Examining the clinical efficacy on the interferential low frequency therapy (IF) for elderly nonneurogenic (idiopathic) overactive bladder patients with urgent urinary incontinence (wet OAB) prospectively, for whom anticholinergics were not effective.Subjects are elderly 80 patients  with urinary incontinence, who are clinically diagnosed with wet OAB without urodynamics (pressure/flow study). For 3 months, they were administered anticholinergics (propiverine hydrochloride 20 mg/once per day in the morning), but all their quality of life (QOL) score were 4 or over due to poor control of storage symptoms and urinary incontinence. We selected patients for whom anticholinergics were not effective (above-mentioned 80 patients) and they were provided with IF alone for 3 months with informed consent. Before and after IF, the followings were examined. (1) frequency of IF treatment required to show optimal effects, (2) average weekly frequency of incontinence, (3) 60-min pad test, (4) frequency and voided volume in the daytime and nighttime, (5) fluid intake volume, (6) International Prostate Symptom Score, quality of life score, (7) Uroflowmetry, (8) postvoid residual urine volume, (9) specific gravity of urine, (10) average hours spent outdoors, (11) average radius of action and activities of daily life score, (12) standing blood pressure (BP) and heart rate, (13) clinical laboratory findings, (14) adverse events, (15) plasma osmotic pressure (OP), and (16) Brain natriuretic peptide (BNP).(1) The patients showed improvements for eight treatments (median). Improvement was observed in the followings: (2), (3), (4) voiding frequency, (6), (7) voided volume, maximum and average flow rate, (10), (11), (12) BP, (15) OP, and (16).The IF has safe and better effects than anticholinergics on the elderly wet OAB patients. Interferential therapy (IF) utilizing effects of low frequency electrical stimulation was first conducted on the lower urinary tract in the treatment of urinary incontinence by McQuire. The util®, Nihon Medix Co. Ltd, Matsudo, Chiba, Japan), using surface electrodes placed at four sites in the lower abdomen and lower buttocks. A 20-min treatment session was conducted twice a week for the first 3 weeks and once every 2 weeks thereafter. Prior to treatment initiation, the patients were telephoned from the hospital once to twice per week to give a brief information, apart from a voiding diary, to their urologists on their subjective and objective findings regarding wet OAB  during the interview held every 2 weeks because of inspecting validity of IF continuation (weak evidence of efficacy of IF for OAB). The following data were compared before and after IF: (1) number of treatment sessions required for improvement to be noted in the assessment made in interviews concerning on subjective symptoms, (2) average weekly frequency of incontinence, (3) 60-min pad test results, (4) frequency and volume of daytime and nighttime voiding, (5) volume of water intake, (6) International Prostate Symptom Score (IPSS) and QOL index score, (7) uroflowmetry test values , (8) postvoid residual urine volume (PVR), (9) specific gravity of urine, (10) average hours spent outdoors in the day, (11) one-day average radius of action and activities of daily life scale score, (12) standing blood pressure (BP) and heart rate (HR), (13) clinical laboratory findings, (14) IF-related adverse events, (15) plasma osmotic pressure (OP) and (16) BNP. The conduct of this study was approved by the Kobe Medical Center Ethics Committee. No behavioral therapy, including water intake instructions, was provided during the study period. Patients were asked to record in their voiding diary 5-day data including data for two successive days. The median was adopted as study data for voiding frequency, single voided volume, and water intake volume. The median of three measurements was adopted for uroflowmetry test data, PVR, BP and HR; PVR was conducted by catheterization at three different times during the day. Changes in hours spent outdoors, radius of action, and ADL scale score were used to assess the patients' lifestyle. As the average hours spent outdoors in one day, the median of daily hours spent outdoors in a 14-day period was adopted. As the one-day average radius of action, the median of the farthest distance traveled from home over a 14-day period was adopted; the distance was measured roughly on a map. For the ADL scale score, a 10-grade visual analogue scale (VAS) from 0 to 9 originally developed at Kobe Medical Center, was also used . In statistical analyses, paired t-test was used for the comparison of paired two samples and Wilcoxon signed rank test for the comparison of ordinal data from two independent samples; P < 0.05 was considered statistically significant.The study subjects were 80 patients aged 65 years or over  with nonneurogenic (idiopathic) “wet OAB” who are clinically diagnosed without urodynamics (pressure/flow study) and no medical history of extra-urological conditions including clinically evident cardiac diseases. None was experiencing serious complications nor undergoing any medical or other therapy that would possibly influence their micturition state. Electrocardiography, echocardiography, and other examinations conducted at the Department of Cardiovascular Internal Medicine of the author's hospital demonstrated the absence of conditions requiring clinical treatment in those patients to exclude potential cardiovascular diseases previously. These patients were given anticholinergic treatment (propiverine hydrochloride 20 mg/day) once a day in the morning for 3 months or longer, with poor control of storage symptoms and urinary incontinence the QOL index scoring at a four (mostly dissatisfied) or more as revealed in the posttreatment interview. All the patients offered cancellation of anticholinergics and hope alternative noninvasive therapy for further improvement of their symptoms. After consultation and written informed consent about IF (including full explanation about weak evidence compared with anticholinergics for OAB treatment) was obtained from each participating patient, a washout period of at least 1 week instituted following the anticholinergic treatment, a 3-month IF alone was performed at 20 Hz and 20 mA  treatment sessions; the improved condition lasted for 3 months without reversal. All patients completed the scheduled course of treatment.P < 0.0001) and the weight of pad in the 60-min pad test, from 17.5 ± 2.1 to 3.1 ± 2.1 g (P < 0.0001).Changes in one-week average frequency of incontinence and 60-min pad test results. Both parameters improved significantly. The frequency of incontinence decreased from 13.3 ± 5.2 (mean ± standard deviation) times/week at pre-IF to 3.6 ± 3.5 times/week in a 3-month period on IF (P < 0.0001) and the nighttime, from 1.8 ± 1.0 to 1.4 ± 1.0 times (P = 0.0004). The daytime voided volume showed an increasing tendency, from 1199 ± 230 to 1220 ± 320 ml while the nighttime volume showed a decreasing tendency, from 514 ± 185 to 464 ± 157 ml; neither changes were significant.Changes in the frequency and volume of daytime and nighttime voiding: The voiding frequency decreased significantly in both the daytime, from 8.3 ± 2.4 to 7.0 ± 1.8 times  from 12.1 ± 5.3 to 6.3 ± 3.3. When the IPSS was subdivided into symptom scores related to storage, voiding and postvoiding, all subdivision symptom scores showed significant improvement: storage, from 7.9 ± 3.6 to 4.3 ± 2.5 (P < 0.0001); voiding, from 2.7 ± 3.8 to 1.0 ± 2.2 (P < 0.0001); and postvoiding, from 1.5 ± 1.9 to 1.0 ± 1.6 (P = 0.0028). The QOL index score decreased by half from 5.2 ± 0.8 to 2.4 ± 1.1 (P < 0.0001).Changes in IPSS and QOL index score . The totmax (from 18.1 ± 6.8 to 25.7 ± 6.6 ml/s) and Qave (from 8.9 ± 4.1 to 12.1 ± 3.5 ml/s) also improved (P < 0.0001 each).Change in the uroflowmetry test values . VoidingChange in PVR: The change (from 17.5 ± 24.0 to 14.5 ± 23.1 ml) was considered nonsignificant.Change in specific gravity of urine: A decreasing trend from 1.019 ± 0.007 to 1.016 ± 0.006 was exhibited, with no statistical significance.P < 0.0001), the one-day average radius of action increased from 400 ± 300 to 1200 ± 500 m (P < 0.0001) and the ADL scale score decreased from 8.0 ± 1.2 to 3.4 ± 1.5 (P < 0.0001); all these improvements were significant. Interviews showed that all patients experienced increases in their amount of outdoor activities, time spent for shopping and hobbies and time spent with their friends and close relatives.Changes in lifestyle . The aveChanging in standing BP, HR and clinical laboratory findings: Both systolic and diastolic BP decreased significantly, from 132.9 ± 8.6 to 127.1 ± 7.4 mm Hg and from 79.7 ± 8.1 to 74.7 ± 8.2 mm Hg, respectively. The change in HR from 73.5 ± 6.8 to 73.0 ± 5.4 bpm was not significant and neither were the changes in laboratory test values.Adverse events: As an IF-related event reported was a sensation of skin stimulation at the time when current was applied to the electrode surface in the initial treatment session. No such adverse events occurred that were deemed clinically significant, including skin stimulation at the site of electrode placement.P < 0.0001) from 295.1 ± 7.8 to 297.8 ± 3.6 mOsm/l while BNP decreased significantly (P < 0.0001) from 41.3 ± 38.7 to 19.2 ± 11.1 pg/ml.Changes in plasma OP and BNP: Plasma OP increased significantly . This is in agreement with improvements achieved in the QOL index and the ADL scale scores. In addition, relatively early manifestation of improvement in subjective symptoms (within about 7 weeks after IF initiation) appears to prompt patients to increase the amount of suggested exercises (including that of the pelvic floor muscles), contributing to even larger therapeutic effects of IF. Additional advantages include: the absence of complications such as increased frequency and (nighttime) voided volume resulting from greater water intake to counter dry mouth, which is an adverse event observed with the use of anticholinergics including propiverine hydrochloride; and satisfactory compliance that can be achieved in patients who are not indicated for oral treatment or are incapable of oral ingestion.Propiverine hydrochloride is presumed to ease OAB by inhibiting afferent transmission of information, undertaken by acetylcholine released from the urothelium via bladder distension stimuli, on bladder tension development control during urine pooling and bladder distension (antimuscarinic action) and by r11The present study demonstrated that even in the absence of clinically evident cardiac disorders, plasma OP and BNP levels can be high and that such levels are lowered in a relatively short period of time. The IF can improve clinical symptoms of “wet OAB” with relatively rapidly and thereby can improve ADL levels, as represented by the greater radius of action accompanying symptom amelioration, together with BNP level decrease, implying a reduced load on left ventricular function. These features suggest that IF is a favorable treatment for the elderly.On the other hand, urine osmotic pressure was isotonic in these patients; although plasma OP significantly increased, the specific gravity of urine did not respond to the change. This indicates impaired urine concentration capacity and decreased cardiac (left ventricular) function in association with advanced age. Given th


