Corylus avellana) is a common cause of lifetime lasting IgE-mediated food allergy. Symptoms range from mild oral allergy syndrome to severe life-threatening anaphylaxis. We aimed to identify allergenic determinants in children living in the Campania region  with hazelnut allergy.Hazelnut  cultivars, with phosphate saline buffer, pH 7.2. The immunoreactive protein components were identified by SDS-PAGE electrophoresis and Western immunoblotting, using patients sera as source of specific IgE. The IgE-binding protein bands were characterized by advanced proteomic strategies and tandem mass spectrometry (MS)-based Four subjects were evaluated . Symptoms were: urticaria (2), angioedema (3), anaphylaxis (2). No significant differences were observed considering the main demographic and clinical characteristics at diagnosis. All children’s sera were immunoreactive to a protein, not previously annotated in database, occurring in hazelnut regardless the variety. The allergen was isolated by combined chromatographic strategies. Only one patient exhibited an additional reactivity to the vicilin-like 7S 48 kDa glycoprotein (Cor a 11). The MS-based characterization provided evidence of a high homology degree between the IgE-binding protein subunit and 11S globulin-like storage proteins expressed in other seeds. The new allergen shares structural traits with the hazelnut 11S globulin-like proteins (Cor a 9) such as the disulfide linkage of two subunits, an acidic (~35 kDa) and an alkaline (~21 kDa) one. Interestingly, only the alkaline subunit exhibits antigenic properties.A previously unrecognized hazelnut allergen was identified. Except for a faint IgE reactivity of Cor a 11 recorded in a single case, the new allergen was the unique IgE-binding protein in our patients. Future study are warranted to better define possible prognostic and immunotherapeutic implications.None declared.


Low molecular weight (LMWH) and unfractionated heparin (UFH) slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition) value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml). By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.The serine protease inhibitor protein C inhibitor (PCI) is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP) is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48×10 Protein C inhibitor (PCI) is a serine protease inhibitor and a member of the serpin superfamily (serpinA5). PCI has originally been described as a plasma inhibitor of activated protein C (APC) Heparin and other glycosaminoglycans can modify the activity and target enzyme specificity of PCI. The heparin-binding site is a basic patch on helix H, which lies close to the reactive center loop By Northern blotting, a wide tissue distribution of PCI has been demonstrated in humans. PCI mRNA is present in the liver, kidney, heart, brain, lung, spleen, reproductive system and pancreas Since PCI has affinity for glycosaminoglycans and phospholipids, both components of the cell membrane, cell membrane association of PCI is not unlikely. We were therefore interested in analyzing the interaction of PCI with serine proteases also present in or on cell membranes. So far there are only a few indications in the literature, suggesting that PCI interacts with type II transmembrane serine proteases in vivo. This function might be executed intracellularly by cathepsin B Activation of trypsinogen is an obligatory step in the pathogenesis of acute necrotizing pancreatitis 1-antitrypsin (A1AT) and AT) and can show that PCI is a strong inhibitor of EP with an apparent 2nd order constant (kapp) comparable to other PCI-protease interactions.We analyzed the interaction of EP with serpin-type protease inhibitors  for the inhibition of EP by human recombinant PCI were calculated under pseudo 1st order conditions as outlined in nd order rate constant of 4.48±0.32×104 M−1 s−1 . In the presence of 10 U/ml UFH, the constant was reduced to 8.83±0.75×103 M−1 s−1 . The SI value for the inhibition of EP by PCI was 10.8±0.7 . In the presence of 10 U/ml UFH, it increased to 17.9±0.6  , as this provides a measure for the overall rate of association app × SI) were 4.84×105 M−1 s−1 in the absence and 1.58×105 M−1 s−1 in the presence of 10 U/ml UFH, respectively.The apparent 2, n = 3) . The kapIn addition to recombinant human EP, the interaction of PCI with two additional EPs from natural sources has been studied. EP purified from porcine intestine (data not shown) as well as bovine EP (bEP) purified from calf intestine formed complexes with PCI, as demonstrated by SDS-PAGE analysis followed by Western blotting . FunctioA dose-response curve for the effect of UFH on the inhibition of bEP by PCI was determined. Increasing concentrations of UFH strongly reduced the inhibitory activity of PCI towards bEP . The intTo investigate if other serpins also interact with EP, activity assays were performed in the presence of either A1AT or AT. After incubation of EP with A1AT for 60 min, no significant reduction of EP activity was observed, even at a molar [A1AT]:[EP] ratio of 1000∶1 (not shown). When EP was incubated with AT for the same time, there was no reduction in EP activity. However, AT slightly inhibited EP when either UFH or LMWH were present . HeparinHuman pancreas lysate was applied to SDS-PAGE and Western blotting. Monoclonal anti-serpinA5 IgG was used to detect PCI. A band at about 57 kDa was seen in pancreas lysate, corresponding to the molecular mass of PCI purified from human plasma. Additionally, two faint bands at about 30 and 37 kDa (possible degradation products) were seen (not shown).app comparable to most protease-PCI interactions which range from 8.00×102 M−1 s−1 for APC inhibition in the absence of heparin to 5.60×107 M−1 s−1 for acrosin inhibition in the presence of heparin In this study, we can demonstrate that PCI is a fairly strong inhibitor of EP, with a kIt has been shown previously that heparin and phospholipids are able to stimulate or to reduce the inhibitory activity of PCI towards several proteases AT, on the other hand, inhibited EP only when heparin was present. So, heparin stimulated the inhibition of EP by AT, but reduced the inhibition of EP by PCI. To our knowledge, this is the first demonstration that heparin led to a reduced inhibition of a particular protease by PCI, but an increased inhibition by AT. This may be due to differences in regulation of serpin activity by heparin, as AT undergoes a conformational change when bound to glycosaminoglycans compared to the bridging mechanism of PCI As mentioned above, native EP is a type II transmembrane serine protease. It contains an N-terminal hydrophobic segment from position 18 to 44, predicted to span the membrane Several publications have shown that PCI mRNA is highly expressed in the pancreas in vivo and, if it does, whether it is really able to damage the pancreas profoundly. However, if duodenopancreatic reflux occurs under some circumstances, inhibition of EP by PCI might have a protective effect. In addition, PCI could protect the pancreas from autodigestion by inhibiting trypsin and chymotrypsin Activation of trypsinogen is a crucial step in the pathogenesis of necrotizing pancreatitis PCI has also been identified in the epidermis, with increasing amounts in its more superficial layers The SI value (10.8) of the PCI/EP-interaction is rather high, especially in the presence of heparin (17.9), suggesting that a significant amount of PCI is cleaved/inactivated by EP. The biological consequences of the PCI/EP-interaction may depend on the local [PCI]:[EP] ratio. At a high [PCI]:[EP] ratio, the inhibition of EP would be nearly complete leading to impaired activation of trypsinogen. Additionally, some active PCI would still be available to inhibit trypsin directly, further reducing its activity. At a low [PCI]:[EP] ratio, the available PCI would not be sufficient to completely inhibit EP. Remaining active EP would still be able to activate trypsinogen and to cleave PCI.For the digestive system, this could mean that in the pancreatic duct, any possibly present EP could be efficiently inhibited by PCI. In the duodenum, where EP is abundantly present, PCI would have only a minor effect on EP activity and would not significantly interfere with the activation of digestive proteases.In the epidermis, where EP is mainly localized in the superficial layers Escherichia coli and purification of recombinant human and mouse PCI was performed as described previously Expression in Phospholipid vesicles were prepared freshly for each experiment as described previously 2, and 0.05% Brij-35, pH 7.5). After 30 min, the reaction was stopped by adding 10 mM 1,10-phenantroline.Recombinant human EP (100 µg/ml) was activated by incubation with thermolysin (3.16 µg/ml) in TCNB buffer ). The absorption was recorded at 405 nm for 5 min to assess the activity of EP. The final concentration of human EP was 0.3 nM in all assays. To study the inhibition of EP by PCI and other serpins, EP was incubated with different concentrations of the respective serpin for different periods of time (5 to 60 min) at 37°C in a total volume of 100 µl TCNB buffer. In some reactions, UFH (0.1–100 U/ml), LMWH (0.5–20 µg/ml), or PAPS (100 µM) were included. The reactions were stopped by the addition of an equal amount of substrate solution. The specific activity of EP was 32.1±2.8 nmol/µg/min .nd order rate constants for the inhibition of EP were determined as described previously for the inhibition of other target enzymes by PCI 0/[EP]0 ratio. Linear regression was used to calculate the SI value (x-intercept of the linear regression line).Apparent 2Lyophilized bovine EP (bEP) was reconstituted to a concentration of 0.5 mg/ml and stored in aliquots at −80°C until further usage. The final concentration of bEP was 0.05 µg/ml and the activity was determined as described for human EP. 1,10-phenanthroline (used to stop the activation of human recombinant EP) had no effect on the inhibition of bovine EP by PCI (data not shown).2HPO4, 0.5 mM KH2PO4, 0.1% Tween-20) either at 4°C overnight or at room temperature for 1 h. Incubations with the primary antibody were also done in 5% milk in PBST at 4°C overnight or at room temperature for 1 h. The membranes were washed 5 times (5 min each) in PBST. The secondary peroxidase-labeled antibody was applied for 1 h in PBST with 5% dry milk. After another washing step, the peroxidase reaction was detected with SuperSignal West Femto. Primary antibodies used in Western blotting were monoclonal mouse anti-serpinA5 IgG (0.5 µg/ml), monoclonal anti-PRSS7 (enteropeptidase) IgG (1 µg/ml) and polyclonal rabbit anti-EP-light-chain IgG (78 µg/ml). The respective secondary antibodies were HRP-conjugated donkey anti-rabbit IgG (0.11 µg/ml), and HRP-conjugated sheep anti-mouse IgG (0.13 µg/ml).SDS-PAGE was performed according to the method of Laemmli


