Without close supervision,

many patients with TB are unable to complete a full course of medication, which results in relapse and acquired drug resistance [17]. China has the second highest burden of TB. The challenge we are facing for the control of TB is a dilemma because of the high incidence of MDR-TB and the lack of funding for the treatment with second-line anti-TB drugs. Previous studies demonstrate that DNA vaccine has a pronounced therapeutic action on TB in mice [8, 9]. In addition, immunotherapy with plasmid DNA encoding mycobacterial antigen in association with conventional chemotherapy is a more rapid and effective form of treatment on reactivation and reinfection of M. tb [10, 11]. In the present study, we test whether immunotherapy with DNA vaccine in combination with RFP or PZA result in effective treatment Autophagy inhibitor purchase of MDR-TB in infected mice. Mycobacterium tuberculosis Ag85A DNA vaccine is a strong immunotherapeutic agent for MDR-TB [14] and TB [8–11]. Th2 response is abundant during M. tb infection; therefore, the therapeutic effect is associated with not only prompt Th1 response but also switching from an improper status to a protective one. In the current study, significantly selleck compound more T cells that secrete IFN-γ are elicited by Ag85A DNA vaccination, and lower

amount of IL-4 are observed in Ag85A DNA vaccine immunized mice, suggesting a predominant Th1 immune response. RFP alone fails to kill the bacteria, but PZA alone is able to kill the bacteria, which suggest that MDR-TB model has been developed successfully. Vaccination with Ag85A DNA vaccine

associated with RFP reduces the pulmonary and splenic bacterial loads by 1.34 and 1.28 logs, respectively, compared with those of the RFP groups, which proves again that Ag85A DNA vaccine is the most efficient immunotherapy for MDR-TB in mice. This is consistent with our previous study [14]. Although Ag85A DNA vaccine associated with PZA treatment reduces the splenic infectious bacterial loads, it fails to reduce the pulmonary infectious bacterial loads when compared with the PZA alone groups. These results suggest that Ag85A DNA L-NAME HCl vaccine fails to strengthen the drug effect of PZA in killing infectious bacteria in lungs, but prevents haematogenous dissemination of M. tb to the spleens. Cai et al. [12] demonstrate that combined DNA vaccine may be a valuable adjunct to shorten the duration of antibacterial chemotherapy. The data of this study indicate that immunotherapy with RFP or PZA results in effective treatment of MDR-TB in infected mice. In conclusion, M. tb Ag85A DNA vaccine has obvious immunotherapeutic effect on TB and MDR-TB in mice. DNA vaccination associated with conventional chemotherapy may have synergistic effect for this treatment. The therapeutic Ag85A DNA vaccine and its combination with anti-TB drugs may be promising and affordable strategies for the treatment of MDR-TB disease in developing countries.

Here, we have studied the role of eDNA in mixed-species microcolony formation in co-culture biofilms. Our study emphasizes the importance of eDNA as a common biofilm EPS component. In summary, we have shown that eDNA behaves as an essential EPS material shared by different species in co-culture biofilms, which facilitates interspecies interactions through the formation of mixed-species compact microcolony structures during biofilm development. Further understanding of mixed-species biofilm formation may provide valuable information

for the diagnostics and therapeutics of biofilm-related problems in medical and industrial environments. This work buy AZD2281 was supported by a grant from the Danish Research Council for Independent Research to L.Y. We would like to thank Dr Matthew Parsek (University of Washington at Seattle) for kindly providing us with Ixazomib ic50 the pDA2 plasmid. Fig. S1. Two-day-old biofilms of P. aeruginosa PAO1–Staphylococcus aureus MN8 co-culture. Fig. S2. Two-day-old biofilms of P. aeruginosa

PAO1–Staphylococcus aureus atl co-culture. 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. “
“Little is known about postpartum immune recovery and relationships of common others dysphoric moods, stress, immunology, and endocrinology. Healthy women (n = 72) were followed for six postpartum months with immune and hormone measures and dysphoric moods and stress scales. A panel of cytokines produced in mitogen-stimulated whole blood assays were measured at each time, along with plasma levels of hsC-reactive protein (hsCRP), Interleukin-6 (IL-6), and a panel of hormones. Cellular immunity, measured by production of Interferon-gamma (IFNγ) and (Interleukin-2 (IL-2) from stimulated whole blood

