In this study, two conserved amino acid residues of CXCR1 on the transmembrane domain (TM) 3 and TM6, Leu1283.43 (L128) and Val2476.40 (V247), respectively, were selectively substituted with other amino acids to investigate the role of these conserved residues in CXCR1 activation. Although two selective mutants on Leu128, Leu128Ala (L128A) and Leu128Arg (L128R), demonstrated high binding affinity to IL-8, they were not capable of coupling to G proteins and consequently lost the functional response of the receptors. By contrast, among the four mutants at residue Val247 (TM6.40), replacing Val247 with Ala (V247A) and Asn (V247N) led to constitutive activation of mutant receptors when cotransfected with Gα15. The V247N mutant also constitutively activated the Gαi protein. These results indicate that L128 on TM3.43 is involved in G protein coupling and receptor activation but is unimportant for ligand binding. On the other hand, V247 on TM6.40 plays a critical role in maintaining the receptor in the inactive state, and the substitution of V247 impaired the receptor constraint and stabilized an active conformation. Functionally, there was an increase in chemotaxis in response to IL-8 in cells expressing V247A and V247N. Our findings indicate that Leu1283.43 and Val2476.40 are critical for G protein coupling and activation of signaling effectors, providing a valuable insight into the mechanism of CXCR1 activation.CXCR1, a classic GPCR that binds IL-8, plays a key role in neutrophil activation and migration by activating phospholipase C (PLC)β through Gα Interleukin-8 (IL-8) is a member of the CXC-chemokine family and is a potent chemotactic factor for neutrophils i and pertussis toxin-resistant Gα153 hydrolysis, intracellular Ca2+ mobilization, and chemotaxis CXCR1 is a member of GPCRs that feature the characteristic seven transmembrane domains. Upon activation, CXCR1 couples to both pertussis toxin-sensitive GαGPCRs possess similar structures that consist of seven transmembrane helices containing well-conserved sequence motifs, which suggests that they are probably activated by a common mechanism 3.43 , located at the region near the second intracellular loop in TM3, is highly conserved (over 70%) Within the structure of GPCR, Leu1283.43 (L128) and Val2476.40 (V247), respectively. We analyzed their features including receptor expression, and ligand binding. The capacity of Gα15 and Gαi to be coupled by CXCR1 mutants is determined by measuring downstream signaling transduction pathways such as PLCβ-IP3 production and cAMP production. To test for functional consequence of activation by CXCR1 mutants, we performed chemotaxis assays with cells that express CXCR1 and its mutants.It is noteworthy that IL-8 and its specific receptor CXCR1 are identified in humans but are not found in the traditional rodent model organisms. As a consequence, we utilized well established cell models which were transfected with CXCR1 and other related cellular components to study the functions of IL-8 and CXCR1 as well as the mechanisms involved in receptor activation by IL-8. In this study, we generated selective mutations on two conserved amino acid residues of CXCR1 on TM3 and TM6, Leu128Wild type (WT) human CXCR1 cloned into pSFFV•neo vector was provided as a gift from Professor Ingrid Schraufstatter at the La Jolla Institute for Experimental Medicine. After digestion with EcoRI, wild type human CXCR1 fragment was subcloned into the pSG5 vector. Oligonucleotides for site-directed mutagenesis were designed to yield several different amino acid substitutions and were synthesized by GIBCO BRL. CXCR1 mutations were generated with the Transformer mutagenesis kit  and were confirmed by Big Dye Terminator Cycle Sequencing (Perkin Elmer) of the final construct. Plasmid DNA for transient transfection was purified with the EndoFree Plasmid Maxi Kit from Qiagen.COS-7 cells and human embryonic kidney 293 cells (HEK293 cells) from the American Type Culture Collection  were maintained at 37°C in humidified air containing 5% CO2 in DMEM with 10% FBS. Cells were grown to 60∼80% confluency prior to transient transfection. Transfection was performed using LipofectAMINE reagent  according to the manufacturer's instruction. COS-7 cells were incubated with the transfection complex for 5 hrs at 37°C. After removal of the transfection medium, the cells were incubated in DMEM with 10% FCS overnight.HEK 293 cells were transfected with either pSG5 plasmid, or CXCR1 WT or mutants. After 48 hr, cells were dissociated and fixed with 4% paraformaldehyde. Cells were then washed three times with cold staining buffer before incubation with mouse anti-human CD128 FITC (CXCR1) from BD Pharmingen (Cat# 555939)  on ice for 20 min. Cells were washed three times and resuspended in 0.5 ml of staining buffer. Stained cell samples were analyzed by flow cytometry COS-7 cells or TSA-201 cells  were transfected with either pSG5 plasmid, or CXCR1 WT or mutants. After 24 hr, cells were fixed with 4% paraformaldehyde followed by blocking with 1% BSA in HBSS. Cells were incubated with primary antibody (mouse anti human CD128a (CXCR1)) from BD Pharmingen (5A12 clone)  at 4°C overnight. After washing three times, the cells were incubated with the secondary antibody  from Invitrogen, CA, or DyLight 549-conjugated goat anti-mouse IgG(H+L) from Jackson ImmunoResearch Laboratories, Inc., PA) at RT for 1 hr and counterstained with Dapi. Expression of CXCR1 or the mutants was observed using confocal microscope.5 cells/ml of DMEM/10% FCS and incubated overnight. The cells were transfected as described before. In brief, the cells were transfected with 0.7 µg DNA plus 2.5 µl of LipofectAMINE reagent and 5 µl Plus Reagent . After 48 hrs from the start of transfection, transfected cells were washed in the binding buffer (HBSS medium containing 0.5% BSA and 25 mM HEPES buffer). The cells were then incubated for 2 hr at 4°C. The final concentration of 125I-IL-8 in the 0.5 ml/well (for 12-well plate) of medium was 0.07 nM (0.05 µCi). The range of unlabeled IL-8 concentrations in the binding assays was 0.1–300 nM . The nonspecific 125I-IL-8 binding was determined by incubating cells with 125I-IL-8 in the presence of 250 nM of cold IL-8. In experiments designed to test the maximal binding of 125I-IL-8 to CXCR1 WT and mutants, unlabelled IL-8 was not added. After washing three times with 2 ml of ice-cold binding buffer, the cells were lysed in 0.5 ml of 1 N NaOH and the lysates were counted using a scintillation counter. The data were curve fitted and affinity constant (Kd) and maximum total binding (Bmax) were calculated using GraphPad Prism . All experiments were carried out in triplicate.COS-7 cells were plated in 12-well plates at a density of 1.6×105 cells per well the day before transfection. The cells were cotransfected with 0.3 µg CXCR1 plasmid DNA (or its mutants) and 0.3 µg Gα15 plus 1.5 µl LipofectAMINE reagent and 0.5 µl Plus reagent. In experiments of cotransfection with WT CXCR1 or its mutants, COS-7 cells were cotransfected with equal amounts of cDNA (0.1 µg per well per component). Plasmids encoding Gαi2 and PLCβ2 were generous gifts from Dr. Kozasa at the University of Illinois at Chicago. Plasmids encoding Gβ1 and Gγ2 were kindly provided by Guthrie Research Institute, PA. . 24 hr after transfection, cells were incubated with inositol-free medium containing 2 µl/ml myo-[2-3H]inositol  in the absence or presence of 100 ng/ml pertussis toxin (PTX) as indicated for 18 hr. At 48 hr from the start of transfection, cells were washed with assay buffer . IP production was measured by incubating the cells for 1 hr at 37°C in 0.2 ml assay buffer containing 10 mM LiCl in the presence or absence of 40 nM IL-8. Perchloric acid was added to each well and samples were then neutralized with a solution of 0.72 M KOH and 0.6 M KHCO3 before being centrifuged. Total IP was measured using Dowex AG1-X8 anion exchange column chromatography . All assays were performed in triplicate, on at least 3 separate occasions with different batches of cells, and always included control cells transfected with WT CXCR1. Data were analyzed using GraphPad Prism  and expressed as fold-increase over basal conditions in cells cotransfected with WT CXCR1 and Gα15 plasmid.For IP assay, COS-7 cells were plated in 24-well plates with a density of 1×10COS-7 cells were transiently transfected with CXCR1 WT or V247N mutant. Preparation of dye-loading solutions of BD calcium assay kits  were performed and fluorescence was measured as described in the manual.i protein which can inhibit cAMP accumulation when transfected with Gαi-coupled receptors such as CXCR1 i2, Gβ1, and Gγ2. TSA-201 cells were grown to 50% confluency in DMEM/10% FBS on 24-well plates and were transfected using lipofectaMINE with 0.2 µg of each DNA encoding human LH receptor, or CXCR1 WT, or V247N mutant per well. 48 hours after transfection, COS-7 cells or TSA-201 cells were washed once with 1×HBSS/0.1% BSA pre-warmed buffer. The cells were incubated with 1×HBSS/0.1% BSA containing 0.5 mM IBMX for 30 min followed by treatment of the cells with the same medium containing either forskolin (10 µM) or hCG (10−7 M) in the presence or absence of IL-8 (40 nM) for another 30 min. Then the treatment buffer was aspirated and the reaction stopped with 0.85 ml of 5% cold perchloric acid (PCA). It was left for 10 min before the transfer of 0.8 ml to the 12×75 glass tubes with 0.18 ml of 3× neutralization buffer . The samples were vortexed and centrifuged at 2,500× g for 5 min. cAMP levels were measured with a RIA Kit  according to the manufacturer's instructions. Data are shown as mean ± SEM for three assays with each condition performed in triplicate.How CXCR1 WT and V247N mutant would affect cAMP formation was assessed in both COS-7 and HEK-293 cell-derived TSA-201 cells. Both cell lines are useful to demonstrate GPCR-triggered cAMP accumulation. However, unlike COS-7 cells, type II adenylyl cyclase I in TSA-201 cells is not stimulated by free βγ subunits released from activated Gαi, Gβ, Gγ, and PLCβ2 was transiently transfected in HEK293 cells. 