Infectious bronchitis virus (IBV) is a very dynamic and evolving virus, causing major economic losses to the global poultry industry. In early 2011, respiratory disease outbreaks were investigated in Iraq, Jordan, and Saudi Arabia. Five IBV isolates  were detected by diagnostic-nested nucleocapsid RT-PCR. Strain identification was characterised by sequencing and phylogenetic analysis of the amplified hypervariable region of the spike 1 (S1) gene. These five IBV isolates were found to be of the IBV strain CK/CH/LDL/97I. Nucleotide identity between these five IBV isolates ranged from 96.9% to 99.7%, and between these isolates and the CK/CH/LDL/97I strain in the range of 96.6–99.1%. The sequenced fragment of the S1 gene of the CK/CH/LDL/97I strain had less than 80% nucleotide identity to the IBV vaccine strains commonly used in the Middle East (M41 and H120). The presence of these CK/CH/LDL/97I-like strains may account for vaccination failure against IBV, since all IBV isolates were from vaccinated chickens. In this paper, we documented for the first time the presence of IBV strain CK/CH/LDL/97I in the Middle East. This strain is known to have originated in China and Taiwan. Coronavirus, group 3 (gamma coronaviruses) [Infectious bronchitis virus (IBV) is a positive sense single-stranded RNA virus, belonging to the genus viruses) . Infectiviruses) –4. IBV wviruses) , 6.IBV strain CK/CH/LDL/97I was first reported in China in 1995 , but in In the Middle East, the common circulating IBV strains are Mass serotype vaccine isolates, H120 serotype vaccine isolates, D274 , IBV varIn the early months of 2011 we noticed IBV outbreaks in spite of massive vaccination, this was due to the presence of a new strain of IBV. In this study, we documented the presence of the IBV strain CK/CH/LDL/97I for the first time in three Middle Eastern countries.Trachea, kidney, ovarian tissues, and cecal tonsils samples were collected from suspected IBV outbreaks in Iraq, Jordan, and Saudi Arabia. These samples were stored at −70°C in RNAlater solution  until RNA extraction was performed. Homogenised tissues were subjected to viral RNA extraction using the Viral Gene-spin Viral DNA/RNA extraction kit . Viral RNA extraction was performed according to the manufacturer's instructions. Viral RNA was stored at −70°C until analysis by RT-PCR. μL MgCl2, 2 μL reverse transcription 10x buffer, 2 μL dNTP mixture (10 mM), 0.5 μL random primers, 0.75 μL AMV reverse transcriptase enzyme, 1 μg RNA, and nuclease-free water to a final volume of 20 μL. Then, the reaction was incubated at 42°C for 60 minutes, then 94°C for 5 minutes. The cDNA was diluted up to 100 μL with nuclease-free water for PCR amplification. Two RT-PCR assays were used. First, a diagnostic-nested RT-PCR assay based on the amplification of the nucleocapsid (N) gene. IBV-specific oligonucleotides for the N gene were obtained according to a published primers sequences [μL GoTaq Green master mix  in which the final concentration of the N gene primers was 0.5 mmol. RT-PCR amplification was performed with a thermal profile of 94°C for 45 sec, 60°C (first step) and 53°C (second step) for 1 min and 72°C for 2 min for 40 cycles.The reverse-transcription (RT) step was performed using an RT system . Briefly, viral RNA was denatured at 70°C for 10 minutes, followed by the addition of a reaction mix including 4 equences ; the IBVn = 100 replicates was performed using the PhyML phylogenetic interface [Next, a phylogenetic RT-PCR was performed using nested spike gene primers according to published protocols , 18. Thenterface . The nucFive IBV isolates were obtained in early 2011. Two IBV isolates were from Jordan (JOA2 and JOA4), two were from Saudi Arabia (Saudi-1 and Saudi-2), and one isolate was from Iraq (Iraqi IBV). The Jordanian isolates were isolated from layer farms at their peak of egg production, the Saudi strains were obtained from broilers and the Iraqi IBV isolate was obtained from a breeder farm as shown in RT-PCR products of the diagnostic N gene assay were detected (380 bp) and for the phylogenetic S1 gene (392 bp). Direct-automated sequencing of the second RT-PCR product of nested S1 (SX3 and SX4 primers) was performed. The reference IBV strains included in the S1 sequence analysis from the United States were M41, Connecticut, DEL072, Ark/15C/96, and Beaudette. The European strains were 4/91, D3896, B1648, and H120. The Australian strains were N1/62 and N1/88. The Chinese strains were IBV LX4, QX IBV, J2, Q1, CK/CH/LDL/97I, and CK/CH/SCYA/10I. Middle Eastern IBV strains were IS/1201, IS/1366, IS/1464, IS/885, variant 1, variant 2, and Sul/01/09 as shown in All five IBV isolates  were found to be of the IBV strain CK/CH/LDL/97I and therefore they are CK/CH/LDL/97I-like strains. The CK/CH/LDL/97I strain is known to be endemic in China and Taiwan. The nucleotide identity between these five IBV isolates ranged from 96.9 to 99.7% with the Saudi-2 isolate being the most divergent. The identity of relatedness between the isolated IBV strains, CK/CH/LDL/97I, and the related strain CK/CH/SCYA/10I  is shownA phylogenetic tree was constructed based on the alignment of the partial S1 sequence. The five IBV isolates were aligned with other reference and related IBV strains as shown in Also, these IBV isolates represent a group distinct from the LX4 IBV type and from QXIBV and IS/1201 (a Middle Eastern strain) related to it. These strains share only 79% nucleotide sequence identity. The lowest nucleotide identity was seen with the DEL072 strain (58%) and with the N1/88 strains (63%) from Australia.In this study, the CK/CH/LDL/97I-like strains were isolated from three different Middle Eastern countries  in 2011. The CK/CH/LDL/97I-like strains were isolated from broilers in Saudi Arabia, layers in Jordan, and from breeders in Iraq. The CK/CH/LDL/97I-like strain was first isolated in China in 1995 [All isolates came from flocks vaccinated with Mass-type H120, and 4/91 vaccines, which implies insufficient protection against these isolates. The poor relationship in the partial S1 sequence between the five IBV isolates and the vaccine strains (average nucleotide identity of 80%) could explain the failure of the Mass 41, H120, and 4/91 vaccination programmes to control IBV in these flocks . There iIn summary, this is the first paper indicting the presence of the IBV CK/CH/LDL/97I strain outside mainland China and Taiwan (in three Middle Eastern countries). In the near future, we expect that this strain might represent a serious problem for the poultry industry and there will be an urgent need to develop a homologous vaccine against this strain.


Insulin stimulated translocation of the glucose transporter GLUT4 from the cytosol to the plasma membrane in a concentration (1 nM–1 μM)-dependent manner and increased glucose uptake in 3T3-L1 adipocytes. Insulin-induced GLUT4 translocation to the cell surface was prevented by the phosphoinositide 3 kinase (PI3K) inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase 1 (PDK1) inhibitor BX912 or the Akt1/2 inhibitor MK2206, and by knocking-down PI3K, PDK1 or Akt1/2. Insulin increased phosphorylation of Akt1/2 at Thr308/309 and Ser473/474, to activate Akt1/2, in the adipocytes. Insulin-induced phosphorylation of Akt1/2 was suppressed by wortmannin and knocking-down PI3K, while no significant inhibition of the phosphorylation was obtained with BX912 or knocking-down PDK1. In the cell-free Akt assay, PI3K phosphorylated Akt1 both at Thr308 and Ser473 and Akt2 at Ser474 alone. In contrast, PDK1 phosphorylates Akt1 at Thr308 and Akt2 at Thr309. The results of this study indicate that PI3K activates Akt1, independently of PDK1, and Akt2 by cooperating with PDK1 in the insulin signal transduction pathway linked to GLUT4 translocation. Akt is a serine/threonine protein kinase bearing multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. Akt includes three closely related isoforms Akt1, Akt2, and Akt3 . Akt2, o3) by phosphorylation of phosphatidylinositol 4,5-bisphosphate P2), and in turn, PIP3 activates PDK1 through its binding. The activation of Akt1/2 is achieved by phosphorylation of Thr308 for Akt1 and Thr309 for Akt2 in the activation-loop of the kinase domain and Ser473 for Akt1 and Ser474 for Akt2 in the carboxy-terminal regulatory region P2 and PIP2, respectively, to attenuate PDK1 activity followed by Akt1/2 phosphorylation at Thr308/309 /phosphatidylinositol 3 kinase (PI3K)/3-phosphoinositide-dependent protein kinase 1 (PDK1)/Akt axis  and under cell-free conditions. We show here that insulin stimulates GLUT4 translocation to the cell surface and increases glucose uptake into 3T3-L1 adipocytes in an Akt1/2-dependent manner; PI3K phosphorylates both Thr308 and Ser473 for Akt1 and Ser474 alone for Akt2; and PDK1 phosphorylates Thr308 for Akt1 and Akt2. This may represent fresh insight into PI3K-regulated Akt1/2 activation in the insulin signal transduction pathway.2 and 95% air at 37 °C. When cells had reached confluence (day 0), medium was changed to DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), 1 μM dexamethasone, 0.5 mM 3-isobutyl-metyhl-xanthine, and 0.1 mg/ml insulin to differentiate from fibroblast to adipocytes (3T3-L1-GLUT4myc adipocytes). At day 3, day 7, and day 11, the medium was changed to DMEM supplemented with 10% (v/v) FBS. At day 14, cells were used for experiments. 