Talin is a large (∼2540 residues) dimeric adaptor protein that associates with the integrin family of cell adhesion molecules in cell-extracellular matrix junctions , where it both activates integrins and couples them to the actin cytoskeleton. Calpain2-mediated cleavage of talin between the head and rod domains has previously been shown to be important in FA turnover. Here we identify an additional calpain2-cleavage site that removes the dimerisation domain from the C-terminus of the talin rod, and show that an E2492G mutation inhibits calpain cleavage at this site in vitro, and increases the steady state levels of talin1 in vivo. Expression of a GFP-tagged talin1 E2492G mutant in CHO.K1 cells inhibited FA turnover and the persistence of cell protrusion just as effectively as a L432G mutation that inhibits calpain cleavage between the talin head and rod domains. Moreover, incorporation of both mutations into a single talin molecule had an additive effect clearly demonstrating that calpain cleavage at both the N- and C-terminal regions of talin contribute to the regulation of FA dynamics. However, the N-terminal site was more sensitive to calpain cleavage suggesting that lower levels of calpain are required to liberate the talin head and rod fragments than are needed to clip off the C-terminal dimerisation domain. The talin head and rod liberated by calpain2 cleavage have recently been shown to play roles in an integrin activation cycle important in FA turnover and in FAK-dependent cell cycle progression respectively. The half-life of the talin head is tightly regulated by ubiquitination and we suggest that removal of the C-terminal dimerisation domain from the talin rod may provide a mechanism both for terminating the signalling function of the talin rod and indeed for inactivating full-length talin thereby promoting FA turnover at the rear of the cell. Cell migration involves a complex cycle of inter-related events initiated by extracellular cues that establish cell polarity and membrane protrusion at the leading edge driven by actin polymerisation Cell-extracellular matrix interactions are typically mediated by members of the integrin family of transmembrane αβ-heterodimers, and both “inside-out" and “outside-in" signalling Talins  are comprised of an N-terminal head (1–400) containing an atypical FERM domain While talin plays a prominent role in FA assembly, calpain2-mediated cleavage of talin Here we report the identification and characterisation of a second calpain2 cleavage site in talin that removes the dimerisation helix at the C-terminus of the talin rod. Expression of GFP-talin carrying mutations in this site singly or in combination with the L432G mutation markedly suppressed protrusion persistence and FA turnover suggesting that both sites are important in regulating the dynamic properties of FA.It is well documented that calpain2 cleaves talin1 in the linker region between the head and rod domain , an evenWe then used TD77 to monitor calpain2-cleavage of purified turkey gizzard talin. Coomassie stained gels of partial talin digests showed two bands , both ofTo map the C-terminal calpain2 cleavage site more precisely, we used the purified recombinant talin1 2300–2541 polypeptide which has an apparent molecular weight of ∼25 kDa by SDS-PAGE. Incubation of this polypeptide with calpain2 in the presence, but not the absence of calcium, generated a proteolytic fragment of ∼20 kDa . If cleaWe have previously determined the structure of the C-terminal region of talin1 15N-labelled 400–480 talin1 polypeptide. The 1H-15N HSQC spectrum was consistent with a peptide that is predominantly unstructured with low signal dispersion in the proton dimension  between the talin F3 FERM domain (residues 309–400) and the 5-helix bundle, VBS1 (482–655) at the start of the talin rod  is also imension . Compariimension , confirmimension . In summWe next compared the relative sensitivities of the N- and C-terminal regions of talin1 to calpain2 cleavage. Interestingly, the N-terminal F3-VBS1 (residues 309–655) talin1 polypeptide was significantly more sensitive to cleavage than the C-terminal polypeptide spanning the C-terminal ABS and DD (residues 2300–2541) . Even atAlthough talin1 and 2 are highly conserved proteins (74% identity), the sequence in the vicinity of the N-terminal calpain2 cleavage site differs, and it has been suggested by Senetar et al. that this may confer different sensitivities to calpain2 cleavage To evaluate the importance of the C-terminal calpain2 cleavage site in talin1 in a cellular context, we first sought to identify mutations that suppress cleavage. Previous studies have shown that substituting the residue at the P2 position upstream of the calpain cleavage site partially inhibits calpain cleavage in vivo, we transfected HEK293 cells with wild-type GFP-tagged talin1 or the L432G or E2492G calpain-resistant mutants as well as a L432G, E2492G double mutant. Western blotting for GFP showed that both the L432G and the E2492G calpain-resistant talin1 mutants accumulated to a significantly greater extent than wild-type GFP-talin1 . Similarly, we were unable to detect GFP-talin1 head in cells transfected with GFP-talin1 . The talTo investigate the possible significance of calpain2-mediated proteolysis of talin at both the N- and C-terminal sites, we co-transfected CHO.K1 cells with paxillin-mCherry, and one of three GFP-talin1 mutants i.e. (L432G), (E2492G), and . Using TIRF microscopy on cells with similar, low, expression levels, we found that all of the various GFP-talin1 mutants localized to focal adhesions (FA) . HoweverWe quantified the dynamics of cell protrusions . The perIn an attempt to detect calpain-mediated cleavage of talin1 in disassembling adhesions at the rear of the cell, we used fluorescence ratio imaging. We compared wild-type talin1 with the double talin1 mutant  dually labelled with GFP and mCherry on opposite ends of the molecule. However, we did not observe any major difference in the ratios between the wild-type talin1 and the double mutant as adhesions disassembled and translocated in retracting regions . This suWe report here the characterisation of a novel calpain2 cleavage site in talin1 that removes the dimerisation helix at the C-terminus of the talin rod. We have identified a mutation (E2492G) that suppresses cleavage at this site in vitro, and show that it leads to an increase in the steady state levels of talin1 in HEK293 cells. Moreover, it reduces FA turnover and cell protrusion in CHO.K1 cells to about the same extent as the well documented L432G mutant that suppresses calpain2 cleavage between the talin head and rod Recent data suggest that the talin1 head liberated by calpain2 cleavage has a function independent of full-length talin1, and promotes FA turnover The proposal that the talin1 head has functions independent of full-length talin1 raises the possibility that the talin1 rod might also have its own unique role in the cell. The talin1 rod contains an integrin-binding site and several vinculin- and actin-binding sites , and in The results of Wang et al., Calpain2 cleavage of FA proteins at the rear of the cell and its role in tail end retraction and cell migration are well documented In conclusion, we report here a novel calpain2-cleavage site in talin that removes the C-terminal helix from the talin rod, and show that it is important in FA turnover and cell protrusion. Whether cleavage at this site is regulated in any way has not been explored, although it is interesting to note that phosphorylation of the C-terminal region of filamin by PKC protects against calpain1 cleavage 2. Analytical gel filtration chromatography of recombinant talin polypeptides was performed using Superdex-75 (10/300) (Amersham Biosciences) at room temperature. The column was pre-equilibrated and run in 20 mM Laflamme Tris pH 8.0, 150 mM NaCl and 2 mM DTT at a flow rate of 0.8 ml/min.Mouse talin1 and human talin2 cDNAs were amplified by PCR, cloned into pET-151/D-TOPO (Invitrogen) and authenticated by DNA sequencing. Recombinant talin polypeptides were expressed as His-tagged proteins in E. coli and purified on Ni-NTA  columns following standard procedures. The tag was removed by TEV cleavage, and the polypeptides purified further on a Resource-Q column . Talin polypeptides were incubated (37°C for 30 minutes) with recombinant rat calpain2  in 25 mM HEPES, pH 7.5, 50 mM NaCl, 1 mM DTT ±3 mM CaCl15N-labeled talin polypeptides were transferred into 20 mM sodium phosphate pH 6.5, 50 mM NaCl and 2 mM DTT, 10% (v/v) 2H2O using a PD10 column , and concentrated to 0.15 mM immediately prior to collection of NMR spectra. NMR spectra of all proteins were obtained at 298 K using a Bruker AVANCE AVII 800 spectrometer equipped with a CryoProbe. Spectra were processed with TopSpin (Bruker Corp.) and analyzed using Analysis For NMR experiments, 2, pH 7.0. Assays were performed using 4 µM talin polypeptides and 10 µM F-actin. The mixture was incubated for 60 min at room temperature and centrifuged