24 hours after transfection, the cells were serum-starved in 0.5% FCS-containing medium for another 24 h. The cells were then trypsinized, treated with trypsin inhibitor (Sigma), resuspended in BSA medium at 5×105 cells/mL, and incubated for 2 h at 37°C. To the bottom wells 28 µl of either 100 ng/ml IL-8 or the solution without IL-8 (control group) were added as chemoattractant. For some indicated wells, cells were pretreated with 200 ng/ml of pertussis toxin for 10 min before being added to the upper chamber for chemotaxis assay. For all the migration assays, 50 µl of 1×106 cells/mL were loaded into the upper chamber fitted with a 10-µm pored membrane  coated with 50 µg/mL rat collagen type 1  for 4 h. This allowed cells to migrate towards the underside of the insert filter. Cells that did not migrate through the pores and therefore remained on the upper side of the filter membrane were gently removed with a cotton swab. Cells on the lower side of the insert filter were quickly fixed with 5% glutaraldehyde solution for 10 minutes and then stained with 1% Crystal Violet in 2% ethanol for 20 minutes. The cells that adhered to the lower surface of the membranes were counted microscopically at 400× magnification. The chemotactic activity was expressed as a chemotactic index, calculated as the number of cells that migrated in response to IL-8, divided by the number of cells that migrated toward the negative control (dilution buffer without IL-8 for control cells). Statistical analysis was performed using Student's t test.IL-8 stimulated chemotaxis through CXCR1 and CXCR1 mutants was assessed using a 48-well microchemotaxis chamber technique . CXCR1 or CXCR1 mutant V247A or V247N as well as GαTo define the G protein coupling and signaling properties of constitutively active forms of the human CXCR1, we generated 7 different mutations at 2 residues that correspond to known sites of activating mutations in TM3 and TM6 of rhodopsin, the human LHR, and several other GPCRs Flow cytometry  utilizedd) of the mutant receptors  of WT CXCR1 for IL-8 obtained by ligand binding assay in transfected COS-7 cells was 4.87×10−9 M, which was similar to those reported by others both in recombinant cell lines and in neutrophils d values similar to the WT, while other mutants including L128A, L128R, V247A and V247Y, demonstrated increased Kd values  was much higher than WT  has been successfully applied to investigate IL-8 signaling pathways in COS-7 cells in a previous study i2 activation in response to IL-8 leads to the release of Gβγ subunits from Gαi2, which results in PLCβ2 activation and subsequent IP accumulation i, totally suppressed Gαi activation and subsequent IP accumulation, suggesting that CXCR1 and its mutants investigated here are indeed coupled to Gαi2. Thus, IP accumulation could be achieved via the PTX-insensitive Gα15 pathway or the PTX-sensitive Gαi2 pathway as described above. Interestingly, L128R and V247F failed to have any effect on IP formation in response to IL-8 in transfected COS-7 cells (data not shown). Among all mutants detected, V247F bound the most poorly to IL-8. Because this mutant showed low expression as indicated by flow cytometry  and the WT CXCR1 or its mutants. The chimeric G protein, Gαqi5, contains main structure of Gαq and the last five C-terminal amino acids of Gαq were replaced with the corresponding amino acids from Gαi2. Mutation of the last five C-terminal amino acids is sufficient to completely switch receptor coupling selectivity and enable Gαi/o-coupled GPCR signaling through phospholipase Cβ (PLCβ). As such, a classical Gαq-mediated output becomes indicative of receptor coupling to Gαi/oqi5 in the COS-7 cell line facilitated the activation of CXCR1 WT and V247N mutant in response to IL-8. Moreover, in the absence of IL-8, transfected cells showed significantly increased IP accumulation in V247N and Gαqi5 coinfected cells, suggesting that CXCR1 WT and V247N are coupled to Gαi protein and that V247N is capable of constitutively activating Gαi protein , suggesting that V247N constitutively activated Gαi-mediated signaling.V247N is a robustly expressed mutant coupled to Gα-7 cells  and TSA--7 cells . To tests protein, allowed us to induce cAMP accumulation in those transfected cells. Again, it was found that in the transfected TSA-201 cells, LH receptor ligand hCG-stimulated cAMP production was inhibited in response to IL-8 in TSA cells expressing CXCR1 WT and V247N mutant  in the absence of IL-8, indicating that V247N mutant constitutively activated Gαi.In order to further verify the ability of CXCR1 WT and V247N mutant to inhibit adenylyl cyclase, we utilized a protocol in which both the CXCR1 receptor and LH receptor were cotransfected. Cotransfection of the LH receptor, meant to stimulate adenylyl cyclase via GαN mutant . As showi protein i, Gβ, Gγ, and PLCβ2. In the absence of IL-8 cells demonstrated disoriented, non-specific migration. We did not observe the increase in chemotaxis in non-IL-8-treated HEK cells expressing these mutants, suggesting that there were no differences in random migration between CXCR1 WT and constitutively active mutants. However, in the presence of IL-8, the cells expressing CXCR1 mutants (V247A and V247N) displayed enhanced migration than those expressing WT CXCR1, suggesting that the constitutively active mutants respond to IL-8 above and beyond their constitutive activity. Both CXCR1 and its constitutively active mutants induced chemotaxis in response to IL-8 , TSH receptor (Leu629), muscarinic receptor (Ile447), and histamine H1 receptor (Ile420) also result in constitutive activation s-coupled TSHR, and Gαi/Gα15-coupled CXCR1) results in constitutive activity of the receptors coupled to specific G proteins . This suggests that the amino acid residue located at TM6.40 is critical for the stabilization of the receptors in the inactive state, which is an intrinsic feature of the receptors and independent of the G protein type. Structural modification of this single amino acid residue affects GPCR-G protein interaction and receptor activation.The amino acid residue located at TM6.40 might be critical in the stabilization of the receptors coupled to various G proteins such as Gα15. Without ligand, mutants V247A and V247N were stabilized in the active state. However, ligand binding caused certain responses including elevated IP accumulation, which might result from conformational changes caused by receptor binding to its ligand, IL-8. There is strong evidence that GPCRs may exist in multiple ligand-specific conformational states 2A receptor structures revealed an intermediate conformation between the inactive and active states to support this model d in this study are constitutively active strongly supports the concept that more than one active receptor state can exist.Both V247A and V247N demonstrated high efficiency in coupling to Gαi2, Gβ1, Gα15 and PLCβ2 in these cells, are likely to be found in neutrophils or related myeloid cells where CXCR1 is expressed 15- PLCβ2 and the Gαi-Gβγ-PLCβ2 pathways are likely to be active in vivo. In addition, chemotactic responses in many leukocytes require activation of Gαi, as indicated by their sensitivity to inhibition by PTX. In agreement with this, our data indicated that chemotaxis mediated by CXCR1 or its mutants is completely abolished in transfected cells pretreated with PTX. Therefore, the increase in chemotaxis in response to IL-8 observed in cells expressing constitutively active mutants V247A and V247N can be attributed to the active signaling through these mutants.Transmembrane signaling through CXCR1 plays a role in many neutrophil functions such as chemotaxis In summary, the two residues in TM3 and TM6 on CXCR1 that correspond to L128 (TM3.43) and V247 (TM6.40), respectively, play distinct roles in receptor activation. Changes in conformational structure by substituting L128 impaired the interaction between the receptor and G proteins without affecting ligand binding. The substitution of V247 impaired the receptor constraint and stabilized an active conformation. The active CXCR1 mutants demonstrated increased chemotaxis in response to IL-8 in a PTX-sensitive manner. These findings regarding the constitutively active form of CXCR1 may contribute to our knowledge of the activation of CXCR1 and may be potentially useful for designing and screening CXCR1-specific antagonists which may serve as targets for prevention and therapy of certain neutrophil-mediated inflammatory diseases such as COPD.


R1ρ (1/T1ρ), when gadofosveset was serum albumin-bound than when unbound. R1ρ values for a nonbinding contrast agent (gadopentetate dimeglumine) in serum albumin were similar to those for unbound gadofosveset. R2 (1/T2) values were also significantly higher at 4.7 T for serum albumin-bound gadofosveset than for unbound. Spin locking at high field generates significantly higher relaxation rates for gadofosveset than conventional contrast agents and may provide a method for differentiating free and bound molecules at these field strengths. Magn Reson Med, 2012. © 2011 Wiley Periodicals, Inc.The contrast agent gadofosveset, which binds reversibly to serum albumin, has a high longitudinal relaxivity at lower magnetic fields (≤3.0 T) but a much lower relaxivity at high fields. Spin locking is sensitive to macromolecular content; it is hypothesized that combining this technique with the albumin-binding properties of gadofosveset may enable increased relaxivity at high fields. In vitro measurements at 4.7 T found significantly higher spin-lock relaxation rates,  The clinically approved contrast agent gadofosveset trisodium  is a small-molecule gadolinium (Gd) chelate that acquires macromolecular properties on binding to serum albumin (SA). The gadofosveset molecule consists of a stable gadopentetate core with a phosphodiester linkage to a lipophilic albumin-binding group . Over 90r1bound) peaks at around 0.5 T and decreases rapidly with increasing field strengths (r1free) is slightly higher than conventional small-molecule Gd-based agents such as gadopentetate and shows only a moderate decrease with field strength  includes contributions from both the bound and free molecules and is influenced by binding fraction and field strength. The nonlinear relationship between r1obs and gadofosveset concentration at low field strengths can be modeled  is characterized by the time constant T1ρ. The SL pulse may be followed by a 90° pulse  and an imaging sequence, or a 180° pulse and readout  range, rather than the main magnetic field (B0). As a result, the image contrast generated by SL is equivalent to image contrast obtained at low magnetic fields, with the advantage that a high signal-to-noise ratio may be maintained , first described as an imaging technique in 1985  but inve readout . T1ρ is intained . It shouSL to TR .The interaction times associated with SL at very low field strengths give this technique an increased sensitivity to proteins and other macromolecules . This coT1ρ.Small-molecule Gd-based contrast agents have been used in combination with SL to provide improved myocardium–blood contrast , 19 and T1ρ for in vitro gadofosveset solutions at a high B0 field strength. It was hypothesized that by combining the macromolecular sensitivity of SL with the albumin-binding affinity of gadofosveset a large contrast shift may be achieved at field strengths where the T1 effects of gadofosveset are very similar to those of conventional Gd-based agents. In addition, it may be possible to use this technique to generate a measurable difference in the relaxation properties of bound and unbound agent.The purpose of this study was to determine the potential impact of albumin binding on T1ρ values fall between T1 and T2, with T1ρ → T2 as B1L → 0  were prepared using phosphate-buffered saline , to replicate the unbound state, and bovine SA , to replicate a combination of bound and unbound molecules similar to that found in humans. Stock solutions were created at a gadofosveset concentration of 10 mM, and serially diluted to produce further solutions at 5.0, 3.0, 2.0, 1.5, 1.0, 0.75, 0.5, and 0.25 mM. Solutions of PBS and BSA