3T3-L1-GLUT4myc adipocytes were used for all the cell experiments in this study.3T3-L1-GLUT4myc fibroblast cell line expressing GLUT4myc is constructed by inserting a human c-MYC epitope (14 amino acids) into the first ectodomain of GLUT4. Cells were cultured in DMEM supplemented with 10% (v/v) calf serum, penicillin , and streptomycin , in a humidified atmosphere of 5% COOil-Red O  was dissolved in isopropanol and left over night at room temperature. The solution was diluted with distilled water (6:4) and filtered through a filter paper. Cells on day 0, day 3, and day 14 from differentiation treatment were fixed with 10% (v/v) formalin in PBS for 10 min and stained with the Oil-Red O solution for 20 min at room temperature. Cells were washed with 60% (v/v) isopropanol and then washed twice with PBS. Fat droplets in adipocytes were visualized with a phase contrast microscope.2, 1.25 mM MgSO4, and 20 mM HEPES, pH 7.5) containing 0.2% (w/v) BSA supplemented with 10 mM glucose for 1 h at 37 °C. Cells were treated with insulin in the presence and absence of inhibitors for 20 min. Then, cells were homogenized by sonication in an ice-cold mitochondrial buffer  containing 1% (v/v) protease inhibitor cocktail  and subsequently, homogenates were centrifuged at 800 g for 5 min at 4 °C. The supernatants were centrifuged at 11 000 g for 15 min at 4 °C and further, the collected supernatants were ultracentrifuged at 100 000 g for 60 min at 4 °C to separate the cytosolic and plasma membrane fraction. The supernatants and pellets were used as the cytosolic and plasma membrane fractions respectively. Whether the cytosolic and plasma membrane components were successfully separated was confirmed in the western blot analysis using antibodies against the cytosolic marker, lactate dehydrogenase, and the plasma membrane marker, cadherin.3T3-L1-GLUT4myc adipocytes were incubated in Krebs–Ringer-HEPES buffer . Plasma membrane fraction proteins were resuspended in the mitochondrial buffer containing 1% (w/v) SDS. Proteins for each fraction were separated by SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with TBS-T  Tween-20, and 20 mM Tris, pH 7.5) containing 5% (w/v) BSA, blotting membranes were reacted with an anti-c-myc antibody (Merck Millipore) followed by an HRP-conjugated goat anti-mouse IgG antibody. Immunoreactivity was detected with an ECL kit (Invitrogen) and visualized using a chemiluminescence detection system . Signal density was measured with ImageQuant software .p-aminobenzoic ethyl ester (ABEE). Then, 5 μl ABEE-labeled solution was injected onto the column (150×4.6 mm) equipped in the HPLC system. ABEE-labeled glucose was detected at an excitation wavelength of 305 nm and an emission wavelength of 360 nm using a fluorescence detector. The concentration of glucose taken up into cells was calculated by subtracting extracellular glucose concentration after incubation for 20–120 min from initial extracellular glucose concentration (10 mM).3T3-L1-GLUT4myc adipocytes were incubated in the Krebs–Ringer-HEPES buffer containing 0.2% (w/v) BSA supplemented with 10 mM glucose for 1 h at 37 °C. Then, cells were left untreated or treated with insulin (100 nM) in PBS supplemented with 10 mM glucose for 20–120 min at 37 °C. After treatment, extracellular solution was collected and glucose was labeled with The siRNA to silence the Akt1/2-targeted gene (Akt1/2 siRNA) was obtained from Santa Cruz Biotechnology, Inc. The siRNAs to silence the PI3K p85α-targeted gene (PI3K KD), the PDK1-targeted gene (PDK1 KD), and the negative control siRNA (NC) were obtained from Ambion . The sequences of siRNAs used were 5′-GCGAAUGAUAUGUAUCAGAtt-3′ and 5′-UCUGAUACAUAUCAUUCGCtc-3′ for PI3K p85α, 5′-CCUCGUUUAUGUUUCUGCGtt-3′ and 5′-CGCAGAAACAUAAACGAGGtc-3′ for PDK1. Each NC siRNA had the scrambled sequence, the same GC content, and nucleic acid composition. siRNAs were transfected into the cells using a Lipofectamine reagent (Invitrogen). Cells were used for experiments 48 h after transfection.g for 5 min at 4 °C. The supernatant was used as total cell lysate.3T3-L1-GLUT4myc adipocytes were incubated in Krebs–Ringer-HEPES buffer containing 0.2% (w/v) BSA, supplemented with 10 mM glucose for 1 h at 37 °C. The cells transfected with and without siRNAs were left untreated or treated with insulin in the presence and absence of inhibitors for 10 min. Next, the cells were lysed in lysis buffer  Nonidet P-40, and 50 mM Tris, pH 7.4) containing 1% (v/v) protease inhibitor cocktail and 1% (v/v) phosphatase inhibitor cocktail , and then centrifuged at 800 For western blotting, proteins were separated by SDS–PAGE and then transferred to PVDF membranes. Blotting membranes were blocked with TBS-T containing 5% (w/v) BSA and subsequently reacted with antibodies against peroxisome proliferator-activated receptor γ (PPARγ) , phospho-Thr308/309-Akt1/2 (pT308(9)), phospho-Ser473/474-Akt1/2 (pS473(4)), Akt1/2 , Akt1 , Akt2 , PI3K (Sigma), PDK1 (Sigma), or β-actin (Sigma). After washing, membranes were reacted with an HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG antibody. Immunoreactivity was detected with an ECL kit (Invitrogen) and visualized using a chemiluminescence detection system . Protein concentrations for each sample were determined with a BCA protein assay kit (Thermo Fisher Scientific).2, 12.5 mM glycerol 2-phosphate, 5 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 2 mM EDTA, 0.25 mM dithiothreitol, and 250 μM ATP at 30 °C for 20 min. Phosphorylated Akt was detected by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2 as described earlier.Human recombinant Akt1 or human recombinant Akt2  was reacted without or with His-tagged human recombinant PI3K (p110β/p85α) (Sigma) or His-tagged human recombinant PDK1 , which was purified by affinity chromatography, in the presence and absence of wortmannin or BX912 in a medium containing 25 mM 3-morpholinopropanesulfonic acid (pH 7.2), 25 mM MgClt-test, Dunnett's test, and ANOVA followed by a Bonferonni correction.Statistical analysis was carried out using unpaired We initially examined whether 3T3-L1-GLUT4myc fibroblasts are differentiated into adipocytes using Oil-Red O. Adipose tissue is stained with Oil-Red O, to detect adipose conversion A. On dayTo obtain further evidence for adipocyte differentiation, we carried out western blotting using an antibody against PPARγ, an adipocyte-specific marker B. This cInsulin promoted GLUT4 translocation from the cytosol to the plasma membrane in 3T3-L1-GLUT4myc adipocytes in a concentration (1 nM–1 μM)-dependent manner A. In theAkt plays a central role in insulin-stimulated GLUT4 translocation to the cell surface. The amount of expression of Akt1 and Akt2 in differentiated 3T3-L1-GLUT4myc adipocytes was calculated from the Akt1 and Akt2 concentration/intensity standard curve, respectively. As previously shown A.The insulin (100 nM)-induced increase in the GLUT4 localization on the cell surface was abolished by the Akt1/2 inhibitor MK2206 (5 μM) B, indicaInsulin (100 nM) increased phosphorylation of Akt1/2 both at Thr308/309 and Ser473/474, indicating that insulin activates Akt1/2 in 3T3-L1-GLUT4myc adipocytes A and B. To knock-down PI3K and PDK1, we constructed siRNAs for PI3K and PDK1. In the western blot analysis, expression of PI3K and PDK1 proteins was significantly reduced in 3T3-L1-GLUT4myc adipocytes transfected with the PI3K siRNA and the PDK1 siRNA, respectively, as compared with that for cells transfected with each NC siRNA A and B, In the cell-free assay, PI3K (1 μg/ml) phosphorylated Akt1 both at Thr308 and Ser473, which was abrogated by wortmannin (20 nM) A. In conFinally, we examined whether PI3K and PDK1 regulate GLUT4 translocation to the cell surface. Insulin (100 nM)-stimulated GLUT4 translocation to the cell surface in 3T3-L1-GLUT4myc adipocytes was significantly inhibited by wortmannin (20 nM) A or knocGLUT4 is a member of the facilitative glucose transporter family, characterized by preferential expression in fat and muscle tissues, where it is responsible for insulin-induced glucose uptake into cells. GLUT4 is unique among other members of the GLUT family in its dynamic cycling within adipocytes and muscle cells. Insulin stimulates GLUT4 translocation to the cell surface by activating insulin receptor, and when insulin receptor is inactivated, GLUT4 is rapidly removed from the plasma membrane into the cytosol and accumulated in intracellular organelles such as the trans-Golgi network, recycling endosome, and diverse tubulo-vesicular bodies .et al. 2008). Akt1/2 is activated through its phosphorylation at Thr308/309 in the activation-loop of the kinase domain and Ser473/474 in the carboxy-terminal regulatory region (Akt is a key regulator for insulin-regulated GLUT4 translocation to the cell surface (y region . PI3K any region . In thisIKBKE and Pak1 have been shown to phosphorylate Akt at Ser473 in a PI3K-independent manner (The three Akt isoforms Akt1, Akt2, and Akt3 have critical and distinct functions in the regulation of metabolism, cell growth, and apoptosis. A growing body of evidence has argued that Akt2 is required for GLUT4 translocation. Insulin activates both Akt1 and Akt2 in 3T3-L1 and primary rat adipocytes . IsoformIn this study, insulin-stimulated GLUT4 translocation to the cell surface in adipocytes was prevented by the Akt inhibitor MK2206 and knocking-down Akt1/2, the PI3K inhibitor wortmannin and knocking-down PI3K, or the PDK1 inhibitor BX912 and knocking-down PDK1. This indicates that insulin stimulates GLUT4 translocation to the cell surface by activating Akt1/2 through a pathway along an IRS/PI3K/PDK1/Akt axis in adipocytes. Akt2 was abundantly expressed in adipocytes and Akt2 activation was achieved by cooperation of PI3K and PDK1 in the cell-free assay. This indicates that insulin dominantly activates Akt2 following activation of PI3K and the downstream effector PDK1 in adipocytes, responsible for GLUT4 translocation. Strangely, insulin-induced phosphorylation of Akt1/2 both at Thr308/309 and Ser473/474 in adipocytes was abrogated by wortmannin and knocking-down PI3K, but otherwise no significant inhibition of Akt1/2 phosphorylation at each site was obtained with BX912 and knocking-down PDK1. A plausible explanation for this is that PDK1 might stimulate GLUT4 translocation to the cell surface by phosphorylating or interacting with an unknown factor, as its downstream target, regardless of Akt1/2 phosphorylation at Thr308/309. To address this, we are currently carrying out further experiments.In conclusion, the results presented here clearly demonstrate that insulin stimulates GLUT4 translocation to the plasma membrane in a PI3K-, PDK1-, and Akt1/2-dependent manner in 3T3-L1-GLUT4myc adipocytes. The results also show that PI3K phosphorylates Akt1 both at Thr308 and Ser473 and Akt2 at Ser474 and that PDK1, a downstream effector of PI3K, phosphorylates Akt1 and Akt2 at Thr308 and Thr309 respectively. This implies that Akt1 is activated by PI3K alone and that Akt2 activation is achieved by cooperation of PI3K and PDK1. The results of the present study, thus, may extend our understanding about Akt1/2 activation pathways linked to insulin signals.A Tsuchiya and T Kanno performed all the experiments, analyzed the data, and prepared the figures. T Nishizaki designed the experiments and wrote the manuscript. All authors have approved the final manuscript.


