines were conducted with a representative sample of 12 care staff from the four homes.

coli strains, was negative for the stcE gene. The presence or absence of the stcE gene in all strains was confirmed by Southern blot (data not shown). Analysis of isolated plasmid DNA by Southern blot demonstrated that stcE was encoded on the large plasmid of the four atypical Shigella B13 strains (data not shown). Sequence analysis of the 2.7-kb stcE gene showed only CHIR-99021 concentration three synonymous substitutions shared among

the atypical Shigella B13 strains and a Q727L substitution in strain 3556-77 compared to the EHEC EDL933 allele (data not shown). Six substitutions within 220 nucleotides of the intergenic region upstream of the predicted stcE promoter are present in the plasmids of all four atypical Shigella B13 strains compared to pO157. To determine whether the StcE protein was expressed and secreted by the atypical Shigella B13 strains, TCA-precipitated supernatants of overnight cultures were analyzed by immunoblot. StcE protein was identified in supernatants from strains 3556-77, 3052-94, and 3053-94, but not from 3557-77 or 5216-70 (Table 2). StcE activity in culture supernatants was assayed for C1-INH proteolysis by immunoblots selleck products and detected with all atypical Shigella B13 strains except 3557-77 and 5216-70 (Fig. 1, Table 2). To determine whether the atypical Shigella B13 plasmid encoding stcE is similar to the large invasion plasmid of Shigella

(pINV), several pINV-encoded virulence factors were sought by PCR amplification (Table 2). None of the pINV-encoded virulence factors could be amplified from the atypical those Shigella B13 strains. PCR analysis using primers specific for pO157-encoded genes resulted in amplification of etpD, but not katP. The gene, traC, which is an F plasmid gene that is also encoded on the large virulence plasmid of E. coli O157:H-, pSFO157, did not PCR amplify from any of the atypical Shigella B13 strains tested. The presence of additional E. coli-specific chromosomally encoded genes was determined by colony PCR (Table 2). The LEE-encoded

regulator (Ler) is a global virulence regulator that has been shown to positively regulate the expression of LEE (Mellies et al., 1999), stcE, and the etp operon in E. coli O157:H7 (Lathem et al., 2002). PCR analysis of the atypical Shigella B13 strains identified the ler gene in the four atypical Shigella B13 strains encoding eae and stcE. An additional LEE-encoded gene, espA, encodes a subunit of the type III secretion system unique to EPEC and EHEC and is encoded by the atypical Shigella B13 strains encoding eae and stcE. PCR analysis of cadA, which encodes lysine decarboxylase and is universally absent in Shigella but present in most E. coli strains (Day et al., 2001), revealed that none of the atypical Shigella B13 strains encoded cadA. The abilities of the atypical Shigella B13 strains to invade HEp-2 cells were determined.