Sparkle (Lee & La Rue, 1992) In Trifolium repens roots, ethylene

Sparkle (Lee & La Rue, 1992). In Trifolium repens roots, ethylene inhibits cortical cell division, a process that is indispensable for nodule primordia formation (Goodlass & Smith, 1979). To obviate some of the inhibitory effects of ethylene in nodule formation, development and function, some rhizobial strains utilize different mechanisms for lowering ethylene levels such as the production of the GSK458 price enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase; this enzyme is responsible

for the cleavage of ACC (the immediate precursor of ethylene in plants) to ammonia and α-ketobutyrate (Honma & Shimomura, 1978), contributing to increase the competitiveness of the strains because of advantages in the processes of nodule formation and occupancy selleck compound (Ma et al., 2003b, 2004). Other rhizobial strains lower ethylene levels by producing the compound rhizobitoxine, an inhibitor of the plant enzyme ACC synthase (Sugawara et al., 2006). The prevalence of ACC deaminase genes in rhizobia has been studied primarily in Rhizobium spp. (Ma et al., 2003a; Duan et al., 2009). In these studies, many Rhizobium spp. have been found to possess an acdS gene and produce ACC deaminase under free-living conditions. For example, in a rhizobia collection of isolates from Saskatchewan (Canada), 27 Rhizobium isolates possessed an acdS gene and were able to produce

ACC deaminase, thus, showing that acdS genes are present throughout Rhizobium isolates (Duan et al., 2009). On the other hand, notwithstanding reports documenting the presence of ACC deaminase in Mesorhizobium spp., not much is known about the environmental distribution of acdS genes in this bacterial genus. The first report on acdS gene presence in Mesorhizobium was obtained following the complete sequencing of Mesorhizobium sp. MAFF303099 (Kaneko et al., 2000). Subsequently, the presence of an acdS

gene in the symbiosis island of Mesorhizobium loti R7A was also reported (Sullivan et al., 2002). However, when Mesorhizobium sp. MAFF303099 and Mesorhizobium ciceri UPM Ca-7 were tested for ACC deaminase activity and the presence of an acdS gene, no activity was detected and the acdS gene was not found in M. ciceri (Ma et al., 2003b). Recently, the genome sequences of Mesorhizobium Phosphatidylethanolamine N-methyltransferase opportunistum WSM2075T (Lucas et al., 2011a), Mesorhizobium australicum WSM2073T (Lucas et al., 2011b), and Mesorhizobium ciceri bv. biserrulae WSM1271 (Lucas et al., 2011c), revealed the presence of an acdS gene in these strains. In some strains of Mesorhizobium, the production of ACC deaminase has been shown to be an important mechanism to promote nodule formation. When compared to the wild-type strain, Mesorhizobium sp. MAFF303099 acdS knockout mutant has a decreased ability to form and occupy nodules, losing both its effectiveness and competitiveness (Uchiumi et al., 2004).

The proximal stump of the nerve was inserted into a silicon tube

The proximal stump of the nerve was inserted into a silicon tube with stimulating electrodes, Selleck SRT1720 through which continuous electrical stimulation was applied for 12 h/day (square pulses of 100 μs, 3.0 V, at 20 Hz) for 4 weeks. Deafferented animals showed significant decreases in cortical evoked potentials, cytochrome oxidase staining intensity (layers II–IV), cortical volume (layer IV) and number of parvalbumin-expressing (layers II–IV) and calbindin-D28k-expressing (layers II/III) interneurons.

These deafferentation-dependent effects were largely absent in the nerve-stimulated animals. Together, these results provide evidence that chronic electrical stimulation has a neuroprotective and preservative effect on the sensory

cortex, and raise the possibility that, by controlling the physical parameters of an artificial sensory input to a sectioned peripheral nerve, chronically deafferented brain regions could be maintained at near-‘normal’ conditions. Our findings could be important for the design of sensory neuroprostheses and for therapeutic purposes in brain lesions or neural degenerative processes. “
“The most spectacular example of oscillations and synchrony which appear in the brain is the rhythmic slow activity (theta) of the limbic cortex. Theta rhythm is the best synchronized electroencephalographic activity that can be recorded from the mammalian brain. Hippocampal Selleckchem DAPT formation is considered to be the main structure involved in the generation of this activity. Although detailed studies of the physiology and pharmacology of theta-band oscillations have been carried out since the early 1950s, the first demonstration of atropine-sensitive theta rhythm, recorded in completely deafferented hippocampal slices of a rat, was performed