without contrast agent (0 mM) were also assessed. In order to separate the influence of the macromolecular solution from that of Gd, an equivalent set of solutions of the nonbinding contrast agent gadopentetate dimeglumine  in BSA (4.5% w/v) were also created using the same method. Prior to scanning at 4.7 T, all solutions were heated to 37°C in a water bath; this temperature was maintained during scanning with warm air flow and verified with a fiber optic temperature probe in an adjacent water tube. At 0.5 T, samples were heated to 37°C and the temperature monitored with an integral heating system.B1L pulse value of 90 μT (3.8 kHz), applied for 14 durations (TSL): 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 12.5, 25.0, 50.0, 75.0, 100.0, 125.0, 150.0, and 200.0 ms. This was followed by a rapid acquisition with relaxation enhancement (RARE) readout, using a coronal  slice of thickness 1 mm. The acquisition parameters were: TR = 2000 ms; TE = 10 ms; field of view = 60 × 60 mm; matrix size = 128 × 128 pixels; RARE factor = 2; averages = 1; centric encoding. No spoiler gradients were applied between repetitions.Tubes were placed vertically in a cylindrical cradle of diameter 60 mm and inserted into a 63 mm quad coil in a horizontal bore 4.7 T magnet with Bruker console running ParaVision 5.1 software . SL was achieved using a T1 and T2 values at 4.7 T were measured using a RARE saturation recovery imaging sequence without the preparatory SL pulse. Tubes were placed horizontally in the cradle and coil described previously and a single axial  slice used. The acquisition parameters for T1 were: recovery times = 57.2, 68.5, 78.5, 88.5, 103.5, 183.5, 283.5, and 383.5 ms; TE = 11 ms. For T2, TE ranged between 11 and 66 ms; TR (BSA) = 2000 ms; TR (PBS) = 8000 ms. Additional parameters common to all: field of view = 60 × 60 mm2; matrix size = 256 × 256 pixels; RARE factor = 2; averages = 1; centric encoding; slice thickness = 1 mm.T1 and T2 measurements at 0.5 T were made on a Maran NMR spectrometer  utilizing a 20-MHz permanent magnet attached to a thermocouple heating mechanism and a PC running standard system software. T1 was measured using an inversion recovery sequence, with 20 log incremental inversion time recovery steps and 16 scans. TR was initially estimated for a test scan and then set to at least five times the expected final T1 value. T2 measurements were made using a standard Carr-Purcell-Meiboom-Gill sequence, with 1000 TE values.http://imagej.nih.gov/ij/, 1997–2011). SI values were adjusted for noise bias using a simple Rician correction (R1ρ followed a nonlinear three-parameter fit suggested by Engelhardt and Johnson (S0) values and relaxation rates (1/T1ρ), along with a parameter (a) to account for residual magnetization in the y axis due to the SL pulse (Eq. R1 (1/T1) and R2 (1/T2) were determined using two-parameter nonlinear fits .Confidence intervals were calculated at the 95% level. Datasets were compared for statistical significance at α = 0.05 using a paired R1 values for solutions of gadofosveset in BSA and in PBS were significantly different at B0 = 0.5 T  should give an indication of the influence of the albumin molecules on SL relaxation. However, the lengthy relaxation times of these blank solutions led to poor model fits. Instead, the influence of albumin is better illustrated by measurements using the nonbinding contrast agent gadopentetate in BSA.Because of the sensitivity of the SL technique to macromolecules, it is not clear from these findings alone the extent to which the difference in −1 s−1, respectively). If the SL relaxivity of gadopentetate in BSA is found to be higher than that of gadofosveset in PBS, this may be attributable to the BSA solution macromolecules. The R1ρ values for solutions of gadopentetate in BSA and gadofosveset in PBS . The outcome of this study suggests that further benefit may be gained by exploiting the albumin-binding characteristics of an agent such as gadofosveset. In addition to increased 0. The ouB1L value of 90 μT was chosen to give improved image quality; although R1ρ increases as B1L decreases, images become increasingly susceptible to artifacts caused by magnetic field inhomogeneities at very low B1L values (T2 values are routinely acquired on clinical scanners, and the effect of gadofosveset is clearly shown by R2 values in the presence and absence of albumin at 4.7 T (R2 and R1ρ demonstrate greater relaxivity for bound gadofosveset than R1. Although T2 measurement may be considered more practical, several studies, in particular those looking at tumor response to cytotoxic treatment, have suggested that T1ρ may be a more responsive early indicator of physiological change than T2 (1ρ rather than T2 (T1ρ-weighted images displayed improved spatial resolution over T2-weighted images and T1ρ maps had a greater dynamic range than equivalent T2 maps.It should be noted that for this in vitro study it was not necessary to optimize SL imaging parameters to take into account potential tissue heating issues resulting from high SAR. A relatively high L values . MethodsL values , were noat 4.7 T b. For th than T2 , 16. It  than T2 . A study than T2  found thT1 values on the 0 mM solutions caused particular problems with model fitting and were excluded from this analysis. In addition, the PBS R2 values at 4.7 T were based on a model fit to just three TE points, rather than the six points used for the BSA solutions, leading to greater imprecision in the calculated PBS R2 values.The scanning parameters at 4.7 T were optimized for physiological contrast agent concentrations. As a result, model fitting was less precise for the solutions containing the lowest and highest concentrations. The long For the purpose of this study, gadopentetate was assumed to be a nonbinding contrast agent, although there is some evidence to suggest that the chelate displays a weak tendency for binding to albumin . At the R1ρ response to gadofosveset in SA at high fields to be significantly larger than to a conventional small-molecule Gd-based contrast agent. This suggests that SL may be a viable method for regaining the T1 relaxivity lost by gadofosveset at high fields and may also provide an opportunity for additional tissue characterization through the differentiation of bound and free gadofosveset molecules. Despite offering potential benefits, implementation of this method in a SAR-limited clinical setting would require further investigation of optimal SL parameters prior to assessment in humans.In summary, this study has shown the


Saccharomyces cerevisiae, its cell cycle functions have not been well appreciated. We found that temperature-sensitive cdc48-3 mutant is largely arrested at mitosis at 37°C, whereas the mutant is also delayed in G1 progression at 38.5°C. Reporter assays show that the promoter activity of G1 cyclin CLN1, but not CLN2, is reduced in cdc48-3 at 38.5°C. The cofactor npl4-1 and ufd1-2 mutants also exhibit G1 delay and reduced CLN1 promoter activity at 38.5°C, suggesting that Npl4-Ufd1 complex mediates the function of Cdc48 at G1. The G1 delay of cdc48-3 at 38.5°C is a consequence of cell wall defect that over-activates Mpk1, a MAPK family member important for cell wall integrity in response to stress conditions including heat shock. cdc48-3 is hypersensitive to cell wall perturbing agents and is synthetic-sick with mutations in the cell wall integrity signaling pathway. Our results suggest that the cell wall defect in cdc48-3 is exacerbated by heat shock, which sustains Mpk1 activity to block G1 progression. Thus, Cdc48-Npl4-Ufd1 is important for the maintenance of cell wall integrity in order for normal cell growth and division.The ubiquitin-selective chaperone Cdc48, a member of the AAA (ATPase Associated with various cellular Activities) ATPase superfamily, is involved in many processes, including endoplasmic reticulum-associated degradation (ERAD), ubiquitin- and proteasome-mediated protein degradation, and mitosis. Although Cdc48 was originally isolated as a cell cycle mutant in the budding yeast  Budding yeast Cdc48 and its metazoan homolog p97, also named as valosin-containing protein (VCP), are abundant and evolutionarily conserved proteins. Cdc48/p97 belongs to the AAA ATPase superfamily and is involved in many aspects of cellular activities, including homotypic membrane fusion of organelles The diverse functions of Cdc48/p97 are mediated by specific cofactors. The binary complex Npl4-Ufd1 is associated with ER membrane and required for degradation of ER proteins Budding yeast Cdc48 was originally isolated as a cell cycle mutant that arrested in mitosis at the restrictive temperature CLN1, CLN2, and CLN3CLN3 constantly exist during the cell cycle and are modestly induced at late G1 CLN1 and CLN2 is mediated through the SCB and MCB sequences in their promoters that bind transcription factors Swi4/Swi6 (SBF) and Swi4/Mbp1 (MBF) complexes, respectively Cell cycle progression is mainly governed by cyclin-dependent kinases (CDK). Coupled with G1 or mitotic cyclins, the CDK activity drives G1/S transition or mitotic entry, respectively. Budding yeast has three G1 cyclins encoded by The Start commitment point in G1 phase of budding yeast is controlled by nutrient availability, cell size, and the presence of mating pheromone In this study, we examine the cell cycle function of Cdc48 and show that Cdc48 and the cofactor Npl4-Ufd1 complex are important for maintaining the cell wall integrity during heat stress to allow G1 progression.cdc48-3 mutant. The mutant was largely arrested at mitosis with 2N DNA content at 37°C as determined by fluorescence-activated cell sorter (FACS) analysis , whereas the temperature-sensitive t 38.5°C . Similar at 37°C . These rcdc48-3, npl4-1, and ufd1-2 cells at 38.5°C may be related to their ERAD function. We thus examined the deletion of two other components of the ERAD system, the ubiquitin-conjugation enzyme UBC7 and the ubiquitin-protein ligase HRD1ubc7Δ and hrd1Δ mutants had only a small delay in DNA replication during a synchronized cell cycle at 38.5°C , Rho1 (a G-protein and a regulator for Pkc1), and Rom2 (GDP/GTP exchange factor for Rho1). The single mutants of these components were growth-defective at elevated temperatures, which can be suppressed by the addition of 1 M sorbitol in the medium up to 35°C  to a degree that over-activates the cell wall integrity pathway and delays G1 progression.Mild heat shock is known to transiently arrest yeast cells at G1 CLN1 and CLN2 are differentially regulated in cdc48-3 at 38.5°C. The activity of CLN1 promoter is lower in cdc48-3 mutant at 38.5°C than that in the wild-type cells, whereas the CLN2 promoter activities are comparable in both strains. Both CLN1 and CLN2 promoters are activated by Swi4/Swi6 (SBF) and Mbp1/Swi6 (MBF) complexes that recognize multiple SCB and MCB sequences, respectively, in the upstream regions of CLN1 and CLN2 CLN1 and CLN2 are known to be regulated similarly during the cell cycle. Our study provides a rare example that these genes can be regulated differently. Heat stress is known to induce the transcription repressor