Cucurbitacins are plant natural products that inhibit activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway by an unknown mechanism. They are also known to cause changes in the organization of the actin cytoskeleton.Dictyostelium discoideum amoebae. Upon treatment of MDCK or B16-F1 cells with cucurbitacin I, there is a very rapid cessation of motility and gradual accumulation of filamentous actin aggregates. The cellular effect of the compound is similar to that observed when cells are treated with the actin filament-stabilizing agent jasplakinolide. However, we found that, unlike jasplakinolide or phallacidin, cucurbitacin I does not directly stabilize actin filaments. In in vitro actin depolymerization experiments, cucurbitacin I had no effect on the rate of actin filament disassembly at the nanomolar concentrations that inhibit cell migration. At elevated concentrations, the depolymerization rate was also unaffected, although there was a delay in the initiation of depolymerization. Therefore, cucurbitacin I targets some factor involved in cellular actin dynamics other than actin itself. Two candidate proteins that play roles in actin depolymerization are the actin-severing proteins cofilin and gelsolin. Cucurbitacin I possesses electrophilic reactivity that may lead to chemical modification of its target protein, as suggested by structure-activity relationship data. However, mass spectrometry revealed no evidence for modification of purified cofilin or gelsolin by cucurbitacin I.We show that cucurbitacin I potently inhibits the migration of Madin-Darby canine kidney (MDCK) cell sheets during wound closure, as well as the random motility of B16-F1 mouse melanoma cells, but has no effect on movement of Cucurbitacin I results in accumulation of actin filaments in cells by a unique indirect mechanism. Furthermore, the proximal target of cucurbitacin I relevant to cell migration is unlikely to be the same one involved in activation of the JAK2/STAT3 pathway. Cucurbitacins are plant triterpenoids with a number of interesting biological properties. They possess antiproliferative and/or cytotoxic activity against various cancer cells  Diversity Set, we identified cucurbitacin I  as a potent inhibitor of cell migration in a Madin-Darby canine kidney (MDCK) cell wound closure assay. Cucurbitacin I has also been shown to inhibit the migration of keloid fibroblasts Dictyostelium discoideum cells. Cucurbitacin I caused actin aggregation in MDCK and B16-F1 cells, yet had no direct effect on purified actin polymerization or depolymerization in vitro. Cucurbitacins A, B, and C lacked anti-migratory activity against MDCK cells, although they still possessed cytotoxic activity. These results indicate that in cells, cucurbitacin I has an indirect effect on actin dynamics that leads to an over-accumulation of actin, most likely by interfering with actin depolymerization.We show that cucurbitacin I inhibits the migration of MDCK cells and B16-F1 mouse melanoma cells, although it has no effect on A high throughput wound closure assay with MDCK cells was used to screen the NCI Diversity Set for inhibitors that slowed the rate of cell migration We imaged the wound closure process in MDCK cells expressing mCherry-actin by time-lapse fluorescence microscopy . The wouDictyostelium discoideum amoebae. When 200 nM cucurbitacin I was applied to migrating B16-F1 cells, translocation rapidly ceased  were prepared from the desiccated solid samples. Stock solutions were stored in a desiccator at −20°C and used within 2 months of preparation.Dictyostelium discoideum strain amoebae were grown in HL5 nutrient media Dictyostelium filamin by PCR using forward primer 5′-CAGTAGGATCCATGGCTGCTGCTCCAAGT-3′ and reverse primer 5′-TACGCTCTAGAGGCATCTGAAGTTTC-3′. These primers added BamHI and XbaI sites, allowing the fragment to be ligated into dRFP-pDXA-3H-Hygro 2. B16-F1 cells stably expressing mCherry-actin were grown in Dulbecco's Modified Eagle's Medium with D-glucose, L-glutamine, and 10% FBS at 37°C and 5% CO2.Wound closure experiments to evaluate migration of MDCK cells in the presence of cucurbitacin I  were carried out as previously described, as were determinations of cell viability at the end of each experiment 5) were plated on Bioptechs chambers (coated with 5 µg/mL laminin) and grown overnight in bicarbonate-free HAMS-F12 medium with 10% FBS at 37°C. The medium was replaced with 500 µL fresh bicarbonate-free HAMS-F12 medium buffered with 10 mM HEPES, and the Bioptechs chambers were placed in a Bioptechs Delta T stage temperature controller set to 37°C. A single scratch wound was created in the monolayer by scraping the surface of the dish with a 200-µL pipet tip. The wound was imaged every 5 min for several hours on an automated Zeiss 200 M inverted microscope with Openlab software (Perkin-Elmer/Improvision), and then cucurbitacin I was added in an equal volume of new medium for a final concentration of 200 µM. Images were taken for several more hours and then analyzed with ImageJ software 2) on a Nikon A1R confocal microscope.For live-cell imaging, MDCK cells  were plated on plastic Petri dishes in HL5 growth medium and allowed to attach for 1 h. Images were captured every 15 s for 1 h, and then cucurbitacin I was added to 200 nM. Imaging was continued for 1.5 h, and then the concentration of cucurbtacin I was raised to 2 µM and imaging continued for another 1.5 h. The captured images were analyzed with ImageJ software with the mTrackJ plugin to manually track the movements of representative cells. The speeds were analyzed with Graphpad Prism by a Mann-Whitney non-parametric U test.For analysis of cell speed, To determine whether cucurbitacin I causes aggregate formation, cells expressing dRFP-FilABD were plated in glass-bottom Petri dishes (Willco Wells) and treated with compound overnight in HL medium. dRFP-FilABD like GFP-FilABD 2, 5 mM EGTA, 5% glycerol, 1% Triton X-100, 0.1% Tween 20, 0.1% 2-mercaptoethanol, 1 mM ATP, 1 mM phenylmethanesulfonyl fluoride, 5 mg/mL N-α-p-tosyl-L-arginine methyl ester, 10 µg/mL N-p-tosyl-L-phenylalanine chloromethyl ketone, 80 µg/mL aprotinin, 20 µg/mL pepstatin, 20 µg/mL chymostatin) and incubated on ice for 30 min. Samples were then centrifuged at 10,000×g for 10 min at 4°C. The supernatant (G-actin) was removed, and the pellet (F-actin) was resuspended in 250 µl lysis buffer. An equal volume of 2x sodium dodecyl sulfate (SDS) sample buffer was added, and samples heated to 100°C for 5 min. Equal volumes of each sample were then electrophoresed on a 10% SDS-polyacrylamide gel and electroblotted onto a polyvinylidene fluoride membrane. The membrane was blocked in non-fat dry milk for 1 h and incubated in a 1∶500 dilution of mouse anti-β-actin antibody for 1 h, followed by incubation with a 1∶5,000 dilution of goat anti-mouse antibody conjugated to alkaline phosphatase for 1 h. Colorimetric detection was achieved with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium as described previously Subconfluent MDCK cells were treated overnight with 0.1% DMSO or 200 nM cucurbitacin I. Cells were harvested into 1.5 mL tubes and centrifuged at 1,000×g for 5 min at 4°C. The media was aspirated, and the cells were resuspended in 250 µl of lysis buffer iodoacetamide (Invitrogen) and used in a pyrene-actin polymerization assay essentially as previously reported 2, 0.2 mM ATP, 0.5 mM dithiothreitol (DTT). Immediately prior to the experiment, ME buffer, consisting of 50 mM MgCl2, 0.2 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), was added to concentration of 1× from a 10× stock of ME buffer to exchange Ca2+ for Mg2+. The sample was incubated in the presence or absence of 50 µM cucurbitacin I for 1 h at 4°C, with pyrene-G-actin at a final concentration of 2 µM. Polymerization was then initiated by addition of a 1/10 volume of 10× KMEI buffer . Fluorescence intensity (340 nm excitation/420 nm emission) was measured over time on a fluorescence microplate reader (BioTek FLx800TBI).Actin was purified from rabbit muscle acetone powder as previously described 2, 0.2 mM ATP. Pyrene-G-actin was polymerized to pyrene-F-actin according to the manufacturer's instructions by addition of a 1/25 volume of 10× actin polymerization buffer . The sample was stored in the dark at room temperature for 1 h. Depolymerization was initiated by diluting the pyrene-F-actin 25-fold with 5 mM Tris, pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, in the presence or absence of cucurbitacin I or phallacidin at the concentrations indicated in Lyophilized pyrene-labeled rabbit muscle actin (∼50% pyrene labeled from Cytoskeleton) was resuspended in water and then diluted to 23 µM in 5 mM Tris, pH 8.0, 0.2 mM CaCl2) in a total volume of 200 µL overnight on ice. G-actin was polymerized to F-actin the next morning at room temperature for 1 h by combining 75 µL actin seeds with 75 µL of 4 µM G-actin in 1× ISAP buffer. 15 µL aliquots of F-actin were added to tubes, and then the actin was diluted by addition of 135 µl of 5 mM Tris, pH 7.8, 0.2 mM CaCl2, 0.2 mM ATP to initiate depolymerization in the presence or absence of 200 nM jasplakinolide or cucurbitacin I. Tubes were incubated overnight at 4°C. Samples were centrifuged in an airfuge for 1 h, and pellets were resuspended in 20 µL of SDS sample buffer. Samples were denatured at 100°C for 5 min and then loaded onto a 10% SDS-polyacrylamide gel. The gel was stained with GelCode Blue G-250 (Thermo Scientific Pierce) and destained in deionized water. Bands were quantified with ImageJ software.As an alternative method to measure actin depolymerization, actin seeds were formed by incubating 4 µM human non-muscle G-actin (Cytoskeleton) in ISAP buffer  on a 10ADvp high-performance LC system (Shimadzu). The proteins were eluted with a linear gradient from 2% acetonitrile/water/0.1% acetic acid to 90% acetonitrile/water/0.1% acetic acid over 20 min at a flow rate of 50 µL/min. The effluent was fed into the ESI source of a QStar Elite mass spectrometer (Applied Biosystems/MDS Sciex), with typical source conditions of IS 5500V, GS1 20, DP 80 V, FP 280 V, DP2 15 V. Acquisition of data was controlled with Analyst software (AB SCIEX).Figure S1Neither cofilin nor gelsolin are alkylated by cucurbitacin I. Mass spectra of human cofilin and human plasma gelsolin following incubation of 15 µM of each protein with 100 µM cucurbitacin I for 6 h at 37°C and LC-ESI-MS. The masses of the cucurbitacin I-treated proteins were identical to those of non-treated protein controls.(1.43 MB TIF)Click here for additional data file.Movie S1Inhibition of wound closure in MDCK cells exposed to cucurbitacin I. mCherry-actin-expressing MDCK cells were grown to confluence on a Bioptechs dish and the monolayer was wounded. The wound edge was imaged at 37°C every 5 min for 5 h in the absence of compound, and then the medium was changed to medium containing 200 nM cucurbitacin I, and the wound edge was imaged for an additional 19 h.(6.52 MB MOV)Click here for additional data file.Movie S2Inhibition of B16-F1 motility by cucurbitacin I. B16-F1 cells were grown overnight on laminin-coated dishes and then imaged at 37°C by phase-contrast microscopy. Images were captured every 3 min for 45 min and then continued following addition of cucurbitacin I to 100 nM for an another 45 min. The cells can be seen to rapidly stop movement and retract protrusions. The compound was then washed out and the cells were imaged over a 5-h period during which movement gradually resumed.(1.89 MB MOV)Click here for additional data file.Movie S3B16-F1 cells exposed to cucurbitacin I immediately stop protrusion and begin retraction of lamellae. B16-F1 cells were imaged at 37°C every 30 s for 3 h by phase-contrast microscopy and then cucurbitacin I was added to 200 nM while imaging. The movie shows the 8 min prior to compound addition and the next 40 min in the presence of compound. The freezing of protrusion is evident within a minute after addition of compound, while retraction took place over a longer time scale.(6.03 MB MOV)Click here for additional data file.Movie S4Dictyostelium amoebae. Dictyostelium discoidium (strain NC4A2) were plated for 1 h in HL5 medium on a Petri dish and then imaged every 15 s at room temperature. After 1 h, cucurbitacin I was added to 200 nM, and the cells were imaged for 1.5 h. The concentration of cucurbitacin I was then raised to 2 µM and imaging was continued for another 1.5 h.Cucurbitacin I does not affect the motility of (5.15 MB MOV)Click here for additional data file.Movie S5F-actin formation in the presence of cucurbitacin I in low-density MDCK cell cultures. MDCK cells expressing mCherry-actin were seeded on a laminin-coated dish at low density and allowed to attach. The cells were imaged at 37°C by DIC and epifluorescence every 1 min before and after the addition of 200 nM cucurbitacin I. The movie begins just after the addition of the compound to the cells. The formation of aggregates of actin over the following 5 h can be seen.(13.96 MB MOV)Click here for additional data file.Movie S6Jasplakinolide causes cessation of migration and lamellipodial collapse in B16-F1 cells. B16-F1 cells were grown on a laminin-coated dish overnight. Cells were then imaged at 37°C every 5 min before and after addition of 200 nM jasplakinolide. After addition, the cells immediately stopped moving and began to retract their lamellipodia. After 100 min, the compound was washed out and imaging continued. The cells recovered and began to move about 4 h later.(0.78 MB MOV)Click here for additional data file.


