By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL.We have established an Moreover, cytosolic IPP is imported into the mitochondria, where it serves as precursor for ubiquinone11.The MVA pathway supplies the biosynthetic precursors for isoprenoids in the majority of eukaryotes . It can be found in several important human parasites, such as19. In plants, the precursors, provided by the plastidial MEP pathway are used for the biosynthesis of essential isoprenoids of the photosynthetic apparatus such as carotenoids, the side-chains of chlorophyll and plastoquinone, as well as for isoprene, tocopherols, phylloquinones and the phytohormones ABA and gibberellin20, with ABA being a cleavage product of carotenoids, like the more recently discovered strigolactones22. In addition to these ubiquitous compounds in plants, the MEP pathway is the route for the biosynthesis of the vast majority of plant terpenoids, including countless secondary metabolites with defensive, allelopathic or signaling properties23 . Furthermore, precursors derived from the MEP pathway are used for the post-translational modification of certain proteins by the covalent addition of a farnesyl-(C15) or geranylgeranyl (C20) residue, a process commonly referred to as protein prenylation (cf. the accompanying paper25 and literature cited therein).The alternative pathway (or MEP-pathway) for the synthesis of isoprenoids occurs in eubacteria, cyanobacteria, and the plastids of phototrophic algae and plants26, it had been observed that blockage of protein farnesylation by chaetomellic acid not only led to a considerable percentage increase in dead cells during the culture period, but also to a specific arrest in the transition from G2 into M phase. By contrast, mevinolin for instance (which inhibits the key-regulatory enzyme HMG-CoA reductase in the cytosolic MVA pathway) arrested cells mainly at the transition from G1 to S phase27. This latter finding is most likely due to the lack of some MVA-derived signal formed at the end of mitotic phase that might be implied in the regulation of the cytoplasmic pH28. These and other observations prompted a series of further studies in which the possibility that the plastidial MEP pathway could complement MVA deficiency was tested. Indeed it was demonstrated that exogenously added deoxyxylulose  could overcome mevinolin-induced growth inhibition, even more efficiently than exogenous MVA29. To enter the plastidial MEP route, this DX needs conversion into its phosphate (DXP) by a cytosolic xylulose kinase30, followed by translocation into plastids. As a logical follow-up, and in view of early reports that in plants geranylgeranylated proteins seem to be present in higher quantities than farnesylated ones32, we embarked on studying this phenomenon more closely. Our interest is not only focused on elucidation of the biosynthetic origin of farnesyl and geranylgeranyl residues, but also on the action of inhibitors on precursor availability and on the specificity of prenyltransferases. For instance, we could demonstrate the incorporation of14C-DX into proteins in BY-2 cells29.In preceding studies investigating the differential effect of a series of inhibitors of isoprenoid biosynthesis and function in cell cycle progression in unsynchronized and synchronized BY-2 cellsin vivo-visualization system based on a stably transformed tobacco BY-2 (TBY-2) cell line for monitoring the prenylation status of a GFP fusion protein. Isoprenylation of proteins, which occurs in all eukaryotic cells, involves the covalent attachment of a C15 (farnesyl) or C20 (geranylgeranyl) group to a C-terminal CaaX motif, followed by a series of post-prenylation reactions. This lipidic post-translational modification plays an important role in the correct membrane targeting of certain proteins and in their interactions with other proteins.The central element of our recent studies was an1. By contrast, inhibition of the MVA pathway (by mevinolin) or protein farnesyltransferases did not affect the localization of the chimeric fusion protein. Among other experiments, complementation assays with pathway-specific intermediates were performed and clearly indicated that the precursors for the geranylgeranylation of the fusion protein in tobacco BY-2 cells were predominantly provided by the MEP pathway1.This system consisted of a dexamethasone-inducible cell line that expressed a reporter protein (GFP) fused to the carboxy-terminal basic domain of the rice calmodulin (CaM61), which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). After induction, the prenylated fusion protein predominantly associated with the plasma membrane. By using pathway-specific inhibitors, we demonstrated that inhibition of the MEP pathway with oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), triggered a shift in the localization of the GFP-BD-CVIL protein from the plasma membrane to the nucleus6-tagged GFP-BD-CVIL fusion protein. In order to prove that the present bioassay was not only able to serve as a qualitative approach for the identification of new drug candidates, but also as a statistical tool to compare the potential drug candidates with other known inhibitorsin vivo, we also performed a quantitative analysis of the intracellular distribution of GFP-DB-CVIL in response to different concentrations of novel prodrugs, targeting the early steps of the MEP pathway.However, at the end of this previous study several questions remained unsolved that will be addressed in more detail in the present work, including the impact of inhibitors of sterol biosynthesis and post-prenylation reactions on the subcellular localization of the His6-GFP-BD-CVIL fusion protein.By blocking the early steps of sterol biosynthesis with pathway-specific inhibitors we tried to determine the impact of sterol depletion in the plasma membrane as well as of the modulation of the pool of endogenously available FPP on the localization of the H33, was used to specifically inhibit the conversion of farnesyl diphosphate (FPP) to squalene by the first committed enzyme in the sterol/triterpene pathway, squalene synthase (SQS). SQ is a competitive inhibitor of SQS and structurally mimics the reaction intermediate presqualene diphosphate37.First of all, squalestatin-1 (SQ), also referred to as zaragozic acid6-GFP-BD-CVIL was induced by addition of 10µM dexamethasone .Seven-day-old BY-2 cells were diluted at a ratio of 1 to 6 in fresh medium and treated with 1µM SQ for 18–24 h (in different independent experiments). 