Xbp1 that shares homology with Swi4 and Mbp1 in the DNA-binding domain CLN1 promoter, and overexpression of Xbp1 can repress the expression of G1 cyclins and lengthen G1. We have found that deletion of XBP1 gene in cdc48-3 promoted budding at 38.5°C, compared to cdc48-3 alone  was added at 1 µg/ml from a 10 mg/ml stock in DMSO and the cells were grown at 25°C for 3 hr. Cells were then shifted to the indicated temperature for 30 min. To release from G1 arrest, the cells were washed 3 times with warm water and then resuspended in pre-warmed medium at the indicated temperature.YEPD medium contained 1% yeast extract, 2% bacto-peptone, and 2% glucose. Complete synthetic medium contained 0.67% yeast nitrogen base without amino acids (YNB), 1× complete supplement mixture (CSM) (Bio 101), and 2% glucose. For induction from the cdc48-1rpt2RF, and rpt5Snpl4-1, ufd1-2, and rho1-104 mutants were backcrossed four times to W303. Gene deletions, epitope tagging, and introduction of pGAL to UBX2 were generated by PCR-mediated integration CLN1 and CLN2 were amplified by PCR from yeast genomic DNA and cloned at SacI and XbaI sites upstream of Renilla reniformis (RLUC) and Pyrophorus plagiophthalamus (spLUC) Luciferase, respectively, in pRS416. pCLN1-RLUC and pCLN2-spLUC regions were then removed with SacI and SmaI and cloned into the cognate sites in vectors pRS404 and pRS405, respectively. pRS404-pCLN1-RLUC and pRS405-pCLN2-spLUC were linearlized with Bsu36I and XcmI, respectively, to integrate into yeast genome at the selection markers.600 ∼1 was collected and washed once with cold TE . Cell pellets were frozen at −80°C if not used immediately. Proteins were extracted by bead-beating the cell pellet in 60–80 µl lysis buffer  with Zirconia beads at 4°C for 1 min. Samples were then centrifuged at 14,000 rpm for 5 min at 4°C, and the supernatants were taken as yeast cell lysates. For Western blot with anti-ubiquitin antibody, the cell pellets were bead-beat in RIPA  containing 10 mM N-Ethylmaleimide, 10 µg/ml each of leupeptin, pepstatin and chymostatin, 1 mM PMSF, and 1× protease inhibitor cocktail (Roche). Protein concentration was determined with DC protein assay kit (Biorad), and then normalized with lysis or RIPA buffer. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was first pre-blocked with blocking solution  for 1 hr at room temperature, followed by incubation with antibodies in blocking solution for 2 hr at room temperature. Antibody dilution: anti-myc , anti-HA , anti-Mad2 (1∶2000) 1.5 ml of yeast culture at OD1 ml of yeast cells were pelleted and resuspended in ice-cold fix solution . Samples were temporarily frozen at −80°C. Cells were then washed with PBS plus 0.5% Triton X-100, and incubated with 100 µg/ml of RNaseA in 50 mM Tris-Cl (pH 8.0) overnight at 37°C. Cells were then resuspended in 100–200 µl of Sytox Green  and briefly sonicated. Samples were diluted in 1 ml PBS and analyzed by FACSCalibur flow cytometer (BD Biosciences).pCLN1-RLUC and pCLN2-spLUC were grown to mid-log phase and arrested at G1 stage by α-factor. The cells were shifted to 37°C or 38.5°C for 30 min before release from the arrest at the same temperature. At each time point, OD600 of the culture was measured and another 1.5 ml of yeast cells were collected. The cells were washed with cold PBS and frozen at −80°C until use. Cell pellets were resuspended to 65 µl in PBS, and 20 µl of the sample was used for each luminescence measurement with Vector3 luminometer plus autoinjector (Perkin Elmer). Through the autoinjector, 100 µl of 1 µM coelenterazine (Promega) and D-Luciferin (Sigma) was added to the sample for R. Luciferase and sp Luciferase, respectively, with 5-sec equilibration and 10-sec integration time for the measurement of luminescence. The results were normalized with OD600.Yeast cells carrying 600 of cells were disrupted by bead-beating in STE buffer . Unbroken cells were pelleted by centrifugation at 300×g for 2 min at 4°C. Total lysate from the supernatant was then subjected to centrifugation at 100,000×g for 60 min at 4°C . The resulting supernatant and pellet represent cytosol and membrane fractions, respectively. The membrane pellet was then resuspended in STE buffer and layered on top of a continuous gradient composed of 20–60% (wt/wt) sucrose in 5 ml of TE buffer . After centrifugation at 100,000×g for 18 hr at 4°C , fractions of 356 µl were manually collected from top to bottom of the gradient. Proteins were resolved by SDS-PAGE for Western blot analysis.Subcellular fractionation was performed as described Figure S1Accumulation of ubiquitin conjugates in cdc48-3 is independent of ERAD components. Wild-type and the indicated mutant strains were grown to mid-log phase and shifted to 38.5°C for 3 hr. pGAL-UBX2 and pGAL-UBX2 cdc48-3 were first grown in YEP containing galactose to mid-log phase and then changed to YPD to suppress Ubx2 expression for 2 hr before shifting to 38.5°C for 3 hr. Cell lysates were prepared for Western blots with anti-ubiquitin (Ub) and anti-Mad2 antibodies. Mad2 serves as a loading control.(TIF)Click here for additional data file.Figure S2Deletion of XBP1 partially rescues budding defect of cdc48-3 at 38.5°C. Wild type, cdc48-3, xbp1Δ, and xbp1Δ cdc48-3 cells were grown as described in (TIF)Click here for additional data file.Figure S3The protein levels of Swi4, Swi6, and Far1 are unaffected by temperature up-shift in cdc48-3. Swi4, Swi6, and Far1 were tagged at the carboxyl-terminus with 3HA at the chromosomal loci in CDC48 and cdc48-3 cells. The cells were arrested at G1 with α-factor and released into the cell cycle at 38.5°C as described in (TIF)Click here for additional data file.Figure S4Ubiquitin conjugates are enriched in the ER fraction of cdc48-3.CDC48 and cdc48-3 cells were grown to mid-log phase and shifted to 38.5°C for 3 hr. Total cell lysates (lanes 1 and 2) were prepared and then separated into cytosol (lanes 3 and 4) and membrane (lanes 5 and 6) fractions. The membrane fractions were further fractionated by continuous 20-60% (wt/wt) sucrose gradient . The fraction numbers from top to bottom of the gradient are indicated. Equivalent to 1/500 of the total lysate, cytosol, and membrane fractions as well as 1/100 of each fraction from the sucrose gradient were subjected to Western blot analysis with antibodies against ubiquitin (Ub), ER protein Sec61, and plasma membrane protein Pma1.(TIF)Click here for additional data file.Figure S5Phosphorylation of Mpk1 is enhanced in cdc48 mutants, but not in proteasome mutants. Cells of indicated genotypes were first grown at 25°C and then shifted to 14°C for 2 days or other indicated temperatures for 3 hr. Cell lysates were prepared for Western blots with anti-phospho-Mpk1 and anti-Mad2 antibodies. Mad2 serves as a loading control.(TIF)Click here for additional data file.


Pachysolen tannophilus is a non-conventional yeast, which can metabolize many of the carbon sources found in low cost feedstocks including glycerol and xylose. The xylose utilisation pathways have been extensively studied in this organism. However, the mechanism behind glycerol metabolism is poorly understood. Using the recently published genome sequence of P. tannophilus CBS4044, we searched for genes with functions in glycerol transport and metabolism by performing a BLAST search using the sequences of the relevant genes from Saccharomyces cerevisiae as queries.P. tannophilus on glycerol and glucose as sole carbon sources. The genes predicted to be involved in glycerol transport in P. tannophilus were expressed in S. cerevisiae to validate their function. The S. cerevisiae strains transformed with heterologous genes showed improved growth and glycerol consumption rates with glycerol as the sole carbon source.Quantitative real-time PCR was performed to unveil the expression patterns of these genes during growth of P. tannophilus has characteristics relevant for a microbial cell factory to be applied in a biorefinery setting, i.e. its ability to utilise the carbon sources such as xylose and glycerol. However, the strain is not currently amenable to genetic modification and transformation. Heterologous expression of the glycerol transporters from P. tannophilus, which has a relatively high growth rate on glycerol, could be used as an approach for improving the efficiency of glycerol assimilation in other well characterized and applied cell factories such as S. cerevisiae. Pachysolen tannophilus is known for its ability to ferment D-xylose, one of the major components of hemicellulose plant residues, to ethanol [P. tannophilus has also been shown to be capable of converting crude glycerol to ethanol under microaerobic conditions [ ethanol . Howevernditions . This abP. tannophilus CBS4044 has been sequenced [P. tannophilus have not been studied so far. P. tannophilus has a relatively high growth rate on glycerol (0.3 h-1) [The whole genome of equenced , and witequenced , but the0.3 h-1) , but impS. cerevisiae, glycerol enters the cell by two different mechanisms: a low affinity transport system (facilitated diffusion) and a high affinity proton symport system (active transport) [FPS1 gene [S. cerevisiae has been described to be glycerol export rather than uptake during hypo-osmotic shock [GUP1 and GUP2 (Glycerol Uptake Protein), were first identified as being involved in active glycerol uptake in S. cerevisiae[S. cerevisiae identified a gene, STL1, involved in active glycerol uptake. Stl1p is localized in the plasma membrane, is glucose repressed and inactivated by growth in glucose [Candida albicans glycerol was actively transported into the cells by a proton symporter encoded by the C. albicans STL1[S. cerevisiae strains harboring the STL1 gene from D. hansenii slightly improved their growth and doubling times on glycerol [Glycerol transport is the first barrier to glycerol utilization in a microbial cell. In ansport) . When glPS1 gene ,9. The pic shock ,10. Two erevisiae. A scree glucose ,12. Impocans STL1. It has glycerol .GUT1 and then by glycerol-3-phosphate dehydrogenase encoded by GUT2, which is located at the outer surface of the mitochondrial membrane [GCY1 followed by dihydroxyacetone kinase encoded by DAK1 and DAK2. In Schizosaccharomyces pombe, it has been reported that glycerol is utilized solely by this pathway [S. cerevisiae, it has been shown that glycerol is degraded by the phosphorylation pathway and that mutants lacking one of the two genes are incapable of utilizing glycerol [S. cerevisiae[S. cerevisiae: redox balance and protection against osmotic stress as a compatible solute (osmolyte) [After glycerol is transported into the cells, two different routes have been identified in yeasts for further assimilation: a phosphorylation route and an oxidation route. Within the first pathway, glycerol is dissimilated by glycerol kinase encoded by membrane . Another pathway ,16. In b pathway ,6. In S.glycerol . Althougerevisiae, the funsmolyte) . Glycerosmolyte) .S. cerevisiae, many industrially used baker’s yeast strains and laboratory strains grow poorly on glycerol. For example, the specific growth rate of the well-studied lab strain S. cerevisiae