To define the primary stimulus perceived by KdpD we focused in this study on the dynamics of the Kdp response. Escherichia coli cells were subjected to severe K+ limitation, and all relevant parameters of the Kdp response, i.e., levels of kdpFABC transcripts and KdpFABC proteins, as well as extra- and intracellular K+ concentrations, were quantitatively analysed over time (0 to 180 min). Unexpectedly, induction of kdpFABC was found to follow a non-monotonic time-course. To interpret this unusual behaviour, a mathematical model that adequately captures the dynamics of the Kdp system was established and used for simulations. We found a strong correlation between KdpD/KdpE activation and the intracellular K+ concentration, which is influenced by the uptake of K+ via the KdpFABC complex. Based on these results a model is proposed in which KdpD/KdpE phosphorylation is inversely correlated with the intracellular K+ concentration. To corroborate this hypothesis an isogenic mutant that produces a defective KdpFABC complex, and the trans-complemented mutant that expresses the KtrAB high-affinity K+ uptake system of Vibrio alginolyticus were quantitatively analysed. Experimental data and simulations for the mutants consistently support the tight correlation between KdpD/KdpE activation and the intracellular K+ concentration. This study presents a striking example of the non-intuitive dynamics of a functional unit comprising signalling proteins and a transporter with K+ as mediator.KdpD and KdpE form a histidine kinase/response regulator system that senses K It is required for the regulation of cell turgor E. coli possesses three K+ uptake complexes. Two of these, TrkG/TrkH and Kup, are constitutively produced and have low affinities for K++ transport complex KdpFABC, is only induced when the other transporters are unable to supply the cell's requirement for K++ limitation (at extracellular K+ concentrations <2 mM), mutation of trk, or under hyperosmotic stress imposed by NaCl An adequate supply of the monovalent cation KkdpFABC operon is controlled by the histidine kinase/response regulator system KdpD/KdpE kdpFABC promoter and thereby triggers transcription of the operon (in vitrokdpFABC expression (phosphatase activity) (Expression of the e operon  [20]. Thctivity)  [21].+] as the control signal for KdpD, and suggested that the primary stimulus might be either the level of intracellular K+, processes associated with K+ transport, or the external K+ concentration kdpFABC expression is at least ten-fold higher under K+ limitation than that induced in response to salt stress, which argues for a specific effect of K+ on KdpD +, which is taken up and significantly reduces the availability of free intracellular K+, induces kdpFABC expression + concentrations seen in E. coli exposed to acid stress is accompanied by an increase in kdpFABC expression In vitro phosphorylation assays based on right-side-out membrane vesicles have revealed an inhibitory effect of K+ on the kinase activity of KdpD + has also been observed during the in vitro reconstitution of the complete signal transduction cascade, consisting of KdpD in proteoliposomes, purified KdpE, a DNA fragment comprising the KdpE-binding site, and a mixture of ATP/ADP. The higher the K+ concentration, the lower the level of phosphorylated KdpE + concentration on the activity of KdpD. However, more recent studies have indicated that KdpD activation cannot be solely attributed to the intracellular K+ content, and that the extracellular K+ concentration also plays a role The nature of the primary stimulus perceived by KdpD remains contentious. Epstein and coworkers proposed that KdpD senses a decrease in turgor or some effect thereof To complete this picture it should be noted here that KdpD activity is also modulated by alterations in the ionic strength in vivo. Quantitative analyses of components of biochemical reaction pathways are generally problematic, and represent a real challenge in the life sciences. Mathematical modelling, which allows one to reconstruct biochemical reaction networks in silico, provides one way around this impasse. Especially in cases where there is uncertainty about interactions between biochemical compounds, model analysis can help to decide whether certain network topologies can explain experimental observations. Given some prior knowledge or assumptions about the reactions they are involved in, the dynamic behaviour of unmeasured parameters can be simulated. Hence such models can also yield virtual “measurements” of these networks.Thus the data collected over the years have not provided a definitive answer to the question of the nature of the primary stimulus for the KdpD/KdpE system. This is largely because it is difficult to determine the various functional states of KdpD and KdpE + affects the KdpD/KdpE system in vivo, we combined a quantitative experimental analysis with mathematical modelling. First we extended and modified an existing model of the Kdp system  at time zero , and declined again thereafter. It is generally accepted that a significant fraction of the intracellular K+, referred to as the “bound” fraction, is associated with anionic macromolecules. This fraction has a much reduced osmotic and ionic activity, and can therefore be distinguished experimentally from the “free” K+ fraction + increased after a lag time, and remained at a plateau level over the next 20 min. Thereafter, this fraction decreased at a constant rate, and was barely detectable at t = 70 min. These results reveal a clear inverse correlation between kdpFABC transcript number and the concentration of free K+ ions in the cell , which encodes a KdpFABC complex with an altered K+-binding site kdpA (Gly345 to Ser), which replaces a glycine in the K+ selectivity filter III by a serine residue. Replacement of this amino acid reduces the affinity for K+ by about 30-fold + under conditions of K+ limitation. The strain also carries a chromosomal point mutation in the rpsL gene, which confers streptomycin resistance and was used for selection during strain construction kdpFABC transcription or KdpFABC synthesis (data not shown).To test this hypothesis further, we wished to analyse a mutant in which KE. coli RH010 cells were subjected to the same severe K+ limitation as the wild type strain. In these cultures the extracellular K+ concentration also decreased over time, but at a much lower rate relative to the wild type , and the transformants were treated and analysed as before. Because expression of ktrAB is constitutive, E. coli RH010 cells producing KtrAB consumed the supply of extracellular K+ much faster than the wild type, which is indicated by a significant drop of the extracellular K+ concentration in the initial phase . As the enzymatic reactions involved occur too rapidly for experimental investigation in vivo, we used mathematical modelling to address this issue. First we had to extend the existing model of the Kdp system +] and intracellular K+ concentrations (free and bound) as well as the total cell volume in our samples. The detailed derivation of the full model is set out in +) and non-stressed (10 mM K+) cells were found to be marginal  either inhibits the kinase or activates phosphatase activity of KdpD (a or b). One can deduce several possible network topologies from these considerations: (i) external K+ (+exK) inhibits KdpD autophosphorylation; (ii) free intracellular K+ (+freeK) inhibits KdpD autophosphorylation; (iii) +exK enhances dephosphorylation of KdpE-P, or (iv) +freeK enhances dephosphorylation of KdpE-P. Moreover, these effects may act in combination: (i) +exK inhibits the kinase activity and +freeK enhances the phosphatase activity of KdpD, or (ii) vice versa. Therefore, we generated several model variants  that depicted these different network topologies and tried to calibrate them against the experimental data for the wild type and the RH010 mutant strain . Initial calibration was performed by manual adjustments made by the modeller during the simulations to improve the fit to the experimental data (see www.mathworks.com) toolbox that integrates different methods for sensitivity analysis and parameter estimation of dynamic models. All parameters used are summarized in Finally, the model parameters had to be tuned to replicate the data reported above for wild type and the RH010 mutant strain , i.e. todata see . Fine-tu+freeK at early time points (t = 0–40) after the shift to K+ limitation for the RH010 mutant, which showed a nearly 2-fold decrease in +freeK compared to the wild type BC, or other unknowns which could not be implemented in the model, might influence the +freeK pool.The final model faithfully reflected the experimental data for ild type . At late+ limitation. However, we also tested the capacity of the model to qualitatively reflect the response of the in vivo system to a broad range of extracellular K+ concentrations. With increasing K+ availability the constitutive K+ uptake system Trk is expected to become more important, and therefore an uptake rate term for this transporter had to be included in the model ) were varied from 0.04 to 8 mM. +exK concentrations, whereas numbers of KdpFABC complexes , whereas under moderate (0.1 to 2.1 mM) or higher K+ concentrations (>2.1 mM), the model captured the decrease in KdpFABC production qualitatively but not quantitatively , K+ uptake constantly compensates for dilution of the internal K+ pools by cell growth.We also predicted the time-courses of free and bound K+ . Under e+ concentrations. Since experimental determination of KdpD and KdpE phosphorylation kinetics in vivo is not possible thus far, the simulations can be seen as virtual measurements. Both KdpD-P and KdpE-P exhibited non-monotonic time-courses for all initial +exK concentrations , reached a peak in less than 20 min, and then declined before rising again at 30 min. This transient dip points to negative feedback regulation of kdpFABC transcription, which was predicted in our earlier in silico and in vivo studies, and was also observed in an independent study + falls significantly, and the cells essentially run out of K+ once more. It is important to note that this loss of K+ is attributed to the onset of growth. Consequently, the KdpD/KdpE signalling cascade and kdpFABC transcription are activated for a second time. At this stage, the level of kdpFABC expression begins to rise again to meet the cell's significantly increased need for K+. Thereafter (t = 60 min) a steady state is attained, marked by continued transcription of kdpFABC and synthesis (and very probably turnover) of KdpFABC, cell division and cell lysis, with concomitant release and re-uptake of K+. Note that, over the whole time-course, the extracellular K+ concentration decreased at a constant rate, and was clearly uncoupled from KdpFABC production and hence from KdpD/KdpE activation.When + on KdpD/KdpE phosphorylation in vitro + environment, the ion's inhibitory effect on the phosphorylation of KdpD/KdpE is relieved. Specifically, according to our simulations, inhibition of the phosphatase activity of KdpD is required to keep KdpE in the phosphorylated state and enable high-level production of KdpFABC, which in turn increases the amount of free K+ in the cell. Our calculations indicated that this increase in the concentration of free intracellular K+ enhances KdpD phosphatase activity and thus reduces the concentration of phospho-KdpE. This is the first indication of an inverse correlation between KdpD/KdpE phosphorylation and the concentration of free intracellular K+.The observed pattern of KdpFABC production, and the known inhibitory effect of K+ concentration modulates KdpD/KdpE phosphorylation is further supported by our finding that no hint of negative feedback regulation is observed when the KdpFABC transport-defective E. coli mutant RH010 is subjected to similar K+ limitation. Moreover, the expression profiles in the RH010 mutant also argue against an effect of the extracellular K+ concentration on KdpD/KdpE activation under these conditions. Thus, although - due to the defect in K+ uptake by the mutant KdpFABC complex - the extracellular K+ concentration declines more slowly than in case of the wild type, levels of kdpFABC transcripts rise to far higher levels. Notably, trans-complementation of this E. coli mutant by the high-affinity KtrAB K+ transporter from V. alginolyticus reduced kdpFABC expression to a level comparable to that in wild type. The experimentally determined and theoretically modelled data for the non-complemented mutant strongly support the notion that the intracellular K+ concentration is an important modulator of KdpD/KdpE activities under conditions of severe K+ limitation. Concomitantly, these results throw light on the mode of negative feedback regulation + by the KdpFABC complex down-regulates its own expression. Regulation of KdpD/KdpE phosphorylation by the intracellular K+ concentration is in line with earlier observations which indicated that both extracellular Cs+ and low pH significantly reduce the availability of free intracellular K+ and lead to induction of kdpFABC expression The hypothesis that the intracellular KkdpFABC expression, and therefore KdpFABC production, is not only induced at very low external K+ concentrations (below 0.1 mM), but gradually increases at moderate K+-limiting conditions (2 to 0.1 mM) + concentrations below 0.1 mM, but deviated from them under moderately K+-limiting conditions (0.1 to 2 mM) and non-stress conditions (above 2.1 mM)  .