at 100,000 rpm for 30 min at 22°C using a Beckman Optima TM ultracentrifuge. Supernatants and pellets were analysed on 12% SDS-PAGE gels and stained using Coomassie blue.G-actin was purified from rabbit skeletal muscle and polymerised in 10 mM Tris, 50 mM NaCl, 100 µM ATP, 1 mM DTT, 1 mM MgClThe GFP-talin1 and GFP-talin1 L432G expression vectors have been previously described To evaluate the stability of the GFP-tagged calpain-resistant talin1 mutants in cells, HEK293 cells (10 cm dishes) were transfected with 5 µg of DNA using Lipofectamine as per manufacturer instructions. For the GFP control 0.2 µg and 5 µg of pcDNA was used. The cells were lysed 24 hours post transfection in 20 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% Deoxycholate, 0.5% NP-40 plus 1% protease inhibitor cocktail set III (Novagen). Proteins (20 µg per lane) were resolved by SDS-PAGE and transferred to a nitrocellulose membrane for the Western blotting using anti-GFP antibodies (Roche). Anti-GAPDH (Santa Cruz) was used as a loading control. All experiments were performed in triplicate.2 atmosphere at 37°C. Transfections with GFP-talin1 and paxillin-mCherry DNA or GFP-talin1-mCh and GFP-talin1 -mCh plasmids were performed using Lipofectamine. CHO.K1 cells were incubated with 5 µl Lipofectamine and 0.1–0.2 µg DNA  for 3–4 hours. For live time-lapse imaging, cells were trypsinised and replated on fibronectin-coated (2 µg/ml) glass bottom dishes in CCM1 medium . Cells were maintained at 37°C using a Warner Instruments heated stage insert .The effects of the calpain-resistant talin1 mutants on FA was studied using CHO.K1 cells  cultured in low glucose DMEM supplemented with 10% FBS, 4 mM L-glutamine, 1% (v/v) non-essential amino acids, and penicillin/streptomycin. Cells were maintained in a humidified 5% COTIRF image time series were acquired on an Olympus IX71 microscope using a 100×1.45 NA Plan Apo TIRFM oil objective, a Ludl controller , and Metamorph Software . The 488 nm and 568 nm lines of an Ar-Kr ion laser  were used to excite fluorescence from GFP- and mCherry-tagged constructs respectively. Laser powers were modified and monitored by an acousto-optical tunable filter (AOTF) unit with digitized power readout . For simultaneous dual-colour imaging, a polychroic mirror (Z488/568 rpc), dual emission filter (Z488/568 nm) , and Dual-View  were used. Image time series were acquired using the QuantEM: 512C EMCCD camera . Quantification of adhesion size and dynamics were performed in Image J . Images were corrected for detector and diffuse background fluorescence. A segmentation algorithm using maximum entropy threshold was used to isolate and measure the size of adhesions from images of GFP-talin1 mutants in CHO.K1 cells. We used the GFP-talin1 channel for adhesion size quantification since variations in fluorescence intensity and adhesion size between the GFP-talin1 and paxillin-mCherry channels were minimal and constant among the different talin1 mutants. Kymography analysis was implemented on image time series to measure the persistence time of cell edge protrusions. We define the persistence time as the time at which a protrusions stop and nascent adhesions mature and grow. For ratio imaging analysis of dual labeled GFP-talin1-mCh and GFP-talin1--mCh, cells were corrected for background intensity effects and the channel 1 (GFP) was divided by channel 2 (mCherry) to yield a ratio coefficient (R). For statistical comparison analysis, we use the Kruskall-Wallis ANOVA on ranks approach. Multiple Pairwise comparisons were performed in Sigma Plot 11.0 following the Dunn's method and the Holm-Sidak method for data presented in Figure S1Epitope mapping of the monoclonal antibody TD77. (A) The recombinant talin polypeptides (2.5 µg) indicated were analysed by SDS-PAGE and stained with Coomassie blue. (B) Western blot analysis of the talin proteins (100 ng) using TD77 (1∶5000) reveals the residues L2515 and R2526 as essential for TD77 recognition. (C) Cartoon of the talin dimerisation domain showing the relative positions of the key residues (L2515 and R2526) of the TD77 epitope.(TIF)Click here for additional data file.Figure S2Fluorescence ratio imaging of wild-type and  calpain resistant talin1 in retracting adhesions in CHO.K1 cells. Image time series of cells expressing GFP-talin1-mCh (left) or GFP-talin1 -mCh (right) were collected every 5 sec for 10 min. (A) Images of summed time series for both construct highlight the location and direction of the retracting adhesions (boxed regions). Intensity time traces and ratios (R) for selected pixels along the highlighted retracting adhesions in (A) for GFP-talin1-mCh (B) and GFP-talin1 -mCh (C). The ratio is channel 1(GFP-black) divided by channel 2 (mCh-red).(TIF)Click here for additional data file.


Anaesthetic propofol, commonly used for general anaesthesia, is known to bind to GABAA receptors . It affeTo answer this question, we built a model of cortical population made of 750 excitatory and 250 inhibitory neurons whose dynamics resemble those found in biological neurons. Excitatory cells are modeled by a leaky-integrate-and-fire model of pyramidal neurons . The celWe show with our minimal model of a cortical assembly that the peak in the power spectrum of the mean membrane voltage of excitatory cells, whose spiking frequency originally is about 11 Hz without tonic inhibition, shifts towards slower frequencies with increasing tonic inhibition. This result is similar to occipital EEG recordings under propofol anesthesia. However, the amplitude of the power spectra diminishes drastically, more so than in biological recordings, in the presence of tonic inhibition compared to without tonic inhibition. This may indicate that tonic inhibition is not the only occurrence responsible for the increase in amount of delta oscillations, but that thalamic inputs may help maintaining slow cortical occipital activity under general anesthesia. We are thus planning for future works to include interactions in our model between cortical and thalamic populations.


Candida albicans, a fungal human pathogen, three histidine kinases were identified, of which CaNik1p is a group III histidine kinase. Heterologous expression of this protein in Sacchromyces cerevisiae conferred susceptibility to different fungicides. Fungicide activity was associated with phosphorylation of the mitogen activated protein kinase Hog1p.Microorganisms use two-component signal transduction (TCST) systems to regulate the response of the organism to changes of environmental conditions. Such systems are absent from mammalian cells and are thus of interest as drug targets. Fungal TCST systems are usually composed of a hybrid histidine kinase, comprising the histidine kinase (HisKA) domain and a receiver domain, a histidine phosphotransfer protein and a response regulator. Among the 11 groups of fungal histidine kinases, group III histidine kinases are of particular relevance as they are essential for the activity of different groups of fungicides. A characteristic feature is the N-terminal amino acid repeat domain comprising multiple HAMP domains, of which the function is still largely unknown. In CaNIK1ΔHAMP in S. cerevisiae led to severe growth inhibition. Normal growth could be restored by either replacing the phosphate-accepting histidine residue in CaNik1pΔHAMP or by expressing CaNIK1ΔHAMP in S. cerevisiae mutants, in which single genes encoding several components of the HOG pathway were deleted. Expression of proteins with non-functional histidine kinase or receiver domains resulted in complete loss of susceptibility to antifungals, such as fludioxonil. Conditions leading to growth inhibition of transformants also led to phosphorylation of the MAP kinase Hog1p.We have constructed mutated versions of CaNik1p, from which either all HAMP domains were deleted (CaNik1pΔHAMP) or in which the histidine kinase or the receiver domains were not-functional. Expression of Our results show that functional histidine kinase and receiver domains of CaNik1p were essential for antifungal susceptibility and for activation of the Hog1p. Moreover, for the first time we show that deletion of all HAMP domains from CaNik1p led to activation of Hog1p without an external stimulus. This phenotype was similar to the effects obtained upon treatment with fungicides, as in both cases growth inhibition correlated with Hog1p activation and was dependent on the functionality of the conserved phosphate-accepting histidine residue. Candida and Aspergillus sp. Candida sp. are the fourth most frequent cause of hospital acquired blood stream infections and up to 90% of HIV patients receive mucosal candidiasis at least once [albicansCandida sp. have emerged in recent years [C. albicans is still responsible for the majority of the cases [Immune-compromised patients are at high risk of becoming infected by opportunistic fungi, such as ast once . Althougnt years , the spehe cases ,4.Several antifungals are available in the market, yet, toxicity and/or development of