CBS 8066 under aerobic conditions was found to be very low, 0.010 ± 0.002 h-1, during shake flask cultivations in minimal medium containing glycerol as sole carbon source [-1 have been achieved [Although there have been several studies on glycerol metabolism in n source . Strategn source ,22. By aachieved . Howeverachieved .S. cerevisiae and P. tannophilus has been performed to study the genes involved in glycerol transport and glycerol metabolism in these two organisms. Quantitative real-time PCR was performed to compare transcript levels during growth on glycerol. The genes involved in glycerol transport in P. tannophilus were heterologously expressed in a S. cerevisiae STL1 knockout strain to assess their function and to evaluate whether they could possibly contribute as transporter genes to improve the growth of S. cerevisiae on glycerol.In this study, a comparison of the genome sequences of P. tannophilus, the genome sequence was searched by BLAST with the sequences of genes known to have these functions in S. cerevisiae as queries. In P. tannophilus, two putative glycerol facilitator and two putative glycerol symporter genes were found and named PtFPS1, PtFPS2, PtSTL1 and PtSTL2, respectively. In the glycerol consumption pathways, the genes with high similarity to S. cerevisiae were found and named PtGUT1, PtGUT2, PtGCY1, PtGCY2, PtDAK  and CGD (Candida Genome Database). Unrooted phylogenetic trees are presented in Figure In order to investigate the phylogenetic relationship among putative glycerol transporters in FPS1, the lengths of PtFps1p, PtFps2p, PpFps1p, YaFps1p and GlpF were relatively short compared to other Fps1p proteins. PtFps1p was shown to be 54% identical to PpFps1p, 45% to YlFps1p, 35% to ScFps1p and only 31% to GlpF. PtFps2p was shown to be 60% identical to PpFps1p, 49% to YlFps1p, 34% to GlpF and only 32% to ScFps1p. PtFps1p and PtFps2p were grouped together with YlFps1p and PpFps1p, while they were in a separate branch than ScFps1p. For STL1, the identity between PtStl1p and ScStl1p was low, 34%, compared to the 56% identity observed between PtStl1p and YlPtStl1p.For PtFPS1, PtFPS2, PtGUT1, PtGUT2, PtGCY1, PtDAK were up-regulated on glycerol to different degrees compared to on glucose, while the genes involved with glycerol production PtGPD and PtGPP were down-regulated on glycerol  glycerol as the sole carbon source was analyzed. The stl1Δ strain expressing different transporter genes PtFPS1, PtFPS2, PtSTL1, PtSTL2, ScFPS1, ScSTL1 and the S. cerevisiae CENPK 113-5D strain were tested under aerobic conditions in shake flasks.The glycerol symporter gene stl1Δ recombinant strain expressing the transporter gene PtFPS2 from P. tannophilus and ScSTL1 from S. cerevisiae showed growth on glycerol after 96 hours of cultivation, while the stl1Δ recombinant strain harbouring PtFPS1, PtSTL1, PtSTL2 and ScFPS1 did not grow  were the two most strongly up-regulated genes, which might lead to the hypothesis that they are most relevant for glycerol assimilation in P. tannophilus. Moreover, the two genes are located closely together in the genome in agreement with a model where the two genes are co-regulated and contribute to the same overall process, i.e. glycerol assimilation. Next, we focused our efforts on understanding the function of the genes involved in glycerol transport as this function may be the rate limiting step for glycerol metabolism. In this paper the functions of the two types of glycerol transporters from P. tannophilus were addressed by transferring them individually to the well characterized yeast S. cerevisiae.With regard to glycerol transport, two glycerol facilitator homologues 3, CO2 or ions without consuming energy. The physiological role of the facilitator Fps1p in S. cerevisiae was previously described to be glycerol export rather than uptake during hypo-osmotic shock and the Fps1p channel closed and retained the glycerol inside the cells in response to hyperosmostic shock [225LYQNPQTPTVLP236 and a C-terminal domain 535HESPVNWSLPVY546 were found to have important roles in controlling Fps1p function. The N-terminus was required for closing of the channels and restricted transport through Fps1p. It was found that the rate of glycerol efflux was higher than that for uptake [ScFPS1 does not suppress the glycerol transport defect of stl1Δ strains as they do not grow on glycerol. Surprisingly, overexpression of PtFPS1 in stl1Δ strains allowed growth and assimilation of glycerol indicating that this homologous transporter is involved in glycerol uptake and recovers the stl1Δ strain growth defect. The similarities of PtFps1p and PtFps2p to ScFps1p are 35% and 32% respectively, but the homology is only restricted to the core of the protein with the six putative TMDs. ScFps1p (669 amino acid residues) is much longer than PtFps1p (389 residues) and PtFps2p (323 residues), the size of which is more similar to the E. coli GlpF (281 amino acid residues). The size difference is mainly due to the long hydrophilic N- and C-terminal domains of ScFps1p, which are absent in PtFps1p, PtFps2p and GlpF  family of channel proteins with six putative transmembrane domains (TMDs). Fps1p is responsible for transporting water, small molecules like glycerol, urea, NHic shock . An N-ter uptake . In agreS. cerevisiae, which is a member of the sugar permease family of the major facilitator superfamily (MFS) [STL1 gene was significantly induced with glycerol as the sole carbon source, and the STL1 gene was subject to glucose repression based on microarray-based transcriptome analysis [STL1 from C. albicans was not affected by the carbon source and salt stress [D. hansenii, it was shown that the active glycerol transport system was constitutively expressed and not subject to glucose repression [PtSTL1 and PtSTL2 in P. tannophilus are constitutively expressed on glycerol and glucose based on qPCR expression analysis. However, the presence of the PtSTL1 and PtSTL2 genes had no obvious effect on the physiology of S. cerevisiae, while the glycerol consumption and growth in strains that overexpressed ScSTL1 was improved compared to stl1Δ strains. The symporter PtStl1p showed a low degree of sequence identities to ScStl1p with 34%. However, the PtStl1p from P. tannophilus exhibited 52% identity to the Stl1p from D. hansenii (DEHA2A12364p) and 56% to the Stl1p from Yarrowia lipolytica (YALI0C16522p). It has been reported that Y. lipolytica can grow on glycerol with a μmax around 0.3 h-1[Pichia pastoris can grow on glycerol with a μmax 0.26 h-1[. P. tannophilus can grow on glycerol with μmax around 0.29 h-1[S. cerevisiae grows relatively slowly on glycerol with μmax of 0.02 h-1 (CEN.PK 113-5D). Both the facilitator and symporter similarities among P. tannophilus, Y. lipolytica and P. pastoris were higher than that compared to S. cerevisiae. However, more protein sequence data with verified functions are needed to provide a definitive conclusion.The high-affinity transporter Stl1p was identified as the glycerol proton symporter in ly (MFS) . It was analysis ,26. Howet stress . In D. hpression . In agred 0.3 h-1 and Pich 0.26 h-1. P. tannS. cerevisiae. Improved glycerol transport has been demonstrated here with an increased glycerol consumption rate and growth rate under aerobic conditions with S. cerevisiae.Since glycerol transport might be the rate-limiting step for glycerol utilization, heterologous expression of the glycerol transporters from yeasts which have relatively high growth rates on glycerol could be used as the approach for improving the efficiency of glycerol assimilation in P. tannophilus. Our studies open new possibilities for further improvement of glycerol fermentation in industrial yeast strains with heterologous expression of glycerol transporters from the glycerol utilizing P. tannophilus. This study thus proposes a possible route to development of glycerol-based bioprocesses in S. cerevisiae.The current study demonstrates the function of the glycerol transporters from P. tannophilus strain used in this study was CBS4044. S. cerevisiae CEN.PK 113-5D was used for the construction of the STL1 knockout strain. The plasmid PUG6 [loxP-kanMX-loxP cassette. The integrative USER vector pXI-5 [TEF1/PGK1. Plasmid pSH47 [kanMX marker gene. All plasmids were propagated in Escherichia coli strain DH5α. All the plasmids and S. cerevisiae strains used in this study are listed in Table The mid PUG6  was utilor pXI-5  was usedor pXI-5  was usedid pSH47  was usedP. tannophilus was grown at 30°C in defined medium, containing (per liter) 0.67 g yeast nitrogen base w/o amino acids, 10 g succinic acid and 6 g NaOH as buffer system , 2% (v/v) glycerol or 2% (w/v) glucose as the carbon source. Cells were harvested at mid exponential growth phase at the same optical density by centrifugation at 5000 g, 4°C for 5mins, resuspended in 1 ml ice cold ddH2O, and the cell pellet was stored at −80°C. S. cerevisiae stl1Δ harbouring different glycerol transporter genes were cultivated at 30°C for 96 hours in agitated flasks with defined medium, containing (per liter) 7.25 g synthetic complete (SC) powder, 10 g succinic acid and 6 g NaOH as buffer system, 1% (v/v) glycerol as the sole carbon source.For quantitative real-time PCR (qPCR) experiments, P. tannophilus CBS4044 was obtained from previous work and is available in the EMBL database [S. cerevisiae FPS1, STL1, GUT1, GUT2, GCY1, Dak1/2, GPD1/2, GPP1/2 were used as queries in a BLAST search against the genome sequence of P. tannophilus. The genes with high similarities and high identities were listed as potential orthologous genes. Gene sequences were registered in GenBank at NCBI. The multiple sequence alignments with the amino acid sequences of FPS1 and STL1 transporters from different yeast strains and E. coli were performed by using ClustalW2 free program at PDBe. Phylogenetic analyses were performed by using PAUP* 4.0b10 [The genome sequence from database . The seq* 4.0b10 . Unweigh* 4.0b10 .18 primer. The cDNA was used as the template for quantitative real-time PCR for determining the transcript levels of the target genes under different growth conditions.Total RNA was isolated from frozen cells using an RNeasy Mini Kit  according to the manufacturer’s protocol. The quantity and quality of the isolated RNA were measured by NanoDrop ND-1000. The total RNA was treated with DNaseI (Fermentas) prior to cDNA synthesis. Five μg total RNA were used to synthesize cDNA employing the RevertAid™ First Strand cDNA Synthesis kit (Fermentas) following the manufacturer’s recommendations using oligo(dT)2O to 20 μl. The PCR program for qPCR was as follows: 10 min of incubation at 95°C, followed by 40 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s, and finally the temperature was increased from 55°C to 94°C to check for unspecific products. The number of fluorescence threshold cycles (Ct) was calculated with the set threshold value by using Mx3005P software. Results presented are mean values of three independent experiments. Suitable primer pairs for all the genes investigated were designed using the Primer Express software v3.0 (Applied Biosystems) software