in the second half of the 1980s. Since the discovery of cholinergically induced in vitro theta rhythm recorded from hippocampal formation slices, the central mechanisms underlying theta generation have been successfully studied in in vitro conditions. Most of these experiments were focused on the basic question regarding the similarities between the cholinergically induced theta activity and theta rhythm examined in vivo. The Org 27569 results of numerous in vitro experiments strongly suggest that cholinergically induced theta rhythm recorded in hippocampal slices is a useful analogue of theta observed in intact animals, and could be helpful in searching for the mechanisms of oscillations and synchrony in the central nervous system neuronal networks. The objective of the present review is to discuss the main results of experiments concerning theta oscillations recorded in in vitro conditions. It is our intent to provide, on the basis of these results, the characteristics of essential mechanisms underlying the generation of atropine-sensitive in vitro theta.

In general,

opacification activity

In general,

opacification activity was evaluated using horse serum (Rakonjac et al., 1995; Courtney et al., 1999; Gillen et al., 2002). We also investigated serum opacification using sera obtained from other sources (horse, pig, cow and human). In the culture supernatants of fish isolates, the strongest reaction was observed when fish serum was used as the substrate. In the opacity reaction, SOF targeted high-density lipoprotein (HDL) particles as the substrate (Courtney et al., 2006). Therefore, the turbidity, which may be attributed to the number of HDL particles, was higher in fish serum than in other sera. Previous studies demonstrated that when the serum agar overlay method using SDS–PAGE was adopted, an opaque band appeared on the serum agar (Rakonjac et al., 1995; Courtney et al., 1999; Gillen et al., 2002). The present study

was able to detect no band on the serum agar with Selleckchem MG 132 SDS-PAGE. Sufficient SOF activity of rSOF-OFD could be determined even if the rSOF-OFD sample was heated for 5 min at 100 °C. Meanwhile, addition of SDS to the sample solution apparently attenuated the opacification reaction in fish serum (data not shown). Labile apoA-1 of HDL has been shown to be required for the opacification reaction in serum (Han et al., 2009). In this study, although we have not determined whether SDS is acting directly on SOF or on fish HDL, it is possible that SDS affects apoA-1 of fish HDL and then prevents the opacification reaction. In addition, apoA-1 of fish HDL could be more labile and sensitive to SDS than that of human or other mammals. The expected size of the immune stained band detected by the Western blotting

with the anti-His tag was approximately half that of the opaque band detected by the serum agar overlay method with a native-PAGE check gel. Previous studies reported that the molecular mass of recombinant SOF was much larger than predicted and might be responsible for a dimer of SOF (Courtney et al., 1999; Katerov et al., 2000). Therefore, rSOF-OFD may also form a dimer, and the SDS disassociated the rSOF-OFD molecules. Further studies are in preparation to investigate the different molecular sizes. The serum opacification activity in S. dysgalactiae has been reported only in strain S2 isolated from bovine (Courtney et al., 1999). In this study, a novel variation of the sof gene, sof-FD, and the SOF activity of GCSD strains isolated from farmed fish were determined. SOF was demonstrated to be a virulence determinant of S. pyogenes and S. suis (Baums et al., 2006; Timmer et al., 2006; Gillen et al., 2008). However, the role of SOF-FD in GCSD isolates was not clear. Further studies on SOF-FD may elucidate the mechanism of the virulence determinant in fish isolates. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports, Japan (21580229).

The use of the TPB provided theoretical underpinning to the empir

The use of the TPB provided theoretical underpinning to the empirical work, identified factors that predicted behaviour (especially intention) and led check details to the collection of respondents’ beliefs underlying the direct constructs (TPB variables) reported. The overall combined response rate of 32% was less than expected and was likely to have been affected by the relatively complex nature of

the questionnaire and its length. However, even with this response rate the data derived from the 927 respondents had sufficient statistical power for all the regression analyses. Hence, the study had sufficient statistical power to achieve its objectives, that is, to examine the theoretical Lumacaftor order predictors of self-reported behaviour, together with respondent characteristics. The study was conducted in Scotland and few respondents were from ethnic minorities. Furthermore, because of the sampling strategy used, more female than males responded and respondents were also more likely to be older and to be married or living with someone. As such, the results might not be generalisable to individuals who are younger, living alone or from ethnic minorities. The sample was derived from the electoral register but