Plasmodium falciparum infected erythrocytes have been associated with protection from clinical malaria. The precise target for these antibodies is unknown. The best characterized and most likely target is the erythrocyte surface-expressed variant protein family Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1).Antibodies targeting variant antigens expressed on the surface of var gene, A4 PfEMP1, the naturally occurring antibody response was assessed, by ELISA, to each domain in serum samples obtained from individuals resident in two communities of differing malaria transmission intensity on the Kenyan coast. Using flow cytometry, the correlation in individual responses to each domain with responses to intact A4-infected erythrocytes expressing A4 PfEMP1 on their surface as well as responses to two alternative parasite clones and one clinical isolate was assessed.Using recombinant proteins corresponding to five domains of the expressed A4 Marked variability in the prevalence of responses between each domain and between each transmission area was observed, as wasa strong correlation between age and reactivity with some but not all domains. Individual responses to each domain varied strikingly, with some individuals showing reactivity to all domains and others with no reactivity to any, this was apparent at all age groups. Evidence for possible cross-reactivity in responses to the domain DBL4γ was found.Individuals acquire antibodies to surface expressed domains of a highly variant protein. The finding of potential cross-reactivity in responses to one of these domains is an important initial finding in the consideration of potential vaccine targets. Plasmodium falciparum infections whilst limiting morbidity and mortality is a feature of the non-sterile immunity acquired by individuals living in malaria endemic areas. Studies whereby antibodies were passively transferred from immune to non-immune individuals suggested this immunity is, at least in part, antibody-mediated [Maintaining mediated ,2. Humanmediated -5. Parasmediated . Followimediated -10 and smediated .var genes, Plasmodium falciparum erythrocyte membrane protein 1, (PfEMP1) [PfEMP1 is a family of extracellular, highly polymorphic and clonally variant adhesion molecules [P. falciparum glycophorin A receptor and the Plasmodium vivax and Plasmodium knowlesi ligands that allow invasion of Duffy blood-group positive erythrocytes [The most extensively characterized of the proteins expressed at the infected red cell surface are the products of the (PfEMP1) . PfEMP1 olecules -16. Theyolecules . They exhrocytes -19. ThesPfEMP1 protein, A4 PfEMP1 from the A4 laboratory parasite line, domain-specific antibodies prior to the transmission season in two communities in Kenya with differing transmission characteristics were measured. The presence of these antibodies was related to the likelihood of experiencing clinical malaria during the subsequent transmission season. It was shown that humans are capable of mounting anti-PfEMP1 domain-specific antibodies and that the prevalence of these antibodies is related to exposure. Furthermore responses to one recombinant domain, DBL4γ, show evidence of cross-reactivity or perhaps are being directed at a more conserved epitope.Using a panel of recombinant proteins corresponding to the domains of one particular This work was carried out at the Kenya Medical Research Institute, Centre for Geographic Medicine Research Coast situated at Kilifi District Hospital, 50 km north of Mombasa on the coast of Kenya. The hospital serves around 240,000 people living north and south of an ocean creek. Individuals investigated during these immuno-epidemiological studies were resident in two sites in Kilifi District (within 20 km of each other), Chonyi and Ngerenya. These study sites have been described in detail elsewhere , whereasSera collection and active surveillance were conducted as part of a study examining the clinical epidemiology of malaria under differing transmission conditions. In briein vitro selection of A4 parasites expressing specifically A4 PfEMP1[PfEMP1 has a well-described and classified sequence already published with established domain boundaries and it displays a common cytoadherent phenotype, binding to both CD36 and ICAM-1 [These experiments were performed using a laboratory clone of parasite, A4. This particular parasite was chosen for a number of reasons. There exists a monoclonal antibody, BC6, which allows A4 PfEMP1. A4 PfEMd ICAM-1 .var gene of between 88–95% in A4U parasites and between 11 and 25% in A4-40 cycle parasites. There was no recognition of either 3D7 or P1 with BC6.The parasite clone A4 having undergone prior selection with BC6 is denoted A4U. In addition two further laboratory clones, A4 40-cycle (parasites of the A4 lineage which have been left in culture for 40 cycles with no selection) and 3D7, and one clinical isolate, P1, obtained from a five year old child admitted to Kilifi District Hospital with moderately severe malaria were used to investigate antibody reactivity against the intact infected erythrocyte by flow cytometry. Using the monoclonal antibody BC6, flow cytometry demonstrated expression of the A4 Escherichia coli (Stratagene UK), vector pMal-c2x (New England Biolabs) was used. Primers for PCR amplification were as follows (Accession number L42244): DBL1α forward 5'-CATGGTAGGGAGGATCCT, reverse 5'-CCCCAAGCTTGCCTATTCCGTATGAGAAAATG including the restrictions sites SmaI and HindIII respectively; CIDR1α forward 5'-TCCCCGGGGCAGGTGGATTATGTATATTCG, reverse 5'-CCCCTGCAGCTATGAATCACCAATAGCATTGG including restriction sites SmaI and PstI respectively; DBL2β forward 5'-CTCCCCGGGACGAACCAATATTCCAATGC, reverse 5'-CCTCTAGACTAGCACACATCCAACTTGGTGTC including restriction sites SmaI and XbaI respectively; DBL4γ forward 5'-GCTCCCCGGGTGCAATACAAAATATTATCCAAC, reverse 5'-CCCGCAAGCTTGCTACGAAGCAAATGTACTGTC including restriction sites SmaI and XbaI respectively; DBL5β forward 5'-GCTCCCCGGGGCTTCGAATTGTGAAC, reverse 5'-CCTCTAGACTAGATTTCGGATCGTTATTACTCG. (Domains DBL3δ and CIDR2 of the A4 var gene were unable to be satisfactorily cloned despite many attempts). Amino acid numbering of each recombinant domain: DBL1α 81–480, CIDR1α 590–841, DBL2β 832–1229, DBL4γ 2006–2437, DBL5β 2435–2802. PCR fragments corresponding to each domain were purified using a QIAquick PCR purification kit  and were ligated into pCR 2.1 vector (Invitrogen), containing the α-peptide of b-galactosidase to allow blue-white selection in the presence of 5-bromo-4-chloro-3-indolyl-b-D-galactoside . Sub-cloning efficiency cells DH5α were transformed and positive recombinant clones were identified by loss of α-complementation. Transformants were checked, by PCR performed with vector-specific primers, for the presence of domain-specific DNA and the plasmid DNA was purified using Wizard Plus SV Minipreps . Purified pCR 2.1 plasmids containing individual domains were digested with the correct restriction enzymes and ligated into pMal-c2x vector (New England Biolabs), which had been previously cut appropriately. DH5α cells were again transformed and blue-white screening as before identified positive recombinant clones. DNA sequencing using an ABI 373 Prism automated sequencing system with a Big Dye terminator sequencing kit  determined nucleotide sequences of the inserts. Sequence identities were confirmed by BLAST analysis. For protein expression BL21-CodonPlus-Ril competent cells (Stratagene) were used. The transformed cells were grown in Luria-Bertani medium with 0.2% glucose, ampicillin (50 μg/mL) to maintain the expression plasmid and chloramphenicol (50 μg/mL), to an optical density at 600 nm (OD600) of 0.3 to 0.5 at temperatures between 25°C and 37°C depending on the individual construct. Each culture was then induced to express the MBP-DBL/CIDR fusion proteins in the presence of 0.1 mM isopropyl-β-D-thiogalactoside (IPTG). The samples were spun and the pellet re-suspended in 1 M Tris lysis buffer. The cells were disrupted by sonication (12 pulses × 1 min). After spinning each MBP-fusion protein was purified by chromatography on amylose resin as recommended by the manufacturer (New England Biolabs), using 100 mM maltose for elution. After purification, the protein obtained was stored in the following buffer: 10 mM Tris-HCl, pH 7.4, 0.2 M NaCl, 10 mM β-mercaptoethanol, 1 mM EDTA at -80°C. Protein purity was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with 10% polyacrylamide and quantity was estimated by a protein assay kit  as recommended by the manufacturer.For secreted, IPTG-inducible expression in BL21-CodonPlus-Ril Plasmodium falciparum was produced and coated onto plates in PBS using the methods described by Ndungu et al [2O2 (Sigma) for 15 minutes in darkness. The mean OD value, taken at 492 nm, was calculated for each sample after correction for binding to MBP alone. A serum was scored as positive if the corrected OD value was higher than the mean + 3 SD of 20 negative control sera from UK residents who had never been exposed to malaria. All sera were screened in duplicate and a selection repeated at a later date. OD values were standardized against a pre-screened high positive standard on each plate. Please see additional files After checkerboard assays to determine the optimal coating concentration, 200 ng of DBL2β and DBL4γ and 400 ng of DBL1α, CIDR1α and DBL5β were coated onto individual wells in Nunc transparent, flat-bottomed 96 well plates (Nunc Technology) in 100 μl phosphate buffered saline (PBS). An equivalent molar concentration of MBP alone was coated as a control. In addition schizont extract of the A4 strain of gu et al . The plaUsing parasite clones A4U, A4-40 cycle and 3D7 and the clinical isolate, denoted P1, cryo-preserved trophozoite-infected erythrocytes at between 1 and 5% parasitaemia were thawed through the sequential restoration of isotonicity . They weet al [Reactivity against the infected erythrocyte surface was scored as mean fluorescent intensity using the method of Williams et al . In detaa = the mean fluorescence intensity (MFI) of uninfected erythrocytes following incubation in negative control plasma; b = MFI parasitized erythrocytes following incubation in negative control plasma; c = MFI uninfected cells incubated in immune plasma or test antibody and d = MFI parasitized erythrocytes incubated in immune plasma or test antibody.All statistical analyses were performed using Stata version 8 . To investigate the relationship between parasite status at the time of cross-sectional bleed and subsequent antibody levels, a multiple linear regression model, controlled for age, expressed as a factor of 6 months duration, location and exposure (estimated by responses to schizont extract) was performed. The results are expressed as coefficients describing the difference in antibody levels attributable to parasites.Significant differences in two or more continuous variables were calculated using the Wilcoxon rank sum test. The Chi-squared test for trend was used to assess a trend across groups.In a randomly selected subgroup of individuals , total IgG responses to erythrocytes parasitized with A4U, A4-40 cycle, 3D7 and P1 were also measured using flow cytometry. The correlation of responses to each individual domain with responses to the parasitized erythrocyte surface was assessed using the Spearman rank correlation coefficient.In order to investigate any association of domain-specific antibody responses with protection from clinical malaria, initially the univariate association between detectable serum IgG to each antigen (measured as OD), and whether or not the individual had an episode of clinical malaria during the subsequent six months was investigated by fitting the continuous variable against a binomial distributed outcome using logistic regression. As antibody reactivity is known to increase with