+ pool predominantly acts as primary stimulus under extreme K+ limitation, and that external K+ influences KdpD/KdpE activation under moderate stress conditions, when a significant drop in the intracellular K+ concentration is unlikely  could be identified which lumps together several kinetic constants for transcription initiation. After re-estimation the accuracy could be improved. It is likely that the Kdp system is not only controlled at the transcriptional level, but also at the post-translational level by proteolysis, and at the activity level + is unclear.During modelling it emerged that the response of the RH010 mutant that produces an inactive KdpFABC was hampered by the fact that the ratio of transcript to protein at steady-state differed from the values for the wild type strain. By means of sensitivity analysis, one parameter , and 3H2O and [14C]sucrose were obtained from Biotrend (Cologne). SYBR Green Mix was from BioRad (Munich), the DyNAmo™ cDNA Synthesis Kit and Protein A magnetic beads were from New England Biolabs (Frankfurt am Main). Goat anti-(rabbit IgG)-alkaline phosphatase was obtained from Biomol (Hamburg). Purified KdpFABC protein was supplied by Marc Bramkamp (Osnabrück University). RNase-free deoxyribonuclease I was from Fermentas (St. Leon-Rot), and silicone oil (DC550) was from Serva (Heidelberg). All other reagents were reagent grade and obtained from commercial sources.sucrose  were added to 1.3-ml samples of growing E. coli cells. After 1 min, cells were centrifuged for 2 min through a 200-µl silicone oil layer of appropriate density (see above). The cell pellets were separated from the supernatants, and cells were lysed by treatment with 1 ml 0.4 M NaOH at 62°C for 1 h. The radioactivity of the two labelled compounds in the cell pellet was quantified simultaneously by liquid scintillation counting. The dual-label protocol was used so the original counts per minute of each nuclide could be converted into disintegrations per minute (dpm) taking the overlap of the energy distribution spectra of the two nuclides into account. For quantification, the radioactivity present in 50 µl of supernatant was also determined. As 3H2O penetrates into every part of the cell, the total cellular water space including the extracellular water layer was calculated as follows: (3H dpm of the pellet) ×50 µl/(3H dpm of 50 µl supernatant). Since [14C]sucrose is only excluded from the cytoplasm, the volume of the extracellular water layer plus the periplasmic space was calculated as follows: (14C dpm of the pellet) ×50 µl/(14C dpm of 50 µl supernatant).The size of the cytoplasmic water space can be used as a proxy for the cytoplasmic volume 3H2O and the space occupied by [14C]sucrose gives the volume of cytoplasmic water, which includes all water layers bound to biological surfaces, the so-called bound water The difference between the space occupied by E. coli strains were determined by quantitative Western blot analysis. The respective E. coli strains were grown as described above. At the time points indicated, aliquots of the cultures were removed and the cells were collected by centrifugation, resuspended in SDS sample buffer and subjected to SDS-PAGE 3, pH 9.5, 0.01% (w/v) nitro-blue-tetrazolium, 5 mg/ml 5-bromo-4-chloro-3-indolylphosphate]. Blots were scanned at high resolution in 256 grey scales and imported as TIF files into ImageQuant 5.0, and the amount of KdpB was quantified by comparison with the standard curve.Levels of KdpFABC in + limitation was analysed in vivo in E. coli MG1655 rpsL150 and E. coli RH010 (MG1655 rpsL150 kdpA4) by labelling the complex with [35S]methionine. Cells were grown to exponential phase and shifted to K+ limitation as described above. After 10 min, 2.5 µCi/ml  [35S]methionine was added, and cells were incubated for an additional 10 min to allow uptake of label into de novo synthesized proteins, in this case predominantly the subunits of the KdpFABC. Then, non-radioactive methionine was added in excess  to block further radiolabeling of nascent proteins. After different times 1-ml culture aliquots were collected, and immediately frozen in liquid nitrogen to stop protein biosynthesis. Before detection of radiolabeled KdpFABC, the protein was concentrated using immunomagnetic separation. For this purpose, cells were sonified  and proteins were solubilized with 1.5% (w/v) n-dodecyl-β-D-maltoside. αKdpFABC antiserum n-dodecyl-β-D-maltoside], and proteins were finally removed from the beads by addition of 30 µl 3-fold concentrated SDS sample buffer 35S, were then subjected to SDS-PAGE The stability of KdpFABC under KkdpFABCDE and the synthesis of its protein products. The first module comprises the rate equations for autophosphorylation (R1) of the sensor kinase KdpD [D], transfer of the phosphoryl group (R2) from KdpD-P [PD] to the response regulator KdpE [E], and the dephosphorylation (R3) of free KdpE-P [PfE] resulting in inorganic phosphate [Pi]:fP] to free sigma factor [fσ] (R4a), the binding of the polymerase complex [P] to promoter DNA [fDNA] ((R4b)+(R4e)) and the binding of two free phosphorylated KdpE response regulator proteins [PfE] to their cognate binding site in the DNA ((R4c)+(R4d)). The order of binding of polymerase and response regulator is mutually independent; however the second reactant binds with a higher affinity, represented by the factor α. The parameters i K denote the equilibrium dissociation constants i = k-i/kiK. Reactions (R4a)-(R4e) are assumed to be very fast in comparison to protein synthesis. Therefore the rapid equilibrium approach was applied to each of the reactions, leading to a set of algebraic equations that describe the protein complexes as functions of the protein concentrations and the reaction constants:fP and fσ is almost impossible, both variables are considered to be constant and are combined in a single parameter K, together with the dissociation constants σK and PK).The mathematical model of the Kdp system PD and PE , two algebraic equations to calculate the amounts of free DNA fDNA, and the unbound response regulator PfE, and two algebraic equations to determine the amount of unphosphorylated sensor kinase D and response regulator E:D0 and E0 are the total concentrations of the sensor kinase KdpD and the response regulator KdpE, respectively. The balance equations for these variables will be derived in the following. The concentration of DNA0 is always taken to be constant.Thus, the mathematical description of the first module comprises two differential equations for F] production, the model was extended to include a second module comprising equations for the transcript dynamics (reflecting synthesis and stability of the mRNA), and the dynamics of the proteins KdpFABC, KdpD and KdpE:trr and tlir  do not depend on the monomer concentration.To describe the overall module up to the level of KdpFABC  was modelled using the following differential equationdegk is the degradation time constant. If protein degradation is actively controlled, degk is expected to vary under different experimental conditions. The specific growth rate μ is also time dependent; therefore it is determined using the empirical equation given above.For the analysis of proteolysis, the degradation of the corresponding protein [Prior to parameter estimation, parameter sensitivities and correlations needed to be analysed in order to detect potential sources of identifiability problems. Parameters that show very little sensitivity compared to others, as well as correlations among parameters , can lead to far-from-optimal results  showed very little sensitivity and were highly correlated with each other. Therefore, these parameters are virtually unidentifiable and could be set to nominal values, and were therefore excluded from parameter estimation. The parameters describing the release of K+ due to cell lysis and the synthesis of the proteins KdpD and KdpE also showed low sensitivities. In addition, we found high correlations among the parameters describing the transcription of kdpFABC.Several parameters that determine the dynamics of the two-component system  concentrations of the sigma factor and polymerase, as well as the equilibrium constants for the reactions in which these two variables are involvedIn order to describe and reproduce the experimental data for the RH010 mutant, the wild type model was used, albeit with appropriate adjustments of several parameters. To account for the lower rate of K changed . Further+ transporter KtrAB from Vibrio alginolyticus + concentrations showed the same qualitative behaviour as in the wild type. However, the intracellular concentrations of free and bound K+ were approximately 30% lower than those measured for the wild type. Hence, the mathematical model of the wild type was modified to match this situation. We added another uptake termmax,KtrV and M,KtrK were adjusted in accordance with the measured K+ data. With these modifications, the model was found to reproduce the experimental findings quite well.The RH010 mutant was also complemented in trans with the high-affinity KFigure S1Proteolysis controls appropriate KdpFABC level.E. coli MG1655 rpsL150 (wild type) and E. coli RH010 were cultivated in phosphate buffered minimal medium containing 10 mM K+ up to the mid-logarithmic growth phase, exposed to extreme K+ limitation (0.04 mM K+) for 10 min to activate kdpFABC expression before proteins were labelled with 35S-methionine. Labelling of de novo synthesized proteins was quenched by adding an excess of non-labelled methionine after 10 min. Protein turnover is determined by the reduction of the labelled protein complex over the time. At different times, samples were taken, immunoprecipitated with αKdpFABC antiserum, subjected to SDS-PAGE, and the amount of KdpFABC was quantified from the autoradiographies of the gels. The data represent one of three independently performed characteristic experiments. Half-life was determined to be 36.7 min for wild type KdpFABC and 28.4 min KdpFA(G345S)BC.(TIF)Click here for additional data file.Table S1Total and viable cell numbers and cytoplasmic volume after exposure of E. coli+ limitation. to KE. coli MG1655 cells were grown in minimal medium containing 10 mM K+ to the mid-logarithmic growth phase and shifted to K+ limitation (40 µM K+). At the indicated times, cells were harvested, and the total and viable cell numbers as well as the cytoplasmic volume of the cells were determined as described in (DOCX)Click here for additional data file.