resistance represent major concerns . Among tIn fungi, two-component signal transduction (TCST) systems have been implicated in osmotic and oxidative stress responses, cell-cycle control, red/far-red light responses, and virulence switches from non-pathogenic to pathogenic states -10. SincS. cerevisiae. In the absence of osmotic stress, the transmembrane HK ScSln1p is active. This HK activity leads to phosphorylation of a histidine residue in the catalytic domain, the so-called HisKA domain, from which the phosphate group is transferred to an aspartic acid residue in an internal receiver domain (REC). Therefore, these HKs are called hybrid HKs. The phosphate group is then shuttled through the HPT protein Ypd1p to the terminal RR proteins Skn7p and Ssk1p [ScSLN1 results in the accumulation of unphosphorylated Ssk1p without external stimulus and thus, constitutive activation of Hog1p, which is lethal [Typical TCST systems in fungi include a histidine kinase (HK), a histidine phosphotransfer protein (HPT) and a response regulator protein (RR). The best understood fungal TCST system is part of the High Osmolarity Glycerol (HOG) pathway in nd Ssk1p ,11. Phosnd Ssk1p -15. Phosnd Ssk1p . Thus, ts lethal .S. cerevisiae has a single HK, namely ScSln1p, C. albicans has three HKs: CaSln1p, CaNik1p  and Chk1p [C. albicans on solid media [While nd Chk1p . CaNik1pnd Chk1p . The prond Chk1p -18. CaNiid media , ScSln1pS. cerevisiae lacks group III HKs and is usually resistant to the fungicides mentioned above. However, fungicidal sensitivity is gained by heterologous functional expression of group III HKs in S. cerevisiae correlating with Hog1p phosphorylation [Several chemical classes of fungicides, such as phenylpyrroles (fludioxonil), dicarboximides (iprodione) and polyketide secondary metabolites of ambruticins, exert their antifungal effects by activating the HOG signaling pathway, resulting in the accumulation of both glycerol and free fatty acids -22. It irylation -28.All classes of HKs share the conserved phosphate-accepting domains HisKA, REC and an ATP-binding domain called HATPase_c domain. Based on homology with other HKs, the histidine residue 510 and the aspartate residue 924 in CaNik1p were identified as the essential phosphorylatable residues for the HisKA and the REC domains respectively  at 30°C. S. cerevisiae transformants were selected and maintained in SD-ura , at 30°Solidified media were prepared by addition of 1.5% bacto agar (Difco).E. coli XL1-Blue growth, transformation and plasmid DNA preparation were performed using standard methods according to the manufacturer’s instructions.CaNIK1-TAG [The plasmid pYES2-NIK1-TAG  was usedIn CaNik1p (1081 aa), all the HAMP domains (63–485 aa) were deleted using the in-fusion HD cloning kit (Clontech). Briefly, the in-fusion enzyme is able to fuse up to four DNA fragments with a linearized vector upon recognizing 15 bp overlapping sequences at their ends. To allow this fusion, the 15 bp overlaps were introduced to the primers which were used to amplify the target fragments.CaNIK1-TAG vector was used as a template for amplification of the gene fragments. The sequence of CaNIK1 upstream of the fragment encoding the HAMP domains (1–186 bp) was amplified using the HMPF1 and HMPR1 primers (Table CaNIK1 fragment located downstream the sequence encoding the HAMP domains and extended by the His-FLAG tag (1454–3243 bp) was amplified using the HMPF2 and HMPR2 primers . The purified fragments were ligated into the digested pYES2 vector using the in-fusion enzyme according to the manufacturer’s instructions.The pYES2 vector was linearized using the restriction enzyme HindIII and the pYES-S. cerevisiae using the lithium acetate method [The existence of the introduced mutations was further confirmed by sequencing the generated constructs  using primers spanning the target fragments. The mutated constructs were used to transform e method . Transfo620nm was again measured after 24 h. The antifungals fludioxonil and iprodione were obtained from Fluka, whereas ambruticin VS3 was produced as described and kindly provided by K. Gerth and R. Jansen  [.In 96 well microtiter plates, working cultures of the transformants were incubated in 180 μl SG-ura supplemented with the appropriate concentrations of the antifungals in triplicates for 24 h. The starting OD at 620 nm was 0.03 and measured using a μQuant scanning microplate spectrophotometer (Biotek). To detect growth inhibitory effects, the ODschweig) .CaNIK1 carrying point mutations as previously described [620nm of 0.2. Cells were harvested 15 min after the start of the working culture by centrifugation. Sorbitol (1 M) was used as a positive control, as it is known to stimulate phosphorylation of the MAPK Hog1p via the induction of osmotic stress [Phosphorylation of the MAPK Hog1 was investigated in transformants with escribed . Brieflyc stress .To avoid Hog1 phosphorylation caused by cold stress , cells wA total of 5 μg protein per sample was separated by SDS-PAGE (12.5%) and proteins were blotted onto a PVDF membrane. Phosphorylated Hog1 was detected using the combination of an anti-phospho p38 MAPK (Thr180/182) 3D7 rabbit monoclonal antibody  with an HRP-conjugated anti-rabbit IgG  as secondary antibody. Incubation of the antibodies was followed by the addition of a peroxidase-specific chemiluminescence substrate . The bound antibodies were removed by treatment with 1xRe-Blot Plus Solution (Millipore) and subsequently total Hog1p was detected using anti-Hog1 (y-215) sc-9079 rabbit polyclonal IgG (Santa Cruz Biotechnology) and the above mentioned secondary antibody followed by visualization with the ECL substrate.620nm = 1. Cells were harvested by centrifugation and transferred to SG-ura (starting OD620nm = 0.2). After incubation at 30°C for 195 and 210 min, samples were centrifuged and treated as previously mentioned for the detection of Hog1p phosphorylation by Western blotting. Fludioxonil was added as an inducer of Hog1 phosphorylation (positive control) after 180 min at a final concentration of 10 μg/ml.Due to the growth inhibitory effect resulting from the expression of CaNik1pΔHAMP in the ΔHa strain, phosphorylation of Hog1p was investigated at an early time point after inducing the expression of CaNik1pΔHAMP. Therefore ΔHa was first cultivated in SD-ura until ODTo confirm the expression of CaNik1pΔHAMP, ΔHa cells were harvested after 180 min incubation in SG-ura, disrupted and the FLAG-tagged protein was detected using a HRP-conjugated monoclonal anti-FLAG antibody (Sigma-Aldrich).S. cerevisiae was transformed with the plasmids carrying the mutated CaNIK1 genes, and the resultant transformants were treated with the antifungals fludioxonil, iprodione and ambruticin VS3. As shown in Figure After alignment with other HKs, in CaNik1p histidine 510 and aspartate 924 were identified as the essential residues for the HisKA and the REC domains respectively  and aspaHis510 and Asp924 are the conserved phosphate-accepting residues in the HisKA and the REC domains, respectively, which are required for kinase function of hybrid HKs. They are phosphorylated by the histidine kinase activity of the protein (His510) and the subsequent phosphate-transfer to the REC domain within the same protein (Asp924). Loss of fungicide susceptibility of the respective mutants suggested that the functionality of both the HisKA and the REC domain was essential for the antifungal activity.Probably the N627D mutation did not completely prevent ATP binding to the HATPase_c domain and as a result only a partial effect was obtained.S. cerevisiae transformed with full-length and truncated forms of CaNIK1[S. cerevisiae transformed with CaNIK1 carrying the H510Q, N627D and D924N point mutations. Phosphorylation of the protein was totally abolished in the mutant strains D924 and H510, while phosphorylation of Hog1p in strain N627 was decreased but not totally inhibited  or components of the Hog1p MAPK module, namely the MAP2K PBS2 (strain Δpbs2) and the MAPK HOG1 (strain Δhog), were transformed with the plasmid pYES2-CaNIK1ΔHAMP. These transformants showed normal growth on SG-ura plates  is a negative regulator of the MAPK Hog1p, as its inhibition leads to activation of the MAPK. However, for group III HKs different effects were reported: Dic1p, the group III HK from of Hog1p , whereasegulator . To the egulator . Previouegulator , so thategulator . It was egulator . Ssk1p rent with  who showD. hansenii[S. cerevisiae transformants. Whereas in the previous reports only selected HAMP domains were deleted, here we deleted all HAMP domains from CaNik1p (CaNik1pΔHAMP) and observed that the synthesis of this truncated protein in the transformed S. cerevisiae strain was associated with severe growth inhibition. This phenotype could be reversed by additional point mutation in the histidine phosphorylation site of the HisKA domain (H510) or by the expression of CaNIK1ΔHAMP in single gene deletion mutants of the response regulator SSK1 or of one of the components of the Hog1 