15 h before observation of the cells by fluorescence microscopy, expression of the prenylable fusion protein H38.The results of this treatment are shown in39), BY-2 cells were treated with terbinafine (Tb), a specific, non-competitive inhibitor of the fungal squalene epoxidase (SQE). In mammals, however, it acts as a competitive inhibitor of this enzyme40. Its inhibitory efficiency in plant cells has been demonstrated with celery (Apium graveolens) cell suspension cultures41, wheat (Triticum aestivum) seedlings42, cat’s claw (Uncaria tomentosa)43 and with our model system, BY-2 cell suspension cultures38. Treatments with Tb were performed at 30µM up to 24 h (as well as 18 h). The cells treated with Tb showed a very slightly stunted growth as compared to the control cells and displayed faint GFP signals in the cytosol, close to the PM. However, no mislocalization of the prenylable fusion protein to the nuclear compartment could be observed under the chosen experimental conditions  in general, including lanosterol synthase (LAS) in mammals and fungi45. The structure of Ro48-8071, which is an orally active inhibitor of human hepatic OSC, has been determined in complex with the squalene-hopene cyclase (SHC), the prokaryotic counterpart of OSCs, responsible for the conversion of squalene into cyclic compounds in bacteria, and it is suggested that Ro reacts with the expected binding site for squalene46. Ro treatments were performed at 2µg/ml under the same experimental conditions as described previously for SQ and Tb applications. Cells treated with Ro for 18/24 h looked morphologically more or less like the control cells. The GFP fusion protein was mainly localized at the level of the plasma membrane, although there were also faint signals  of fluorescence near the PM like those observed for the previous Tb treatments. However, no mislocalization of the GFP fluorescence to the nucleus/nucleolus was observed  was used to inhibit one of the key steps of sterol biosynthesis, the conversion of the linear oxidosqualene into cycloartenol, the first cyclic precursor of phytosterols. This reaction is catalyzed by cycloartenol-synthase (CAS) in plantsbserved . When coed here , the effVarious complementation experiments were performed with SQ-treated cells. Whereas DX (0.5mM) and geranylgeraniol  were able to re-establish the membrane localization in 100% of the cells (better than the control), neither geraniol  nor mevalonolactone  and its open carboxylic form mevalonate  could complement the mislocalization under the chosen experimental conditions. Finally, squalene was added at 2–4mM, but did not overcome the SQ-induced effect either .6-tagged GFP-BD-CVIL fusion protein revealed that it was geranylgeranylated and carboxyl-methylated in BY-2 cells1. In the past the study of post-prenylation inhibition has become a very attractive topic, as both reactions are essential for the localization of many prenylated proteins by mediating their attachment to membranes and protein-protein interactions.As mentioned earlier, the purification and analysis of the His29 and requires adjustment of the experimental conditions. Nonetheless, the results obtained after various treatments indicated that both inhibition of RCE1 and ICMT affected the localization of H6-GFP-BD-CVIL (N-acetyl-S-farnesyl-L-cysteine (AFC)) strongly changed the distribution pattern of the H6-GFP-BD-CVIL and besides the PM, both the cytosol as well as the nucleolus displayed strong GFP signals , using bombardedPetunia leaves. Long-term treatments of BY-2 cells (> 10 h) however led to loss of GFP fluorescence and cell death.In preliminary experiments we tested inhibitors of RAS converting enzyme 1 (RCE1), and of isoprenylcysteine carboxyl methyltransferase (ICMT), but most of them induced cell death when used in commonly cited concentrations and time-scales in our model system. This is most likely due to the efficient uptake and high metabolic activity of BY-2 cellsBD-CVIL . For inssignals . InteresN-tosyl-L-phenylalanine chloromethyl ketone (TPCK48) led to a partial mislocalization of the fusion protein to the nuclear compartment. However, optimal conditions to observe the induced effects still need to be identified.Inhibition of the –AAX-proteolysis byD-xylulose 5-phosphate synthase) catalyzes the first step of the alternative MEP pathway, the condensation of glyceraldehyde 3-phosphate (GAP) and pyruvate, yielding 1-deoxy-D-xylulose 5-phosphate (DXP). DXS enzymes are highly conserved in bacteria and plants and analyses of their sequences revealed a weak homology with other thiamine-dependent enzymes, such as transketolases, and the pyruvate dehydrogenase E1 subunit52. These enzymes all catalyze similar biochemical reactions54 by using thiamine diphosphate (TPP) as a cofactor and pyruvate as a substrate. In addition to TPP, DXS also requires a divalent cation (Mg2+ or Mn2+) for optimum enzyme activity56. More recently, efforts were successful to partially crystallize DXS fromE. coli andDeinococcus radiodurans (in complex with TPP), afterin situ proteolysis of the purified enzyme by a fungal protease58, which led to a better understanding of the catalytic mechanism of DXS and possibly paves the way for the design of novel active inhibitors. As to date, only two inhibitors of this enzyme are known: oxoclomazone (OC), for the plant DXS59 and fluoropyruvate, for the DXS ofE. coli60. OC, sometimes also referred to as 5-ketoclomazone, was only recently reported to have exhibited antibacterial activity against a pathogenic bacterium,Haemophilus influenza61.DXS (1-deoxy-64). These compounds were then tested with our bioassay for possible effects on the localization of the H6-GFP-BD-CVIL  is the second enzyme of the MEP pathway and catalyzes the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP). Part of this transformation is an intramolecular rearrangement, yielding 2-C-methyl-D-erythrose 4-phosphate, which is then reduced to MEP in an NADPH-dependent reaction step68. Several studies have revealed detailed information about the structure and biochemical properties of the DXR enzyme fromE. coli69. Those results – including the three-dimensional structures of DXR in a ternary complex with DXP/fosmidomycin (Fos) and the co-factor NADPH – suggest a physiologically active homodimer, with each subunit consisting of three distinct domains71. In addition to NADPH, DXR requires a divalent metal cation for activity, such as Mn2+, Mg2+ or,in vitro, Co2+, which is bound by three highly conserved amino acid residues72. This metal cation is chelated by DXP before the first step of the conversion to MEP – an intramolecular rearrangement – takes place. According to the structural data, binding of DXR to its substrate (or to Fos) involves a major conformational rearrangement of the enzyme in the presence of NADPH. Fos acts as a competitive inhibitor, chelating a bivalent cation with its hydroxamate group and binding slowly but very tightly to the catalytic site of DXR70. The substrate binding site of the DXR enzyme can be divided into three distinct regions: first of all, a positively charged pocket which interacts with the phosphonate function of Fos (“phosphate-recognition site”), a hydrophobic region covering the backbone of the molecule, as well as an amphipathic region that binds the hydroxamic acid moiety of the molecule. This results in a conformation with a flexible loop covering the central catalytic site, thus forming a barrier with the surrounding solvent71.1-Deoxy-74, and experiments with various plant species have demonstrated its potential as herbicide, as approved by the chlorotic and bleaching phenotypes that have been observed after its application75. In addition, Fos successfully inhibited the isoprenylation of the GFP-BD-CVIL fusion protein in our fluorescent bioassay at concentrations in the micromolar range1. Fos and its methylated derivative FR-90098 are phosphonohydroxamic acids. In both compounds a hydroxamate function is linked to a phosphonic acid function by a propyl chain. Fos and FR-90098 as well as two hydroxamate derivatives 4-(hydroxyamino)-4-oxobutyl-phosphonic acid (LK1) and 4-[hydroxy(methyl)amino]-4-oxobutyl-phosphonic acid (LK276) have been tested on the transgenic BY-2 cell line expressing the H6-GFP-BD-CVIL marker protein using the well-established standard conditions. At 100µM, the majority of fluorescence emitted by the GFP marker protein of the cells treated with Fos, with FR-900098 or LK2 accumulated in the nucleus, indicating an efficient inhibition of the MEP pathway (Fos is known to inhibit the DXR enzyme from higher plantspathway . At this77). A common strategy to overcome this problem is masking the phosphonic acid moiety by esterification. For instance, double ester drugs of FR-900098 have shown a 2 to 3 fold higher biological activity compared to their unmodified models in experiments with malaria-infected mice78.A major disadvantage of phosphonate drugs is that the phosphonate group is highly deprotonated at physiological pH. Because of the resulting high polarity (and/or low lipophilicity), the transport across biological membranes and the general bioavailability are restricted  and divided into three major groups-according to their respective phenotype – to facilitate a statistical evaluation  and LK2 (derived from FR-900098) were recently synthesizedis work . The goaluation :1. Cells in which the fluorescence emitted from the nucleus was clearly dominant;2. Cells with significant intensity of fluorescence still emitted from the PM ;3. Cells in which the fluorescence was mainly associated with the PM and looking like the untreated control (the important part being that no fluorescence was visible in the nucleolus).76) as well as those obtained with Fos and FR-900098 (cf.78).The results obtained at a final concentration of 50µM indicated that most of the prodrugs, especially SP2 and SP4, caused a fluorescence shift comparable to that observed after treatment with 50µM Fos . Among tHowever, at 50µM it was not possible to determine whether the prodrug SP2 was a better inhibitor than Fos. To address this issue, we therefore conducted a second set of experiments, using the inhibitors at a final concentration of 5µM. At this concentration, two major results are noteworthy : First o6-GFP-BD-CVIL, reported earlier. The fact that MVA, at high concentrations, was able to rescue the fluorescence at the PM, whereas farnesol (Fol) only partially complemented the inhibition by Fos suggests (among other possibilities) the existence of a bottleneck for the synthesis of GGPP from FPP in the cytosol , or the incapacity of FPP to be translocated into the plastid, where the formation of GGPP could take place.The inhibition of the sterol biosynthetic pathway was used as a tool to check whether a possible re-direction of the metabolic flux in BY-2 cells, in particular FPP molecules non-incorporated into sterols, could lead to a change in the GFP fluorescence pattern or even contribute to overcome the Fos-induced mislocalization of the H2 and plants80. This is supported by the fact that tobacco plants over-expressing HMGR were shown to produce higher amounts of sterol intermediates and end products82, whereas the over-expression of a FPP synthase inArabidopsis did not result in an increased production of sterols83.This bottleneck is perhaps HMG-CoA reductase (HMGR), which is generally considered to catalyze the rate-limiting step in sterol synthesis, for both animals38. SQ was shown to be a very efficient inhibitor of SQS (IC50 = 5.5nM), almost completely inhibiting SQS activity after treatment for 24 h at 50nM. In addition, the same concentration of SQ led to a 97% decrease in total radioactivity incorporated into sterols, when radiolabeled [14C] sodium acetate was added for 2 h before cell harvesting. At 0.5µM, the cells showed a significant decrease in cell mass (50%), which correlates very well with the stunted growth observed during this study. In a preceding study it was demonstrated that the presence of SQ led to a growth arrest of BY-2 cells in the early G1 phase, but did not cause any cytotoxic effect or apoptosis26. Inhibition of SQS by SQ was also shown to trigger a stimulation of HMGR expression and activity38.Due to previous experiments with sterol biosynthesis inhibitors we had some experience on the effective concentrations of SQ and Tb in BY-2 cells38.Inhibition by Tb was revealed to cause a significant decrease in the cell