Therefore, this enzyme is an attractive target for antimalarials. However, biochemical investigations of the parasite enzyme needed for the study of new drugs are challenging. In order to facilitate the study of new compounds targeting the enzyme, we are modifying the inhibitor binding sites of the yeast Saccharomyces cerevisiae to generate a complex that mimics the P. falciparum enzyme. In this study we focused on its Qo pocket, the site of atovaquone binding which is a leading antimalarial drug used in treatment and causal prophylaxis. We constructed and studied a series of mutants with modified Qo sites where yeast residues have been replaced by P. falciparum equivalents, or, for comparison, by human equivalents. Mitochondria were prepared from the yeast Plasmodium-like and human-like Qo mutants. We measured the bc1 complex sensitivity to atovaquone, azoxystrobin, a Qo site targeting fungicide active against P. falciparum and RCQ06, a quinolone-derivative inhibitor of P. falciparum bc1 complex.The data obtained highlighted variations in the Qo site that could explain the differences in inhibitor sensitivity between yeast, plasmodial and human enzymes. We showed that the yeast Plasmodium-like Qo mutants could be useful and easy-to-use tools for the study of that class of antimalarials.The  P. falciparum are compromising the treatment of malaria patients. Thus there is an urgent need for new antimalarial drugs. The complex III or bc1 complex is an attractive target for antimalarial drugs. This respiratory chain complex is essential for P. falciparum proliferation as its catalytic activity is critical for the maintenance of the mitochondrial membrane potential and for the reoxidation of ubiquinol, which is needed for the ubiquinone-dependent dihydroorotate dehydrogenase, and by consequence for the biosynthesis of pyrimidine  as antimalarial drug in treatment and causal prophylaxis. The compound is also used to treat Pneumocystis jirovecii pneumonia, Toxoplasma gondii toxoplasmosis, and other infections. In the USA, Malarone® prescription accounted for more than half of all antimalarial prescriptions. However, the cost of atovaquone is so far prohibitive for more general use. The patent for Malarone® expires this year, which might result in lower cost generics. With the possibility of more extensive use of atovaquone, the risk of resistance to atovaquone would likely increase.The hydroxynaphthoquinone atovaquone is currently the sole P. falciparum atovaquone-resistant parasites have been reported to emerge during atovaquone-proguanil therapy, leading to typical treatment failure H)-pyridones, acridones, acridinediones, and 4(1H)-quinolones  is provided by the main subunit of the bc1 complex, cytochrome b. Cytochrome b is mitochondrially encoded in all eukaryotes while the other subunits of the complex are nuclearly encoded. The Qo site is a relatively large domain formed from components encompassing amino acid residues 120–150 and 260–280 of the cytochrome b. Hydrogen bonds form a ‘loose stitching’ that allows a degree of expansion of the site on occupant binding o site, such as components of the ‘PEWY’ motif , demonstrate considerable conformational flexibility on inhibitor binding o site forms a bifurcated volume. On the basis of structural and mutational analysis, Qo inhibitors can be separated into two classes by their mode of binding bl proximal’ domain of the Qo site whereas stigmatellin binds in the ‘haem bl distal’ domain, occupying the region of the Qo site close to the interface with another catalytic subunit of the complex, the iron-sulphur protein (ISP). Crystal structures for several inhibitor-liganded bc1 complexes are available, such as azoxystrobin- and stigmatellin inhibited enzymes  model. By modifying the inhibitor binding sites of the yeast native bc1 complex, new versions can be generated that mimic the P. falciparum enzyme. In this study, we constructed a series of yeast mutants harbouring variants of the Qo site where yeast residues have been replaced by P. falciparum equivalents, or, for comparison, by the human equivalents. The yeast mutants with Plasmodium-like (and human-like) Qo site were used to investigate the structural determinant of the differential sensitivity to atovaquone and azoxystrobin and to explore the binding mode of the novel molecule RCQ06. We also constructed a mutant harbouring the atovaquone resistance mutation Y279S  in the Plasmodium-like enzyme to study the effect of this mutation on new inhibitors such as RCQ06. The yeast mutants could be useful tools for the study of new drugs with selective activity and for the analysis of resistance mutations.In order to facilitate the study of drugs targeting the c, decylubiquinone, azoxystrobin and atovaquone were obtained from Sigma. RCQ06 was kindly supplied by Prof. Paul O’Neill .Equine cytochrome b gene.The mutants were generated by side-directed mutagenesis and mitochondrial transformation as described in c reductase activity measurements were performed in 10mM potassium phosphate pH 7 and 20µM equine cytochrome c at room temperature. Mitochondria were diluted to 5–30 nM bc1 complex. Concentrations of monomeric bc1 complex were determined from the cytochrome b α-band in dithionite-reduced optical spectra, using ε = 28.5 mM−1.cm−1 at 562 nm minus 575 nm. Activity was initiated by the addition of 20µM decylubiquinol. Cytochrome c reduction was recorded at 550 nm versus 540 nm over a 3-min time-course in a Beckmann DU 640 spectrophotometer. Initial rates were measured. From these rates, turnover numbers (TN) were calculated as cytochrome c reduced per bc1 complex per second.Yeast mitochondria were prepared as in c reduction activity was measured as described above in presence of increasing concentrations of inhibitors (six to ten different concentrations). Each measurement was repeated at least twice and averaged. The errors did not exceed 10%. The mid-point inhibition concentrations (IC50) were determined from the titrations. As the titrations were performed using mitochondrial samples containing different concentrations of bc1 complex (5–30 nM), the results were presented as ratio of IC50 on the concentration of bc1 complex.Cytochrome o site of yeast cytochrome b (3CX5.PDB) as described in b residue E272 in the Qo site of 3CX5.PDB using EADock DSS via SWISSDOCK Atovaquone was docked into the Qbc1 complex (cytochrome c reductase) activity towards atovaquone, azoxystrobin and RCQ06 is presented in bc1 complex, as P. falciparum enzyme, is highly sensitive to atovaquone (IC50 of 4 (molar ratio)) while the bovine enzyme was less reactive (IC50 of 75). Azoxystrobin has been shown to be a potent inhibitor of P. falciparum proliferation with an IC50 in the nanomolar range (15 nM bc1 complex with an IC50 of 20 while it is less active against the bovine enzyme (IC50 of 180). RCQ06 was also reported as potent inhibitor of the plasmodial bc1 complex 50 of around 1.3 nM, which is in the same range than the atovaquone IC50 (around 3 nM) 50 of 40). The differential sensitivities to RCQ06 and atovaquone between the Plasmodium and mammalian enzymes are thus similar. By contrast, the yeast enzyme was highly resistance to the RCQ06 (IC50>500).The sensitivity of the ge 15 nM . The druo site of the yeast bc1 complex, single mutations and combined Plasmodium- or human-like changes.In order to study the structural determinants that could explain the differential sensitivity to these drugs, we monitored the impact of mutations introduced in the Qbc1 complex (as compared to the sensitivity of the yeast enzyme) was investigated. A computed energy-minimised structure for atovaquone liganded to the yeast bc1 complex suggested that residue 275 plays a key role in the differential sensitivity. The presence of a phenyalanine at position 275 in the bovine enzyme would hinder the drug binding. F275 being replaced by L in the yeast enzyme, the steric constraint is lessened and the sensitivity to atovaquone increases. When the mutation L275F was genetically introduced in the yeast bc1 complex, its sensitivity was significantly decreased (over 20-fold) In previous works, the low atovaquone sensitivity of the bovine b of P. falciparum (and of other Plasmodium and of Toxoplasma), the F275 variant is naturally present. However the parasite bc1 complex is highly sensitive to atovaquone. Thus other structural variations should explain the differential sensitivity between the parasite and the mammalian enzymes.In the cytochrome b sequences . None of the modifications introduced in the Qo site severely affected the overall assembly of the complex.In order to investigate their impact, we constructed a series of yeast strains harbouring multiple changes in the Qents see : in posio site, the sequence comparison . The study of the deletion and of its possible compensations will be reported elsewhere.In addition to substitutions in the Qmparison  showed a Qo site . The delPlasmodium sp., T. gondii and P. jirovecii) are located at positions 139, 147, 148, 150, 152, 266, 269, 275, 278, 279, 282, 283, 286, 291 and 295 P. falciparum, with the exception of residues 275, 283 and 295. Thus the Plasmodium-like mutations studied here are located at residues or closed to residues involved in acquired-atovaquone resistance.It should be noted that the acquired atovaquone-resistance mutations found after drug exposure of parasites  whereas F275 alone in the yeast Qo site (mutant L275F) caused a strong atovaquone resistance (IC50 of 150). A similar behaviour was observed with PF2 and PF8 but the atovaquone sensitivity was less marked . In Rhodabacter capsulatus cytochrome b, a phenylalanine is naturally present at this position. The IC50 of atovaquone was estimated to be around 30 nM. Interestingly, the sensitivity of the bacterial complex to atovaquone was increased (IC50 decreasing from 30 nM to 10 nM) by the introduction of two mutations replacing bacterial residues by the plasmodial equivalents, namely I281M and R283K (yeast numbering) Plasmodium-like mutations, the impact of L275F is weakened. From these data, it appears that the atovaquone sensitivity of P. falciparum bc1 complex could be explained by a combination of variations (variations from the yeast Qo site) that cancel the resistance effect of F275. We could hypothesise that the P. falciparum Qo site and the yeast Plasmodium-like Qo site have a larger volume that could accommodate atovaquone despite a phenylalanine in position 275. The modified yeast bc1 complex harbouring a Plasmodium-like Qo and highly susceptible to atovaquone (PF11) could be useful model to study the effect of resistance mutations.Interestingly, PF11 was highly sensitive to atovaquone despite the presence of F275   .o site of yeast bc1 complex and introduced