module namely the MAP2K PBS2 and the MAPK HOG1. This proved that the inhibition of growth of the transformant upon expression of CaNIK1ΔHAMP was dependent on the functionality of both the histidine kinase activity of CaNik1p and the functionality of the Ssk1 - Pbs2 - Hog1 branch of the HOG pathway. We could also unambiguously show that the inhibited growth of the strain ΔHa expressing CaNIK1ΔHAMP correlated with the constitutive phosphorylation of Hog1p without any external stimulation, which was also abolished after mutation of the conserved phosphate-accepting histidine residue (CaNIK1ΔHAMP(H510Q)). By analogy to the previous discussion this leads to the conclusion that the expression of CaNIK1ΔHAMP decreased the phosphate transfer activity to Ssk1, whereas the presence of the mutant CaNik1pΔHAMP(H510Q), which cannot be phosphorylated on the conserved histidine residue, did not affect the endogenous phosphorylation state of the Ssk1p. Thus, in summary, deletion of all HAMP domains had the same effect on the phosphate transfer activity to Ssk1p as treatment with fungicides. Additionally, the presence of mutated proteins, which are assumed not to possess histidine kinase activity and thus are not phosphorylated on either histidine in the HisKA domain or on aspartate in the REC domain, did not inhibit growth of the transformants and did not activate the Hog1p MAPK module. As a consequence of these results, it seems to be unlikely that in the transformed S. cerevisiae strains the histidine kinase activity of CaNik1p was inhibited by fungicide treatment, because inhibition of the kinase activity will lead to an enrichment of the non-phosphorylated form of the protein, similar to the protein variants carrying point mutations. The mutated proteins, however, did not influence growth whereas fungicide treatment did. Thus, our results support a model, in which the wild-type CaNik1p protein is not phosphorylated without external stimuli, and Ssk1p is kept in a phosphorylated form via indirect phosphate transfer from Sln1p. Upon deletion of all HAMP domains from CaNik1p or fungicide treatment CaNik1p is phosphorylated and this form prevents phosphate transfer to Ssk1p , respectively, was detected in the protein extracts via Western Blot using an anti-Flag antibody. The bands of CaNik1p, CaNik1pΔHAMP and CaNik1pΔHAMP(H510Q) appeared at 121, 75 and 75 kDa respectively. The strain YES with the empty vector was used as control.Click here for file


E24) and molecular docking method. Then the identified epitope of IgE was determined theoretically, which showed the key role of IgE-Cɛ3 in interacting with both FcɛRIα and MAE11. By normal protocols, we immunized mice with purified protein E34 and screened six anti-E34 monoclonal antibodies. Purified antibodies could identify E34 by Western blot; furthermore, all of them could bind IgE by ELISA, in which QME5 seemed to be the best. Flow cytometry analysis displayed that only QME5 could bind membrane IgE and it could compete with membrane FcɛRIα to bind soluble IgE. Meanwhile, QME5 couldn’t bind FcɛRIα-attached IgE, which suggested no hypersensitivity in triggering the target cells (mast cells or basophils) by crosslinking or inducing the release of a variety of chemical mediators.Using computer-guided homology modeling method, the 3-D structure of the Fv fragment of a functional anti-IgE antibody (MAE11) was constructed and the spatial structure of E24-MAE11 complex was modeled based on the crystal structure of IgE-Fc ( IgE acts a key role in the allergic response and anaphylactic diseases such as asthma, allergic rhinitis, atopic dermatitis and food allergies. Unlike other immunoglobulin classes, IgE bind specifically and with a very high affinity to its receptor FcɛRIα on the surface of human basophils and mast cells (Ka=109 M−1)  contributes to the permanent sensitization of target cells. IgE cross-linking of FcɛRIα+ cells by specific antigens results in the release of a variety of chemical mediators  and cytokines, which show their effects by interacting with specific receptors on target organs (Immunoglobulin E (IgE) was the last of the immunoglobulins discovered by Ishizaka Several strategies were mentioned to treat IgE-mediated allergic diseases by down-regulating IgE levels. The basic idea was that humanized anti-IgE antibodies could be used for the isotype-specific control of IgE . The antE.coli expression system   in n system  and IgE n system . For Fcɛn system , then a lizumab) . And theStable cell line FI5F10 with extracellular part of FcɛRIα was established using CHO cell line (CRL-2092) and conserved in our lab; SKO-007, a B lymphocyte cell line which was identified to express IgE  and SP2/0 (P3-X63-Ag8.653) were also conserved in our lab.http://www.expasy.org/spdbv/) (http://www.expasy.ch/). To ensure proper packing of the variable domains of the heavy chain (VH) and the light chain (VL) in the resulting models, the surface accessible solvent area and surface electrostatic potential of MAE11-VH and MAE11-VL were analyzed using InsightII 2005 software . Using molecular docking method, the 3-D structure of VH-VL complex (Fv) was constructed. After structural optimization of Fv, the 3-D complex structure of MAE11-Fv and E34 was obtained with molecular docking method.The heavy and light chain variable domains of MAE11 were constructed according to the canonical structures methods using the Swiss-PDB Viewer program (version 3.7) (/spdbv/)  and the o-phenylene-diamine hydrochloride (OPD) and 8.5 mM H2O2. The light absorbance was measured at 492 nm with ELISA reader.ELISA plates were coated at 4°C overnight. Then after being blocked with 1.5% BSA in PBS at 37°C for 1h, 100 μL specific protein (e.g. culture media supernatant) was added and incubated at 37°C for 1 h, followed by 100 μL HRP_conjugated polyclonal antibody for 45 minutes at room temperature . The peroxidase reaction was developed with color development solution containing 5.5 mM 5 per sample were incubated separately with diluted ligand (E24 or E34 etc.) in 100 μL PBA  for 30 minutes at 4°C. FITC conjugated polyclonal antibody was added to determine the amount of membrane-binding ligand. Samples were analyzed on a Becton Dickinson FACSCAN flow cytometer.Cells at 5 × 106 cell/mL, and 200 μL cells was plated in each well on five 96-well plates. Hybrids were selected in RPMI 1640 Medium supplemented with 20% fetal calf serum and 5 × 10−3 M hypoxanthine, 2 × 10−5 M aminopterin and 8 × 10−4 M thymidine (HAT). After about 10 days, cell clones secreting antibodies against E34 were screened by ELISA, and the positive clones were selected and subcloned until all clones were positive.Immunization and production of MAbs were carried out using standard protocols. Five 4-week-old female BALB/c mice were subcutaneously immunized with 100 μg purified truncated protein E34 in complete Freund’s adjuvant per animal. The animals were then boosted twice at 4-week intervals using 100 μg antigen in incomplete Freund’s adjuvant. Three days after the final booster using 100 μg antigen, one mouse was sacrificed, and its splenocytes were fused with NS-1 at a 5:1 ratio to a final concentration of 2 × 106 hybridoma cells of all five clones were expanded and injected into the peritoneal cavity of mice. Usually, 5mL ascites was collected from each mouse. After 14 days, ascites were withdrawn and centrifuged at 1500 rpm for 5 minutes. The binding capacity of the ascites was analyzed by ELISA. The supernatant was collected and applied to a column of protein A-sepharose 4B which had been pre-equilibrated in PBS. The bound MAb was eluted with citric acid (pH 4.0) and dialyzed against PBS overnight. The purified proteins were analyzed by SDS-PAGE and Western Blot.To produce the MAb in large quantity, 3 × 10E34 was mixed with SDS-PAGE loading buffer . Proteins were separated with electrophoresis, and then they were transferred to a nitrocellulose sheet. After blocking with 5% skim milk at RT for 1 h, 10 μg/mL MAbs were added separately for 2 h at RT. 5 μg/mL HRP_GAM IgG (Biodesign) was used to display the specific E34 line by an enhanced chemilunimescence (ECL) method using ECL Western blotting detection reagents (Amersham Life Science). 5 μg/mL HRP_Omalizumab and A HRP_conjugated mouse anti-E34 monoclonal antibody (HRP_1095) were set as positive controls.0 means the antigen concentration; A is the absorbance value in this specific condition; A0 means the absorbance value of positive control with no antigen in the pre-incubated complex. The mean value of all K values was the final affinity constant.To evaluate the affinity constant of QME5 and IgE, a modified ELISA method was used. In brief, diluted QME5 (from 4 to 0.0625 μg/mL) was added to IgE-coated plate, then after three washes, GAM_HRP was added for detecting the bound MAbs and OPD reaction system was used as described above. From the binding curve, 1 μg/mL QME5 was chosen to carry out the detection of affinity constant. QME5 and diluted IgE were mixed and incubated at 4°C overnight, and then the complexes were added to IgE-coated plate. Subsequent procedures were the same as described before. We used the following formula to calculate the affinity constant K,Mouse Mab Isotyping test kit  was used to identify the subclass of QME5. 