sterol content as well as a massive accumulation of squalene, which accumulated in cytosolic lipid bodies. However, the cell growth was not impaired as indicated by the analysis of the cell mass after treatment with 30µM Tb for 24 h. Interestingly, Tb treatment also stimulated HMGR activity, but not the corresponding transcript levelsKeeping all this in mind, how can the particular phenotype observed with SQ-treated cells, i.e. the partial mislocalization of the GFP fusion protein be explained? In fact, in a first set of experiments, we added 20µM exogenous Fol to BY-2 cells treated with both Fos and SQ. In cells treated with SQ only (control experiment), a partial mislocalization of the fluorescence was observed in the majority of treated cells (> 85%), whereas neither Tb nor Ro-treated cells displayed a similar effect. In cells treated with both SQ and Fos and complemented with Fol, the same pattern of partial mislocalization of the fluorescence was observed. It needs to be pointed out that this mislocalization was clearly visible, but less pronounced at the level of a single cell, as compared to Fos- or OC-treatments.84) or other organelles or converted to Fol, which might become cytotoxic86. Feeding experiments with radiolabeled farnesol showed that all these scenarios could occur. However, inhibition with SQ did not lead to an additional incorporation of FPP into the ubiquinone side-chain84.Hypothetically, due to SQS inhibition, the redirection of the metabolic flux might provide much more FPP to other FPP-metabolizing enzymes, such as GGPP synthase or protein farnesyltransferase. FPP in excess could also be transported to mitochondria , but also contains a putative nuclear localization sequence (NLS), it is possible to imagine that the farnesyl moiety is not sufficient either to guarantee an efficient integration in the PM (because of a shorter hydrophobic domain) or to mediate the transport to the PM (which is still an open question for H6-GFP-BD-CVIL). In addition, the occurrence of several farnesylated, nuclear proteins have been recently reported90. Very recently Chandraet al.91 identified a role for the GDI-like solubilizing factor (GSF) photoreceptor cGMP phosphodiesterase δ subunit δ(PDEδ) in modulating signaling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. In this study it was shown that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm91. This would allow more effective trapping of  Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane91.Thus, a protein farnesyltransferase (PFT) might use the excess FPP to non-specifically farnesylate the H92. PFT and PGGT-1, purified from bovine brain, were inhibited by SQin vitro with an IC50 of 216 and 620nM, respectively92. As we applied comparably high concentrations of SQ (500nM to 1µM) to BY-2 cells, it seems quite reasonable to assume that the treatment with SQ might also have affected the PGGT-1 enzymein vivo, responsible for the prenylation of the H6-GFP-BD-CVIL, even thoughin vitro data are not yet available. In addition, by using lower concentrations of SQ , the protein prenyltransferase should theoretically not be inhibited, allowing us to more or less rule out the cross-reactivity and the farnesylation scenario. In order to investigate the capability of isoprenols like geraniol (Gol), farnesol (Fol) and geranylgeraniol (GGol) and intermediates of both isoprenoid biosynthetic pathways  and its open-acid form MVA; MEP-pathway: DX) to overcome the  mislocalization induced by SQ, different chemical complementation experiments were performed. Interestingly, both DX and GGol completely complemented the SQ-induced effect. As discussed extensively before, the most likely explanation is an efficient uptake and metabolization of both compounds: DX, after being converted to DXP by cytosolic xylulose kinase30 enters the plastid and is incorporated into IPP, GPP and GGPP, which might then be exported towards the cytosol, where it serves as a substrate for prenylation of the H6-GFP-BD-CVIL. For GGol, again, two scenarios are possible: either its import into the plastid, phosphorylation and export to the cytosol, or direct phosphorylation in the cytosol. The existence of a system for phosphorylation of prenyldiphosphates was mentioned previously93.Studies with SQ in mammalian systems revealed that besides acting as a specific inhibitor of SQS, SQ was able to inhibit mammalian prenyltransferases, in particular protein farnesyl transferase (PFT)6-GFP-BD-CVIL.SQ is known to be an irreversible, competitive inhibitor of squalene synthase (SQS). Therefore, the DX-induced recovery of the PM fluorescence itself cannot result from an effect on SQS. As SQ structurally resembles FPP, it would be more likely that, by increasing the pool of bio-available GGPP, we succeeded in replacing SQ by GGPP at the substrate-binding site of PGGT-1 (or even both prenyltransferases) and thereby restored the correct geranylgeranylation of H94. Therefore, if the tobacco BY-2 mevalonate-kinase were also sensitive to the feedback-regulation by FPP and GGPP, this would very conveniently explain why externally fed MVA could not complement the SQ-induced phenotype due to a large pool of FPP. Moreover, the resulting decrease in cytosolic IPP due to the block in the MVA pathway could also prevent a sufficient supply of IPP for the putatively cytosolic conversion of FPP to GGPP.Interestingly, mevalonate kinase is down-regulated at the post-transcriptional level by high levels of FPP and GGPP88 and suggests that Gol is not accepted as a substrate for the phosphorylation system that exists in plants93. Intriguingly, exogenously applied Fol was previously able to restore the PM localization of H6-GFP-BD-CVIL to over 60% in Fos-treated cells88. However, as there are very specific inhibitors that act either on PFT or on PGGT-1, and are thus useful for “calibration”, in a follow-up experiment an unknown inhibitor giving a “hit”, but then applied at much lower concentration and carrying out similar complementation assays, would easily distinguish between inhibition of sterol biosynthesis or protein prenylation.The fact that Gol does not complement the effects of SQ is in agreement with previous results88. However, the transport of GFP-BD-CVIL was not significantly affected by any treatment targeting either components