culture, was low in the early postpartum with changes by 3 months. Tumor necrosis factor alpha (TNFα) showed a similar pattern. Plasma levels of CRP and Interleukin-6 (IL-6) showed higher levels in the early postpartum. Mood disturbance scores dropped across the postpartum with a change in slope at 3 months. No significant relationships were found between immune, endocrine, and psychosocial measures. Return to normal cellular immune function may take 3–4 months in the postpartum. Some aspects of early immunology (hsCRP and IL-6) probably reflect the latter stage of pregnancy, the stress of birth and the inflammation associated with involution. Dysphoric moods are higher in the early postpartum but are not related to immune factors or hormones. “
“We have previously shown that in differentiated T-helper (Th)1 and Th2 cells, polycomb group (PcG) proteins are associated differentially with the promoters of the signature cytokine genes.

a. We check details recommend that CKD be diagnosed in all individuals on at least two occasions for a period of at least 3 months, irrespective of the underlying cause and on the basis of: (1C) an estimated or measured GFR <60 mL/min per 1.73 m2 and/or evidence of kidney damage (albuminuria, proteinuria, haematuria after exclusion of urological causes, or structural abnormalities on kidney imaging tests) Note: These diagnostic criteria are the same for all races and gender. b. We recommend that the stages of CKD should be based on the combined indices of kidney function (measured

or estimated GFR) (Table 2) and kidney damage (albuminuria/proteinuria) (Table 3), irrespective of the underlying diagnosis (1C). The following diagnostic evaluation tests for CKD are always indicated: Full blood count Repeat (within 1 week) serum urea/electrolytes/creatinine/eGFR/albumin Urine ACR (preferably Napabucasin mw on a first morning void, although a random urine is acceptable) Fasting lipids and glucose Urine microscopy and culture Renal ultrasound scan The following diagnostic evaluation tests for CKD are sometimes indicated: If patient: Then carry out the following test: Has diabetes HbA1C Has eGFR <60 mL/min per 1.73 m2 Serum calcium, phosphate, PTH, 25-hydroxy-vitamin D and iron studies Is >40 years old Serum and urine electrophoresis Has rash, arthritis or features of connective tissue disease Anti-nuclear antibodies, Extractable nuclear antigens, Complement studies

Has pulmonary symptoms or deteriorating kidney function Anti-glomerular basement membrane antibody, Anti-neutrophil cytoplasmic

antibody Has risk factors for HBV, HCV and HIV HBV, HCV, HIV serology Has persistent albuminuria >60–120 mg/mmol (approximately equivalent to 24 h urinary protein >1–2 g/day) Refer to Nephrologist for consideration of renal biopsy We recommend referral to a specialist renal service or nephrologist in the following situations: Stage 4 and 5 CKD of any cause (eGFR < 30 mL/min per 1.73 m2) (1C) Persistent significant albuminuria (ACR ≥ 30 mg/mmol, approximately equivalent to protein creatinine ratio (PCR) ≥50 mg/mmol, or urinary protein excretion ≥500 mg/24 h) (1C) A consistent decline Dynein in eGFR from a baseline of <60 ml/min per 1.73 m2 (a decline > 5 ml/min per 1.73 m2 over a 6-month period which is confirmed on at least three separate readings) (1C)* We suggest referral to a specialist renal service or nephrologist in the following situations: Glomerular haematuria with macroalbuminuria (2C) CKD and hypertension that is hard to get to target despite at least three anti-hypertensive agents (2C). We suggest discussing management issues with a specialist by letter, email or telephone in cases where it may not be necessary for the person with CKD to be seen by the specialist (2D). Once a referral has been made and a plan jointly agreed, routine follow-up could take place at the patient’s General Practitioner surgery rather than in a specialist clinic.

02). Comparison of Kaplan–Meier

curves of PM patients with risk scores of >22 (n = 27) vs. ≤22 (n = 48) confirmed a significantly higher rate of mortality within 28 days of initial PM presentation (HR 8.2, 3.6–18.9, P < 0.0001) and higher cumulative 28-day mortality (14.6% vs. 78%, P < 0.001). The estimated median survival time in PM patients with a risk score >22 was 7 days. Dorsomorphin The majority of patients [47 (73%)] received Mucorales-active antifungal therapy, either amphotericin B formulation [54 (72%)] (as monotherapy or in combination with other antifungal regimens) or posaconazole [10 (13%)] (as monotherapy or in combination with other antifungal regimens) within 5 days after symptoms initiation. Administration of appropriate therapy (over 5 days) was delayed in 28 patients (37%). Immune augmentation therapy included white blood cell (WBC) transfusions, and administration of haematopoietic growth factors (granulocyte-macrophage colony-stimulating factor/granulocyte colony-stimulating factor) or interferon-γ. Thirty-one per cent of the patients received a colony-stimulating factor during treatment, 8% WBC transfusions and 7% interferon-γ. Surgical management, including debridement and wedge resection, was performed in 28 patients (37%). Overall, 28 of 75 patients (37%) died