human variations, single or combined (o site. The mutant enzyme combining eight human changes (HSc) showed nearly WT activity (89%).To investigate further the observed resistance, we modified the Qcombined . The sub50 increased 35-, 9- and 2.5-fold in L275F, F278A and M295L, respectively. The combination of these mutations (HSa and HSb) caused a further increase of resistance. When combined with amino-acid changes at positions 133 to 141 (HSc), the resistance to atovaquone was decreased, as observed with Plasmodium-like mutants. However, the human-like bc1 complex remained 15-fold more resistant than the WT enzyme, with an IC50 value of 60, similar to the IC50 value of 75 obtained with the bovine enzyme. It can thus be suggested that the combination of F275, A278 and L295 in human bc1 complex is responsible for the natural resistance towards atovaquone.The three human-like mutations, L275F, F278A and M295L, conferred some level of resistance towards atovaquone. The IC50 of 140). Thus the 9-fold resistance of the mammalian enzyme to azoxystrobin (IC50 of 180), compared to yeast enzyme (IC50 of 20) could be explained by the combination of F275, A278 and L295 in the binding site.In the human-like mutants, F278A caused a 6-fold increase in azoxystrobin resistance, M295L and L275F had a weaker effect. The triple mutation (HSb) conferred 8-fold resistance. Changes in position 133 to 141, when combined with that triple mutation (HSc), did not affect the resistance , but the changes in position 133 to 141 lowered the resistance by 5 fold (IC50 of 40 for PF11). That combination of residues might explain the susceptibility of P. falciparum to azoxystrobin.In the i site inhibitor antimycin of the bc1 complex of the human- and the Plasmodium-like mutants, HSc and PF11, with that of the WT control. No or very little difference was observed (data not shown).As a control, we compared the sensitivity to the QP. falciparum isolated from patients after treatment failure, three mutations have been reported, Y279S/C/N . Mutations Y279S and C are the most frequent mutations in the parasite. Their consequences on the catalytic activity and the stability of the complex have been characterized in the P. falciparum (Y268S) In P. falciparum resistant enzyme. As presented in o site caused a 70% decrease of the bc1 complex activity. In the parasite bc1 complex, the acquired resistance mutation resulted in a low activity and unstable ISP o site via tyrosyl-benzoquinone hydrophobic packing, facilitating quinol deprotonation and electron transfer to the ISP We introduced the mutation Y279S in PF11 to obtain a model mimicking Plasmodium-like Qo site (PF12) or in the yeast Qo site, Y279S caused a high level of resistance to atovaquone (IC50>850), similar to that observed with the Y268S plasmodial enzyme. By contrast, the sensitivity to azoxystrobin was little affected by the atovaquone resistance mutation . This result was not unexpected since azoxystrobin has been shown to bind in the Qo site at a position proximal to the haem blblWhen introduced in the bc1 complex is highly resistant to RCQ06. Strikingly, the introduction of mutation L275F rendered the enzyme over 50-fold more sensitive to RCQ06 than WT. The sensitivity to RCQ06 was observed in all the mutants with L275F in the Qo site: PF2, PF8, and PF11. Thus RCQ06 binding is likely to be stabilised by an aromatic residue at position 275, whereas the binding of atovaquone is hindered by the bulkier residue. The chemical structures of RCQ06, atovaquone and azoxystrobin are presented in As shown in Plasmodium-like Qo site (PF12) caused a high level of resistance to RCQ06, as it did for atovaquone. This suggests that the two inhibitors have similar binding mode. On the basis of this mutational analysis, a model of RCQ06 has been build. o site of the yeast bc1 complex. As mentioned in the introduction, a crystal structure of the atovaquone-bound enzyme is not available but atovaquone is predicted to interact with the Qo site in a manner similar to that of stigmatellin via hydrogen bonding from its naphthoquinone moiety via a bridging water molecule to E272 (yeast numbering) of cytochrome b, with a second hydrogen bond formed to the imidazole group of the ISP [2Fe2S] cluster ligand H181 The introduction of the atovaquone resistance mutation Y279S in b residue E272. This association would require that the glutamyl is acting as the hydrogen bond acceptor, and is thus present in the deprotonated (carboxylate) form, which is thermodynamically accessible when haem lb is oxidised b. Inspection of the model presented in Our model suggests that RCQ06 binds in a manner reminiscent to that of stigmatellin with a 3.5Å hydrogen bond between its quinolone amine moiety and the glutamyl sidechain of cytochrome b residue Y279 . New drugs are needed that can circumvent the acquired atovaquone resistance: for example, drugs targeting the Qi site o site in the haem bl-proximal region such as azoxystrobin or in the distal site but with different binding interactions than atovaquone Acquired resistance to atovaquone developed readily, which is caused by the mutation of the cytochrome b sequences from yeast, human and P. falciparum are highly conserved, making yeast an attractive model to investigate sensitivity and resistance to drugs targeting the bc1 complex. Crystal structures of yeast bc1 complex are available. Most importantly, genetic tools have been well developed to introduce designed mutations in yeast mitochondrially-encoded cytochrome b gene. Yeast is the only organism, with the green algae Chlamydomonas reinhardtii, amenable to mitochondrial transformation. By modifying the yeast enzyme, useful tools could be created for the discovery and the analysis of new drugs with potential antimalarial activity and for the study of the development of resistance mutations.Cytochrome o site Plasmodium-like Qo sites that mimic the site of the plasmodial enzyme. We tested the sensitivity to atovaquone and azoxystrobin to validate the model. We showed that the differential sensitivity to atovaquone and azoxystrobin, and the high resistance caused by Y279S (Y268S) could be reproduced in the yeast models.In previous reports, we studied acquired resistance mutations found in parasites and introduced in yeast Qo site, indicated that the drug shares the same binding pocket than atovaquone. Even though the binding mode of RCQ06 might not be identical to that of atovaquone, the efficiency of binding is affected by the Y279S mutation in the yeast enzyme.We then used the mutants to study the binding mode of a new molecule RCQ06. The experimental data, together with the model of RCQ06 binding in the Qo and Qi sites will be performed to obtain easy-to-use models that mimic more accurately the plasmodial enzyme. These mutants with a P. falciparum-like enzyme could be useful tools for the discovery and the analysis of new drugs with potential anti-malarial activity and for the prediction of sensitivity/resistance phenotype associated with naturally occurring cytochrome b polymorphisms segregating in parasite populations.More remodelling of the yeast Q


In further study, we examined the mode of interaction of NMK-TD-100 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. NMK-TD-100 induced arrest in mitotic phase of cell cycle, caused decline in mitochondrial membrane potential and induced apoptosis in HeLa cells. Immunofluorescence studies using an anti-α-tubulin antibody showed a significant depolymerization of the interphase microtubule network and spindle microtubule in HeLa cells in a concentration-dependent manner. However, the cytotoxicity of NMK-TD-100 towards human peripheral blood mononuclear cells (PBMC) was lower compared to that in cancer cells. Polymerization of tissue purified tubulin into microtubules was inhibited by NMK-TD-100 with an IC50 value of 17.5±0.35 µM. The binding of NMK-TD-100 with tubulin was studied using NMK-TD-100 fluorescence enhancement and intrinsic tryptophan fluorescence of tubulin. The stoichiometry of NMK-TD-100 binding to tubulin is 1:1 (molar ratio) with a dissociation constant of ~1 µM. Fluorescence spectroscopic and molecular modeling data showed that NMK-TD-100 binds to tubulin at a site which is very near to the colchicine binding site. The binding of NMK-TD-100 to tubulin was estimated to be ~10 times faster than that of colchicine. The results indicated that NMK-TD-100 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided insights into its potential of being a chemotherapeutic agent.Thiadiazoles are one of the most widely utilized agents in medicinal chemistry, having a wide range of pharmacologic activity. Microtubules (MTs) have always remained a sought-after target in rapidly proliferating cancer cells. We screened for the growth inhibitory effect of synthetic 5-(3-indolyl)-2-substituted-1,3,4-thiadiazoles on cancer cells and identified NMK-TD-100, as the most potent agent. Cell viability experiments using human cervical carcinoma cell line (HeLa cells) indicated that the IC Worldwide, cervical cancer is considered to be the second most common form of cancer as far as mortality and incidence are concerned and India contributes to about 20–30% of the global burden . CervicaMicrotubules are cytoskeletal hollow fibers present in most eukaryotic cells, are among the most successful targets for anticancer therapeutics . These dCruciferae Camelina sativa infected with Alternaria brassicae. Analogues of camalexin were evaluated for their cytotoxic activity against human breast tumor cell lines. A thiazolyl indolequinone, BE 109881b isolated from culture broths of Streptomyces strain, is known to increase DNA-topoisomerase complex formation and displayed significant anticancer activities , where [P] was the tubulin concentration. Free ligand concentration /Fmax, where F0 is the fluorescence intensity of tubulin in the absence of NMK-TD-100, F is the corrected fluorescence intensity of tubulin in the presence of NMK-TD-100, and Fmax is calculated from the plot of 1/(F0-F) versus 1/[NMK-TD-100] graph and extrapolating 1/[NMK-TD-100] to zero. The dissociation constant (Kd) was determined using the relationship, (1/X) =1 + Kd/Lf, where Lf represents free NMK-TD-100 concentration, and Lf =C -X[Y], where C is total concentration of NMK-TD-100 and [Y] is the molar concentration of ligand-binding sites, assuming a single binding site per tubulin dimer [Tubulin (1 µM) was incubated with varying concentrations of NMK-TD-100 (0-50 µM) at 37 °C for 30 min. The fluorescence measurements were performed using 295 as the excitation wavelength to specifically excite the tryptophan residues of tubulin. When excited at 295nm, tubulin displayed a typical emission spectrum with a maximum at 335 nm, and NMK-TD-100 reduced