with the following parameters: product size 140 - 180 bp and melting temperature (Tm) 57-59°C. The primers used in this work are listed in Additional file qPCR was performed on a Stratagene Mx3005P using the SYBR Green technology. The qPCR reaction mixture was prepared with 5 μl of 5 times dilution of cDNA as template, 10 μl SYBR Green master mix, 2 μl of 1 μM forward primer and 2 μl of 1 μM reverse primer and ddH-ΔΔCT, where ΔΔCT =  sample A -  sample B. CT represents the cycle number at which a sample reaches a predetermined threshold signal value for the specific target gene. Relative expression data were normalized to the relative expression value of the housekeeping gene TAF12 in each sample, thus giving normalized relative expression for a target gene.The relative expression levels were calculated approximately based on 2STL1 knockout strain was done through the loxP-kanMX-loxP /Cre recombinase system [loxP-kanMX-loxP fragment from pUG6 were: KanMX_fw_STL1 (sequence 5′- AAAGCTGAAAATAGAGGGTTGCTGGTCAATTTAGAAGGTTCCACAATTGCCTGAAGCTTCGTACGCTG -3′) and KanMX_re_STL1 (sequence 5′- AAGCGTTTGTTGATGCACGAACTTTCATTGATGCAATTTCTGGTGGGTATCTAGTGGATCTGATATCAC -3′). The loxP-kanMX-loxP fragment with 50 bp homologous sequences in both ends to the gene STL1was transformed into CENPK-113-5D [The construction of the e system . The priK-113-5D  for obtaK-113-5D .P. tannophilus and S.cerevisiae were constructed. The USER vector pXI-5 was digested with AsiSI and then with the nicking endonuclease Nb.BsmI for making the AsiSI/Nb.BsmI USER cassettes [PtFPS1, PtFPS2, PtSTL1, PtSTL2, ScFPS1, ScSTL1 and bidirectional promoter TEF1/PGK1 with appropriate USER tails for insertion into the designated USER cassette AsiSI/Nb.BsmI. Primers used are listed in Additional file TEF1/PGK1, 5× Phusion HF buffer (Fermentas), and 1 U of USER enzyme (Fermentas), adjusted to 10 μl by adding ddH2O. The mixture was incubated at 37°C for 20 min, followed by 25°C for 20 min. The 10 μl reaction mix was used directly to transform chemically competent E. coli DH5α cells. All the glycerol transporter genes were cloned under control of the TEF1 promoter. Constructs were named as: pXI-5-PtFPS1, pXI-5-PtFPS2, pXI-5-PtSTL1, pXI-5-PtSTL2, pXI-5-ScFPS1, pXI-5-ScSTL1 . Samples were taken periodically from the flasks and filtered through a 0.22  μM syringe filter, and supernatants were preserved at −20°C for later HPLC analysis. Concentrations of the substrate glycerol in supernatants were measured by HPLC refractive index detector RID-10A using an Aminex87H column . Separations were performed at 60°C, flow rate of 0.6 ml/min and 5 mM H2SO4 as mobile phase.Growth was monitored by measuring ODThe authors declare that they have no competing interests.XL carried out the experimental work, analysis and drafted the manuscript. MW, supervised the experimental work and drafting of the manuscript, and has critically reviewed the text. UHM participated in the design and discussion of the experimental work and critical reviewing of the manuscript. All authors have read and approved the manuscript.Primers used for Semi-quantitative RT-PCR and USER Cloning.Click here for file


In recent years, the reported infection cases by coxsackievirus (CV) have been on the rise. In order to reveal the relationship between the nucleotide and amino acid sequences and the viral virulence of the CVB3/MKP strain causing myocarditis, we initially confirmed the virulence of the strain in myocardial tissue and then carried out the whole genome sequencing of CVB3/MKP strain and performed a phylogenetic analysis among different CVB3 strains.CVB3/MKP infected mouse model was established to check lesions of myocardial tissue in mice using immunohistochemical detection at different periods. RT-PCR analysis was used to amplify seven fragments covering the whole viral sequence and comparable analysis was performed.The immunohistochemical results showed that particles of CVB3/MKP virus persisted in the cardiac tissue and caused severe pathology. The length of whole genome sequence of CVB3/MKP strain was 7400 bp. CVB3/MKP had 99.7% and 99.6% homology in nucleotide sequence with CVB3/28 and non-virulent CVB3/0, respectively. The former can induce pancreatitis and myocarditis. The nucleotide sequence in the 5′untranslated region of CVB3/MKP strain shared 99.6% and 99.5% homology with CVB3/20 and CVB3/Nancy, respectively.We confirmed in our animal experiments that CVB3/MKP had cardiotoxicity. CVB3/MKP, CVB3/28, and CVB3/0 may share evolutionary convergence and the 5′untranslated region (5′UTR) may be associated with virulence phenotype. Our findings will provide a basis for identifying the genomic determinant of viral virulence of CVB3/MKP strain and phylogenetic relationship among different CVB3 strains. In recent years, the reported infection cases by coxsackievirus (CV) have been on the rise and the most commonly known viral infectant of the heart is coxsackievirus B3 (CVB3). It is a member of the genus Enterovirus, which is within the family Picornaviridae. CVB3 RNA, can be detected in the heart muscle of 40–50% of patients with dilated cardiomyopathy (DCM) ,2. In adAs the whole-genome sequencing techniques have become more advanced in recent years, more and more whole genome sequencing and amino acid sequence for CVB3 strains has been completed ,7, Some Of note, the 5′untranslated region of CVB3 strains is the main region determining viral virulence, and specific nucleotide variation in the 5′untranslated region may be closely related to different viral virulence, In fact, the mutation of nucleotide U at nucleotide position 234 in the 5′untranslated region of CVB3 strain to C caused its cardiac toxicity significantly reduced. The internal ribosome entry site (IRES), a S-D-like region in configuration and rich in pyrimidine, exists in the 5′untranslated region and binds to ribosome to start viral replication , nt88-18The myocardial tissue of mice infected with CVB3/MKP was seriously affected compared with the uninfected mice. In the early viral infection stage, severe congestion appeared in myocardial vascular and interstitial edema was observed with larger fissures. Cloudy swelling appeared in the mouse cardiomyocytes at 7–21 days post infection, with blurred cell stripes, enhanced eosinophilic staining in cytoplasm, condensed fragmented or disappeared nuclei, and spottily distributed infiltrating inflammatory cell, and focal myocardial necrosis was obvious, the infiltrating inflammatory cells in the myocardial tissue was significantly reduced at 40 days post infection, but the myocardial necrosis, calcification of muscle fibers or dissolved absorption still existed . The full-length of the genome was 7400 bp, of which, the length of the open reading frame was 6555 bp that encodes a polyprotein precursor with 2185 amino acids. ClustalW (1.83) software was used to compare CVB3/MKP genome sequence with full-length nucleotide sequences of other eight CVB3 strains in GenBank. The genome sequence identity of the strain with CVB3/28 and CVB3/0 reached 99.7% and 99.6%, respectively. The nucleotide sequence difference between CVB3/MKP and other CVB3 strains was within 0.4% to 0.8%  and CVB3 (CXAB3CG) was within 91.9?~?99.6% and the sequence identity among CVB3/MKP, CVB3/GA and CVB3/CO were only within 83.3% and 83.6% , and the corresponding amino acid in CVB3/GA, CVB3/20 and CVB3/28 was lysine (K); the 512 amino acid in the VP3 “knob” area of CVB3/MKP strain was Valine (V), while the corresponding amino acid of CVB3/GA, CVB3/20 and CVB3/28 was Alanine (A). We also found that non-pathogenic strains CVB3/GA had 58 different amino acids from CVB3/MKP, CVB3/20 and CVB3/28. Although it is not clear whether these variations have any impact on the function of these strains, it is likely that the changes in the nucleic acid or amino acid can influence the ability of CVB3/MKP strain to produce persistent infection. It is necessary to further investigate what role(s) these mutations play in the autoimmune response during the pathogenesis of myocarditis.In summary, we demonstrated that CVB3/MKP strain had cardiotoxicity and a very close genetic relationship with CVB3/28 strain, which can induce pancreatitis and myocarditis , and furE. coli JM109 and Hela cells were kept at the Department of Pathogen Biology of Jilin University. CVB3/MKP strain was from Shanghai epidemic prevention station. Healthy, 3-week-old Swiss male mice were obtained from the Experimental Animal Center of Jilin University. The animals were maintained and handled according to standard animal experimental protocol approved by Jilin University.Cell culture medium IMDM and fetal calf serum were from Hyclone (USA). Reverse transcriptase Superscript III was from Invitrogen(USA). Taq DNA polymerase, restriction endonuclease Pvu II and pMD18-T vector were from TaKaRa Co (Japan). QIAamp viral RNA Mini Kit was from Qiagen. Mouse anti-CVB3 monoclonal antibody was from Bio Chemicon, Immunohistochemistry kit was from MaiXin New Biotechnology Product Development Company of Fuzhou, China.2O2 at room temperature for 5-10 min, washed with distilled water and PBS for 5 min, and blocked with 5-10% normal goat serum for 10 min. Then, the sections were incubated with mouse anti-CVB3 monoclonal antibody at 4°C overnight, washed with PBS three times, followed by incubation with biotinylated goat anti-mouse secondary antibody at 37°C for 30 min and washed with PBS three times again. Horseradish peroxidase-labeled streptavidin was added to the sections and incubated at 37°C for 30 min; the color was developed with DAB, followed by hematoxylin nucleus staining.80 healthy Swiss mice were randomly divided into CVB3/MKP infection group  in which each mouse was intraperitoneal injected 0.2 ml 400TCID50 CVB3/MKP and normal control group  in which each mouse was intraperitoneal injected 0.2 ml saline, all virus injected mice were separated from normal control mice. At day 3, 7, 21, 40 and 55 post infections, eight mice at each post infection day were sacrificed, and their hearts were removed fixed with 10% formalin and paraffin sectioned, followed by routine hematoxylin-eosin staining (HE staining). For immunohistochemistry, the myocardial sections were treat with 3% H+ Plus) 2.5 μl, dNTP Mixture (10 mM) 2 μl, upstream primer CS (10 pmol/μl) 2 μl, downstream primer CS (10 pmol/μl) 2 μl, Taq DNA polymerase (5 u/μl) 0.5 μl, and sterile H2O 15 μl. The PCR condition was 94°C for 2 min; 94°C 45 s, 55°C 45 s, 72°C 2 min for 35 cycles; 72°C 10 min. Based on the published sequences from GenBank, seven pairs of primers were designed and synthesized .Whole genome sequence was obtained by DNASTAR software. Nucleotide and amino acid sequences analysis and sequence identity comparison with other CVB3 strains from GenBankTable  were comThe authors declare that they have no competing interests.LB carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. LZ carried out the immunoassays. XFY participated in the sequence alignment. LF participated in the design of the study and performed the statistical analysis. WGQ and YZ conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