exposure, a multiple logistic regression was then performed with age, location and reactivity to schizont extract, as a marker of exposure, included as co-dependent variables. Results are expressed as odds ratios per 10 fold increase in antibody levels.Evidence for age-associated acquisition of domain-specific IgG was apparent for responses against recombinant domains DBL1α and DBL4γ in both Ngerenya and Chonyi  and 32.5% (95% CI 27.9 – 37.3%) of individuals recognizing DBL1α and DBL4γ respectively, compared to 18.8% (95% CI 13.9 – 24.3%), 21.2% (95% CI 16.4 – 26.7%) and 19.6% (95% CI 14.7 – 25.2%) for CIDR1α, DBL2β and DBL5β respectively .Noticeable differences could be seen in the response to the different There was a clear increase in proportion of responders and rate of acquisition of responses in Chonyi compared to Ngerenya. For example with DBL4γ the maximum response in Chonyi occurs in the 16 – 20 year age group with 61.8% (95% CI 43.6 – 77.8%) of individuals in this age group recognizing this domain. This is compared to the maximum response in Ngerenya against the same domain, which did not occur until the 51 – 85 year age group and was lower at 56.5% (95% CI 34.5 – 76.8%). Acquisition of antibody responses against DBL4γ was more rapid in Chonyi where, by the age of 8 – 9 years 45.2% (95% CI 32.5 – 58.3%) of children responded compared to 18.6% (95% CI 9.70 – 30.9%) of children resident in Ngerenya (p = 0.0019 Wilcoxon ranksum). For responses against DBL1α, the situation was different with acquisition in both areas continuing throughout adulthood. The maximum response in both areas did not occur until the 51 – 85 year age group although as with DBL4γ, the maximum response was greater in Chonyi, 67.6% (95% CI 50.2 – 81.9%) than in Ngerenya at 43.5% (95% CI 23.2 – 65.5%) although this difference was not significant. Overall a greater proportion of individuals resident in Chonyi showed evidence of reactivity against DBL1α and DBL4γ but not CIDR1α, DBL2β or DBL5β. The two-sample Wilcoxon rank sum for the differences in response between the two locations gave the following significance values: DBL1α p = 0.0128, CIDR1α p = 0.0594, DBL2β p = 0.0774, DBL4γ p = 0.0000 and DBL5β p = 0.3848.461 individuals recognized none of the domains tested and of these 57.7% were resident in Ngerenya and 42.3% in Chonyi. Of these 461 individuals, 61.2% were aged less than 10 yrs. 350 individuals only recognized 1 domain to the exclusion of all the others, 160 of these individuals (45.7%) were aged less than 10 years. Of those only recognizing one domain, 26.6% recognized DBL1α only, 11.7% recognized DBL2β, 12.7% recognized CIDR1α, 37.4% DBL4γ only and 11.4% DBL5β only, making DBL4γ significantly more likely to be the single domain recognized  p = 0.001). Only 38 individuals recognized all 5 domains tested (3.12% of the whole cohort), of these 24 (63.2%) were aged over 10 yrs.Overall the number of domains recognized increased with age in both areas Figure . In six It has previously been noted that antibody responses to a number of parasite antigens are enhanced in younger individuals who are parasitized at the time of blood sampling -30. UsinUsing a multiple linear regression model, the effect of asymptomatic parasitaemia on the OD value obtained for each domain after controlling for age, location and exposure was determined. Having detectable parasites at cross-sectional bleed was associated with significantly higher OD values for domains DBL1α, DBL2β and CIDR1α  In an attempt to control for exposure accounting for any positive correlation, a multiple linear regression model was performed with age, location, parasite status and exposure (estimated by responses to schizont extract) as independent variables, for each of the pairs. Using this model, only the correlation between responses to DBL4γ and A4u reached significance (Coefficient 0.332 (95% CI 0.101 – 0.563) P = 0.005).Figure PfEMP1 domain were correlated against antibody responses to two further parasite clones and one clinical isolate as measured by flow cytometry; A4-40 cycle, 3D7 and a clinical isolate P1. Additional file In addition individual responses to each recombinant A4-PfEMP1 recombinant domains was analysed as a whole within Chonyi, no association was found between antibody response and subsequent disease experience , b) and c) respectively, using flow cytometry. The proportion of individuals in each age category, with upper 95% confidence interval, scoring positive for antibody recognition are shown. Positivity was scored as defined in the text. The dark grey bars represent individuals resident in Chonyi and the light grey bars represent individuals resident in Ngerenya.Click here for file


Human papillomavirus type 16 (HPV16) infects anogenital epithelia and is the etiological agent of cervical cancer. We showed previously that HPV16 infection regulates the key splicing/alternative splicing factor SF2/ASF and that virus late transcripts are extensively alternatively spliced. hnRNP A1 is the antagonistic counterpart of SF2/ASF in alternative splicing. We show here that hnRNP A1 is also up-regulated during differentiation of virus-infected epithelial cells in monolayer and organotypic raft culture. Taken together with our previous data on SF2/ASF, this comprises the first report of HPV-mediated regulation of expression of two functionally related cellular proteins during epithelial differentiation. Further, using electrophoretic mobility shift assays and UV crosslinking we demonstrate that hnRNP A1 binds the HPV16 late regulatory element (LRE) in differentiated HPV16 infected cells. The LRE has been shown to be important in temporally controlling virus late gene expression during epithelial differentiation. We suggest that increased levels of these cellular RNA processing factors facilitate appropriate alternative splicing necessary for production of virus late transcripts in differentiated epithelial cells. HPV infDuring a normal infection, transcription of the 7.9 kb HPV16 genome yields polycistronic RNAs, which are processed by alternative splicing to produce mature messenger RNAs . The virCis-acting regulatory elements have been identified in the coding regions and the late 3′ untranslated regions (UTRs) of several papillomaviruses (aviruses . It is paviruses . The 5′ aviruses  that areaviruses . Inhibitaviruses .The best characterised 3′ UTR regulatory element is the bovine papillomavirus type 1 (BPV1) inhibitory element, which is located in the BPV1 long control region. It is 53 nucleotides in length and contains a good consensus 5′ splice site. This 5′ splice donor site base-pairs with the 5′ end of U1 snRNA, allowing U1 snRNP to assemble on the element . A direcA similar element is present in the HPV16 late 3′ UTR. This 79-nt late regulatory element (LRE) overlaps the 3′ end of L1 gene and extends into the late 3′ UTR . The eleIf these elements regulate viral RNA processing during the virus life cycle then the proteins they bind may be regulated during epithelial differentiation. So far only SF2/ASF levels have been shown to respond to virus infection and epithelial differentiation . Increasin vitro. HPV16-induced changes in expression of both hnRNP A1 and SF2/ASF may facilitate alternative splicing of virus late transcripts.In this study, we demonstrate a significant specific increase in expression of nuclear hnRNP A1 during differentiation of HPV16 genome-containing epithelial cells in both monolayer and organotypic raft culture. The timing and level of increase is similar to that of SF2/ASF, the counterpart of hnRNP A1 in splicing regulation. Further, we demonstrate that hnRNP A1 binds the HPV16 LRE in the nucleus of differentiated W12 cells and GST-hnRNP A1 can bind the element directly 22.15 cells/100 mm dish (The 20863 (W12E) cell line is a subclone of the W12 line, an HPV16 positive cell line derived from a low-grade cervical lesion , which c mm dish . The W122.2For W12 cells, feeder cells were removed by treatment with 0.1% trypsin–0.5 mM EDTA prior to utilising for extraction. Nuclear and cytoplasmic extracts were prepared as described . In some2.3Proteins were separated on 12% SDS-PAGE before electroblotting onto polyvinylidene fluoride (PVDF) membranes (Amersham). The membranes were blocked in 5% (w/v) dried milk powder in phosphate-buffered saline (PBS) overnight at 4 °C. Primary antibodies were diluted in PBS with 5% (w/v) dried milk powder, 0.05% (v/v) Tween-20 (PBS-T) and incubated with the membranes for 1 h at room temperature with shaking. The anti-hnRNP A1 antibody, 4B10 , was used at a dilution of 1/500, monoclonal antibodies against SF2/ASF , involucrin , keratin 10 , filaggrin  and GAPDH  were used at a dilution of 1/1000. After washing with PBS-T, the membranes were incubated with an anti-mouse secondary antibody–horseradish peroxidase (Sigma) at a dilution of 1/1000 in PBS-T with 5% (w/v) dried milk powder for 1 h at room temperature. Following washing in PBS-T, antibody–protein complexes were visualised using ECL reagents (Amersham) according to the manufacturer's instructions.2.4in vitro transcription, riboprobes were synthesised with the Stratagene RNA transcription kit with addition of 25 μCi [α-32P]UTP  following the manufacturer's protocol. The full-length riboprobes were purified from a 5% denaturing polyacrylamide gel. EMSAs were carried out as described (in vitro transcribed with the addition of unlabeled UTP substituting for [α-32P] UTP. Non-specific competitor RNA was transcribed from pBluescript KS(+) (Stratagene) polylinker linearised with EcoRI. Supershift EMSA experiments were carried out by preincubation of nuclear extracts with the anti-hnRNP A1 antibody for 30 min on ice. The EMSA reactions were electrophoresed on a 5% non-denaturing gel.Primer sequences used to generate riboprobe templates by PCR amplification of pCATPE445  are showescribed . For com2.5GST-tagged hnRNP A1 expression plasmid  was transformed into BL-21 cells with induction of protein expression by adding 250 μl IPTG (100 mg/ml) to the cell culture. Transformed cells were lysed in 1 ml lysozyme (10 mg/ml) on ice for 15 min before incubating with 100 μl Triton X-100 for another 15 min. The supernatant was collected by spinning at 9500 rpm and mixed with 1 ml of 50% glutathione sepharose (Amersham) for 1 h before washing with PBS. The protein was eluted in 5 mM reduced glutathione in 50 mM Tris pH 8.0 three times and the concentration of the purified hnRNP A1 was determined by Bradford assay.2.65 J2 3T3 cells were mixed with 80% collagen type IV, 10% reconstitution buffer (2.2% (w/v) NaHCO3, 4.77% (w/v) HEPES in 0.062N NaOH), 10% 10× DMEM, and 0.024N NaOH. The collagen mixture was left to solidify in a 6-well cell culture plate before seeding 1.0 × 106 epithelial cells suspended in E-medium (Organotypic raft culture was carried out as described . BrieflyE-medium  onto thel-lysine (Sigma) coated slides. Immunohistochemistry was carried out using the ABC Elite kit (Vector Laboratories) according to the manufacturer's instruction. Briefly, sections were dewaxed by incubating in xylene and rehydrated in a graded series of alcohol . Antigen retrieval was achieved by heating in citrate buffer  for 15 min before blocking the sections with normal blocking buffer for 30 min. Monoclonal antibody was added for 1 h at room temperature. Diaminobenzidine tetrahydrochloride (DAB) (Vector Laboratories) was used as the chromagen to visualise the sections. Monoclonal antibodies against involucrin  and hnRNP A1  were used. For immunofluorescence, after being dewaxed and rehydrated, the sections were blocked in PBS-20% calf serum for 1 h before incubating with primary antibody for 1 h at 4 °C. Following washing with PBS, the sections were incubated with FITC conjugated secondary antibody for 1 h and mounted in VECTASHIELD mounting medium with DAPI (Vector Laboratories), and