Elkind, H. Rechnitzer, T. Vaisid, J.D. Kornspan, S. Barnoy, S. Rottem & N.S. Kosower, unpublished data). In conclusion, the fact that an appreciable

proportion of human cell cultures is contaminated by mycoplasmas, specifically by M. hyorhinis (Timenetsky et al., 2006), renders the results presented here significant and relevant to studies using human cell cultures. Because the calpain–calpastatin system plays important roles in cell functions, the altered calpain–calpastatin buy RG7422 system in the mycoplasma-infected cells may influence the response of the infected cells to stress-inducing conditions. The results may also be relevant to mycoplasma-associated diseases. In addition, the mycoplasma-infected cells provide a system for studying the factors and pathways involved in the regulation of cellular calpastatin. This work was performed in partial fulfillment of the requirements for a PhD degree (Esther Elkind), Sackler School of Medicine, Tel Aviv University. “
“Rainbow trout gastroenteritis has been related to the accumulation of segmented filamentous bacteria in the digestive tract of fish, which presents lethargy, reduced appetite and accumulation

of mucoid faeces. Some authors Atezolizumab order associate the comparison of illness with the presence of viable filaments, which produce and release strings of endospores in the lumen of the gut. The segmented filamentous bacteria that could not be cultured in vitro have been related to Clostridium group I, and they have been named Candidatus arthromitus. Despite the various strategies that have been used to detect unculturable microorganisms, molecular methods have facilitated studies on culture-independent microorganisms. Direct DNA

extraction from samples and subsequent study of 16S rRNA genes represent a tool for studying unculturable microbial flora. As direct detection of specific microorganisms is possible through the utilization of primers or probes annealing specific DNA sequences, the aim of this work was to design specific primers for the direct detection of C. arthromitus in fish using a nested PCR. Gram-positive, endospore-forming, segmented filamentous bacteria (SFB) have been observed in the small intestine of many animals (e.g. rats, pigs, insects) and Megestrol Acetate in the intestinal content of trouts (Oncorhynchus mykiss) affected by diarrhoea. Intensive fish-farming systems have been actively developed during recent decades. This intensification has resulted in an increase in the number of pathogens reported from these intensive aquaculture production systems. An enteritic syndrome affecting farmed rainbow trout [rainbow trout gastroenteritis (RTGE)] has been described and related to the accumulation of the SFB in the digestive tract of fish (Goodwin et al., 1991; Klaasen et al., 1993).

In particular, Gram-positive bacteria, in contrast to Gram-negative bacteria or endotoxin, seem to induce a considerable IL-6 response and less so of TNF-α and IL-1β as reviewed by Opal & Cohen (1999); this is in line with our results. IL-6 has been implicated as a marker of the severity of disease and a target for therapy (Ziegler-Heitbrock et al., 1992; Oda et al., 2005). Pigs are normally hypercoagulable compared with other species (Jankun et al., 2009). The decrease in the platelet counts and the increase in TEG MA correspond well with the development of an increasingly hypercoagulable state of the blood clotting system, which could contribute towards the development of thrombosis,

unrelated to suppurative inflammation, as was observed in the heart of one Selleckchem Ruxolitinib of the pigs

(McCrath et al., 2005; Kashuk et al., 2009). The assays of liver function (serum bilirubin, AST and creatine kinase) and the histological lesions showed the liver as dysfunctional or failing by 48 h. Within the SOFA scoring system, a distinction has been made between organ dysfunction (SOFA score ≤2) and failure (SOFA score ≥3) (Vincent et al., 1998), and although the assignment of the change in the level of the organ-specific variable to the SOFA score has been decided on by consensus (Bone et al., 1992), later studies have proven the robustness of the scoring system in predicting mortality (Vincent et al., 1998; Moreno et al., 1999). In pigs, the relationship between the levels of serum bilirubin and the degree of liver disease is not known. However, GSK J4 the bilirubinaemia observed did seem to have a hepatic origin that was not directly