1 mL of the supernatant of QME5 was collected, freeze-dried and re-dissolved to a final volume of 100 μL. All procedures were carried out following the protocols.330, Ser331, Leu429 and Arg427) were involved in the van der waals interaction with Cɛ2, which indicated that lacking Cɛ2 domain might permit E34 to be more flexible. However, E34 keeps essential residues to exhibit the function of binding FcɛRIα or MAE11.Using Homology and Discover modules in InsightII 2005 software , the optimized 3-D structures of E34 Fig.  was obtaBesides, MAE11 variable domain Fig.  was alsoELISA plates were coated with 5 μg/mL Omalizumab, then diluted E24 or E34 were added, then samples were incubated 100 μL HRP_conjugated goat anti-mouse antibody (GAM_HRP) for 45 minutes at RT. As shown in Fig. −5 before fusion. Hybrids were growing up in about 10 days and ready for screening. After ELISA analysis of the culture media, six positive cell clones  were identified as secreting antibodies against E34 and further subcloned to generate stable hybridoma.At 14 days of each immunization, blood was collected from mouse-tail and the titration of antibody against E34 was detected in serum. Usually, the titration was at least 1:10−5 (data not shown). Purified MAbs were analyzed by SDS-PAGE. As shown in Fig. The capacity of the ascites was analyzed by ELISAs, and the titration was about 1:10To test whether MAbs had the capacity of recognizing antigen, E34 protein was resolved on SDS-PAGE and then transferred to a nitrocellular membrane. Western blot analysis showed that all six MAbs at 10 μg/mL could recognize the antigen E34 Fig. ; FurtherQME5 could bind membrane IgE. SKO-007 was incubated with 10 μg/mL QME5 for 30 minutes at 4°C, then FITC_GAM IgG was added to determine the amount of membrane-binding Mab. A mouse IgG1 Mab 4C13 .After 24 hours’ incubation, QME5 interacted specifically with anti-IgG1 and anti-κ antibody on the reagent paper sheet, which formed a visible brown annulus Fig. , suggest+ cells (basophils or mast cells) or to induce the release of a variety of chemical mediators and cytokines, although more functional experiments about QME5 are necessary in future study.FI5F10 were incubated with 20 nM IgE, then after washing twice, cells were incubated with 75 nM QME5. FITC_GAM IgG was finally added in order to investigate the binding of QME5 and FcɛRIα-bound IgE. Cells incubated with 37.5 nM mouse IgG1 4C13 were set as negative control, and 37.5 nM mouse anti-IgE MAb 1095 was set as positive control. Results of this investigation are giving in Fig in vivo.750 nM QME5 and 7.5 nM IgE (molar ratio=1:100) were mixed and then the complex was added into FI5F10 cells, cells incubated with 7.5 nM IgE was set as a positive control, while equimolar Omalizumab pre-incubated with IgE was set as negative control. After washing, 5 μg/mL FITC_GAH IgE was added to determine the amount of membrane-binding IgE. Comparing to the positive control . IgE-Fc (E24) contains Cɛ2-4 of IgE, in which Cɛ2 replaces the hinge region of IgG. E24 has the capacity of binding FcɛRIα with nearly the same affinity of IgE . The cryThere are four distinct regions within the IgE-Fc for FcɛRIα binding, including the Cɛ2-Cɛ3 linker (residues 334−336), the BC loop (362−365), the DE loop (393-396) and the FG loop (424−427). The 1:1 complex showed that three residues  involved in IgE Cɛ3 domains are observed as contact residues to bind FcɛRIα; besides, Y131 from the receptor projects into a pocket on the IgE-Fc formed by the BC and FG loops of Cɛ3 and the Cɛ2-Cɛ3 linker.The extracellular part of FcɛRIα contains two domains (D1 and D2), in which the C-terminus D2 domain interacts directly with IgE. It contributes residues to bind IgE from three regions: the D1-D2 linker region (residue 85−87), the D2 BC loop (residues 110 and 113), and the D2 FG loop (residue 156-158). Eight residues including Y131 which located in the D2 domain of FcɛRIα interact directly with IgE; meanwhile residues from the D1 domain of the receptor do not form direct interactions with the IgE-Fc. It was also show that P426 from the IgE-Fc inserts receptor residues W87 and W110, forming a hydrophobic proline sandwich. All information above suggests new approaches with anti-IgE antibodies included to inhibit the binding of IgE to FcɛRIα for the treatment of allergic diseases.Ka=5 × 106 M−1 when binding to the receptor (Wurzburg  determinreceptor . Previoureceptor . Therefoabbr. E24) and molecular docking method, the spatial structure of the interaction complex IgE (or E24)-MAE11 was modeled, and the identified epitope of IgE was determined theoretically, which showed that Cɛ3 in IgE was very important to interact with FcɛRIα and MAE11. Experiment results indicated that E34 could mainly retain the 3-D structure of IgE-Fc and the capacity to bind FcɛRIα or MAE11. According to our modeling results, the flexibility of E34 was possibly affected by lacking Cɛ2 domain, which might be a reason why E34 bound Omalizumab or membrane receptor FcɛRIα at a higher concentration  could replace human IgE, which is scarce in blood serum or hard to be purified from cell supernatant, for preparing neutralizing anti-IgE antibodies target allergic diseases.In this study, the 3-D structure of the functional anti-IgE antibody MAE11 Fv fragment was constructed using computer-guided homology modeling method. Based on 3-D crystal structure of IgE-Fc fragment abbr. E4 and molion Fig. ; meanwhiion Fig. , the keyeg. histamine, leukotriene and prostaglandins) and cytokines  in order to hinder the formation of membrane IgE/FcɛRIα complex, for the affinity of IgE to bind its high affinity receptor FcɛRIα was about 10−10 ∼10−11 M  seemed to be the best Fig. . Moreove1 M Fig. . Anyway,


Escherichia coli and Saccharomyces cerevisiae have been extensively subjected to Metabolic Engineering for the fermentative production of biorenewable fuels and chemicals. This often entails the introduction of new enzymes, deletion of unwanted enzymes and efforts to fine-tune enzyme abundance in order to attain the desired strain performance. Enzyme performance can be quantitatively described in terms of the Michaelis-Menten type parameters Km, turnover number kcat and Ki, which roughly describe the affinity of an enzyme for its substrate, the speed of a reaction and the enzyme sensitivity to inhibition by regulatory molecules. Here we describe examples of where knowledge of these parameters have been used to select, evolve or engineer enzymes for the desired performance and enabled increased production of biorenewable fuels and chemicals. Examples include production of ethanol, isobutanol, 1-butanol and tyrosine and furfural tolerance. The Michaelis-Menten parameters can also be used to judge the cofactor dependence of enzymes and quantify their preference for NADH or NADPH. Similarly, enzymes can be selected, evolved or engineered for the preferred cofactor preference. Examples of exporter engineering and selection are also discussed in the context of production of malate, valine and limonene.Microbial biocatalysts such as  Escherichia coli was first engineered to produce ethanol as its major fermentation product . Th. ThE. coThe Michaelis-Menten parameters described above all relate to an active enzyme, its affinity for the substrate and its speed in forming product. However, many enzymes have at least some degree of post-translational allosteric regulation which serves to fine-tune enzyme activity in response to the abundance of key metabolites. This activity control occurs in the form of both activation and inhibition; here we focus on examples of enzyme inhibition.As with the Michaelis-Menten model of enzyme activity, there also exist quantitative models for enzyme inhibition. These describe both competitive and non-competitive inhibition. In standard cases of competitive inhibition the inhibitor (I) competes with the substrate for binding to the active site, resulting in the additional reaction (rxn 2)i, which reflects the affinity of the enzyme for the inhibitor according toto the simplified schematic described above. This reversible binding is described with the inhibition parameter Kand the overall velocity v of the reaction is represented by the modified Michaelis-Menten equationi is the concentration of the inhibitor.Note that cBy contrast, in standard cases of non-competitive inhibition, the inhibitor binds to a site distinct from the active site and this binding induces a conformational change in the enzyme that decreases enzymatic activity. Thus, in addition to Rxn 2, it is possible for the inhibitor to bind to the E-S complex; this E-S-I complex can revert to E-I by dissociation of the substrate or possibly proceed to product formation, though at a much slower rate than the E-S complex in the absence