visualised using a Zeiss LSM510 laser scanning confocal microscope.Organotypic raft tissues were fixed in 10% neutral-buffered formalin overnight, paraffin-embedded, cut into 4 μm cross sections, and placed on poly-33.1Y-axis scale) demonstrating that levels of SF2/ASF and hnRNP A1 were specifically increased in differentiated W12 cells.We reported previously that the key splicing regulator SF2/ASF is up-regulated during differentiation of HPV16 infected epithelial cells. The antagonistic counterpart of SF2/ASF in regulating alternative splicing is hnRNP A1 . HPV16 l65 confirmed the results of the remaining Western data (data not shown).In case the changes in hnRNP A1 levels we observed were specific to differentiation in monolayer culture, we carried out immunofluorescence studies in W12E organotypic raft culture tissue. Involucrin expression commenced in the cell cytoplasm of the suprabasal layers as expected, indicating that these cells were differentiated A. Simila3.2hnRNP A1 has been shown to bind an exonic sequence silencer in the 5′ portion of the L1 open reading frame . However3.3E. coli and then incubated with [α-32P]-labelled full-length LRE probe in an EMSA experiment. Direct binding of hnRNP A1 to the LRE began to be observed at 90 nM  as well as several SF2/ASF binding sites but none of these overlap. Although both proteins bind the LRE, SF2/ASF does not bind directly  , locatedcis-acting RNA sequence in the HPV16 L1 coding region in HeLa cells transfected with an expression construct for the entire HPV16 coding region (hnRNP A1 has been demonstrated to bind a g region . This big region . It is p5We have demonstrated an increase in hnRNP A1 levels in HPV16 infected, differentiated epithelial cells, which parallels that seen for SF2/ASF . Binding


Influenza A virus (IVA) exploits diverse cellular gene products to support its replication in the host. The significance of the regulatory (β) subunit of casein kinase 2 (CK2β) in various cellular mechanisms is well established, but less is known about its potential role in IVA replication. We studied the role of CK2β in IVA-infected A549 human epithelial lung cells.CK2β gene silencing led to increased virus titers, consistent with the inhibition of CK2β during IVA replication. Notably, virus titers increased significantly when CK2β siRNA-transfected cells were inoculated at a lower multiplicity of infection. Virus titers also increased in cells treated with a specific CK2 inhibitor but decreased in cells treated with a CK2β stimulator. CK2β absence did not impair nuclear export of viral ribonucleoprotein complexes (6 h and 8 h after inoculation) or viral polymerase activity . The enhancement of virus titers by CK2β siRNA reflects increased cell susceptibility to influenza virus infection resulting in accelerated virus entry and higher viral protein content.Activation of CK2β was observed in A549 cells during virus binding and internalization but appeared to be constrained as replication began. We used small interfering RNAs (siRNAs) targeting CK2β mRNA to silence CK2β protein expression in A549 cells without affecting expression of the CK2α subunit. This study demonstrates the role of cellular CK2β protein in the viral biology. Our results are the first to demonstrate a functional link between siRNA-mediated inhibition of the CK2β protein and regulation of influenza A virus replication in infected cells. Overall, the data suggest that expression and activation of CK2β inhibits influenza virus replication by regulating the virus entry process and viral protein synthesis. CK2β activation in uninoculated cells was assigned a value of 100% (baseline control). CK2β activation did not differ between inoculated and control cells at any point during hours 2 to 20 after inoculation Figure , suggestWe next assessed CK2β activation during the 1-h inoculation period, when virus binds to cells and is internalized. Cells were incubated with virus-containing medium (MOI = 5) at 4°C for 20 min. The medium was then replaced with pre-warmed medium and cells were incubated at 37°C. CK2β activation was analyzed 0, 30, 45, and 60 min after start of inoculation period. At 45 min and 60 min after infection, CK2β activation was approximately 65% and 80%, respectively, greater than that in control cells Figure . TherefoTo evaluate the effect of CK2β on IVA replication, we silenced CK2β expression in A549 cells by using a gene-specific siRNA. To ensure that the amount of siRNA duplex used did not negatively influence cell viability, we first performed a cell proliferation assay 24 h, 48 h, and 72 h post-transfection (p.t.). CK2β siRNA did not reduce the number of viable cells or affect cell morphology, as compared to those of control cells, at any time point (data not shown), suggesting that the amount of siRNA used is not toxic to the cells.Total amount of the CK2β protein was assayed 24 h, 48 h, and 72 h p.t. by Western blot analysis. In cells transfected with CK2β siRNA, CK2β expression was substantially less than that in control-transfected cells at 24 h p.t. and was almost completely absent at 48 h p.t. Figure , demonst2O before and during virus inoculation. At 24 h p.i., the mean virus titer in DMAT-treated cells increased as compared to controls  or withs Figure . Converss Figure . These rHaving shown that 1) CK2β activation is inhibited during IVA replication and 2) CK2β silencing results in increased virus titers, we next studied the effect of CK2β gene silencing on the initial step of the viral replication cycle: virus entry. We transfected cells with either control or CK2β siRNA for 48 h. Cells were then inoculated with a high dose of virus (MOI = 50), incubated at 4°C for 30 min to block virus entry while binding to the cell surface, and incubated at 37°C for 30 min to allow virus entry. The cell membranes were then stained with an antibody specific for epidermal growth factor receptor (EGFR) and examined by confocal microscopy. Virus entry was slowed in control siRNA-transfected cells but not in CK2β siRNA-transfected cells, where they had accumulated at the nuclear membrane (predominantly) and in the nucleus 30 min after temperature shift  for 9 h or 24 h, and lysed for Western blot analysis. Total amount of viral protein was detected by using an antibody specific for IVA of the H1N1 subtype. As NP was the most heavily expressed of the viral proteins, we quantified the NP bands. The value obtained at 9 h p.i from cells transfected with control siRNA was assigned a value of 100%. NP production increased in both control and CK2β siRNA-transfected cells between 9 h and 24 h p.i. Figure .However, a substantially larger amount of viral NP protein was synthesized in CK2β siRNA-transfected cells than in control-transfected cells at both time points Figure . While t+  by FACS. Markedly more NP was produced in cells transfected with CK2β siRNA than in control siRNA-transfected cells Figure . 64% of + Figure . This fiNext, we investigated the contribution of CK2β to the intracellular localization of the viral RNP complexes. Cells were transfected with either control or CK2β siRNA for 48 h and then inoculated (MOI = 1) for 6 h and 8 h, the time points at which optimal nuclear export of RNP usually occurs ,32,42. VWe used a minigenome (RNP-like) system to evaluate whether CK2-mediated phosphorylation of PA and NP influences viral polymerase activity. Cells were transfected with either control or CK2β siRNA for 48 h and then co-transfected with the pol I-driven plasmid encoding the luciferase gene and pol I/pol II-responsive plasmids that express the viral PB2, PB1, PA, and NP proteins. After incubation for 24 h, luciferase activity  was assayed in cell extracts. Transfection without the PB1 plasmid revealed negligible background luciferase expression. Inhibition of CK2β expression did not substantially influence viral polymerase activity, which was only slightly greater after CK2β siRNA treatment than after control siRNA treatment Figure . The resThe molecular mechanisms of influenza virus multiplication and the interactions between viral and cellular gene products determine host susceptibility to infection and disease. We have demonstrated that the regulatory β-subunit of the cellular protein kinase CK2 inhibits IVA replication in A549 human epithelial lung cells. Importantly, pulmonary cells are among the first to be targeted by respiratory-tract pathogens, such as influenza viruses. When activated by infection, they can participate in lung inflammation by producing various soluble factors, including cytokines and by recruiting other cells .in vitro and in vivo. We found that this tool efficiently diminished CK2β expression in human lung cells without affecting the expression of CK2α protein. Cellular CK2β activation (phosphorylation) was upregulated during virus binding and internalization but diminished when virus replication began. Interestingly, inhibition of CK2β translation increased virus titers, suggesting that expression of this cellular kinase is inconsistent with influenza virus replication. It is noteworthy that virus titers in the absence of CK2β were inversely proportional to the MOI used for inoculation ; titers showed the greatest increase in cells inoculated at an MOI of 0.001. At this low MOI, the multiple replication cycles are required to infect the entire cell population leading to stronger differences in virus titers. Prevention of CK2β phosphorylation with a CK2 inhibitor also resulted in increased virus titers. In contrast, stimulation of CK2β with polyamine reduced virus titers, strengthening the evidence that CK2β activation negatively influences virus replication. This finding will facilitate further studies to understand the role of CK2β in viral biology. We hypothesize that the absence of CK2β reduces the activity of the CK2α subunit, thereby wholly or partially inactivating the heterotetrameric holoenzyme and providing a viral replication advantage. Our findings are compatible with a model in which influenza viruses have acquired the ability to exploit or impede host cell responses to support viral propagation. Several host factors other than CK2β have also been shown to possess anti-influenza virus activity. For example, inhibition of JNK signaling reportedly increases cellular virus yields [siRNAs are widely used to study the function of cellular genes s yields .Moreover, Mx1-induced expression of type I interferons efficiently protects mice against highly pathogenic H5N1 influenza virus by inhibiting viral polymerase activity, thereby reducing virus titers in the lung and liver . Thus, tBiochemical and genetic evidence indicates that CK2 participates in the maintenance of cell viability and plays a global anti-apoptotic role . TreatmeExport of viral RNPs from the nucleus is a key step in the influenza virus infectious cycle. Inhibition of CK2β expression did not affect the nuclear export of viral RNP complexes at 6 h and 8 h p.i.; thus, nuclear RNP export does not appear to be responsible for the increase in virus titers. Instead, influenza virus interacts with and employs other host factors, such as ERK ,32, CRM1CK2β gene silencing appeared to promote influenza virus entry by reducing the inhibitory effect of CK2β. However, further analysis is required to fully decipher how and at what level CK2β inhibits this early stage of viral replication.Many viruses use receptor-mediated endocytosis to enter a target cell ,57. ActiCK2β gene silencing on accumulation of viral RNA, cRNA, and mRNA in infected cells should be examined in more detail. Overall, siRNA-mediated CK2β gene silencing appears to increase cell susceptibility to influenza virus infection resulting in accelerated virus entry process and larger viral protein quantity, and these effects in turn contribute to higher virus titers. However, the molecular mechanisms remain to be