associated with bacterial growth as indicated by the drastic increase Benzatropine in serum bilirubin, combined with an increase in AST and normal creatine kinase levels (pig no. III-1), and by the nature of the histological lesions and low bacterial counts in the liver. In large domestic animals, ALT is not a specific marker of hepatocyte damage. In these animals, an increase in AST indicates hepatocyte damage or damage of the skeletal muscle; the latter will, however, also induce an increase in creatine kinase (Tennant, 1997). No dysfunction of the kidneys was evident. The microbiological results conform to previous experimental S. aureus intravenous-inoculation studies in pigs (Nielsen et al., 2009b; Jensen et al., 2010), indicating a tremendous blood-clearing action of the lungs and a gradually increasing bacterial load in bones. The finding of macroscopic pulmonary abscesses at 12–48 h PI shows that S. aureus can establish itself early in the lungs, causing focal lesions. The absence of acute microabscesses at 48 h in the lungs and spleen as well as the decreasing concentration of bacteria in soft tissues indicate that the infection does not progress in these sites, in contrast to the bones. In conclusion, we were able to induce sepsis and severe sepsis in pigs.

Further year-round, large-scale study from multiple sites in Southeast Asia would provide more precise information. Second, we could not analyze the incidence of travelers’ diarrhea separately in each individual country. Although we could report the country selleck where diarrhea occurred and could compare with the number

of visitor to that country (Table 5), it could not be interpreted as an incidence. Since the duration of stay (exposure time) in each country could not be obtained and the number of cases were too small to enable separate analysis. Moreover, some backpackers with travelers’ diarrhea had complex itineraries, which crossed multiple international borders. In those cases, the country where diarrhea the occurred may not have been the country of exposure. Therefore, we reported the incidence of diarrhea in a region-specific manner. We were able to conclude that backpackers in Southeast Asia were at risk of developing travelers’ diarrhea. The incidence rate among this group was much higher than among general travelers in the same region. Specific attention should be paid to this particular group, to minimize the risk and lessen its impact. We would like to thanks Ms. Phatcharee Danwiwatdecha for assistance with data collection. We also thank Mr. Paul Adams of the Faculty of Tropical Medicine, Mahidol University, Y-27632 supplier for reviewing the manuscript. The authors declare no conflict of interest in this study.

“
“Background. In 2009, 58.6 million UK residents traveled abroad. Of these, 49.5 million (84.5%) visits were to Europe and North America and 9.1 million Farnesyltransferase (15.5%) were to other parts of the world. Rabies is widely distributed and continues to be a major public health issue in many developing countries. The UK is free of rabies in carnivore host species, although cases

of rabies in bats have been reported. This study examined the rabies postexposure prophylaxis (PEP) service from 2000 to July 2009 at the Liverpool School of Tropical Medicine. Methods. Medical records of patients who attended the clinic for rabies PEP were reviewed. Results. During the study period, 139 patients were treated for possible rabies exposure. The mean age was 35 years. Thailand and Turkey each accounted for 31 (22.3%) cases. Sixty-nine (49.6%) of those seen were due to dog bites. Most injuries involved a lower limb (n = 67, 48.2%) or hands (n = 26, 18.7%). Eighty-six (61.9%) cases had initiated rabies PEP overseas, but only 3 of the 78 (3.8%) meeting UK criteria for rabies immunoglobulin (RIG) received it while overseas. Only an additional 11 patients received RIG on return to the UK; most were seen more than 7 days after initiation of PEP. The median time from exposure to receiving rabies PEP was 1 day (range: 0–1,720). Only 14 (10.1%) had received preexposure rabies vaccination. Conclusions. The majority of travelers seeking PEP at this clinic initiated treatment overseas.

Their median age (interquartile range) was 9.1 (6.8–11.0) years, the median duration of their NNRTI regimens was 23.7 (15.7–32.6) months, their median CD4 percentage was 12% (4–20%), and their median plasma HIV RNA at the time of genotype testing was 4.8 (4.3–5.2) log10 HIV-1 RNA copies/mL. The nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations found were as follows: 85% of the

children had M184V/I, 23% had at least four thymidine analogue mutations, 12% had the selleck inhibitor Q151M complex, 5% had K65R, and 1% had the 69 insertion. Ninety-eight per cent of the children had at least one NNRTI resistance mutation, and 48% had etravirine mutation-weighted scores ≥4. CD4 percentage <15% prior to switching regimens [odds ratio (OR) 5.49; 95% confidence interval (CI) 2.02–14.93] and plasma HIV RNA>5 log10 copies/mL (OR 2.46; 95% CI 1.04–5.82) were independent predictors of at least four thymidine analogue mutations, the Q151M complex or the 69 insertion. In settings without routine viral load monitoring, second-line antiretroviral therapy regimens should be designed assuming that clinical or immunological failure is associated with high rates of multi-NRTI resistance and NNRTI resistance,

including resistance to etravirine. The widespread LY2109761 use of antiretroviral therapy (ART) for the treatment of HIV-infected children has dramatically changed the course of HIV infection, leading to reductions in morbidity and mortality [1–3]. A triple drug combination including two nucleoside reverse transcriptase inhibitors (NRTIs) plus one nonnucleoside reverse transcriptase inhibitor