excluded individuals registered with the Mail Preference Service. While this is likely to have introduced bias into the sample, it was the most inclusive method available for this survey. The additional belief items were included in only half the sample to minimise the impact on the overall response rate. In general, respondents had positive perceptions regarding giving information to MCAs during consultations for NPMs. Previous research has shown that the extent of communication in terms of information exchange between patients and MCAs during consultations is often minimal, and that MCAs perceive that the public fail to realise

their role (the MCA) differs from that of general shop assistants[22] and that patients are reluctant to provide information.[4] Nevertheless, Clomifene family doctors and the NHS rather than MCAs, were identified as likely to have more influence on people’s behaviour, as indicated by the significant difference in these beliefs between those who did and did not give information. Patients’ desire to receive information during counselling for NPMs has been demonstrated previously,[23] as has awareness of the need to provide specific information during consultations.[8] Based on the results of this study, it seems that patients may be more positive about providing information during consultations than MCAs realise and the behaviour of MCAs may actually inhibit rather than facilitate information exchange. Patient demographics, such as age and gender have previously been shown to influence health professional communication behaviour.

In terms of surfaces, the labial surface of the molars and inciso

In terms of surfaces, the labial surface of the molars and incisors was the most frequently affected. The occlusal surface was the least affected but presented the most severe lesions in terms of treatment needing, as in other studies[6, 30, 31]. The reason is probably its morphology, the ease with which it accumulates traces of plaque and is therefore more likely to trigger caries, and its greater involvement in mastication compared with other tooth surfaces. Authors have classed the defect according to its extent and the degree to which it affects the teeth[5, 15, 27,

30], but there is no consensus on classifying the severity of the lesion. MS-275 price Consequently, to find out the social impact of MIH, the present study estimated the treatment need for each molar and incisor of each child with MIH in accordance with the WHO criteria[24], classified into checkups, non-urgent need and immediate need of care. This resulted in 3.8% of children with MIH needing urgent treatment, which implies severe

involvement and 27.9% needing some kind of treatment other than urgent which could be interpreted as moderately affected, which is similar to the figures reported by Ghanim et al.[31], Lygidakis et al.[37], Kusku et al.[38] One possible bias in PI3K inhibitor this study concerns teeth with atypical restorations mafosfamide with sound margins, with post-eruptive loss of enamel, or with opacities without retentive surface areas or caries, which have been considered at the time of study as needing checkups or preventive treatment where other authors, considering the severity of the lesion, would have classed them as moderate. However, in epidemiological studies, it is usual to estimate severity on the basis of treatment need, and this would allow greater agreement among researchers. Some studies have classed the severity

of MIH according to the number of teeth affected[6, 13, 20, 25, 37]. In the present study, in agreement with these authors, the number of teeth affected increased significantly as the need of treatment rose in terms of urgency. It was also observed, in agreement with Jasulaityte et al.[25] and Chawla et al.[32], that the children with both molars and incisors affected (the MIH group) presented more affected molars and more serious defects which demands treatment than those that only had hypomineralized molars (the MH group). Because of the greater porosity of the tooth structure and, consequently, its lower mechanical resistance, MIH is considered a risk factor for dental caries in low caries prevalence populations[1, 6, 20, 41].

1a–d) To increase the resolution of the IFA observations, C bur

1a–d). To increase the resolution of the IFA observations, C. burnetii-infected Vero cells were analyzed by IEM using C. burnetii IcmT-, IcmV-, and DotH-specific antibodies. IEM

analyses revealed polar localization of the gold particle-conjugated secondary antibodies to one or both poles of C. burnetii cells when using antibodies against IcmT and DotH (Fig. OSI-744 in vitro 2). Interestingly, based on the length of a C. burnetii LCV (0.5–1.0 μm), the IEM staining of IcmT and DotH indicates that the polar localization is primarily observed on LCVs using this methodology (Fig. 2), (McCaul & Williams, 1981; McCaul, 1991; Heinzen et al., 1999). Attempts to obtain conclusive IEM results for IcmV localization were unsuccessful; however, both IcmT and DotH clearly localized to the bacterial pole(s), thus supporting the IFA observations. Using Vero cell cultures infected with C. burnetii NMII for 3 weeks