within 4-week follow-up [median time of death, 34 days after diagnosis (range, 0–94 days)]. No treatment variables were found to be independently associated with improved survival when patients Romidepsin datasheet were stratified by the mortality risk score. Mucormycosis has emerged as the second most common invasive mould infection after aspergillosis in patients with haematological malignancies and allogeneic HSCT.[2] In this 12-year retrospective study, we identified 75 such patients with PM. The most important conditions predisposing to mucormycosis,

Protirelin according to various studies, include malignant haematological diseases with or without HSCT, prolonged and severe neutropenia, poorly controlled diabetes mellitus with or without diabetic ketoacidosis, iron overload, major trauma, prolonged use of corticosteroids, illicit intravenous drug use, neonatal prematurity and malnourishment.[3, 11, 12] Not surprisingly, 57% and 64% of our patients, respectively, were profoundly neutropenic and lymphocytopenic. Prior corticosteroid therapy (55%) and diabetes mellitus (31%) appeared to be common additional risk factors for PM. Moreover, 57% of the patients had refractory haematological disease and thus received intensive cytotoxic chemotherapy. Also, 48% of the patients underwent HSCT, 81% of whom were allogeneic transplant recipients. We stratified our patient population according to the probability of death using easily available clinical, laboratory and radiological variables at the time of diagnosis.

RYUGE AKIHIRO, OZEKI TOSHIKAZU, MINATOGUCHI SHUN, MURAI YUKARI, KAWATO RUI, OZEKI TAKAYA, OYAMA YUKAKO, NOMURA ATSUSHI, TOMINO TATSUHITO, SHIMIZU HIDEAKI, FUJITA YOSHIRO Chubu-Rosai Hospital Introduction: There are few reports concerning tumor lysis syndrome arising from autolysis Doxorubicin purchase of solid cancers.

We describe a recently encountered case of tumor lysis syndrome detected during detailed examination of lung cancer with liver metastasis. Methods & Results: The patient was a 79-year-old male. He was being managed at the Department of Nephrology of our hospital because of chronic kidney disease (Cr: 2.5 mg/dl). Early in April of XXXX, he developed pain involving the right hypochondrial region and anorexia. Because of intense malaise, he visited the outpatient critical care unit of our hospital on April 6. At that time, blood tests revealed marked elevation of

hepatobiliary enzymes, and CT scan disclosed a tumorous lesion approximately 13 cm in size in the right lobe of the liver. He was thus hospitalized to undergo detailed examination. Liver biopsy was performed on the 11th hospital day. Around April 15, his urine volume began to decrease, and blood tests the following day revealed elevation of BUN (60.0 mg/dl) and Cre (3.67 mg/dl), accompanied Veliparib by uric acid elevation (22.2 mg/dl). Renal function did not improve despite fluid therapy. Hemodialysis was thus started on April 18. Thereafter, the uric acid level decreased but urine volume showed no improvement and his general condition gradually deteriorated. The biopsy results allowed a diagnosis of small-cell carcinoma, suggesting that the nodular shadow noted in the right lung represented the primary Bacterial neuraminidase tumor. Treatment

was judged to be difficult in view of his general condition, and the patient was followed without active treatment. He died on April 23. Conclusion: We thus encountered a case of tumor lysis syndrome probably arising from autolysis of small-cell lung carcinoma and an associated metastatic hepatic lesion. RYU HAN JAK1, HAN IN MEE1, HAN JI SUK1, PARK JUNG TAK1, YOO TAE-HYUN1,2, KANG SHIN-WOOK1,2, MOON SUNG JIN3, OH HYUNG JUNG1 1Department of Internal Medicine, College of Medicine, Yonsei University, Seoul; 2Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul; 3College of Medicine, Kwandong University, Gyeonggi-do, Korea Introduction: Platelet size has been demonstrated to reflect platelet activity and to predict poor clinical outcomes in patients with cardiovascular disease. However, the prognostic value of platelet size for mortality has not been studied in patients with acute kidney injury (AKI). Methods: A total of 349 patients who received continuous renal replacement therapy (CRRT) for AKI between August 2009 and October 2011 were divided into two groups based on the median mean platelet volume (MPV) at the time of CRRT initiation.