(NNRTI) or one Smoothened protease inhibitor (PI) [4] is widely recommended as first-line therapy. For resource-limited settings, the World Health Organization (WHO) recommends an NNRTI-based regimen, which is preferred because it is effective, well tolerated and inexpensive. Plasma HIV RNA monitoring after initiation of ART is usually not available through treatment programmes in resource-limited settings [5]. For example, the Thai national AIDS programme provides antiretroviral drugs for HIV-infected patients and CD4 monitoring every 6 months. Annual plasma HIV viral load monitoring was only recently incorporated into the national programme in 2008. Thus, in the past, the majority of Thai children were diagnosed with treatment failure when they had clinical or immunological failure, that is a long time after virological replication had resumed while they were still on treatment. Consequently, resistance-associated mutations may have occurred in these children as a result of persistent viral replication under drug pressure. The goal of second-line treatment is to fully suppress HIV replication; therefore, the new regimen should comprise at least two, but preferably three, fully active drugs.

Their median age (interquartile range) was 9.1 (6.8–11.0) years, the median duration of their NNRTI regimens was 23.7 (15.7–32.6) months, their median CD4 percentage was 12% (4–20%), and their median plasma HIV RNA at the time of genotype testing was 4.8 (4.3–5.2) log10 HIV-1 RNA copies/mL. The nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations found were as follows: 85% of the

children had M184V/I, 23% had at least four thymidine analogue mutations, 12% had the selleck chemicals llc Q151M complex, 5% had K65R, and 1% had the 69 insertion. Ninety-eight per cent of the children had at least one NNRTI resistance mutation, and 48% had etravirine mutation-weighted scores ≥4. CD4 percentage <15% prior to switching regimens [odds ratio (OR) 5.49; 95% confidence interval (CI) 2.02–14.93] and plasma HIV RNA>5 log10 copies/mL (OR 2.46; 95% CI 1.04–5.82) were independent predictors of at least four thymidine analogue mutations, the Q151M complex or the 69 insertion. In settings without routine viral load monitoring, second-line antiretroviral therapy regimens should be designed assuming that clinical or immunological failure is associated with high rates of multi-NRTI resistance and NNRTI resistance,

including resistance to etravirine. The widespread Vorinostat price use of antiretroviral therapy (ART) for the treatment of HIV-infected children has dramatically changed the course of HIV infection, leading to reductions in morbidity and mortality [1–3]. A triple drug combination including two nucleoside reverse transcriptase inhibitors (NRTIs) plus one nonnucleoside reverse transcriptase inhibitor

(NNRTI) or one of protease inhibitor (PI) [4] is widely recommended as first-line therapy. For resource-limited settings, the World Health Organization (WHO) recommends an NNRTI-based regimen, which is preferred because it is effective, well tolerated and inexpensive. Plasma HIV RNA monitoring after initiation of ART is usually not available through treatment programmes in resource-limited settings [5]. For example, the Thai national AIDS programme provides antiretroviral drugs for HIV-infected patients and CD4 monitoring every 6 months. Annual plasma HIV viral load monitoring was only recently incorporated into the national programme in 2008. Thus, in the past, the majority of Thai children were diagnosed with treatment failure when they had clinical or immunological failure, that is a long time after virological replication had resumed while they were still on treatment. Consequently, resistance-associated mutations may have occurred in these children as a result of persistent viral replication under drug pressure. The goal of second-line treatment is to fully suppress HIV replication; therefore, the new regimen should comprise at least two, but preferably three, fully active drugs.