elucidated.Reduced or absent CK2β expression substantially enhanced the cellular viral protein content, suggesting that CK2β may interfere with viral protein synthesis at the molecular level, e.g., replication , transcription (cRNA → mRNA), or translation. On the other hand, a considerably greater quantity of viral NP was detected at 24 h p.i. than at 9 h p.i., indicating the progressive spread of infection during that time. We hypothesize that while the viral protein synthesis rate per cell remains constant, loss of the CK2β protein seems to increase the susceptibility of A549 cells to influenza virus infection in the whole cell population. However, further investigation is needed to determine how CK2β absence increases cellular viral protein and why this increase does not significantly affect viral polymerase activity. The effect of CK2β gene silencing significantly increases virus titers in A549 cells, explaining the advantage of viral inhibition of CK2β. Treatment of cells with a specific CK2 inhibitor also increased virus titers, whereas stimulation of CK2β reduced virus titers, suggesting that CK2β activation negatively affects virus replication. CK2β gene silencing impaired neither nuclear export of viral RNP complexes nor viral polymerase activity. However, it appears to increase cell susceptibility to influenza virus infection and thereby accelerates virus entry process and boosts the total viral protein content. siRNA-mediated CK2β gene silencing may be useful in improving the yield of vaccine-strain influenza virus. Clinically, it may be possible in the future to introduce a construct expressing a constitutively active CK2β into influenza-infected hosts as a therapeutic intervention.Our results are the first to demonstrate a functional link between the CK2β protein and regulation of IVA replication. In contrast to virus-induced upregulation of many cellular kinases, IVA appears to constrain CK2β activation during viral replication. We found that 2. The H1N1-subtype human influenza virus A/Puerto Rico/8/34 (PR/8) was generated by 8-plasmid reverse genetics [The human epithelial lung cell line A549 was purchased from the American Type Culture Collection  and was cultured in FK12 medium containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) antibiotics (penicillin/streptomycin). Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS) and 1% (v/v) antibiotics (penicillin/streptomycin). All cells were cultivated at 37°C in a humidified atmosphere containing 5% COgenetics  and usedConfluent monolayers of MDCK cells in 35-mm dishes were inoculated with 10-fold dilutions of PR/8 influenza virus in DMEM with 3% bovine serum albumin (BSA) and antibiotics and incubated at 37°C for 1 h. The inoculum was removed, and cells were washed with PBS and overlaid with 2-fold DMEM containing 1.8% bacto-agar, 0.2% BSA, and 2 μg/ml TPCK trypsin. After 3 days of incubation at 37°C, cells were stained with 0.1% crystal violet in 10% formaldehyde solution and the plaques were counted.2O 1 h prior to infection. Cells were then inoculated at MOI = 5 in the presence of DMAT or DMSO. Virus-containing supernatants were harvested 24 h post-infection (p.i.) and virus titers were determined by plaque assay.To determine the effect of CK2 inhibition, confluent monolayers of A549 cells in 35-mm dishes were treated with 5 μM 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole  or with solvent (DMSO)  1 h prior to infection. To determine the effect of CK2β activation, cells were treated with 300 μM spermine (Sigma-Aldrich) or with HHuman CK2β siRNA  is a pool of 3 target-specific 20- to 25-nt siRNAs designed to silence CK2β gene expression in human cells. A549 cells (grown in 35-mm dishes) were transfected with siRNA duplex (1 μg) by using Lipofectamine 2000 . The control siRNA (Santa Cruz Biotechnology) is a nontargeting 20- to 25-nt siRNA that does not cause specific degradation of any known cellular mRNA. Cells were transfected for 48 h, washed with FK12 medium without supplements, then inoculate at the indicated MOIs and incubated for different time periods, depending on the purpose of the experiment. Transfection efficiency monitored by fluorescein-conjugated control siRNA (Santa Cruz Biotechnology) varied up to 85%  according to the maximum protein level reduction.2. The cell medium was then replaced with fresh medium. After further incubation for 1 h, the medium was replaced with 200 μl of FK12 medium containing 10% (v/v) FBS and tetrazolium bromide . After 90 min, the incubation medium was replaced with PBS/4% paraformaldehyde (PFA), and cells were fixed for 30 min at room temperature (RT). Cells were air dried and, after addition of isopropanol (150 μL/well), plates were vigorously agitated for 10 min. The plates were photometrically analyzed at 550 nm in an enzyme-linked immunosorbent assay reader . Sixteen samples from each group were measured, and the mean and standard error was calculated. Cell morphology was analyzed by confocal laser scanning microscopy after transfection with either siRNA.A549 cells were grown in 96-well dishes and transfected with 1 μg of CK2β siRNA or control siRNA for 24 h, 48 h, or 72 h at 37°C, 5% COCell lysate was cleared by centrifugation, and total protein concentration was determined by the Bradford assay before separation by SDS-PAGE. A rabbit polyclonal antibody that detects phosphorylated CK2β (P-S209)  was used to determine CK2β activity. Expression of CK2α and CK2β were detected with anti-CK2α  and anti-CK2β  mouse monoclonal antibody, respectively. Anti-β-actin mouse monoclonal antibody  was used to detect β-actin expression as a protein-loading control. Proteins recognized by mAbs were further analyzed with horseradish peroxidase-conjugated, species-specific secondary antibodies  and a standard enhanced chemiluminescence reaction . Specific bands were quantified by using the PC-BAS software package . To detect synthesis of viral proteins, cells were incubated with a goat anti-IVA (H1N1) polyclonal antibody .To detect ERK activation, cells were transfected with either control or CK2β siRNA for 48 h and were then infected at an MOI of 5 and further incubated for 4, 6, or 8 h. After Western blot analysis, ERK activation was measured with a mAb specific for phosphorylated ERK . ERK2  served as a protein loading control.After 48 h of transfection with either siRNA, cells were infected at MOI = 1 and incubated for 24 h. Cells were detached with trypsin, fixed in PBS/4% PFA, permeabilized with 1% Triton X-100, and incubated with FITC-conjugated mouse anti-NP mAb  in PBS/3% BSA for 30 min on ice. The percentage of NP-expressing cells was determined by FACS analysis using .A549 cells grown on collagen-coated glass coverslips  were transfected with either control or CK2β siRNA for 48 h, then infected at MOI = 1 and incubated further for 6 h and 8 h. Cells were washed with PBS at the indicated time points p.i. and fixed with 4% PFA in PBS for 30 min at room temperature or overnight at 4°C. Cells were permeabilized with 1% Triton X-100 in PBS for 10 min and incubated with a goat anti-NP antibody, clone G150  and anti-CK2β mouse monoclonal antibody  in PBS/3% BSA for 1 h for analysis of the nuclear export of RNP complexes and the amount of expressed CK2β protein. The AlexaFluor488-conjugated goat anti-mouse antibody and AlexaFluor594-conjugated donkey anti-goat antibody  were used as secondary antibodies. Cells were washed with PBS and then with double-distilled water and mounted with para-phenylene diamine (Sigma-Aldrich) containing 500 nM TO-PRO-3  for nuclear staining. Fluorescence was visualized with a confocal laser scanning microscope .To study virus entry, cells were infected at a MOI = 50, chilled at 4°C for 30 min, and incubated at 37°C for 30 min. Cells were permeabilized, fixed, and incubated with a combination of goat anti-IVA (H1N1) polyclonal antibody (1:100)  and mouse anti-epidermal growth factor receptor (EGFR) mAb . Alexa594-conjugated donkey anti-goat and AlexaFluor488-conjugatedgoat anti-mouse antibodies (Molecular Probes) were used as secondary antibodies.5 cells in 35-mm dishes) were transfected with CK2β siRNA or control siRNA for 48 h. Cells were then transfected with 2 μg of luciferase reporter plasmid (enhanced green fluorescent protein (EGFP) open reading frame in pHW72-EGFP substituted with the luciferase gene[Subconfluent monolayers of A549 cells (7.5 × 10rase gene) and a mThe authors declare that they have no competing interests.HM performed most of the experiments and wrote the manuscript. H–LY made the luciferase reporter plasmid. CS and RGW contributed to scientific ideas and analysis of the data. All authors reviewed the manuscript, and all author approved the final version.


Type-1 (CB1) and type-2 (CB2) cannabinoid receptors belong to the rhodopsin family of G protein-coupled receptors, and are activated by endogenous lipids termed “endocannabinoids”. Recent reports have demonstrated that CB1R, unlike CB2R and other receptors and metabolic enzymes of endocannabinoids, functions in the context of lipid rafts, i.e. plasma membrane microdomains which may be important in modulating signal transduction. Here, we present novel data based on cell subfractionation, immunoprecipitation and confocal microscopy studies, that show that in C6 cells CB1R co-localizes almost entirely with caveolin-1. We also show that trafficking of CB1R in response to the raft disruptor methyl-β-cyclodextrin (MCD) is superimposable on that of caveolin-1, and that MCD treatment increases the accessibility of CB1R to its specific antibodies. These findings may be relevant for the manifold CB1R-dependent activities of endocannabinoids, like the regulation of apoptosis and of neurodegenerative diseases. They biAPE-PLD)  or degraAPE-PLD)  AEA, theAPE-PLD)  or hydroAPE-PLD)  2-AG, anAPE-PLD) .Lipid rafts are subdomains of the plasma membrane that contain high concentrations of cholesterol and glycosphingolipids, and are well-known modulators of GPCR-dependent signaling and membrane trafficking in central and peripheral cells . Not surIn order to ascertain whether CB1R could reside within caveolae, here we investigated the colocalization of CB1R with CAV1 in C6 cells, by using subfractionation, immunoprecipitation and confocal microscopy techniques. We also investigated the trafficking of CB1R and CAV1 in response to MCD treatment, in the light of its potential relevance for the regulation of apoptosis and neurodegenerative diseases.22.1Rabbit anti-CB1R polyclonal antibodies were from Affinity BioReagents, Inc. . Rabbit anti-CAV1, anti-TRPV1, and anti-CAV1 mouse monoclonal antibodies were from Santa Cruz Biotechnologies . Anti-NAPE-PLD rabbit polyclonal antibodies were from Cayman Chemical . Anti-FAAH polyclonal antibodies were elicited in rabbits against the conserved FAAH sequence VGYYETDNYTMPSPAMR  conjugat2.22 and were fed every 2 days. Lipid raft disruption was performed by preincubating C6 cells for the indicated periods of time at 37 °C with 2.5 mM MCD , followed by a washing step in phosphate-buffered saline , supplemented with 10% fetal bovine serum. Cells were maintained at 37 °C in a humidified atmosphere with 5% COd saline .2.36 cells/test) were placed at the bottom of a discontinuous sucrose density gradient and were spun in an SW41 Ti rotor  at 188,000 × g and 4 °C for 22 h. Twelve 1 ml fractions were collected from top to bottom, saving also the heavy pellet. Proteins from each fraction were precipitated with 7.2% (v/v) trichloroacetic acid, and were solubilized in SDS–PAGE sample buffer  were sonicated in immunoprecipitation buffer  and centrifuged at 4 °C for 15 min at maximal speed in a microcentrifuge, and extracts were prepared as reported (C6 cells (100 × 10reported . Immunopreported . Cell ly2.54 cells/cm2. After 24 h C6 cells were treated with 2.5 mM methyl-β-cyclodextrin for the indicated time or left untreated (control cells). Cells were then fixed for 10 min at room temperature with 4% paraformaldehyde and processed for immunofluorescence (C6 cells were settled on a glass coverslip at a density of 2 × 10rescence . Rabbit 33.1Membrane fractionation studies were carried out to access the distribution of CB1R and CAV1 in C6 cells. CB1R was found exclusively in membrane fractions enriched with CAV1 A, and itAdditional confocal microscopy analysis further supported the colocalization of CB1R and CAV1 in C6 cells, demonstrating that CB1 receptors are predominantly localized intracellularly C. It sho3.2CB1R has been shown to permanently and constitutively cycle between plasma membrane and endosomes . Therefo4Lipid rafts are subdomains of the plasma membrane that contain high concentrations of cholesterol and glycosphingolipids, and are well-known modulators of GPCR-dependent signaling and membrane trafficking in central and peripheral cells .We have demonstrated that, in rat C6 glioma cells, raft perturbation by MCD treatment enhances CB1R binding and signaling . ShortlyHere, we supplement novel information that demonstrates that CB1R resides almost entirely in a specialized type of lipid rafts, the caveolae. This observation suggests a strong link between CB1R and CAV1, that seems interesting because caveolae play a role in neurodegenerative diseases, like Parkinson's disease, Alzheimer's disease and dementia with Lewy's bodies , as wellCaveolae represent a subset of lipid rafts, characterized by high caveolin content and formation of 50–100 nm flask-shaped membrane invaginations . Caveoli