Southern blotting analysis demonstrated that 20 strains showed a two-copy arrangement of the capb locus (45-kb), two strains showed three copies (63-kb), and the other two showed four copies (81-kb) (Fig. 1). The incidence of multiple-copy strains (>two copies) among examined strains was 16.7% (4/24). PF-6463922 ic50 All of the strains with the dominant PFGE pattern (A1) possessed two copies, while one with the closely-related A2 subtype harbored four copies. The other three strains with multiple copies showed minor PFGE patterns (B, G or I). All the patients infected by strains with multiple

copies were treated successfully without neurological or physical sequelae. Amplified capb sequences were detected more frequently among strains from children with true vaccine failure MAPK Inhibitor Library than among those from unvaccinated children (24% vs. 10%) in the United Kingdom (8). Furthermore, the proportion of strains with multiple copies of the capb locus increased over time in Italy (9). Amplification of the capb locus is associated with decreased susceptibility to complement-mediated lysis and decreased complement-mediated opsonization (11). Thus, amplification of the capb locus may result in the overcoming of host defenses and contribute to vaccine failure. We have found that Hib strains with multiple (three or four) copies of the capb locus were present in Japan before the introduction of the Hib conjugate vaccine.

The incidence of 16.7% (4/24) of multiple-copy strains found in our study is slightly higher than that found in the UK between 1991 and 1992 before routine immunization was introduced (10.1%, 9/89) (8). In our study, most of the multiple-copy strains showed rare PFGE patterns. Thus these strains might be selected and involved in vaccine failure after the introduction of Hib conjugate vaccination in Japan. Sequence typing of the capb locus is based on the considerable sequence divergence in the hcsA and hcsB genes, which are involved in the transport of capsular polysaccharides across the outer membrane (18). Schouls et al. have reported that type

II strains display less expression of capsular polysaccharide than do type I, and were isolated only during the pre-vaccination era in the Netherlands (12). The greater polysaccharide expression may have provided Methamphetamine a selective advantage for type I strains, resulting in the rapid elimination of type II. In addition, there have been remarkable differences in the geographic distribution of type I and type II; with a higher incidence in the United States (73%) than the Netherlands (5%) of type II among Hib strains isolated from patients (12). While we did not find type II strains in this study, more Hib strains should be evaluated to clarify the exact incidence. To our knowledge, this is the first study to investigate capb locus copy number in invasive Hib strains isolated in Japan.

This minimal invasive see more surgical approach was reported to be successful even in cases where the posterior wall of the frontal sinus was already affected.[42] In a study by Hachem et al. [43], 39 cases of invasive Aspergillus sinusitis were analysed regarding the outcome between the group of 13 patients who received sinus surgery and the group of the remaining 26 patients, who received systemic antifungal therapy alone. Overall response among neutropenic patients with invasive

Aspergillus sinusitis was 53.2% (7/13) in those who underwent sinus surgery and 19.2% (5/26) in the control group (P = 0.06). Among the subgroup of patients with neutropenia at the onset of infection, the response rate in the sinus surgery group was significantly better than in the non-surgery group (57% vs. 11.8%; P = 0.028). Similar results were reported by Chen et al. [44] in 2011, who found that surgical debridement was an independent good prognostic factor (P = 0.047) in multivariate analysis in 46 patients with invasive fungal sinusitis. In the discussions section

of that study, aggressive surgical debridement was recommended despite the poor immune status of the host and the bleeding tendencies of many patients with this infection. Eliashar and colleagues reported optimal outcome in 2007, when they analysed 14 patients with invasive Aspergillus sinusitis. All 14 patients received selleck products surgery; however, seven patients needed two or more surgical interventions. In all 14 cases, eradication of invasive Aspergillus sinusitis was achieved. However, none of these cases presented with an intraorbital or an intracranial extension, so no excessive surgery from an open external