the intrinsic fluorescence of tubulin. The apparent decrease in the fluorescence values in the presence of varying concentrations of NMK-TD-100 were corrected for the inner filter effect from in dimer .The kinetics of the association of NMK-TD-100 with tubulin was measured under pseudo-first-order conditions, where the ligand was present in a large excess. Concentrations of NMK-TD-100 were 10, 20, and 30 µM, whereas the tubulin concentration was 1 µM. The ligand was added to the tubulin solution, and emission at 335 nm was measured upon excitation at 295 nm. The value ln (Qmax – Qt) was plotted against time, where Qmax is the maximum amount of quenched fluorescence, and Qt is the quenched fluorescence at time t. The biphasic plot was analyzed according to the method of Lambeir and Engelborghs ,on) were calculated as kon= α /c, where α is the slope of the plot of ln (Qmax – Qt) versus time t and c is the concentration of ligand.where A and B are amplitudes and α and β are observed rate constants of the fast and slow phases, respectively. The amplitude of the slow phase (B) was low relative to that of the fast phase (A). The apparent association rate constants (kThe ability of NMK-TD-100 to bind at the vinblastine binding site of tubulin was monitored by checking the change in NMK-TD-100- tubulin complex fluorescence . TubulinTubulin (2 µM) was incubated with varying concentrations of colchicine  for 1 h at 37°C. Then, 5 µM NMK-TD-100 was added to the reaction mixtures and incubated for an additional 30 min. The dissociation constants for colchicine interaction with tubulin are reported to be 0.5 µM . Therefochem draw software and converted to mol file. The docking was performed using algorithm-based ligand docking program Discovery Studio 2.5. The energy minimized structures of tubulin (1JFF.pdb) and the ligand NMK-TD-100 were generated by using prepare protein module (using CHARMm force field) and prepare ligand module of Discovery Studio 2.5 respectively. In both cases all parameter were set to default values (prepare ligand module) was then docked to its binding site (previously determined by using 1JFF.pdb file) on tubulin surface and the binding energy value in that site was calculated.The crystal structure of αβ-tubulin (PDB ID: 1JFF) was utilized as template for docking studies . The molt values . The lowData are presented as the mean of at least three independent experiments along with Standard deviation (SD). Statistical analysis of data was done by one-way analysis of variance (ANOVA) with a Student’s t test, by using MS Excel, and two measurements were statistically significant if the corresponding p value was <0.05.50 value of NMK-TD-100 in HeLa cells was 1.42±0.11 µM for 48 h of treatment and the maximum proliferation inhibition of ≈ 90% occurred at the concentration of 100 µM. NMK-TD-100, being the most potent one in killing cancer cells was chosen for further work.The anti-proliferative effect of some synthetic 5-(3-indolyl)-2-substituted-1,3,4-thiadiazoles was screened using human cervical epithelial carcinoma (HeLa) by MTT assay (data not shown) and it was found that NMK-TD-100 . The IC550 value of NMK-TD-100 in PBMC from healthy volunteers was 50±2.66 µM for 48 h, which was approximately 30-fold higher than the IC50 value of NMK-TD-100 in HeLa cells  for 48 h. After removal of the media, the cells were maintained for 10 days with triweekly change in media. After 10 days, the cells were fixed with 4% glutaraldehyde and stained with crystal violet. Each plate was photographed after removing the excess stain . Colony To assess whether NMK-TD-100 induced apoptosis, treated cells were labeled with Annexin V-FITC/PI and analyzed by flow cytometer . The AnnChemically promoted apoptosis mediated by the mitochondria activation pathway is often associated with the collapse of ΔΨm as a result of depolarization and leakiness of the inner mitochondrial membrane. Therefore, to investigate whether mitochondrial membrane integrity is damaged by NMK-TD-100, change in mitochondrial membrane potential (ΔΨm) was measured using JC-1, a sensitive fluorescent probe for ΔΨm. When ΔΨm is low, JC-1 exists mainly in a monomeric form, which emits green fluorescence whereas it exists in aggregates and emits red fluorescence in high ΔΨm. As shown in The effect of NMK-TD-100 on the mitochondrial membrane potential intrigued us to study the major protein component of the mitochondrial apoptotic pathway. As shown in 2/M arrest in HeLa cells in a dose-dependent manner  to HeLa cells caused a substantial reduction in the number of interphase microtubules at the periphery and disorganization of central networks of the cells was observed  occurred at NMK-TD-100 concentration of 17.5±0.35 µM.Since we observed that NMK-TD-100 affects the cellular architecture of HeLa cells by perturbing the intracellular microtubular network, we examined the effect of NMK-TD-100 on tubulin polymerization using purified tubulin and determined the polymerization inhibitory potential of the ligand. We investigated the concentration-dependent effect of NMK-TD-100 on polymerization ability of purified tubulin into microtubule. Purified tubulin (12 µM) was polymerized in the presence of different concentrations of NMK-TD-100 (0-50 µM) as described in the Experimental Procedures. NMK-TD-100 inhibited the rate and extent of tubulin polymerization in a concentration-dependent manner . For exaThe inhibition of tubulin polymerization in the presence of NMK-TD-100, as monitored by light scattering assay, could be due to the formation of very short microtubules, altered polymer morphology, or the formation of small aggregates of tubulin. To differentiate among these possibilities, reaction products formed in the absence and presence of NMK-TD-100 were examined with a transmission electron microscope . In the cell-free system. We monitored decrease of absorbance at 350 nm as a measure of depolymerization of preformed microtubules. Tubulin (1.2 mg/ml) was polymerized in the presence of 1 M glutamate, 1mM GTP for 20 min at 37 °C, different concentrations of NMK-TD-100 were added to the preformed microtubule suspensions and the change in optical density was studied. NMK-TD-100 induced rapid depolymerization of preformed microtubules in a concentration-dependent fashion, as indicated by the decrease in the absorbance values at 350 nm  of the tubulin-NMK-TD-100 interaction had been estimated by measuring NMK-TD-100 fluorescence enhancement. From the titration curve (Binding of NMK-TD-100 (2 µM) to tubulin enhanced its fluorescence intensity and showed a blue shift in the emission maxima with an increase in the concentration of protein(0-15 µM) . Enhanceon curve  of a cond) of the tubulin-NMK-TD-100 interaction was estimated by measuring the effects of NMK-TD-100 on the intrinsic tryptophan fluorescence of tubulin. The double-reciprocal plot of the binding data  of NMK-TD-100. Under the same experimental condition, the apparent association rate constant (kon) calculated for association of colchicine with tubulin using the biexponential equation was 155.9±23.32 M-1s-1 at 37°C.The kinetics of the NMK-TD-100-tubulin association reaction was studied monitoring the quenching of intrinsic protein fluorescence under pseudo-first-order condition, with NMK-TD-100 used at least 10-fold molar excess. We used the NMK-TD-100–tubulin complex fluorescence to determine whether NMK-TD-100 could bind to the colchicine or vinblastine site on tubulin. Tubulin (2 µM) was incubated with varying concentration of colchicine for 1 h at 37°C. NMK-TD-100 was added to the tubulin-colchicine complex and fluorescence of NMK-TD-100 in different samples was monitored at 450 nm after exciting at 390 nm. We checked the fluorescence values of tubulin and colchicine complexes at 450 nm after excitation at 390 nm and found them to be very low as compared to tubulin and NMK-TD-100 complex, and those values were subtracted from those of the corresponding fluorescence values of tubulin-NMK-TD-100- colchicine complex. We hypothesized that if the ligand could inhibit the binding of NMK-TD-100 to tubulin, it should cause decline in the development of NMK-TD-100–tubulin complex fluorescence. The extent of NMK-TD-100 binding at various concentrations of colchicine is shown in To gain further insights into the binding site of NMK-TD-100 in tubulin, computation based docking analysis was performed with tubulin–NMK-TD-100 complex. For determination of the binding site of NMK-TD-100 on tubulin surface, the ligand was docked on tubulin surface by using discovery studio 2.5. Based on the highest negative value of binding energy , the most stable tubulin-NMK-TD-100 complex was found to be formed when the ligand was docked on site 1 (according to the software), located between α and β subunit of tubulin . NMK-TD-For determining the effect of colchicine on tubulin-NMK-TD-100 binding, NMK-TD-100 was docked on its binding site (previously determined by using 1JFF.pdb file) in 1SA0.pdb file (which is colchicine bound tubulin). Subsequently binding energy at the preferred site was calculated using Discovery Studio 2.5. and it was found the binding energy  is less negative compared with when docked to site 1 of pdb 1JFF. This indicates that binding of colchicine can partly interfere with binding of NMK-TD-100 to tubulin.It was also previously reported that colchicine binding on tubulin surface was stabilized by amino acids present in H7, H8 and T7 loops of β subunits of tubulin. The binding pocket of NMK-TD-100 also constitutes of the H8 and T7 loop of the β subunit of tubulin. Furthermore Lys 254 of β subunit which was found to be hydrogen bonded with NMK-TD-100, also interacted with colchicine by noncovalent interactions other than hydrogen bond. Moreover, during NMK-TD-100 and tubulin interaction, Lys 254 also formed hydrogen bond with Asn 258 and non-covalently interacted with other amino acid residues like Cys 241, Leu 242, Ala 250. These amino acid residues were also important for stabilization of colchicine into its binding sites. So it can be inferred that NMK-TD-100 binds to tubulin very near to the colchicine binding site and also colchicine interferes with NMK-TD-100 binding to tubulin.In this work, a group of 5-(3-indolyl)-2-substituted-1,3,4-thiadiazoles  was scre2/M phase effectively after 24 h of treatment  and for inhibition of purified tubulin polymerization (around 1 µM). The probable reason is that solution environments are different in cellular system and in a cell-free system.NMK-TD-100 not only depolymerized microtubules and inhibited reassembly of depolymerized microtubules in HeLa cells but also inhibited tissue purified tubulin assembly in a e system . The IC5d value of 1.26±0.32 µM Click here for additional data file.