allowed for ready identification of T4BSS polar localization by IEM; however, biological questions remain about the ultrastructure of the T4BSS. Does the C. burnetii T4BSS initially localize medial to the polar region(s) and then migrate laterally to the poles during the course of cellular development or does it initially nucleate at the Ribociclib purchase pole(s) and recruit other T4BSS components to the nucleation site? The IEM images show immunoreactivity at sites somewhat medial to the C. burnetii cell pole (Fig. 2b), making this a possibility. Another observation is that the T4BSS of C. burnetii may localize on one or both pole(s) of the bacterium. The bipolar localization of the T4BSS on C. burnetii cells may correlate to cells that are approaching cell division. In such a case, the bipolar localization would ensure that daughter cells are equipped with the

components for a functional T4BSS after binary fission, hence the observation of bacteria with T4BSS localized at a single pole (Figs 1 and 2). The utility of having the T4BSS localized on the pole(s) of C. burnetii cells and how this relates to the pathogen’s interactions Rutecarpine with the host cell is not clear. The observation that A. tumefaciens intimately interfaces with a host cell at the bacterial pole (Matthysse, 1987) and that this T4ASS machinery then secretes effector molecules into the host (Christie & Vogel, 2000) would indicate that direct association of a T4SS with a membrane may allow effector secretion across/into the membrane. An analogous interface between pathogen and membrane has been observed in the intravacuolar pathogen Chlamydia trachomatis, which secretes effector proteins into the host cell via a T3SS (Fields et al., 2003) and closely associates with the PV membrane during infection (Matsumoto, 1988; Hackstadt et al., 1997). An obvious and consistent interface between a pole of C. burnetii and the PV membrane has not been demonstrated. However, a study of published EM micrographs (McCaul, 1991; Coleman et al.

While overall tone-evoked response magnitudes were comparable bet

While overall tone-evoked response magnitudes were comparable between the two structures, tone signal : noise was significantly greater within the OT than in the PCX. Z-VAD-FMK chemical structure No effect of tone frequency (1–55 kHz) was found within either structure, with most units being narrowly tuned to a single frequency. These results suggest that a major portion of odor-evoked output from the olfactory bulb (i.e. that entering the OT and PCX) is subject to auditory sensory input in a manner that may modulate odor information processing,

odor-guided behaviors and perception. “
“Behavioral rhythms induced by methamphetamine (MAP) and daily restricted feeding (RF) in rats are independent of the circadian pacemaker in the suprachiasmatic nucleus (SCN), and have been regarded to share a common oscillatory mechanism. In the present study, in order to examine the responses of brain oscillatory systems to MAP and RF, circadian rhythms in clock gene, Period2, expression were measured in several brain selleck chemicals llc areas in rats. Transgenic rats carrying a bioluminescence reporter of Period2-dLuciferase were subjected to either daily injection

of MAP or RF of 2 h at a fixed time of day for 14 days. As a result, spontaneous movement and wheel-running activity were greatly enhanced following MAP injection and prior to daily meal under RF. Circadian Per2 rhythms were measured in the cultured brain tissues containing one of the following structures: the olfactory bulb; caudate-putamen; parietal cortex; substantia nigra; and SCN. Except for the SCN, the circadian Per2 rhythms in the brain tissues were significantly phase-delayed by 1.9 h on average in MAP-injected rats as compared with the saline-controls. On the other hand, the circadian rhythms outside the SCN were significantly phase-advanced by 6.3 h on average in rats under RF as compared with those under ad libitum feeding. These findings indicate that the circadian rhythms in specific brain areas of the central dopaminergic system respond differentially to MAP

injection and RF, suggesting that different oscillatory mechanisms in the brain underlie the MAP-induced behavior and pre-feeding activity under RF. “
“Glutamate is the major excitatory neurotransmitter of the central nervous system in vertebrates. Excitotoxicity, caused by over-stimulation Reverse transcriptase of the glutamate receptors, is a major cause of neuron death in several brain diseases, including epilepsy. We describe here how behavioural seizures can be triggered in adult zebrafish by the administration of kainate and are very similar to those observed in rodent models. Kainate induced a dose-dependent sequence of behavioural changes culminating in clonus-like convulsions. Behavioural seizures were suppressed by DNQX (6,7-dinitroquinoxaline-2,3-dione) dose-dependently, whilst MK-801 (a non-competitive NMDA receptor antagonist) had a lesser effect.