Differences between guidelines reflect different understandings and use of terms for the components of the HIV testing process, as well as, most importantly, possible differences in the appraisal

GSK2118436 in vivo and use of the limited evidence base regarding barriers and facilitators of HIV testing. While a substantial body of research regarding the benefits of expanding HIV testing in a wider range of settings exists, studies are mostly descriptive and typically focus on only a few demographic, potential barriers that are largely assessed in isolation, and may not translate to all settings and populations at risk. There currently is a lack of published HIV testing protocols and, in particular, a lack Obeticholic Acid of evidence regarding the performance of different HIV testing models used across health services on a range of indicators of efficacy and cost-effectiveness, and how informed

consent and pre- and post-test counselling are addressed in these models. Based on this, HIV in Europe suggests studying the development and implementation of best practice service models that contribute to increasing the uptake and frequency of HIV testing as well as making optimal use of opportunities to promote risk reduction. A discussion forum will be launched on http://www.hiveurope.eu with the aim of presenting and discussing different definitions of counselling for different health care settings and test situations. A draft guideline for routine HIV testing in indicator conditions was presented to conference participants for feedback. The guidance document was published in

October 2012 [13, 14]. Findings from the HIV Indicator Diseases across Europe Study [15] contributed to the evidence base of conditions that should trigger a routine offer of an HIV test in specific health care settings. Other studies Janus kinase (JAK) have also demonstrated the cost-effectiveness and broad acceptance of routine testing for all health care clients in a wide variety of settings, including emergency departments and primary care clinics [16-21]. Recent data demonstrate that indicator condition-guided HIV testing is an effective method of identifying undiagnosed HIV infection, potentially at an earlier stage of disease [15], which is currently being further studied through the HIV Indicator Diseases across Europe Study phase 2 (HIDES 2). It is also likely to be more cost-efficient than other methods, as it is opportunistically offering an HIV test at a time when patients are already accessing services for another reason. However, despite the evidence and new European guidelines, this strategy is not being widely implemented. This is, to a large extent, attributable to operational and health care worker (HCW) barriers to offering HIV testing.

46 years ± 2.97. More than 70% of athletes had visible untreated decay. Almost 30% (29.8%) of the athletes had gingival inflammation. Pain in the oral cavity was reported by 28.6%. Athletes who had untreated decay reported 6.67 times (95% CI OR; 4.00–11.14) more pain compared to those who did not have untreated decay. Athletes

living in provinces on Java Island had 1.54 times (95% CI OR; 1.15–2.07) more untreated decay compared to the athletes who live in provinces in outer this website Java Island. 21.63% of the screened athletes were referred to the dentist for urgent treatment. The results suggest that there is an elevated oral treatment need in Indonesian Special Smiles population. “
“To evaluate the impact of traumatic dental injury (TDI) among Brazilian adolescents on their families’ quality of life (QoL). A cross-sectional study was carried out with a BIBW2992 supplier population-based sample of 1122 schoolchildren aged 11–14 years selected using a multistage sampling procedure. Parents/caregivers answered the Brazilian version of the 14-item Family Impact Scale (B-FIS) to assess the impact on family’s QoL. The main independent variable was TDI, which was diagnosed using the Andreasen classification. Malocclusion, dental caries, gender and socio-economic

classification were the other independent variables. Poisson regression analyses were carried out (P < 0.05). The prevalence of TDI was 14.8%. The multivariate model demonstrated that families of adolescents diagnosed with fracture involving the dentine or dentine/pulp were more likely to report a negative impact on the overall B-FIS score [rate ratio (RR) = 1.44; 95% confidence interval (CI): 1.10–1.88] as well on the Parental/Family Activity (RR = 1.45; 95% CI: 1.09–1.94), Parental Emotions (RR=1.45; 95% CI: 1.03-2.04) and Family Conflict (RR = 1.46; 95% CI: 1.01–2.11) subscales in comparison with those who had no signs of TDI. Families of adolescents with more severe TDI were more likely to report a negative impact on QoL, affecting family activities and emotions, which can result in family conflicts. "
“International Journal of Paediatric Dentistry 2010; 20: 391–399

Background.  An enhanced frequency of cognitive and behavioural disturbances has been reported in preterm children. It is not known if this affects Afatinib clinical trial their perceptions of or behaviour in the dental care situation. Hypothesis.  The hypotheses were that preterm (PT) children aged 12–14 years more often exhibit dental fear and anxiety (DFA) than full-term controls (C), while no differences were expected regarding oral health behaviour. Methods.  One hundred and nine PT and 108 C children took part in the present questionnaire study. DFA was assessed using the Children’s Fear Survey Schedule – Dental Subscale (CFSS-DS). In addition the questionnaire covered items including satisfaction with received dental care, oral health behaviour and medical health. Results.