of the cytoskeleton  or thetrans-Golgi-network (TGN: brefeldin A88), suggesting that GFP-BD-CVIL was not transported to the PMvia the classical secretory pathway. This result was surprising, as this vesicular transport route is used by the majority of identified prenylated proteins95, such as HRAS, NRAS and KRAS4A in mammals. But the correct targeting of Ras proteins is successfully accomplished only when the proteins contain a second “signal” in addition to their prenylation. In the case of HRAS, NRAS and KRAS4A this second signal consists of one or moreS-acylation sites 97, whereas KRAS4B contains a lysine-rich polybasic (hypervariable) domain, located nearby the prenylation side100. The same combination, a polybasic domain with a cluster of basic amino acids, in addition to the CAAX motif is also found in the H6-GFP-BD-CVIL fusion protein.Some previous studies in our laboratory focused on the intracellular transport of the prenylated GFP-fusion protein from the ER to the PM in BY-2 cells102 that requires, at least in yeast, mitochondrial function and vacuolar sorting proteins (VPS)103.Besides this structural resemblance, KRAS4B is also insensitive to inhibitors of vesicular transport and transits to the PM by a largely unknown, non-vesicular (Golgi-independent) pathway88, the study of molecules that could interfere with the transport of the H6-GFP-BD-CVIL fusion protein might be a promising tool to observe the effects of post-prenylation inhibition in plants in general, and this knowledge might even be transferred to other models.Despite the fact that corresponding experimental efforts with transformed BY-2 cells were not crowned by much successin vivo isoprenylation (more precisely: geranylgeranylation) of the chimeric H6-GFP-DB-CVIL protein.Overall, two sets of experiments have been conducted in the context of validating the test system. Two groups of novel inhibitor candidates, each targeting a specific enzyme of the MEP pathway, DXS in the first and DXR in the second case, were tested to assess their capacity to inhibit thein vivo assay, despite the fact that one of them, TJ150, induced cytotoxic effects at extreme concentrations  can be measured by spectrometry at 340nm (cf.57). None of the tested compounds exhibited an inhibitory effect, in agreement with the results obtained with our bioassay .First, six compounds that were inspired by known inhibitors of pyruvate decarboxylases and pyruvate dehydrogenases were tested. In summary, all tested pyruvate derivatives were inefficient in ourrations . At thisin vitro76. Those compounds (here referred to as LK1 and LK2) successfully inhibitedE. coli DXR. The IC50 values measured were 170nM and 48nM for LK1 and LK2, respectively. The IC50 value of LK2 was even comparable to that of Fos (32nM). In addition, both molecules inhibited the growth ofE. coli, but with a lower efficiency than Fos76. Interestingly, the N-methylated compound (LK2 – derived from FR-900098) also successfully inhibited the growth of anE. coli strain that was cross-resistant to Fos and fosfomycin, which bears some faint structural similarity, but acts on another molecular target, UDP-N-acetylglucosamine enolpyruvyltransferase104. Among other reasons, this could be due to a difference in the uptake or detoxification of these two antibiotics, which are known to share a common import mechanism via theL-3-glycerolphosphate and the glucose 6-phosphate pathway inE. coli105. As the N-methylated compound LK2 nearly equaled Fos in terms ofin vitro inhibition of the DXR enzyme76, changing the bioavailibility of this compound was a promising strategy to further enhance the performance of the hydroxamic drugs and provide new active compounds against resistant pathogen strains.The second set of experiments focused on inhibitors and novel prodrugs targeting the second enzyme of the plastidial pathway, DXR. Only recently, analogs of Fos and FR-900098 with a rearrangement of the hydroxamate group had been testedin vivo experiments with transformed BY-2 cells were conducted under similar conditions: clonal cells tested under pre-defined standard conditions were counted and examined by visual inspection after different treatments . The results obtained at 100µM clearly proved that Fos and FR-900098 as well as LK2 were applied at saturating concentrations. We therefore decided to better start our direct comparison at 50µM. At this concentration, Fos, SP2 and SP4 were very active, causing a partial mislocalization in nearly 100% of the cells. The other tested prodrugs did not act that well at this concentration, even though they were able to induce a partial mislocalization in at least 50% of treated cells. At 5µM, only SP2 and SP4 proved to be more efficient than Fos, whereas the other inhibitors only induced a barely detectable effect. At 500nM final concentration, SP2 and SP4 were still able to induce a mislocalization of the GFP fluorescence in more than 50% of the cells (data from a single experiment).The test of the six new prodrugs107. The efficiency of a prodrug in anin vivo assay indeed depends on several parameters, such as transfer through biological membranes and the stability of the prodrug against hydrolysis or esterases.Obtaining only two out of the six compounds with a higher efficiency than the respective model drugs might appear deceiving at first view. However, it is a fine example confirming the prodrug concept 106,107All the six prodrugs tested in these experiments were able to induce a localization shift of the prenylable GFP fusion protein to some extent, which indicates that the active drug at least partially entered the plant cell, where it could inhibit the DXR enzyme . This point is important to mention, as it indicates that:i) The prodrugs were soluble in the BY-2 medium;ii) They successfully crossed the plant cell wall;iii) They were imported inside the plant cell across the PM.108. Until now, a similar transporter has only been identified in the mitochondrial membrane of plant cells109.How this transport took place and which transporters were involved remains unclear, even though data from bacteria suggest that a cAMP-dependent glycerol 3-phosphate transporter could be a likely candidate110. Interestingly, the best performing methylated prodrug SP4 also shared the same pro-moieties with the best performing