In a retrospective chart review of patients with HIV infection, P

In a retrospective chart review of patients with HIV infection, Pugliese et al. [83] found the incidence of PAH to be higher in individuals who received HAART compared with those

individuals who only received NRTIs. This result may have arisen from differences in the cohort populations: the HAART cohort may have had more progressed HIV disease as it was defined as individuals selleck screening library with a CD4 count of <300 cells/μL and a viral load of >30 000 copies/mL, whereas the NRTI cohort was defined as individuals with stage C2 (CD4 count between 200 and 499 cells/μL and AIDS-defining illness) or greater disease. The reason that HAART does not favourably impact the prevalence of HIV-related PAH is unclear at the present time. We do know from studies that HIV does not directly infect vascular endothelial cells or smooth muscle cells [24]. Hence, the association between HIV and PAH may not be related to viral load or immune status, partially explaining why HAART does not prevent PAH. The results of the case reports reveal that HIV-related PAH selleck compound is most common in male patients with an average age of 35 years who contracted HIV via injection drug use or male-to-male sexual activity. Although purely speculative, the increased frequency found in individuals who have used intravenous drugs might be related to foreign

body emboli, the use of amphetamine-based drugs, or the presence of portal hypertension, which might be under-diagnosed given the relatively high prevalence of concomitant hepatitis B and C found in this population [92]. The average CD4 count was around 354 cells/μL and 53% Tolmetin of the patients had been diagnosed with AIDS. There was no relationship identified between CD4 cell count and HIV-related PAH, which is corroborated by other studies [8]. The average time from diagnosis of HIV infection to developing PAH was 4.3 years. These results indicate that HIV-related PAH is a chronic disease that occurs gradually. The physical examination, chest X-ray, ECG and echocardiographic findings mentioned above in HIV-related PAH

are similar to those in other causes of PAH. There are no distinct physical or imaging findings that distinguish HIV-related PAH from other causes of PAH. Furthermore, the histopathological examination reveals that HIV-related PAH is characterized by plexogenic pulmonary arteriopathy similar to primary pulmonary vascular disease. The histopathology of primary pulmonary vascular disease is classified as primary pulmonary arteriopathy (plexiform arteriopathy, thrombotic arteriopathy, isolated medial hypertrophy, and medial hypertrophy with intimal fibrosis), pulmonary veno-occlusive disease and pulmonary capillary haemangiomatosis [93]. Plexogenic pulmonary arteriopathy has also been found in other causes of PAH including cirrhosis of the liver, portal hypertension, connective tissue disorders and congenital cardiac disorders [94].

To identify proteins other than gingipains secreted by the PorSS,

To identify proteins other than gingipains secreted by the PorSS, we compared the proteomes of P. gingivalis strains kgp rgpA rgpB (PorSS-proficient strain) and kgp rgpA rgpB porK (PorSS-deficient strain) using two-dimensional gel Bleomycin in vivo electrophoresis and peptide-mass fingerprinting. Sixteen spots representing 10 different proteins were present in the particle-free culture supernatant of

the PorSS-proficient strain but were absent or faint in that of the PorSS-deficient strain. These identified proteins possessed the C-terminal domains (CTDs), which had been suggested to form the CTD protein family. These results indicate that the PorSS is used for secretion of a number of proteins other than gingipains and that the CTDs of the proteins are associated with the PorSS-dependent secretion. The Gram-negative bacterium Porphyromonas gingivalis, a major pathogen of check details periodontal disease, possesses a number of virulence factors, including fimbriae, hemagglutinins, lipopolysaccharides

and proteinases. Extracellular and surface proteinases with high hydrolytic activities named gingipains are of particular importance as they have the ability to destroy periodontal tissue directly and/or indirectly (Potempa et al., 2000; Andrian et al., 2007). Gingipains are encoded by three separate genes, rgpA, rgpB and kgp, on the P. gingivalis chromosome (Curtis et al., 1999). The kgp and rgpA genes encode polyproteins comprising the signal peptide, propeptide, Lys-