, 1997). This polyP accumulation is due to the inhibition of polyP degradation by ppGpp rather than the loss of PhoU function (Kuroda & Ohtake, buy SCH772984 2000; Kuroda, 2006). To determine whether YjbB reduces the levels of polyP under conditions of amino acid starvation, we introduced pMWyjbB into the wild-type strain and then subjected the transformant to amino acid starvation. The levels of polyP in the transformant were lower than those of the strain carrying a control vector plasmid (Fig. 2a). Escherichia coli also accumulates polyP when its growth is blocked by antibiotics that inhibit nucleic acid synthesis

(Kuroda & Ohtake, 2000; Kuroda, 2006). When treated with rifampicin, the levels of polyP in the transformant were also lower than those in the strain carrying a control vector plasmid (Fig. 2b). These results also supported the hypothesis that the reduction of polyP was not due to the suppression of the expression of Pho regulon genes including pstSCAB. As noted above, the N-terminal half of YjbB shows homology with Na+/Pi BMS-907351 price cotransporters, indicating the possible involvement of YjbB in the Pi flux. Escherichia coli possesses four Pi transporters (PitA, PitB, PhnCDE, and PstSCAB). Here, we constructed a mutant strain, MT2006, which lacks all four Pi transporters (Table 1). This mutant lost the ability to grow on a medium containing

Pi as the sole source of phosphorus (Pi medium) (Fig. 3a). To test whether YjbB is involved in Pi import, we introduced pMWyjbB into MT2006. However, this transformant still failed to grow on the Pi medium (Fig. 3a). Escherichia coli can utilize glycerol-3-phosphate as the sole source of Pi (Hayashi et al., 1964; Schweizer et al., 1982). The transformant could grow on a medium containing glycerol-3-phosphate as the sole source of phosphorus (GP medium) (Fig. 3b), indicating that YjbB has no or little Pi-uptake activity. On the other

hand, the transformant released approximately 1 mM Pi into the supernatant very when it grew for 8 h on the GP medium. To exclude the possibility that Pi was due to the degradation of glycerol-3-phosphate by an elevated alkaline phosphatase activity in the phoU mutant, we constructed MT2013 (phoA, yjbB, pitA, pitB, phnC, pstSCAB-phoU). Similar to MT2006, MT2013 and its transformant harboring pMWyjbB lost the ability to grow on Pi medium, but could grow on GP medium (Fig. 3). MT2013 carrying pMWyjbB still released a large amount of Pi into the GP medium, while MT2013 carrying a control vector plasmid only released a small amount of Pi during the lag phase (Fig. 4a and b). Escherichia coli can take up glycerol-3-phosphate via glycerol-3-phosphate transport systems (Ugp and GlpT) (Hayashi et al., 1964; Schweizer et al., 1982). The GlpT transport system can function in the exchange mode, so that glycerol-3-phosphate is taken up in exchange with internal Pi, while the Ugp system does not release Pi.

In particular, the nature of the polysaccharides

available for fungal growth induced a specific transcriptional response aiming at the targeted enzymatic degradation of the given polysaccharides. “
“Natural and anthropogenic impacts such as terrestrial runoff, influence the water quality along the coast of the Great Barrier Reef (GBR) and may in turn affect coral reef communities. Associated bacterial biofilms respond rapidly to environmental conditions and are potential find more bioindicators for changes in water quality. As a prerequisite to study the effects of water quality on biofilm communities, appropriate biofilm substrates for deployment in the field must be developed and evaluated. This study investigates the effect of different settlement substrates (i.e. glass slides, ceramic tiles, coral skeletons and reef sediments) on bacterial biofilm communities grown in situ for 48 days at two locations in the Whitsunday Island Group (Central GBR) during two sampling times. Bacterial communities associated with the biofilms were analysed using terminal restriction fragment length polymorphism