of bound inhibitor. This non-competitive inhibition is modeledi, where a higher value indicates decreased sensitivity to inhibition.Competitive and non-competitive inhibition can be distinguished by the use of Lineweaver-Burk plots, which are not discussed here. The relevance of these equations to the current work is the fact that enzyme sensitivity to inhibition can be quantified by the parameter KThis regulatory control of enzyme activity presumably serves to balance metabolic flux distribution and can be problematic when one desires to produce a single metabolic product at high concentration and yield, as this can conflict with the microbial need to balance production of biomass constituents. Thus, enzyme inhibition is a problem that often needs to be addressed in the fermentative production of biorenewable fuels and chemicals.et al . Si. Siet alpropanol  and enabt shown) . Note thE. coli KO11 was limited in defined growth media due to NADH-mediated inhibition of citrate synthase, resulting in limitation of the biomass precursor alpha-ketoglutarate and limitation of overall growth and therefore product formation [E. coli citrate synthase with an NADH-resistant isozyme from Bacillus subtilis resulted in a 50% increase in growth and ethanol production in the desired growth condition [Biomass formation by ethanologenic ormation . Replaceondition .et al [+ through fermentative pathways. The lack of PDH activity during fermentative growth, when NADH is abundant, has resulted in reliance on recombinant expression of the Zymomonas mobilis PET pathway for ethanol production by E. coli [Z. mobilis PET pathway  was first demonstrated as useful for malate uptake by S. cerevisiae [S. cerevisiae [Transporters that are discovered when searching for importers can also be useful as exporters. The revisiae , but wasrevisiae .E. coli and is necessary for protein translation, the branched-chain amino acid valine has long been known to be toxic to E. coli [et al's strain design for valine production included a means to mitigate intracellular valine accumulation via overexpression of the ygaZH transporter [E. coli but was not previously recognized as a valine transporter; Park et al identified it as a potential valine exporter due to its homology with the brnFE branched-chain amino acid exporter encoded by Corneybacterium glutamicum [Product export becomes increasingly important when the target compound is inhibitory to the microbial biocatalyst. Here we discuss two examples of the selection of appropriate exporters in order to improve the microbial production of an inhibitory compound. Despite the fact that it is naturally produced by  E. coli . Thus, Pnsporter . This stutamicum .et al [E. coli's tolerance of limonene, among other biofuels. Introduction of the most useful pump, YP_692684 from Alcanivorax borkumensis, enabled an approximately 50% increase in limonene titer when expressed in an E. coli strain engineered for limonene production.Many biorenewable compounds are not naturally produced by the microbial biocatalyst and thus there is an absence of effective export systems. Dunlop et al  generateThese three examples highlight the use of native transporters, recombinant transporters and engineered/evolved transporters to increase production of biorenewable fuels and chemicals.m, kcat and Ki, can improve the fermentative performance of a microbial biocatalyst. Each of the examples that we have described represent improved biocatalyst performance in the context of production of biorenewable fuels and chemicals. While the Michaelis-Menten is a simplified model of enzyme kinetics [Here we have highlighted recent examples of how improvement of enzyme parameters, as reflected in the Michaelis-Menten-type parameters Kkinetics , 65–68, kinetics  are a usThere are some enzymes that are tantalizing targets for improvement in order to increase production of biorenewable fuels and chemicals, yet these enzymes remain remarkably intractable to such improvement. The most well-known example is photosynthesis pathway enzyme Rubisco, which has a low catalytic efficient and poor substrate specificity , 70. A ret al [et al [While this work demonstrates the impact that improved enzyme properties can have on biocatalysts, it is apparent from the literature that additional collaboration between protein engineers and metabolic engineers could result in further advances. For example, Campbell et al  and Machl [et al  have botP. stipitis transaldolase resulted in a 5-fold increase in its kcat/Km for F6P, but less than a 30% increase in xylose consumption and ethanol production [L. lactis alcohol dehydrogenase resulted in a 30-fold increase in its kcat/Km for isobutyraldehyde, the final isobutanol titer was increased less than 2-fold [It is interesting to note that while improvement of enzyme parameters can improve strain performance, the magnitude of these improvements often differs . For exaoduction . Similarn 2-fold . This iscat, Km and kcat/Km.While it makes sense that the fold improvement in enzyme parameters will not result in the same fold improvement in strain performance, the impact that mitigation of enzyme inhibition can have on strain performance is particularly striking . Work wi


V7 potassium channels, particularly KV7.2, KV7.3, and KV7.5 controls neuronal excitability. Numerous neurotransmitters acting via G protein-coupled receptors signaling via Ca2+/calmodulin or depletion of membrane phosphatidylinositol-4,5-bisphosphate (PIP2) tightly regulate KV7 channel function. Moreover, the phosphorylation of KV7 channels has been proposed to play a crucial role. However, in vivo phosphorylation sites and their functional implications need to be determined.The family of KV7 channel phosphorylation, superior cervical ganglion (SCG) neurons were pretreated for 30 min with different kinase inhibitors  which block CDK5, GSK3, p38 MAPK, and PKC as well as PKA, respectively. Thereafter, oxotremorine M (OxoM) or bradykinin-induced inhibition of the M-currents (primarily through KV7.2/7.3 heterotetramers) was tested.To investigate the role of steady-state KV7.2 channels and M1 receptors, caused a leftward shift of the OxoM concentration-response relation. In mass-spectrometric studies, several phosphorylated amino acid residues in the C-terminus of native and heterologously expressed KV7.2 channels were detected, 5 of them are located within the putative PIP2 binding site. CDK5 was predicted to target serines S427 and S446. In contrast to S446, mutation of S427 to alanine significantly increased KV7.2 channel sensitivity towards inhibition via M1 receptors. Additionally, treatment with GW8510 failed to cause any further effect. Nevertheless, these alanine mutations did not influence the channel-voltage dependence.Inhibition of CDK5 shifted the concentration-response curve for OxoM to the left, but not that of bradykinin. Similarly, GW8510 treatment of tsA201 cells, heterologously expressing KV7.2 modulation by M1 muscarinic, but not by B2 bradykinin receptors, suggesting that the phosphorylation state of S427 regulates the affinity of the KV7.2 C-terminus for PIP2.Hence, phosphorylation of C-terminal serine residue 427 determines K


The type I cGMP-dependent protein kinases (PKGIα and PKGIβ) are splice variants that differ in their first ~100 amino acids, giving each isoform unique dimerization and autoinhibitory domains. The unique coiled-coil dimerization domains mediate isoform specific protein-protein interactions, and we have previously identified the amino acids that are important in mediating the interaction between PKGIβ and its two known interaction partners, TFII-I and IRAG [in vitro and in intact cells. Phosphorylation on serine 12 or a phospho-mimetic S12E mutation significantly reduced the interaction between CaD and myosin IIA. We found that siRNA mediated caldesmon depletion increases the migration of MDA-MB-231 cells, and that reconstitution with wild-type or phospho-deficient S12A caldesmon slowed migration. In contrast, migration was not slowed by reconstitution with caldesmon containing an S12E mutation. We also found that PKG activation leads to indirect phosphorylation of mouse and human CaD in 293T and MDA-MB-231 cells. The indirect phosphorylation seen in 293T cells is accompanied by a shift in the apparent molecular mass of CaD during SDS-PAGE. The observed migratory shift is similar to that previously seen when purified platelet CaD was directly phosphorylated in vitro by PKA [Using wild-type and mutant PKGIβ D/D domains as affinity probes in a proteomic screen and we identified the actin/myosin associated protein caldesmon as a PKGIβ specific interacting protein . Using iSince serine 12 is not conserved in mouse or rat, our results indicate that PKGIβ regulates caldesmon in a species-specific manner. While the PKGI-mediated indirect phosphorylation site(s) have not been determined, our preliminary data suggests that PKGI could potentially regulate all CaD isoforms, albeit in a cell type and species specific manner.