E.coli. We confirmed the source of ATPase activity using various deletion constructs of p53 and an In-gel ATPase assay followed by LC-ESI-MS/MS analysis of the activity band. The activity was associated with Hsp70 homologue in E.coli, DnaK, a known interactor of p53. We clarify that wildtype human p53, expressed in E. coli BL21 (DE3) strain, carries no ATPase activity.p53 is one of the most well studied tumor suppressor proteins and regarded as the guardian of the genome. The protein mediates cell-cycle arrest, apoptosis in response to myriads of cellular stresses including DNA damage via its trascriptional as well as non-transcriptional roles. ATP binding/hydrolysis by p53 had been implicated in its DNA binding functions. However, till date, no ATP binding/hydrolysis domains have been mapped in p53. In the current study, we have reinvestigated the ATP hydrolysis activity associated with recombinant human p53 protein expressed and purified from  The transformed cells, grown at 37°C till A600 of 0.5 in LB medium containing 50 μg/ml kanamycin, were induced with 0.5 mM IPTG at 25°C and harvested after 12 hours. The cells were resuspended in 25 mM HEPES-KOH (pH 7.6), 0.1 M KCl, 2 mM EDTA, 2 mM DTT, 20% glycerol, 1 mM Benzamidine, 0.25 mM PMSF and protease inhibitors cocktail (Roche), incubated with lysozyme (1 mg/ml) on ice for 30 minutes and sonicated after adding 0.1% NP-40. The cell lysate was centrifuged at 18,000 rpm for 45 minutes at 4°C. The supernatant was diluted five times in volume with 50 mM NaH2PO4 (pH 8.0), 1 mM DTT, 1 mM Benzamidine, 0.1 mM PMSF and protease inhibitors cocktail (Roche), followed by incubation with pre-equilibriated Glutathione S sepharose beads  for 2 hours at 4°C. The beads were then packed into an Econo-column (Bio-Rad Laboratories). The resin was washed with 50 mM NaH2PO4 (pH 8.0), 0.3 M KCl, 1 mM DTT, 1 mM Benzamidine and 0.1 mM PMSF. The protein was eluted with 20 mM reduced glutathione in 50 mM NaH2PO4 (pH 8.0), 0.3 M KCl, 1 mM DTT, 1 mM Benzamidine and 0.1 mM PMSF and dialyzed against 40 mM NaH2PO4 (pH 8.0), 50 mM KCl, 2 mM DTT and 5% glycerol. The dialysed protein was stored at −80°C. The dialysed protein was further purified by FPLC-gel filtration (size exclusion) chromatography using GE healthcare AKTA system and HiLoad 16/60 Superdex 200 pg. The flow rate was maintained at 1 ml per minute. The protein fractions were eluted in buffer containing 40 mM NaH2PO4 (pH 8.0), 50 mM KCl, 2 mM DTT and 5% glycerol. 120 fractions (1 ml/fraction) were collected in 2 hours. The equal volume of peak fractions were analysed for ATP hydrolysis activity.The protein was expressed in E.coli cells , except that the cells were cultured at 30°C instead of 37°C till 0.6 O.D., as the cells are temperature sensitive.Similarly, the GST tagged full length p53 was expressed and purified from ΔDnaK BL21(DE3) Clones 3C, 24, 25 and 35, inserted into the expression vector pET11GST  encode GST fused to amino acids 155–393, 94–269, 94–293 and 155–299 of p53, respectively. All the deletion constructs were purified as per the protocol followed for full length wildtype p53-GST (described above), except the cultures were grown in LB media containing 100 μg/ml ampicillin instead of kanamycin.Proteins were analyzed by SDS-PAGE The p53 protein was subjected to electrophoresis on a 12% SDS polyacrylamide gel and was transferred to Immun-Blot PVDF membrane (Bio-Rad Laboratories). The primary antibody used to probe the blot in this study was mouse monoclonal anti p53 pAb122 . The antibody was diluted 1∶3000 in 1% casein in 1× TBST solution and incubated overnight at 4°C. Horseradish peroxidise linked secondary antibody was anti mouse IgG (Roche diagnostics). The secondary antibody was diluted 1∶2000 in 1% casein in 1× TBST solution and incubated for 1 hour at room temperature. The blot was incubated with SuperSignal West Dura (Thermo Scientific) chemiluminescent substrate. The blot was exposed to Super Rx blue sensitive X-ray film (Fujifilm) and developed using Optimax 2010 X-ray film processor.NADH coupled microtiter plate assay was performed as described in published protocol 32P] ATP  in 25 mM Tris–HCl (pH 7.6), 1.8 mM DTT and 13 mM magnesium acetate at 30°C for 30 minutes. The reactions were stopped with 0.2% SDS, heated at 100°C for 3 minutes and spotted on polyethyleneimine sheet (Merck) that was developed in 0.75 M KH2PO4 (pH 3.6). The dried PEI sheet was scanned using Molecular Dynamics Storm 820 PhosphorImager. The percentage of [α-32P] ATP hydrolyzed to [α-32P] ADP was quantified by Image J software.Radioactive ATPase assay was performed using thin layer chromatography (TLC) with 2 μM each of GST tagged full length wildtype p53 (p53-FL), p53-3C, p53-24, p53-25 or p53-35 incubated with 0.25 μM of 50 nCi/μl  containing 50% isopropanol at 30°C for an hour with mild shaking to remove SDS. The gel is then incubated at 30°C for about 24 hrs in Buffer A containing 20% methanol and 0.075% lead nitrate till the white ATPase activity band appeared as a result of lead phosphate precipitate formation within the gel. The buffer was changed after 12 hrs in between the incubation. The ATPase activity stained band was excised and prepared for LC-ESI-MS/MS following trypsin digestion.Purified human p53 protein was treated with AcTEV protease (Invitrogen) using manufacturer's protocol at 30°C for 2 hours, wherever mentioned. TEV treated and untreated p53 protein samples resuspended in 1× Laemmli buffer with reduced SDS (1.25% instead of 2.5%) without heating and was electrophoresed on SDS-PAGE (12%) gel containing reduced SDS (0.05% instead of 0.1%) in both stacking and resolving portions using SDS-PAGE buffer with reduced SDS (0.05% instead of 0.1%). After electrophoresis, the gel was either stained with coomassie brilliant blue stain or analysed for ATPase activity using a modified protocol from H.sapien and E.coli databases from NCBInr’ using an in-house Mascot server.For the preparation of samples for mass spectrometric analysis, the gel was serially sliced, minced and digested with trypsin and peptides were extracted BRC1-8 on hRAD51 mediated ATP hydrolysis We have studied the effect of p53 and BRC repeats domain of BRCA2, BRCA2E.coli BL21(DE3). Using NADH-coupled ATPase assay, we detected ATP hydrolysis whose rates increased as a function of p53 concentration. This real-time assay showed distinct slopes of absorbance decline as a function of reaction time that revealed the rates of ATPase reactions  observed in coomassie stained gel are clipped products of p53 or not, we cleaved GST tag in p53-GST using TEV protease before performing immunostaining with anti p53 C-terminus antibody. A single p53 band at ∼53 kDa position was evident on the western blot  E.coli cells that expressed DnaK but were still cultured at 30°C. The coomassie stained SDS-PAGE gel revealed that the DnaK protein was co-purified with p53 from normal BL21(DE3) cells, but not from ΔDnaK BL21(DE3) E.coli cells  li cells . We studli cells  and realli cells . The p53li cells , lane 1.li cells , lane 2.li cells .E.coli DnaK is a homolog of eukaryotic chaperone, Hsp70 with overall homology of about 50% E.coli. We surmise that all the p53 deletion mutants 3C, 35, 25 and 24 showing varied ATPase activity (E.coli proteins is high. We conclude that the ATPase activity detected in purified p53 was entirely associated with DnaK, where p53 by itself does not possess ATPase activity. In lieu of this finding, we suggest that one has to be circumspect about the conclusions drawn on p53 functions based on its supposedly demonstrated ATPase activity. p53 interaction with DNA modulated by ATP/ADP must therefore be an indirect consequence of an interactor protein dependent ATPase activity. We strongly believe that a protein hub such as p53 that dynamically interacts with a large variety of cellular protein machineries might exhibit high propensity to facilitate co-purification of its strong interactor protein, compounding the biochemical characterization of intrinsic functions associated with p53 itself. The current study is an important alert in that direction, which is worth noting, given the “high profile” nature of p53 protein in higher eukaryotic cells.activity  might be


Here, we reported a unique member of the family, Mth10b from Methanobacterium thermoautotrophicum ΔH, whose amino acid sequence shares high homology with other Sac10b family proteins. However, unlike those proteins, Mth10b is an acidic protein; its potential isoelectric point is only 4.56, which is inconsistent with the characteristics of a nucleic acid-binding protein. In this study, Mth10b was expressed in Escherichia coli and purified using a three-column chromatography purification procedure. Biochemical characterization indicated that Mth10b should be similar to typical Sac10b family proteins with respect to its secondary and tertiary structure and in its preferred oligomeric forms. However, an electrophoretic mobility shift analysis (EMSA) showed that neither DNA nor RNA bound to Mth10b in vitro, indicating that either Mth10b likely has a physiological function that is distinct from those of other Sac10b family members or nucleic acid-binding ability may not be a fundamental factor to the actual function of the Sac10b family.The Sac10b protein family is regarded as a group of nucleic acid-binding proteins that are highly conserved and widely distributed within archaea. All reported members of this family are basic proteins that exist as homodimers in solution and bind to DNA and/or RNA without apparent sequence specificity  Sulfolobus acidocaldarius by Grote et al. in the 1986 et al. found that the gene sac10b, which encodes Sac10b, has one or two homologs in each of the archaeal organisms whose genomes had been sequenced Sulfolobus shibataeSulfolobus solfataricusSac10b, generally regarded as a chromosomal DNA-binding protein, was first isolated from the hyperthermophile in vitroin vivoin vitroIn vivo UV cross-linking and co-immunoprecipitation experiments using S. shibatae cells, however, have shown that Ssh10b binds exclusively to RNA in vitroin vivo, which results in a reduction in its nucleic acid-binding affinity in vivoSac10b binds cooperatively to DNA with no significant compaction and protects DNA against degradation by the nuclease DNase I et al.Sulfolobus solfataricusSulfolobus shibataeMethanocaldococcus jannaschiiArchaeoglobus fulgidusAeropyrum pernix K1Pyrococcus horikoshii OT31α1β2α2β3β4 topology. Although the lengths of the flexible β-hairpin arms vary, the central presumed DNA-binding surfaces are all distributed with positively charged residues predominantly.In 2002, the crystal structure of Sso10b was determined by Wardleworth Methanobacterium thermoautotrophicum ΔH, is a lithoautotrophic, thermophilic archaeon that grows at temperatures in the range of 40–70°C, with an optimal temperature of 65°C The most extensively studied methanogen, Escherichia coli cells and developed an efficient protocol for Mth10b purification. Biochemical characterization suggested that Mth10b has a structure similar to the structures of other typical Sac10b