approach was necessary, thanks to the early diagnosis of the Aspergillus sinusitis. Suslu et al. [45] reported on 19 patients with acute rhinosinusitis. Early diagnosis and treatment, including aggressive surgical debridement was found essential for recovery in that study. Surgical interventions are also of paramount importance for establishing a microbiologic and histologic diagnosis.[41-44] This demonstrates that surgery is a key factor in the treatment of this disease, however, early diagnosis to allow prompt surgery is necessary.[41, 42, 46] Resection of devitalised tissue, stabilisation of bones that are at risk of fracture, as well as prevention and Erythromycin treatment of neurological complications due to compression are indicated in Aspergillus osteomyelitis. Surgical intervention can also help to increase penetration of antifungal agents into the bone (in case of failure of conservative therapy).[47-52] Vertebral aspergillosis can lead to catastrophic destruction of the spine, resulting in destabilisation and kyphosis, requiring surgical fusion and/or fixation of vertebrae. In the thoracic spine, the osteomyelitis is mostly caused by haematogenous spread from a pulmonic focus of Aspergillus infection.

In contrast to T cells, activation of the BCR in blood B cells was not associated with changes in RhoH levels. These data suggest that RhoH function might be regulated by lysosomal degradation of RhoH protein following TCR complex but not BCR activation. This newly discovered regulatory pathway of RhoH expression might limit TCR signaling and subsequent T-cell activation upon Ag contact. RhoH (also known as

TTF) is a member of the Rho (ras homologous) GTPase subfamily of the Ras (rat sarcoma) superfamily of small GTP-binding proteins 1. RhoH mRNA expression was reported to be restricted to hematopoietic cells 1. Protein expression data are not available, click here except for one recent report, which demonstrated increased RhoH protein SAHA HDAC expression in GM-CSF-stimulated neutrophils 2. Rho GTPases are important intracellular

signaling molecules regulating the organization of the cytoskeleton, cell polarity, activation, proliferation, and survival (for review: 3). They usually cycle between an active, GTP-bound, and an inactive, GDP-bound, state. In contrast, RhoH has no measurable intrinsic GTPase activity and resides always in the active form 4. As a consequence, regulation of RhoH function appears to be only possible at the expression level, e.g. by modulating RhoH transcription 4 and/or alternative splicing 5, or by modifying its subcellular localization. Mice lacking RhoH have been independently generated by two research groups 6, 7. The phenotype of these mice revealed that RhoH is an important regulator of T-cell activation since deficiency of RhoH results in reduced T-cell differentiation and proliferation, and consequently in reduced numbers of T cells in the thymus, lymph nodes, and spleen 6, 7. Although the exact molecular mechanisms remain to be determined, Gu Y et al. suggested that RhoH recruits Zap70, a crucial tyrosine kinase in TCR signaling, to the immunological synapse 7. In contrast, Dorn T et al. proposed that RhoH regulates TCR signaling downstream of Zap70 6. In contrast to T cells,

the functional role of RhoH in primary B cells remains unknown. It is possible, however, that RhoH might Protirelin play a role in the pathogenesis of B-cell lymphomas since dysregulated RhoH expression has been reported in a number of B-cell malignancies 1, 8. T cells play central roles in all adaptive immune responses against pathogens. Since RhoH activity was shown to be crucial for T-cell activation 6, 7, it is important to study its regulation. We hypothesized that besides transcription 4 and alternative splicing 5, additional mechanisms might play a role that contribute to the regulation of RhoH expression and function. In this manuscript, we report RhoH protein expression levels in different blood cells and a new pathway of regulating RhoH protein expression in T cells, based on lysosomal degradation of the protein.

Recently, in attempts to prolong allograft survival, the possibility of targeting alloreactive memory cells via their IL-7Rα was postulated [38]. Cyclopamine Our current data indicate that this approach would attack only part of the alloreactive memory cells, leaving unaffected the IL-7Rα- cells which, on the contrary, seem

the most harmful alloreactive memory/effector cells. In conclusion, using the multi-parameter MLC–CFSE assay we have shown that allostimulated cells have a highly activated and differentiated phenotype with increased expression of chemokine receptors relevant for migration of T cells into the graft and high expression of effector molecules. In addition, our analysis of patients before transplantation

who are at risk for experiencing an acute cellular rejection episode, versus those who are not, revealed a higher dsp CD8pf and lower percentage of alloreactive IL-7Rα+ CD8+ T cells. However, given the retrospective nature of our present study and the overlap in results of rejectors compared to non-rejectors, it is not possible to predict the outcome of the transplantation with respect to the occurrence of acute rejection on a per-patient basis. Our data point to quantitative and qualitative differences between T cells of a group of patients who will experience acute cellular rejection episodes and those who will not. The predictive value of these parameters needs to be established in a large prospective study. All authors declare no conflicts of interest. This Pritelivir study was supported financially by grants from the Dutch Kidney Foundation (grant C05·2141), the RISET consortium (Sixth Framework Programme of the European Commission) and Novartis Pharma BV. “
“Citation Doncel GF, Joseph T, Thurman AR. Role of semen in HIV-1 transmission: inhibitor or facilitator? Am J Reprod Immunol 2011; 65: 292–301 Sexual transmission of human immunodeficiency virus type 1 (HIV-1) accounts for 60-90%