An experiment was conducted to investigate the effect of screw speed, inclination angle and variety on the required power, and conveying capacity of a screw conveyor. The experiment was designed with four levels of screw speed , five levels of inclination angle , and three levels of variety . The Length, diameter, and pitch of screw were 2, 0.78, and 0.5 m, respectively. The experimental design was a randomized complete block (RCB) with factorial layout. Maximum and minimum power requirements of tested screw conveyor were 99.29 and 81.16 Watt corresponding to conveying capacity of 3.210 and 1.975 ton/hour obtained for khazar and Alikazemi varieties, respectively. The results indicated that as screw inclination angle increased from 0 to 80°, the conveying capacity decreased significantly from 3.581 to 0.932 t/h. It can be concluded that the most conveying capacity was 4.955 t/h at tests with khazar variety and conveyor inclination angle zero degree. Rice is one of the commonly consumed cereals and food staples for more than half of the world's population. It is an important source of energy, vitamins, mineral elements, and rare amino acids. World rice production increased from 520 million tons in 1990 to 605 million tons in 2004, while Iran's rice production increased from 1.3 million tons in 1980 to 3.4 million tons in 2004 [http://www.crri.nic.in/Research/Divisions/Engineering.htm). Efficient rice conveying implements are to be used to achieve energy savings. Screw Conveyers (augers) are used to convey free flowing materials such as grain to more difficult fibrous materials such as straw and alfalfa. At gain elevators and on the farmstead, augers are used to move grain to and from storage. In these applications, the conveyor consists of a rotating shaft which carries a helicoid flighting through a stationary tube. Factors affecting capacity include auger dimensions , shear-plane flighting orientation, auger speed, angle of inclination, commodity being conveyed, and entrance-opening configuration [Application of engineering principles for reducing energy requirement in the form of mechanical and electrical power is necessary to reduce cost of production , a facility of University of Mohaghegh Ardabili, to investigate the effect of screw speed, inclination angle and variety on the required power and conveying capacity of a screw conveyor. The experiment was designed with four levels of screw speed , five levels of inclination angle , and three levels of variety . The experimental design was a randomized complete block (RCB) with factorial layout at four replications. For means comparison, Duncan's Multiple Range Test was used. The length of screw, screw pitch, and diameter were 2000, 50, and 78 mm, respectively. The clearance between screw and tube is 6 mm, which was developed by Iranian Ashtad cooperative company . A threeAn inverter, a rotational speed gauge, and a digital wattmeter were used for continuously determining the variable transmission of screw shaft, the screw rotational speed, and the electromotor electrical power, respectively. The wattmeter was connected to a personal computer for recording and displaying the power data during the tests so that power consumption variations were graphically visible on monitor during the experiments. Each test was started by pouring 30 kg paddy grain into the input section. After a few seconds of unloading grain at outlet section, a container was directed towards the discharge, and then discharged grain mass was weighted. The time of purring of paddy grain into the container was between 10 and 12 seconds. Conveying capacity data was recorded every second for future analyzing.P < 1%).The analysis of variance of power requirement and conveying capacity is presented in Means comparison results of the main effects revealed that power requirement of Khazar variety was more than others . The reaMean comparison results of interaction effects of variety and inclination angle are shown in Mean comparison results of interaction effects of variety and screw speed are showed in Mean comparison results of interaction effects of inclination angle and screw speed are showed in  Mean comparison results of interactions between three factors revealed that the most power requirement mean was 156.88 W at tests with Khazar variety, screw speed of 1000 rpm, and inclination angle of 60°.P < 1%). These results conform to the obtained findings of the researchers in the study of screw conveyor performance [Analysis of the variance of data obtained from measuring conveying capacity was presented in formance , 9. Theoformance .P < 5%). The highest and lowest conveying capacity was allocated to Khazar and Alikazemi varieties, respectively. By increasing screw speed from 600 to 1000 rpm, conveying capacity was increased significantly and then decreased at test with screw speed 1200 rpm. Similarly, this case had happened for power requirement. By increasing screw inclination angle from 20 to 80°, conveying capacity decreased significantly from 3.352 to 0.932 t/h, respectively. In horizontal state, the screw conveyor had been the greatest amount of 3.581 t/h of conveying capacity. Similar results were obtained by Ray [Means comparison results of variety effects on conveying capacity was significant (d by Ray  and SrivMean comparison results of interaction variety and conveyor inclination angle revealed that at tests with all varieties, conveying capacity reduced significantly by changing of conveyor inclination angle from 0 to 80°. In tests with Khazar variety, by changing screw speed from 800 to 1200 rpm, conveying capacity was not significantly changed .Mean comparison results of interaction effects of variety and screw speed were shown in Mean comparison results of interaction effects of three factors showed that the most conveying capacity of 4.955 t/h was obtained at tests with Khazar variety, conveyor inclination angle 0 degree, and screw rotational speed 1000 rpm.The main effects of variety, conveyor inclination angle, and screw rotational speed and their interactions were significant on power requirement and conveying capacity. Maximum and minimum power requirements of tested screw conveyor were 99.29 and 81.16 Watt corresponding to conveying capacity of 3.210 and 1.975 t/h obtained for khazar and Alikazemi varieties, respectively.The most and least power requirement, and conveying capacity were obtained at screw rotational speeds of 1000 rpm and conveyor inclination angle of 40°, respectively. By increasing the conveyor inclination angle from 0 to 80°, conveying capacity was decreased significantly from 3.581 to 0.932 t/h. Generally, the most conveying capacity was 4.955 t/h at tests with khazar variety and zero inclination angle degree.


This and the fact the basic properties of VDAC, such as single-channel conductance and selectivity, remained unaltered by phosphorylation allowed us to suggest the phosphorylation regions positioned on the cytosolic loops of VDAC and establish channel orientation in our reconstitution experiments. Experiments on human hepatoma cells HepG2 support our conjecture that VDAC permeability for the mitochondrial respiratory substrates is regulated by dimeric tubulin and channel phosphorylation. Treatment of HepG2 cells with colchicine prevents microtubule polymerization, thus increasing dimeric tubulin availability in the cytosol. Accordingly, this leads to a decrease of mitochondrial potential measured by assessing mitochondrial tetramethylrhodamine methyester uptake with confocal microscopy. Inhibition of PKA activity blocks and reverses mitochondrial depolarization induced by colchicine. Our findings suggest a novel functional link between serine/threonine kinase signaling pathways, mitochondrial respiration, and the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation.Tubulin was recently found to be a uniquely potent regulator of the voltage-dependent anion channel (VDAC), the most abundant channel of the mitochondrial outer membrane, which constitutes a major pathway for ATP/ADP and other metabolites across this membrane. Dimeric tubulin induces reversible blockage of VDAC reconstituted into a planar lipid membrane and dramatically reduces respiration of isolated mitochondria. Here we show that VDAC phosphorylation is an important determinant of its interaction with dimeric tubulin. We demonstrate that  The role of mitochondria in energy production, calcium signaling, and promoting apoptotic signals is well established. There is also emerging evidence of the involvement of mitochondria in multiple other cell signaling pathways with the intimate and dynamic relationship between mitochondria function and cytoskeleton organization and microtubule (MT) network remodeling Previously, we demonstrated that tubulin induces reversible blockage of VDAC reconstituted into planar lipid membrane and dramatically reduces respiration of isolated mitochondria mK for ADP, thus dramatically decreasing the availability of ADP to adenine nucleotide translocase. It was concluded that by blocking VDAC permeability for the respiratory substrates tubulin may selectively regulate metabolic fluxes between mitochondria and the cytosol and, therefore, control mitochondrial respiration.We suggested a model in which the negatively charged C-terminal tail (CTT) of tubulin permeates into the channel lumen, interacting with VDAC with high specificity and partially blocking channel conductance for small ions in vivo experiments, phosphorylation of VDAC was reduced Protein phosphorylation is a key element of the complex network of regulatory and signaling pathways. It is commonly known as an on/off mechanism for numerous cellular processes. Therefore, it is reasonable to expect that phosphorylation of VDAC