proteins. However, an electrophoretic mobility shift analysis (EMSA) showed that neither DNA nor RNA bound to Mth10b in vitro, suggesting that either Mth10b likely has a physiological function that is distinct from those of other Sac10b family members or nucleic acid-binding ability may not be a fundamental factor to the actual function of the Sac10b family.To answer this question, we cloned and expressed Mth10b in Methanobacterium thermoautotrophicum ΔH, encoded by the gene MTH1483, which we termed Mth10b. The amino acid sequence of Mth10b shares identities with Sso10b, Sso10b2, Ssh10b, Mja10b, Afu10b, Ape10b2, and Pho10b of 50.5, 30.9, 50.5, 56.0, 61.5, 45.1, and 61.3%, respectively  cells as described in methods. Analysis by 15% SDS-PAGE  is a common method to study protein secondary structure because different types of regular secondary structure found in proteins give rise to characteristic CD spectra in the far UV region. Using Ssh10b, a typical member of the Sac10b family, as a control, we investigated the secondary structure of Mth10b with far-UV CD spectroscopy. The far-UV CD spectrum of Mth10b, recorded at pH 7.5 and 25°C and presented in 15N-13C CBCA(CO)NH, HNCACB, we tried to introduce some mutations in Mth10b to improve its quality for NMR study. Fortunately, a triple mutant Mth10b[M15QL19TV22Q], in which Met15, Leu19 and Val22 are replaced by Gln15, Thr19 and Gln22 respectively, was found no longer aggregate in higher concentrations and can give qualified three-dimensional spectra. Using the three-dimensional spectra of this triple mutant, we finished the backbone assignment. The 2D 1H-15N HSQC spectrum of the Mth10b variant Mth10b[M15QL19TV22Q], presented in 15N resonances are distributed from approximately 107 ppm to 130 ppm, and the chemical shifts of 1H resonances are distributed from 6.5 ppm to 9.5 ppm. Due to the amino acid sequence of Mth10b sharing highly identity with its archaeal homologs, not surprisingly, those conserved residues in Mth10b and other typical Sac10b family members, such as Ssh10b 1H-15N HSQC spectrum suggest that the three-dimensional structure of Mth10b is similar to the three-dimensional structures of typical Sac10b family proteins.A preliminary study on the tertiary structure of Mth10b was carried out by using NMR. Due to the wild type Mth10b tending to aggregate at concentrations beyond 0.4 mM which results in seriously less signals than needed in three-dimensional spectra, such as The association state of Mth10b in solution was analyzed by analytical ultracentrifugation. According to the experimental results, as shown in Escherichia coli DH5α in binding buffer, followed by separation of bound and free species using an agarose gel and visualization by ethidium bromide staining. Escherichia coli DH5α were visibly shifted at protein quantities greater than 1 µg. At higher quantities of Ssh10b, the RNA became progressively retarded until it apparently saturated the RNA at recombinant protein levels greater than 5 µg, preventing the RNA from entering the gel. The total RNA samples incubated with Mth10 showed no significant band shift, even at protein quantities of 50 µg. These results indicated that DNA and RNA can bind to Ssh10b with similar affinities in vitro, in agreement with previous reports in vitro. These results distinguish Mth10b from other members of the Sac10b protein family, indicating that Mth10b may have a physiological function that is distinct from the functions of other members of the Sac10b protein family.The nucleic acid-binding affinity of Mth10b was investigated by agarose gel electrophoresis using Ssh10b as a positive control. Increasing quantities of recombinant proteins were incubated with supercoiled plasmid pAS22 or total RNA from et al. found that Sac10b is located exclusively in the cytoplasm rather than in the nucleus in vitroet al. found that it binds exclusively to RNA in vivo using ultraviolet irradiation et al. found that Sso10b is associated with all investigated chromosomal regions but was released from the insoluble chromatin-containing pellet by treatment with either DNase I or RNase A. This result indicated that Sso10b is associated with both DNA and RNA in vivoet al. used a bioinformatics analysis to show that the Sac10b protein family is related to two eukaryotic protein families involved in RNA metabolism Although originally identified as DNA-binding proteins, there is evidence that Sac10b protein family members can also bind RNA. Using immunogold electron microscopy, Bohrmann Methanobacterium thermoautotrophicum ΔH, a unique member of the Sac10b protein family whose amino acid sequence shares high homology with other members of the Sac10b protein family. Interestingly, Mth10b has many more acidic amino acid residues than basic residues, and its isoelectric point is only 4.56 . The plasmid pET11a from Novagen was used to make the vector-DNA construct. Escherichia coli DH5α and BL21 (DE3) cells were used for plasmid cloning and protein expression, respectively. The expression plasmid pET11a-ssh10b containing the ssh10b gene was obtained from our laboratory stocks. Enzymes and reagents for DNA manipulations were purchased from TAKARA. Plasmid miniprep kits were obtained from OMEGA. Yeast extract and tryptone were purchased from OXOID. Isopropyl β-D-thiogalactoside (IPTG) was obtained from MERCK. 15N-labeled ammonium chloride and 13C-labeled glucose were purchased from Cambridge Isotope Laboratories, Inc. All chemicals were of analytical grade for biochemical use. All apparatus and chromatography materials were purchased from GE.The mth10b gene (ID code: MTH1483) was amplified from Methanobacterium thermoautotrophicum ΔH using polymerase chain reaction (PCR) with the forward primer CATATGTCAGAGGAGAATGTAG-3′5′-TA and reverse primer GGATCCTATTATTAATCCTTTCGGAGCTGA-3′5′-CC. PCR introduced NdeI and BamHI restriction sites (underlined and in boldface) at the 5′ and 3′ ends, respectively, of the amplified fragment. The PCR product was digested with NdeI and BamHI and ligated into the expression plasmid pET11a, which was linearized with the same two enzymes. The constructed plasmid pET11a-mth10b was confirmed by DNA sequencing. Using the parental plasmid pET11a-ssh10b, the expression plaimid of the Mth10b variants were constructed. All mutations were introduced by site-directed mutagenesis through overlap extension PCR and verified by DNA sequencing.The Escherichia coli BL21 (DE3) expression strain and purified as described previously Recombinant Ssh10b protein was expressed in the mth10b was also transformed into the Escherichia coli BL21 (DE3) expression strains. A single colony was picked and grown in 100 ml of LB media containing 100 µg/ml ampicillin with shaking (approximately 200 rpm) at 37°C overnight. The cultures were diluted 1∶50 in fresh antibiotic-containing LB media, and the cells were grown at 37°C until reaching an OD600 of 0.8. Protein expression was induced by the addition of IPTG at a final concentration of 0.3 mM and incubating at 25°C overnight. The cells were harvested by centrifugation at 4,000 rpm for 30 min.For expression of Mth10b and its variants, the constructed plasmid pET11a-4)2SO4 by the addition of buffer C (3 M (NH4)2SO4, 20 mM Tris-HCl/pH 7.5). After centrifugation at 16,000 rpm for 30 min, the supernatant was loaded onto a 20-ml Phenyl Sepharose High Performance column that was equilibrated with buffer D (1 M (NH4)2SO4, 20 mM Tris-HCl/pH 7.5), and proteins were eluted with a 0–100% gradient (120 ml) of buffer A. Fractions containing the target protein, identified by SDS-PAGE, were dialyzed against buffer A overnight. After centrifugation at 16,000 rpm for 30 min, the dialyzed sample was loaded onto a 6-ml Resource-S column equilibrated with buffer A, and proteins were eluted with a 0–50% gradient (60 ml) of buffer B. Fractions containing the recombinant protein, identified by SDS-PAGE, were dialyzed against buffer E (50 mM NH4HCO3) and lyophilized. All chromatography experiments were performed on an AKTA Purifier-10 system.For purification, the harvested cell pellets from 3-L cultures were resuspended in 75 ml of buffer A (20 mM Tris-HCl/pH 7.5) and disrupted by ultrasonication in ice bath. After centrifugation at 16,000 rpm for 30 min, the supernatant was loaded onto a 15-ml Q Sepharose Fast Flow column equilibrated with buffer A. The bound proteins were eluted with a 0–50% gradient (120 ml) of buffer B . Fractions containing Mth10b were identified by 15% SDS-PAGE and dialyzed against buffer A overnight. The sample was adjusted to a final concentration of 1 M (NH2HPO4-NaH2PO4/pH 7.5) at a concentration of 0.25 mg/ml. Measurements were carried out by using a rectangular quartz cuvette with a path-length of 1 mm over the 190- to 250-nm wavelength range with a 2-nm bandwidth. Each spectrum was the average of five scans and was corrected for spurious signals generated by the solvent.The far-UV CD measurements were performed on a PiStar-180 spectrometer  at 25°C. The protein samples were prepared in buffer F (50 mM Na15N/13C-labeled proteins were expressed in Escherichia coli BL21 (DE3) in M9 minimal medium containing 15NH4Cl and 13C-glucose as the sole nitrogen and carbon sources, respectively. The labeled proteins were purified as described above. The purity of the protein was demonstrated by its visualization as a single band on SDS-PAGE and an ultraviolet absorbance ratio of A280/A260≧1.7. Samples for NMR measurements contained 1.0–2.0 mM labeled proteins, 90% H2O/10% D2O, 200 mM NaCl, 5 mM DTT, 0.02% NaN3, 1 mM EDTA and 0.01% sodium-2,2-dimethyl-2-silapentane-5-sulfomate (DSS) in 20 mM Tris-HCl buffer (pH 7.5).The uniformly 1HN, 15N, 13Cα and 13Cβ nuclei were indentified using 2D 1H-15N HSQC, 3D CBCA(CO)NH and HNCACB experiments. All NMR spectra were processed and analyzed using FELIX98 software (Accelrys Inc.). Proton chemical shifts and 15N and 13C chemical shifts were referenced to internal DSS and indirectly to DSS, respectively All NMR experiments were carried out at 37°C on a Bruker Advance DMX 600 MHz spectrometer equipped with a triple resonance cryo-probe. Resonance assignments for backbone Sedimentation velocity experiments were performed on a Beckman-Coulter XL-A analytical ultracentrifuge using two-channel centerpieces with an An60Ti rotor at 60000 rpm and 20°C. Sedimentation was monitored using absorption at UV 238 nm with protein concentrations of 0.6 and 1.0 mg/mL in buffer F. UV absorption was scanned every 30 s for 6 h. Data were analyzed with software provided by Beckman Instruments .Escherichia coli DH5α cells were incubated with varying quantities of purified recombinant protein in buffer G  in a total volume of 15 µl at room temperature for 15 min. The samples were resolved by electrophoresis in 1.2% agarose gels in 1×TAE buffer at constant voltage. After electrophoresis, the gels were stained with ethidium bromide and visualized under UV light.The nucleic acid-binding affinity of Mth10b was analyzed by EMSA using Ssh10b, a typical member of the Sac10b protein family, as a positive control. Approximately 370 ng of supercoiled plasmid pAS22 (3789 bp) or 1.1 µg total RNA from