The Kv7.1 pore-forming subunit can interact with various KCNE auxiliary subunits to form K+ channels with very different gating behaviors. In an attempt to characterize the nature of the promiscuous gating of Kv7.1 channels, we performed a tryptophan-scanning mutagenesis of the S4 sensor and analyzed the mutation-induced perturbations in gating free energy. Perturbing the gating energetics of Kv7.1 bias most of the mutant channels towards the closed state, while fewer mutations stabilize the open state or the inactivated state. In the absence of auxiliary subunits, mutations of specific S4 residues mimic the gating phenotypes produced by co-assembly of Kv7.1 with either KCNE1 or KCNE3. Many S4 perturbations compromise the ability of KCNE1 to properly regulate Kv7.1 channel gating. The tryptophan-induced packing perturbations and cysteine engineering studies in S4 suggest that KCNE1 lodges at the inter-VSD S4-S1 interface between two adjacent subunits, a strategic location to exert its striking action on Kv7.1 gating functions.Kv7 potassium channels whose mutations cause cardiovascular and neurological disorders are members of the superfamily of voltage-gated K However, much less is known about the impact of KCNE β subunits on the voltage sensor properties of Kv7.1. Recently, it was shown that the unique S4 charge deficit of Kv7.1 compared to other Kv channels underlies its unique conversion to a leak channel by auxiliary subunits such as KCNE3 Kv7 channels (Kv7.1-5 or KCNQ1-5) comprise a subfamily of voltage-gated KShaker and human Kv1.2 sequences, the S4 segment of Kv7.1 contains only 4 basic residues, where R3, K5 and K7 have been replaced by polar amino-acids, Q234, H240 and G246, respectively. Assuming that S4 moves upon membrane depolarization, mutation of a residue to tryptophan is expected to produce a marked gating perturbation if it lies at a protein-protein interface but a weaker alteration if located at a protein-lipid interface. Indeed, tryptophan is assumed to interact favorably with membrane lipids because of its hydrophobic and bulky nature. All mutants were analyzed in CHO cells using the whole-cell patch-clamp technique. The strength of the energetic perturbation produced by each mutation was deduced from conductance-voltage relations, expressed as the free energy difference between wild-type and mutant channels and then corrected for the differences in side chain volume ΔΔG0c = ΔG0mut−ΔG0wt  and V50 = −29.2±1.3 mV (n = 20) for A226W and WT, respectively . Another S4 mutation, V241W, produces a similar left-shift of the activation curve (ΔV50 = −21.8 mV). However, the perturbation does not reach significance because of a lower z value , τdeact = 80±6 ms (n = 8) and τdeact = 89±12 ms (n = 9), respectively, in comparison to WT Kv7.1   .Reflecting the multiple facets of perturbations produced by tryptophan substitutions in Kv7.1 S4, two mutations stabilize the inactivated state. Mutants L233W and Q244W, which shift the gating equilibrium towards the closed state, also stabilize the channel in the inactivated state and in fact exhibit a voltage-dependent macroscopic inactivation . As meas0c values  or copper-phenanthroline . We found that upon external exposure of the oocyte to Cd2+ (100 µM) or Cu-Phen (100 µM), the time- and voltage-dependent outwardly-rectifying shape of the R228C current is switched to a nearly linear leak character with currents devoid of time and voltage dependence . We posit that KCNE1 lodges between S1 and S4 segments of two adjacent subunits. KSI current. Instead, mutant channels open instantaneously and do not close normally in mutants G229W, R231W, I235W and R237W. For example, the mutant G229W at the S4 N-terminus may be in close proximity to residue E43 at the N-terminal boundary of the KCNE1 transmembrane segment and therefore stabilizes the channel open state conformation. In contrast, a perturbation in the S4 C-terminus, R243W, dramatically prevents KCNE1 to open the mutant channel. Residue R243 resides at the boundary of the S4 helix and the S4-S5 linker, a probably critical interface for the functional interaction between Kv7.1 and KCNE1. When expressed alone, R243W has an apparently normal sensor function and expresses functional K+ currents. However, co-expression of R243W and KCNE1 dramatically suppresses the current, suggesting that KCNE1 prevents mutant channel opening by disrupting either S4 movement or the coupling between S4 and the activation gate. It is possible that residue R243 may interact with the cytoplasmic boundary of the KCNE1 transmembrane segment. The importance of residue R243 for proper KCNE1 function is underscored by the existence of naturally occurring long QT mutations  leading to profound perturbations in channel gating By which mechanisms KCNE1 could alter this inter-VSD interface between S1 and S4? We suggest that KCNE1 affects the packing of S4 residues. The results of the tryptophan scan perturbation identified a series of S4 residues for which the ability of KCNE1 to properly regulate Kv7.1 channel gating has been severely impaired. Thus, when the normal packing of S4 is disturbed by bulky tryptophan substitutions, KCNE1 is unable to reproduce the characteristic slow + currents and leads to a gain of function The role of the S1 C-terminus in proper KCNE1 function and its importance in channel gating is underscored by the recently described familial atrial fibrillation mutation in S140G. This Kv7.1 mutation in the presence of KCNE1 generates large instantaneous, voltage-independent leak KIn all, this study indicates that despite the high degree of conservation of the S4 sequence among Kv channels, Kv7.1 channels exhibit a significant uniqueness. It also suggests that for exerting its striking effect on channel gating, KCNE1 needs to be strategically located close to S1 and S4 of two adjacent VSDs, thereby constraining sensor movement.Xenopus Laevis frogs were purchased from Xenopus 1 . The procedures followed for surgery and maintenance of frogs were approved by the animal research ethics committee of Tel Aviv University and in accordance with the Guide for the Care and Use of Laboratory Animals . Frogs were anaesthetized with 0.15% tricaine (Sigma). Pieces of the ovary were surgically removed and digested with 1 mg/ml collagenase  in Ca2+-free ND96 for about one hour, to remove follicular cells. Stage V and VI oocytes were selected for cRNA injection and maintained at 18°C in ND96 , supplemented with 1 mM pyruvate and 50 µg/ml gentamycin. The human Kv7.1 cDNA (in pGEM vector) was linearized by Not1. This vector served also as a template to generate the Kv7.1 mutants, using site-directed mutagenesis performed by the QuikChange (Stratagene) method. All mutant sequences were verified by DNA sequencing. Capped complementary RNA was transcribed by the T7 RNA polymerase, using the mMessage mMachine transcription kit (Ambion Corp). The cRNA size and integrity was confirmed by formaldehyde-agarose gel electrophoresis. Expression of WT and Kv7.1 mutants was performed by injecting 40 nl per oocyte (5 ng cRNA) using a Nanoject injector . Standard two-electrode voltage-clamp measurements were performed at room temperature (22°C–24°C) 2–5 days following cRNA microinjection. Oocytes were placed into a 100 µl recording chamber and superfused with a modified ND96 solution (containing 0.1 mM CaCl2) using a fast perfusion system which operates under controlled N2 pressure allowing constant perfusion velocity of 3.9–4.2 ml/min. The exchange of solutions was performed by computer-controlled pinch valves . A home made manifold having virtually no void volume and very narrow connecting tubes prevented backward flow upon valve switch. The bath solution was completely replaced within 1.5 seconds, allowing a solution exchange time of about 25 ms around the oocyte. Whole-cell currents were recorded using a GeneClamp 500 amplifier (Axon Instruments). Stimulation of the preparation, and data acquisition were performed using the pCLAMP 6.02 software and a 586 personal computer interfaced with a Digidata 1200 interface (Axon Instruments). Glass microelectrodes  were filled with 3 M KCl and had tip resistances of 0.2–0.5 MΩ. Current signals were digitized at 1 kHz and low pass filtered at 0.2 kHz. Errors introduced by the series resistance of the oocytes were not corrected and were minimized by keeping expression of the currents below 10 µA.Female A pcDNA3-based vector encoding human KCNQ1 served as a template to generate the S4 mutants using standard PCR techniques. The PCR-amplified regions of all mutants were verified by DNA sequencing. CHO cells were plated on poly-D-lysine-coated glass coverslips and grown in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 10% fetal calf serum and antibiotics. Transfections were carried out with pIRES-CD8 (0.5 µg) as a marker for transfection and with KCNQ1 wild-type (WT) and mutants (0.5 µg), using FuGENE (Roche Diagnostics) 2ATP, 5 EGTA, 10 HEPES, adjusted with KOH at pH 7.4 (290 mOsm). The external solution contained (in mM): 140 NaCl, 4 KCl, 1.8 CaCl2, 1.2 MgCl2, 11 glucose, 5.5 HEPES, adjusted with NaOH at pH 7.4 (310 mOsm). Channel expression into Xenopus oocytes and two-electrode voltage-clamp recordings were performed as described Recordings were performed 40–48 h following transfection, using the standard whole-cell patch-clamp technique. Signals were amplified using an Axopatch 200B patch-clamp amplifier, sampled at 2 kHz and filtered at 800 Hz. Data were acquired using pClamp 8.1 software with a DigiData 1322A interface (Axon Instruments). The patch pipettes were pulled from borosilicate glass (Warner Instrument. Corp) with a resistance of 2–5 MΩ and were filled with (in mM): 130 KCl, 1 MgCl2, 5 Krev) where the calculated reversal potential Vrev was −90 mV. G was then, normalized to the maximal conductance value, Gmax. Activation curves were fitted by a single Boltzmann distribution G/Gmax = 1/{1+exp[(V50−V)/s], where V50 is the voltage at which the current is half-activated and s is the slope factor. For the thermodynamic analysis, the difference in Gibbs free energy between closed and open states at 0 mV (ΔG0) was calculated according to ΔG0 = 0.2389 zFV50. The change in free energy difference between WT KCNQ1 and the mutant was computed by ΔΔG0 = ΔG0mut−ΔG0wt. To take into account the differences in side chain volume ∥Δvol∥ produced by the tryptophan mutations, ΔΔG0c values were corrected according to the relative change in side chain volume ΔΔG0c = ΔΔG0 [∥Δvolav/Δvol∥], with an average change volav = 68 Å3, as previously described Conductance (G) obtained from tail current amplitudes or from steady-state currents (when deactivation was very fast) was calculated by the following equation G = I/. The membrane-embedded segment of the KCNE1 auxiliary subunit was folded as a classical alpha helix. Then, this helix was docked to the open state by enabling sufficient proximity of KCNE1-Glu43 to Kv1.7-Ala226, so as to account for the findings of Nakajo and Kubo


Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes.Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex.Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion. This model would allow the SM protein to prevent futile reformation of SNARE complexes following the action of the ATPase NSF, and allow the SNARE proteins to recycle for further rounds of membrane fusion. Since different syntaxin/SM pairs are likely subject to different levels of regulation, the rate-limiting step in the SNARE assembly/disassembly cycle may differ for different membrane trafficking pathways. Thus, in the case of Sx1A/Munc18a the SNARE complex controls a tightly regulated fusion pathway (regulated exocytosis), and Munc18a binds preferentially to Sx1A in the closed conformation with high affinity in vitro possibly reflects the highest affinity interaction, with the lower affinity interactions overlooked.In striking contrast to the interaction between Munc18a and Sx1A, the extreme N-terminal region of other syntaxins are both necessary and sufficient to capture their cognate SM proteins. For example, the N-terminal 44 residues of Sed5p are sufficient to bind the SM protein Sly1p This may be exemplified by Munc18a that cradles monomeric Sx1A in its closed conformation, as shown by the crystal structure described above The insulin-dependent delivery of GLUT4-containing vesicles to the plasma membrane is a specialised example of regulated membrane trafficking in vitro liposome fusion assay offers a powerful tool with which to finely dissect the mechanistic basis of SM protein function We set out to define the role of Munc18c in membrane fusion driven by Sx4/SNAP23/VAMP2. The Two different SM proteins have recently been shown to stimulate SNARE-mediated liposome fusion Bilayer fusion catalysed by this SNARE complex was found to exhibit similar rates of fusion to that reported for the neuronal SNARE complex Sx1A/SNAP25/VAMP and the yeast exocytic SNARE complex Sso1p/Sec9p/Snc2p  [12], [1Although most research on SM protein function has focussed on their interaction with their cognate syntaxins, it is becoming apparent that interactions with non-syntaxin SNAREs also plays an important role in a conserved feature of SM protein function. We have previously shown that the yeast SM protein Vps45p not only binds directly to its cognate syntaxin, Tlg2p, but also to the assembled SNARE complex and also the v-SNARE Snc2p Munc18c has been reported to bind directly to monomeric Sx4, and also to the assembled Sx4/SNAP23/VAMP2 SNARE complex The SM:v-SNARE interaction between Vps45p and Snc2p can be disrupted by the presence of the syntaxin Tlg2p SM proteins clearly play a central role in SNARE-mediated membrane traffic. Here we have focussed on the SM protein, Munc18c, which regulates fusion mediated by the Sx4/SNAP23/VAMP2 complex. This complex regulates the delivery of GLUT4-containing vesicles to the surface of insulin-sensitive cells. In this study, we have shown that Munc18c inhibits membrane fusion catalysed by its cognate SNARE complex. Our findings are consistent with the observation that homozygous knockout of Munc18c in mice results in an increased sensitivity of GLUT4 exocytosis in response to insulin, suggesting that Munc18c negatively regulates GLUT4 exocytosis in vitro, whereas the proteins used to demonstrate a stimulatory effect of Munc18a and Sec1p favour an active conformation. It is possible that Munc18c, like other SM proteins However, others have reported a positive role for Munc18c in the insulin-stimulated delivery of GLUT4 to the plasma membrane of adipocytes D-glucopyranoside (OG) were from Sigma. A mutant of VAMP2 lacking the first 30 amino acids was generated by PCR and expressed in pGEX4T-1 as an N-terminal GST-fusion protein. Protein A-tagged Sx16 cytosolic domain was generated by PCR from human syntaxin 16A (obtained from H. Stenmark) Rat GST-tagged Sx4 and GST-VAMP 2 and 3 were from R. Scheller. Human VAMP8 and human VAMP4 were from A. Peden. Anti-Sx4, VAMP2, VAMP4 and VAMP8 and Munc18c were from Synaptic Systems. Lipids were from Avanti Polar Lipids and Triton ×100 and n-octyl-β-To purify the syntaxin 4/SNAP23 complex, plasmids containing the entire coding sequence of syntaxin 4 (pQE30) and an N-terminal GST-SNAP23 fusion (pET41a) were co transformed into BL21 DE3 cells and selected on dual antibiotic plates. Colonies were used to start an overnight culture, and the next day a further culture was grown (containing 500 µg/ml ampicillin and 50 µg/ml kanamycin). This overnight culture was then used to inoculate 12 L of Terrific broth containing 200 µg ampicillin and 25 µg kanamycin, and was grown at 37°C with shaking at 250 rpm. An additional 100 µg ampicillin was added each hour. Protein expression was induced by the addition of IPTG to 1 mM when the cells reached an OD600 of approximately 0.6, and incubated overnight at 25°C with shaking at 250 rpm.The protein complex was purified using glutathione sepharose (Amersham). The cells were broken by two passes through a French press at 950 p.s.i. in buffer A200  glycerol and 2 mM β-mercaptoethanol) containing 4% Triton, complete protease inhibitors (Roche) and 2 mM PMSF. Insoluble matter was removed by centrifugation at 30,000×g for 1 h. The supernatant was incubated with 5 ml of pre-equilibrated glutathione sepharose overnight at 4°C. The beads were washed with 100 ml of A200 containing 1% Triton. The Triton was then exchanged for OG by washing the 10 times with 15 ml of A200 containing 1%OG. The beads were resuspended in an equal volume of A200 containing 1% OG and 125 Units of thrombin were added. The beads were incubated with rotation for 4 h at room temperature, following which the supernatant was collected, aliquoted, snap frozen and stored at −80°C until use.6-myc fusion protein. The culture was grown to an OD600 of 0.8. Protein expression was then induced by the addition of 1 mM IPTG for 3 h at 37°C. Cells were resuspended, broken and the cell lysate centrifuged as outlined for syntaxin 4/SNAP23. The VAMP2 was then purified using Ni-NTA agarose. The beads were then washed once with 100 ml of A200 containing 1% Triton ×100 and 15 mM imidazole. The Triton ×100 was then exchanged for OG by washing the beads 10 times with 10 ml of A200 containing 1% OG and 15 mM imidazole. The protein was eluted from the beads with 3 ml of A200 containing 1% OG and 500 mM imidazole for 30 minutes at 4°C. The supernatant was collected, aliquoted, snap frozen and stored at −80°C until use.VAMP2 was expressed as a C-terminally tagged His6-fusion protein from the vector pQE30 in M15 cells co-transformed with a vector encoding GroEL. Cells were grown to an OD600 of 0.6 and expression of Munc18c was induced by adding 0.2 mM IPTG overnight at 25°C. Cells were broken by sonication in A400 buffer  glycerol, 2 mM β-mercaptoethanol) containing 10 mM Imidazole, EDTA-free complete protease inhibitors and 2 mM PMSF. Insoluble matter was removed by centrifguation. The supernatant was incubated with pre equilibrated Ni-NTA Agarose for 2 h. The beads were washed with 150 ml of A400 containing 15 mM Imidazole. Protein was eluted in A400 buffer with 500 mM imidazole for 1 h then dialysed against A200 without glycerol overnight.Munc18c was expressed as an N terminal HisLipid stocks were prepared in chloroform and stored at −80°C under nitrogen. For t-SNARE liposomes, a 15 mM lipid stock was made up in chloroform containing 85 mol% POPC and 15 mol% DOPS. For v-SNARE liposomes a 3 mM lipid stock was made up in chloroform containing 82 mol% POPC, 15 mol% DOPS, 1.5 mol% NBD-DPPE and 1.5 mol% rhodamine-DPPE 100 µl of 15 mM unlabelled lipid stock, for t-SNARE liposomes, or 500 µl of 3 mM labelled lipid stock, for v-SNARE liposomes was placed at the bottom of a 12×75 mm glass test tube. The chloroform was then evaporated using a stream of nitrogen for 15 min in a fume hood. To ensure that the lipid films were completely dry the samples were then dried for a further 30 min under vacuum. Purified t- or v-SNARE (500 µl) purified as outlined above was then added to each tube. The lipid film was then resuspended, by vortexing for 15 min. After the lipid film was completely resuspended the detergent was diluted below its critical micellar concentration by the addition of 1 ml of Buffer A200 containing 1 mM DTT, drop-wise while the sample was continuously vortexed. To remove any remaining detergent, the samples were placed into pre-equilibrated 3 ml Float-a-Lyzers with a molecular weight cut off of 10,000 and dialysed against 4 L of Buffer A200 containing 1 mM DTT and 4 g of Bio-Beads, with stirring at 4°C overnight. Samples were recovered the following day, and placed at the bottom of a SW60 tube on ice for subsequent separation using gradient centrifugation.Proteoliposomes were recovered by floatation on a Nycodenz gradient. An equal volume of 80% nycodenz in buffer A200 containing 1 mM DTT was mixed with the recovered dialysate to produce a 40% nycodenz mixture. This was overlaid with 1.5 ml of 30% nycodenz in buffer A200 containing 1 mM DTT. This layer was then overlaid with 250 µl of glycerol free A200 and centrifuged at 65,000×g for 4 h at 4°C. Proteoliposomes were recovered by removal of 400 µl from the top of the gradient, snap frozen and stored at −80°C.Typically fusion assays were set up by mixing 5 µl of v-SNARE liposome with 45 µl of t-SNARE liposome directly in a well of a 96 well microtitre plate, on ice. This was then sealed and incubated overnight at 4°C. For assays requiring the addition of soluble v-SNARE, 2 µl of purified protein in buffer A200 was added to the t-SNARE liposomes on ice for 10 minutes prior to the addition of v-SNARE liposomes. To correct for the resulting difference in volume 2 µl of A200 was added to all other wells in that run. The fluorescence was measured for 2 h with the excitation set to 460 nm and the emission recorded at 520 nm at 2 min intervals at 37°C. After this period, the plate was removed and 10 µl of 2.5% (w/v) n-dodecylmaltoside was added to each well. The plate was gently mixed for 2 min and then fluorescence was recorded for 40 min at 2 min intervals.2, 0.05% Tween 20, 20 mM HEPES pH 7.4) in 200 µl for 1.5 h at 4°C with rotation. Unbound protein was removed by 3 washes with 1 ml binding buffer. 5 µg of HIS-tagged Munc18c protein and binding buffer was added to each tube  and incubated at 4°C overnight with rotation. 20 µl was removed in order to examine protein input. Unbound protein was removed by 3 washes with 1 ml binding buffer plus 0.2% fish skin gelatin (Sigma), followed by 3 washes with 1 ml of binding buffer plus 5% (w/v) glycerol and 4 washes with 1 ml of binding buffer alone. After the final wash, all remaining supernatant was removed and 15 µl of 1× SDS-PAGE sample buffer (with 20 mM DTT) was added to and samples boiled for 5 min. After centrifugation at 14,000×g for 5 min, the supernatant was removed and analysed by SDS-PAGE and/or immunoblotting as outlined in 5 µg of GST-tagged proteins , were incubated with 10 µl of glutathione sepharose in binding buffer (150 mM Potassium acetate, 1 mM MgCl