by the cytosolic kinases might also be involved in regulation of VDAC permeability by tubulin. It was recently shown that VDAC could be phosphorylated by glycogen synthase kinase-3β (GSK3β), and that this phosphorylation is modulated by Akt-dependent inactivation of GSK3β in vitroCyclic AMP-dependent protein kinase (PKA) is another serine-threonine kinase with established key roles in cell growth and differentiation. It is also one of the targets in cancer therapy. Phosphorylation of cytoplasmic substrates mediated by PKA is crucial for multiple cell functions, including metabolism, differentiation, synaptic transmission, ion channel activity, and growth and development in vitro by either GSK3β or PKA increased the on-rate of tubulin binding by up to two orders of magnitude. At the same time, the basic properties of VDAC, such as single-channel conductance and selectivity, remained unaltered by phosphorylation. This, and the finding that VDAC phosphorylation increases tubulin binding at the cis side of the channel only, suggest that protein regions that are phosphorylated are presumably positioned on the cytosolic loops of VDAC. Modulation of mitochondrial potential by colchicine and PKA inhibitor H89 in human hepatoma HepG2 cells support our conclusion that VDAC phosphorylation by serine/threonine kinases could regulate mitochondrial respiration by affecting VDAC blockage by tubulin.Here, using mammalian VDAC reconstituted into a planar lipid membrane, we show that VDAC-tubulin interaction appears to be extremely sensitive to the state of VDAC phosphorylation. VDAC phosphorylation cis side of the membrane induced strong blockage of phosphorylated VDAC at −25 mV , vigorous VDAC blockage was observed only at negative polarities of the applied voltage . To achieve a similar level of VDAC blockage at the opposite, positive potentials, the concentration of tubulin in the trans side had to be increased hundred times up to 30 nM . Thus, the sensitivity of VDAC to tubulin was increased by two orders of magnitude, but only for VDAC-tubulin interaction at the cis side of the channel. The interaction from the trans side was virtually unchanged by phosphorylation with PKA.Another remarkable effect of VDAC phosphorylation is a distinct asymmetry of VDAC blockage by tubulin. cis side and 20 nM tubulin in the trans side induced comparable blockage. When more than one channel was inserted, the asymmetry in tubulin binding to phosphorylated VDAC was maintained off and τ(2)offted VDAC . The offNot only the times of tubulin residence in the channel, but also two other basic properties of VDAC, single-channel conductance and selectivity, remained practically unaltered by VDAC phosphorylation  that is usually called mitochondrial potential. To address this question, we measured ΔΨ in HepG2 human hepatoma cells by assessing mitochondrial tetramethylrhodamine methyester (TMRM) uptake using confocal microscopy ΔΨ by 60%. The amount of free tubulin measured in cancer cells by Western blot rises or drops after colchicine or paclitaxel treatment, respectively ΔΨ could be explained by modulation of VDAC permeability to mitochondrial metabolites ΔΨ. In support of this hypothesis, 1 µM of PKA inhibitor, H89, increased mitochondrial TMRM fluorescence by 60% in HepG2 cells . VDAC was isolated from the membrane fraction by the standard method 2) containing 50 µM MgATP for 30 min at 37°C in the presence of phosphatase inhibitors (Pierce). For dephosphorylation, 1.0 mg of the mitochondrial membrane fraction was incubated with ∼100 units of alkaline phosphatase (AP) or 1 µg of PP2A in 5 µl of Tris HCl at pH 8.0 at 37°C for 30 min. After phosphorylation or dephosphorylation the samples of membranes with kinase in the ATP cocktail were centrifuged for 10 min at 12,000 rpm to collect membrane fraction. The supernatant containing free kinase was discarded and the membranes were processed for running on 4–12% NuPAGE gels (Invitrogen). VDAC bands were verified by Western blotting using a primary anti-VDAC antibody which was a generous gift from Marco Colombini (University of Maryland at College Park). Phosphorylation of VDAC was detected using Pro-Q Diamond phospho-protein stain (Invitrogen). The bands were imaged at 532–560 nm excitation on a Fuji gel scanner, and the difference in staining between control and de- and phosphorylated VDAC was quantified using “Multi Gauge V3.0” software. The VDAC band intensities with gel background subtracted were normalized versus intensity of the untreated samples in each corresponding gel. All gels were analyzed in raw, unmodified state.For VDAC phosphorylation 1.0 mg of a wild type mouse mitochondrial membrane fraction was incubated with 1000 units of GSK3β or PKA in 10 µl of kinase buffer . After phosphorylation or dephosphorylation the sample of VDAC with kinase in the ATP cocktail was directly used in the channel reconstitution experiments. Phosphorylation of the isolated VDAC was verified as described above and in Supplemental cis compartment while stirring. Potential is defined as positive when it is greater at the side of VDAC addition (cis). After VDAC channels were inserted and their parameters were monitored, tubulin was added to one or both sides of the membrane under constant stirring for 2 min. Current recordings were performed as described previously Planar lipid membranes were formed on 70–80 µm diameter orifices in the 15-µm-thick Teflon partition that separated two chambers as previously described 2/air at 37°C. For confocal microscopy, cells were cultured on glass bottom culture dishes  for 48 h. Cells in modified Hank's balanced salt solution (HBSS) containing (in mM): NaCl 137, Na2HPO4 0.35, KCl 5.4, KH2PO4 1, MgSO4 0.81, Ca2Cl 0.95, glucose 5.5, NaHCO3 25 and HEPES 20, pH 7.4 were loaded 30 min at 37°C with 200 nM of tetramethylrhodamine methyester (TMRM). After loading and washing, subsequent incubations were performed with 50 nM TMRM to maintain equilibrium distribution of the fluorophore 2/air at 37°C in modified (HBSS). Colchicine and H89 were purchased from Sigma  and TMRM from Invitrogen . All other reagents were analytical grade. TMRM-loaded cells were placed on the stage of a Zeiss LSM 510 inverted laser scanning confocal microscope  in humidified 5% CO2/air at 37°C. Fluorescence of TMRM was imaged with a 63× 1.4 N.A. planapochromat oil immersion lens using 543-nm HeNe laser (0.5–1.5% full power) excitation, respectively. Emitted red fluorescence of TMRM was detected through a 560-nm long-pass filter and a one Airy unit diameter pinhole. Differences between groups were analyzed by the Student's t-test using p<0.05 as the criterion of significance. Results were expressed as means ± SEM of three or more experiments.HepG2 cells  were grown in Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin in 5% COFigure S1Times between successive tubulin-induced VDAC blockages, open times, strongly depend on VDAC phosphorylation. Statistical analysis of the open times, τon, in the experiments, examples of which are presented in τon equal to 415, 52, and 519 ms for VDAC untreated, phosphorylated with PKA, and dephosphorylated with PP2A, respectively.(TIF)Click here for additional data file.Figure S2On-rate of VDAC-tubulin binding strongly depends on the level of VDAC phosphorylation, while residence times remain unaltered. On-rate, on,k, (A) and two residence times, τ(1)off and τ(2)off, (B) are the average values obtained in multiple experiments with VDAC phosphorylated with PKA or GSK3β and dephosphorylated with AP or PP2A in comparison with untreated VDAC. The applied voltage was −20 mV. Experimental conditions were as in onk values. Data are means ± S.E. Statistical analysis of onk values was done using two-tailed t-test (a = 0.05) as a comparison with untreated VDAC samples (P<0.2).(TIF)Click here for additional data file.Figure S3The phosphorylation-induced asymmetry of blockage is maintained after simultaneous insertion of many channels. Current records of 5 channels obtained with VDAC phosphorylated with GSK3β. 2 nM of tubulin in the cis side induce more frequent closure events at −20 mV applied voltage than 20 nM of tubulin added to the trans side at +20 mV. Simultaneous closure of more than one channel at −20 mV is seen. Experimental conditions as in (TIF)Click here for additional data file.Figure S4Detection of phosphorylation of isolated VDAC. (A) Pro-Q Diamond phospho-protein gel stain image (i) of untreated VDAC isolated from mouse liver mitochondria and phosphorylated with PKA or dephosphorylated with AP and subsequent Comassie blue staining of VDAC. To improve Coomassie blue and phospo-stain gels resolution, isolated VDAC was concentrated by cold chloroform/methanol procedure before applying to SDS-PAGE gels and treated to the phosphoprotein gel stain (see Invitrogen protocol). The dry sample was resuspended in SDS sample buffer and run on 4–12% SDS Page Gels (Invitrogen). The gel was treated with Pro-Q Diamond Phospho-protein stain as described in the Methods. The band of auto-phosphorylated PKA (39 kDa) is clearly seen. VDAC band at 36 kDa is seen in Commassie blue gel (ii). (B) Relative band intensities shown in Inset, normalized versus untreated VDAC samples (control). Number or repeats 3.(TIF)Click here for additional data file.Figure S5PKI blocks depolarization induced by colchicine in HepG2 cells. Cells in HBSS were loaded with TMRM, as described in upper panel) or after treatment with PKI, a PKA peptide inhibitor (20 µM) for 30 min (bottom panel). (B), TMRM fluorescence is plotted for the different treatments.* p<0.05 vs other groups.(TIF)Click here for additional data file.Table S1Characteristic channel properties of VDAC remain unaltered after phosphorylation.(PDF)Click here for additional data file.