non-methylated prodrug SP3. Although the better performance of the methylated drugs, such FR-900098 or LK2, could be attributed to the hydrophobic interaction of the methyl-group at the catalytic site of the DXR enzyme, the methyl group may also have an influence on the lipophilicity of the prodrug and be responsible for its overall better import rate in tobacco BY-2 cells . The peptidomimetic protein geranylgeranylation inhibitor GGti-2133112 was purchased from Calbiochem (Merck). Oxoclomazone (OC) was kindly supplied by Dr. Klaus Grossmann . Please seeIf not indicated otherwise, chemicals used were provided by Sigma-Aldrich/Fluka. Squalestatin (Sq) was obtained from Glaxo  and dissolved in 0.1M Tris-HCl (pH 7.4) to give a 0.1mM stock solution. Terbinafine (Tb) was kindly provided by Dr. N.S. Ryder (Vienna) and dissolved in dimethyl sulfoxide. MVA lactone, Gol, Fol, and GGol were purchased from Sigma-Aldrich. Mevinolin (MV) was a kind gift from Dr. Alfred Alberts . Before use, the lactones of MV and MVA were converted to their open acid forms. Fosmidomycin (Fos)Nicotiana tabacum L.) cv Bright Yellow-2 (BY-2) cell line used in this work was provided by Professor Toshiyuki Nagata . It was initially isolated by Katoet al.113 and is derived from calli induced from young plants114. The cell suspension was maintained by subculturing the 7-day-old cells (in stationary phase) as described85.The original untransformed tobacco .Routinely, 0.75ml of a 7-day-old culture was diluted into 40ml of modified Murashige and Skoog medium116) has been adjusted to the 7-day growth cycle of TBY-2 cells. After 6-fold dilution in fresh medium, the cells were treated in commercial multi-well plates. Total volume of treated cells was scaled to 3ml. In general, initial screenings started between 50 and 100µM of a given inhibitor/prodrug and were refined if a delocalization occurred. Induction of the fluorescent marker proteins took place 15 to 24 h before observation; the treatments were scheduled 3 to 6 h after the induction. Observation and image acquisition were made by laser scanning microscopy as described . For statistical observations, such as counting cells after various treatments, a 10x Zeiss objective (“Plan-Neofluar”) was used. At confocal resolution, images were taken using a 63x, 1.2 numerical aperture water immersion objective (“C-Apochromat”). In both cases, differential interference contrast (DIC) images were recorded.Zeiss LSM Image Browser software  and exported as Tiff or JPEG files. Final image analysis and adjustments was performed using either Photoshop version 8.0  or theImageJ software version 1.31 .Epifluorescence microscopy was performed using a Nikon E 800 fluorescence microscope equipped with a Nikon DXM1200 CCD high-resolution color camera (20 x 0.45 objective) and ACT-1 image software. Images were processed with the latest version of the I believe the authors have successfully addressed the reviewer comments and thus, I again approve the publication of this article.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors have answered my questions and I have no further comments.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells” by Hartmannet al., describes the use of an in vivo system to study the effectiveness of known inhibitors of MEP and MVA pathways and also to screen for new chemicals that affect these pathways with the hope of eventually being able to use them as herbicides, antibacterial/ant-parasitic drugs and as inhibitors of early sterol biosynthesis and of post-prenylation reactions.The article entitled “ The experiments were well designed and executed and showed that this is a promising system that can be used not only for the screening of new inhibitors of the MEP pathway, but also to study the possible channelling of the intermediates to different compartments and/or to follow the interplay between the two pathways. The latter can also help with applying metabolic engineering to terpenoid production in plants or microorganisms.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We are quite grateful for the very positive comments by this colleague, who has provided a broader and deeper perspective to our article than might at first be obvious by a superficial read of the manuscript.  It is clear that he is a specialist in the realm of plant isoprenoid research. This is an interesting chemical genetics study addressing the parallel biosynthetic pathways for isoprenoids in plants . As such, this work provides a paradigm for the discovery of bioactives that might be applied in several contexts. Of course, the unique characteristics of plant cell walls / membranes, contrasted with bacteria, may make this screen less general than might be desirable. Being based on imaging, this screen is relatively low throughout, but cell-based assays typically have some of this sort of limitation. A few new chemotypes have been investigated and found to have significant activity. This study follows well-established processes for examining the sequence of steps in a biosynthetic pathway, with opposing effects of inhibitors and molecules downstream of the site of inhibition. A slight ambiguity about the chemical structures of the pro-drugs and their active forms hinders understanding of the structure-activity relationships. It should be emphasized that the most active SP compounds have N-methyl groups on their hydroxamate nitrogens. Also, structures presented on p. 20 are in error, in that all but TJ290 are missing a carbonyl group.