and Arg-specific proteinase domains, adhesin domains and C-terminal Thiamine-diphosphate kinase domain (CTD). The rgpB gene encodes a protein comprising the signal peptide, propeptide, Arg-specific proteinase domain and CTD. These proteins are synthesized as polyproteins in the cytoplasm, are translocated across two membranes, inner and outer membranes, and secreted onto the bacterial cell surface. In our previous studies (Sato et al., 2010; Shoji et al., 2011) we found that gene products of rgpA, rgpB and kgp were translocated across the outer membrane by the Por secretion system (PorSS) in which porK, porL, porM, porN, porO, porP, porQ, porT, porU, porV (PG27, lptO), porW and sov genes were involved. Expression of some of these genes is regulated by a two-component system, the PorX response regulator and PorY histidine sensor kinase (Sato et al., 2010). Primary gene products of rgpA, rgpB and kgp have common motifs in their CTD regions. The P. gingivalis genome encodes a number of putative CTD-containing proteins (Seers et al., 2006). Nguyen et al. (2007) showed that CTD-containing proteins were also found in predicted proteins of other bacteria in the Bacteroidetes phylum, such as Prevotella intermedia and Tannerella forsythia. Among P.

5% Difco yeast extract, 1% NaCl), and 01% arabinose (Sigma) was

5% Difco yeast extract, 1% NaCl), and 0.1% arabinose (Sigma) was used to induce traJ in pBAD constructs. Colonies were grown on LB agar (1.5% Difco Bacto agar) or 2% water agar [5 mM MgSO4, 1.5% Difco Bacto agar, double-distilled (dd) water] with 1 × M9 minimal salts (5 mM Na2HPO4·7H2O, 22 mM KH2PO4, 8 mM NaCl, 19 mM NH4Cl) and 0.4% lactose. The final concentrations of the LDE225 ic50 antibiotics were as follows: ampicillin (Ap) 100 μg mL−1, chloramphenicol (Cm) 20 μg mL−1, kanamycin (Km) 25 μg mL−1, streptomycin (Sm) 200 μg mL−1 and spectinomycin (Sp) 100 μg mL−1. All DNA restriction and ligation enzymes were from Fermentas as well as DNA marker ladders. Vent polymerase

was from NEBiolabs. Formaldehyde was purchased from Anachemia, whereas disuccinimidyl suberate (DSS) was purchased from Pierce. Protein A beads were purchased from Roche. DNA (QIAprep Spin Miniprep Kit) and PCR purification (QIAquick PCR purification kit) solutions and columns were purchased from Qiagen. Primer synthesis

and DNA sequencing find more were carried out by The Molecular Biology Services Unit at the University of Alberta. Mutagenesis of selected residues was performed using the QuikChange kit (Stratagene) according to the manufacturer’s directions. All point mutations were constructed using complementary double-stranded oligonucleotides of 30 base pairs with the mutation in the center Bcl-w of the oligonucleotide. Details of the mutagenic primers are available upon request.

Deletions were constructed by entering an ochre codon at the desired location within the mutagenic primer or by a PCR using primers specific to the 5′ and the appropriate 3′ end of traJ (Table 2). Mating assays have been described elsewhere (Frost & Manchak, 1998). Briefly, MC4100/Flac traJ90 containing pBADTraJ or mutants derived from it were used as donor strains and ED24 was used as the recipient. Overnight cultures of both donor and recipient cells (0.15 mL) grown with appropriate antibiotics were diluted (1 : 50) into 3 mL of LB broth with no antibiotics. Cultures were grown at 37 °C, and after 1 h, 0.1% arabinose (final concentration) was added to donor cells in order to induce TraJ production. At an OD600 nm of approximately 1.0, 100 μL each of donor and recipient cells were incubated together for 1 h at 37 °C in 1 mL of fresh LB with 0.1% arabinose. Mating efficiency was determined as the ratio of transconjugants per 100 donors and expressed as a percent. Simultaneously, another 100-μL aliquot of donor cells was pelleted for immunoblot analysis. Immunoblot analysis was performed as described previously (Gubbins et al., 2002). Polyclonal anti-TraJ antiserum and the secondary antibody (horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin G, GE Health Care) were used at a 1 : 20 000 dilution. Anti-H-NS antiserum was used at a 1 : 100 dilution.