of new infections, especially in developing selleck compound countries. During male-to-female transmission, the virus is typically deposited in the vagina as cell-free and cell-associated virions carried by semen. But semen is more than just a carrier for HIV-1. Evidence from in vitro and in vivo studies supports both inhibitory and enhancing effects. Intrinsic antiviral activity mediated by cationic antimicrobial peptides, cytotoxicity, and blockage of HIV–dendritic cell interactions are seminal plasma properties that inhibit HIV-1 infection. On the contrary, neutralization of vaginal acidic pH, enhanced virus–target cell attachment by seminal amyloid fibrils, opsonization by complement fragments, and electrostatic interactions are factors that facilitate HIV-1 infection. The end result, i.e., inhibition or enhancement of HIV mucosal infection, in vivo, likely depends on the summation of all these biological effects.

Immortalized hPDL cell lines provided by Dr Takada (Hiroshima University) were cultured in α-minimum essential medium (α-MEM; Invitrogen, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) plus penicillin G solution (10 U/ml) and streptomycin (10 mg/ml) in a humidified atmosphere of 5% CO2 at 37°. Telomerase catalytic subunit hTERT gene-immortalized human periodontal ligament (HPDL) cells were derived by transfecting primary cultured hPDL cells from a healthy premolar extracted for orthodontic treatment, as described previously [25,26]. These immortalized hPDL cells are similar to those in primary PDL-derived cells, and could be a model for the investigation of factors contributing to inflammation and differentiation of PDL

cells GSK2126458 [17,22]. For experiments, the cells were seeded into culture dishes and then cultured in DMEM containing 10% FBS for 3 days until 70% confluent. Subsequently, the cells were exposed to MS. All treatments were performed in triplicate. Human PDL cells (3 × 105/well) were subcultured into six-well, 35-mm flexible-bottomed

Uniflex culture plates with a centrally located this website rectangular portion (15·25 mm × 24·18 mm) coated with type I collagen designed to provide a uniform uni-axial strain, and subjected to an intermittent deformation of 3, 6, 12 or 15% of maximum stretch for 2·5 s followed by 2·5 s of relaxation (12 cycle/min 24 h) with a Flexercell FX-4000 Strain Unit (Flexcell Corporation, Hillsborough, NC, USA), according to the manufacturer’s instructions. siRNA-annealed oligonucleotide duplexes for SIRT1 (sequence 5′->3′ sense: GAUGAAGUUGACCUCCUCAtt; anti-sense: UGAGGAGGUCAACUUCAUCtt) and negative control (catalogue no. SN-1003) were purchased from Bioneer (Daejeon, South Korea) and PDL cells were transfected using lipofectamine 2000 (Gibco BRL), following the manufacturer’s instructions. After applying the MS, Loperamide total RNA was isolated from the cells using Trizol reagent (Invitrogen Life Technologies, Gaithersburg, MD, USA), according to the manufacturer’s instructions. Briefly, 1 µg of RNA isolated from each culture was reverse-transcribed using oligo(dT)15

primers (Roche Diagnostics, Mannheim, Germany) and AccuPower RT PreMix (Bioneer), according to the manufacturer’s protocols. An amount of cDNA equivalent to 25 ng of total RNA was then subjected to PCR. The primers used for cDNA amplification are listed in Table 1. PCR products were subjected to electrophoresis on 1·2% agarose gel and were stained with ethidium bromide. An equal volume of ×2 sodium dodecyl sulphide (SDS) sample buffer was added and the samples were then boiled for 5 min. Samples (40 µg) were subjected to electrophoresis on 12% SDS-polyacrylamide gels for 2 h at 20 mA and then transferred onto nitrocellulose. The membrane was incubated for 1 h in 5% (wt/vol) dried milk protein in phosphate-buffered saline (PBS) containing 0·05% (vol/vol) Tween-20, washed in PBS and then incubated for 1 h in the presence of primary antibody (1:1000).