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The referee went quite deeply into the details of the manuscript and we are very grateful for his sharp eyes, detecting a mistake in the supplementary figure summarizing some (synthetic) inhibitors checked in our study. The corresponding figure will of course be replaced. ad extremum to get into the plastids. It is obvious and also a major result that “pro-drugs” with increased lipophilicity enter more easily, like is known for a large number of pesticides and pharmaceuticals. It is somewhat ambiguous to go too deeply into the field of structure-activity relationships, but Dr. Pirrung is right in ascribing the highest activity to a certain group of compounds, like those that have N-methyl groups and their hydroxamate nitrogens.The colleague points to some limits of such a system, but we have prepared a sequel manuscript also to be sent to F1000research, in which all has been “miniaturized” to the use of 96-well plates and a semi-automatic analysis by confocal microscopy. It was necessary however, to shown that the reporter system as such, based on this GFP fusion protein works sufficiently well before publishing the second manuscript. As to the efficacy of compounds that interact with the MEP pathway or with downstream reactions of protein isoprenylation the first criteria is to penetrate into the BY-2 cells, and E. coli, but not into the humano-pathogenicMycobacterium. Once an inhibitor has been found efficient in our plant cell-based test system, it is clear that in follow-up experiments one has to investigate whether it acts similarly upon a whole collection of such bacteria. Human pathogens are not even limited to bacteria. Simply pragmatically speaking, a plant cell is somewhat representative of different pathogenic cells: protistans, bacteria and fungi.There is no “ideal” system for testing inhibitors, and this concerns those whose research is based around bacteria, as well.  Let us take for instance the well-known fosmidomycin that penetrates into This manuscript by Bach, Rohmer and colleagues describes a new and powerful plant screening system for chemical inhibitors of the MEP or Rohmer pathway. The basic concept as well as the underlying chemistry of the manuscript is excellent, but the presentation of the results and conclusions driven from the biological experiments, as well as a number of paragraphs need carefull revision. A few points that shall help the authors to improve the manuscript are:None of the inhibitors the authors measure show their uptake. This should be mentioned/discussed.The size of the cells should be shown with exactly the same magnification in all figures. Otherwise it is hard to compare the morphology of the cells upon treatment with different inhibitors. To my mind it seems that the treatment does result in changes in cell size and shape i.e. Figure 1, unlike what is stated for Ro in the 7th paragraph of page 4.  In Figure 2 only the cells in the top line show stunting; the cells shown in the lines below look normal to me.The authors should carefully check again whether parts/paragraphs can be moved into the method section and vice versa.In Figure 6 fluorescence intensity is given. How was this measured, do the authors not mean distribution?I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. This was the first comment to be delivered (thank you), but we waited until now, so as to respond to all reviews at the same time. The criticism on the organization of the paper is certainly justified, but in fact it is the result of frequent exchanges of e-mails and versions with the editorial office of F1000. The initial version was a long manuscript with a very large supplement.  Upon the advice received, everything was restructured and finally two separate manuscripts were prepared, and to tell you the truth, what was a major part of the so-called supplement is now this first manuscript that describes the effects of a series of MEP pathway inhibitors and some that putatively interfere with protein isoprenylation and processing of correspondingly modified proteins.  The follow-up manuscript will provide information on the establishment of a medium-throughput system and what measures need to be taken to miniaturize the system and to facilitate microscopic analysis of rather large numbers of samples, based on BY-2 cells grown and treated in small volumes (96-well plates). We think that by then all questions that remain open in this paper will become clear, including when it comes to statistics and so forth. Only if a compound is absorbed, meaning that it had passed the thin cell wall and the plasma membrane of BY-2 cells it can exert some effect in the cytoplasm , or beyond after having surmounted the barrier of the double membrane that makes up the plastidial envelope to target the MEP pathway. Thus specific uptake studies, for instance with radiolabeled compounds would go far beyond the scope of this paper, not to speak of costs! Dr. Feussner is right in pointing to the different size of cells as they appear in the figures. But for that reason the (high resolution) photographs made with the confocal microscope are automatically completed with white bars for scaling. We know from earlier studies with BY-2 cells treated with sterol biosynthesis inhibitors that cells become more stunted, but in this series of experiments here we searched for putative effects on the insertion of the isoprenylated GFP reporter protein and found none.   Treatment of BY-2 cells with squalestatin  and by terbinafin  led to similar morphological adaptations (reference #38): After cell division sterols are needed for cell elongation. However, a clear effect on the GFP reporter protein was seen with squalestatin, but this could be attributed to its direct inhibition of protein isoprenylation, as already described in literature. Indeed, the treatment with Ro at the concentrations applied did not really affect the cell shape  and the statement criticized in the main text needs to be weakened.Dr. Feussner is right: In Fig. 6 we mean fluorescence distribution, not intensity. This will be changed accordingly. In Fig. 2 chemical complementation assays are displayed, with 1 µM squalestatin as the inhibitor.    The only compound that really counteracted the apparent side effect on protein geranylgeranylation besides DX was geranylgeraniol (GGol). However, in previous studies we had already demonstrated that under certain conditions, when the cytoplasmic MVA pathway is blocked, in the BY-2 cell system exogenous DX can override the inhibition of sterol biosynthesis, which would result in less stunted cells. GGol, at the concentrations tested might prevent the binding of squalestatin to protein geranylgeranyl transferase. This does not hold true for MVA (open acid or lactone form), which would reconstitute sterol biosynthesis to some extent resulting in some cell elongation, but not entirely the inhibition of PGGT type I. The slight effect of geraniol might be due to its conversion into farnesyl-PP, but that is more speculation.  We think we can leave this as it is described and discussed now.