(T-RFLP) and clone library analyses of 16S rRNA genes. Findings revealed that substrate type had little influence on bacterial community composition. Of particular relevance, glass slides and coral skeletons exhibited very similar communities during both sampling BAY 80-6946 datasheet times, suggesting the suitability of standardized glass slides for long-term biofilm indicator studies in tropical coral reef ecosystems. Similar to coastal regions worldwide, local natural and anthropogenic impacts such as land runoff from agriculture deliver inorganic nutrients, sediments, freshwater and pesticides to the coastal and coral reef waters of the Great Barrier Reef (GBR) (Bell, 1991), and thereby influence the

water quality of this ecosystem. L-NAME HCl Coral reefs harbour abundant bacterial biofilms that are crucial catalysts of biogeochemical nutrient cycling (Battin et al., 2003) and are therefore critical to reef ecosystem functioning. This underlines the necessity to understand community composition and function of microorganisms within coral reef-associated biofilms. Marine biofilms are complex microbial communities comprising of surface-attached microorganisms embedded in an extracellular polymeric matrix (Mihm et al., 1981). The bacterial communities within biofilms respond rapidly to changing environmental conditions, and therefore bacterial community composition of artificially and field grown biofilms have previously been used as bioindicators for water quality in freshwater (Campbell et al., 2011), estuarine (Jones et al., 2007; Nocker et al., 2007) and temperate and polar coastal marine environments (Moss et al., 2006; Webster & Negri, 2006; Dang et al., 2008). In addition, biofilms may also be potential bioindicators for water quality in tropical coastal coral reef ecosystems (Kriwy & Uthicke, 2011).

For expression of proteins, the transformed yeasts were grown at 30 °C with shaking at 200 r.p.m. in 300 mL of YPD medium in 500-mL baffled shake flasks. For fermentation, recombinant strains were allowed to grow aerobically at 30 °C with shaking at 200 r.p.m. in 800 mL of SD medium in 1-L Erlenmeyer flasks to an OD600 nm value of 1.5–2.0. The inoculum culture was harvested by centrifugation, washed twice with sterile distilled water, and then inoculated

into 20 mL of CMC medium in a 50-mL closed bottle to an OD600 nm value of 20. These cultures were cultivated at 30 °C with shaking at 100 r.p.m. Yeast transformants containing the chimeric endoglucanase CelE with the altered dockerin domain were screened for CMC-degrading ability by patching on YPD plates containing 1 g L−1 CMC. After 48 h of growth, colonies on the plate were washed, and the remaining CMC was stained with 1 g L−1 Congo red and destained with 1 g L−1 Protein Tyrosine Kinase inhibitor NaCl (Den Haan et al., 2007). To confirm the secretion of endoglucanase, halos were detected on YPD–CMC plates that adsorbed 5 μL of culture supernatant. β-Glucosidase activity was detected by screening on YPD plates containing 5 mM p-nitrophenyl-β-d-glucopyranoside, as described previously (Jeon et al., 2009). For the production and secretion of proteins, recombinant selleck chemical yeasts were grown at 30 °C

for 48 h in YPD medium. Medium supernatant was then obtained by centrifugation. The supernatant was concentrated by ultrafiltration using an Ultrafree Biomax centrifugal filter unit (Millipore Co.) with a 10-kDa cut-off membrane. The concentration of secreted proteins was measured using the Bradford method (Bradford, 1976) with a Quick Start™ protein assay kit (Bio-Rad Laboratories Inc.) using bovine serum albumin as the standard. Purification was performed using cellulose (Sigmacell Type 50) at a concentration 2-hydroxyphytanoyl-CoA lyase of 10 mg protein per 1 mg cellulose and binding was performed at room temperature for 1 h with continuous shaking (Shpigel et al., 1999). The CBD-fusion protein

bound to the cellulose was centrifuged at 1600 g. Nonspecific proteins bound to cellulose were removed by washing cellulose samples three times, once with 1 M NaCl in 20 mM Tris, pH 8.0, and twice with 20 mM Tris, pH 7.5. Subsequently, bound proteins were eluted with 50 mM Tris, pH 12.5. Proteins in the cellulose-bound fraction were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The assembly of minicellulosomes was confirmed by native PAGE and zymogram analysis as described previously (Murashima et al., 2002). A zymogram with CMC was obtained by incorporating 0.2% of the substrate into the polyacrylamide gels used for native PAGE (Zhou et al., 2008). After electrophoresis, the gel was washed at room temperature in solution A (50 mM sodium acetate buffer, pH 5.0, containing 30% isopropanol) for 1 h and then solution B (50 mM sodium acetate buffer, pH 5.0) for 1 h.