Protein kinase C (PKC)-θ is a serine/threonine kinase belonging to the calcium-independent novel PKC subfamily; its expression is restricted to certain tissues and cell types, including T cells. The signals delivered from T cell receptor (TCR) and CD28 costimulatory molecules trigger PKC-θ catalytic activation and membrane translocation to the immunological synapse, leading to activation of NF-κB, AP-1, and NF-AT. These transcription factors are important for T cell survival, activation, and differentiation. Phosphorylation of PKC-θ at multiple Ser/Thr/Tyr residues is induced in T cells during TCR signaling. Some phosphorylation sites play critical roles in the regulation of PKC-θ function and downstream signaling. The regulation mechanisms for PKC-θ phosphorylation sites are now being revealed. In this review, we discuss the current understanding of the regulation of PKC-θ function by phosphorylation during TCR signaling. Protein kinase C (PKC)-θ was originally cloned in 1993 as a novel member of the PKC gene family . BecauseFigure 1. Like other PKC family members, PKC-θ consists of an N-terminal regulatory region (amino acids 1–378) and a C-terminal catalytic region  may be involved in PKC-θ T538 phosphorylation in Jurkat T cells upon PMA/ionomycin stimulation  was proposed to be the kinase directly phosphorylating PKC-θ based on the following evidence: (i) PKC-θ T538 phosphorylation is impaired in PDK1-knockdown Jurkat T cells and PDK1-deficient primary T cells upon anti-CD3/CD28 stimulation ; (ii) PDmulation . In addimulation . Howevermulation .in vitro . How such a complex regulation of PKC-θ phosphorylation is integrated with TCR/CD28 signaling is the key for the future understanding of PKC-θ function and T cell activation. Specifically, what kinases or phosphatases regulate the important PKC-θ phosphorylation sites in T-cell signaling? Does the phosphorylation of PKC-θ at different residues occur in a sequential and spatial manner during T cell signaling? How does PKC-θ phosphorylation at a specific site regulate its catalytic activity and membrane translocation? Future studies of the regulatory mechanisms of PKC-θ phosphorylation are important not only for the understanding of the underlying mechanism of PKC-θ-mediated cell activation, but also for the design of novel therapeutic targets for PKC-θ-mediated inflammatory diseases.In conclusion, phosphorylations play critical roles in regulating PKC-θ catalytic activity and membrane translocation, both of which are required for the proper function of PKC-θ in T cell activation. The regulation of PKC-θ phosphorylation is much more complex than we initially thought. The individual PKC-θ phosphorylation sites can be induced by autophosphorylation or upstream kinases depending on different cell types or cell stimuli. In TCR signaling, T538 and Y90 are phosphorylated on PKC-θ by GLK and LCK, respectively. Although phosphorylations of T219, S676, S685, and S695 are all induced during TCR signaling, the direct kinases for the phosphorylation of these sites on PKC-θ remain unclear. T538 phosphorylation is most critical for PKC-θ catalytic activation, whereas S676, S685, and S695 may be required for optimal catalytic activation of PKC-θ during TCR signaling. Phosphorylations of Y90 and T219 regulate PKC-θ membrane translocation and T-cell activation. PKC-θ is recruited to different locations in effector T cells and regulatory T cells and performs opposite functions in these cells . For exaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila mixed lineage kinase protein, Slpr, a mitogen-activated protein kinase kinase kinase (MAP3K) in the Jun Kinase (JNK) pathway. Genetic analysis of the function of non-phosphorylatable (PXAP) and phosphomimetic mutant (PXEP) Slpr transgenes in several distinct contexts revealed minimal effects in JNK-dependent tissue closure processes but differential requirements in heat stress response. In particular, PXAP expression resulted in sensitivity of adults to sustained heat shock, like p38 and JNK pathway mutants. In contrast, PXEP overexpression conferred some resistance. Indeed, phosphorylation of the PXSP motif is enriched under heat shock conditions and requires in part, the p38 kinases for the enrichment. These data suggest that coordination of signaling between p38 and Slpr serves to maintain JNK signaling during heat stress. In sum, we demonstrate a novel role for JNK signaling in the heat shock response in flies and identify a posttranslational modification on Slpr, at a conserved site among MAP3K mixed lineage kinase family members, which bolsters stress resistance with negligible effects on JNK-dependent developmental processes.Defining context specific requirements for proteins and pathways is a major challenge in the study of signal transduction. For example, the stress-activated protein kinase (SAPK) pathways are comprised of families of closely related transducers that are activated in a variety of tissues and contexts during development and organismal homeostasis. Consequently, redundant and pleiotropic effects have hampered a complete understanding of the individual contributions of transducers in distinct contexts. Here, we report on the function of a context-specific regulatory phosphorylation site, PXSP, in the  Drosophila mixed lineage kinase, Slipper (Slpr), which is responsive to environmental stress and required for optimal stress signaling.Cellular responses to environmental cues require the appropriate spatial and temporal coordination of signaling events. In fact, the sensitivity, amplitude, and duration of signaling activity in response to external stimuli can dictate distinct cellular outputs Upon exposure to stressful conditions like increased temperature, cells experience an accumulation of misfolded and aggregated proteins 2-terminal Kinase (JNK) and p38 MAPKs, are highly conserved transducers of cellular information in response to a variety of distinct signals. Cumulative evidence shows that these pathways are used during animal development, yet they are also inducible to reestablish homeostasis in an unstable environment Drosophila genome encodes three p38 MAPK genes, p38a (Mpk2), p38b and p38c, which can be activated by the MAPK kinases Mkk3/licorne and Mkk4 Drosophila MAP3Ks, Tak1, Ask1, and Mekk1, have been implicated in p38 signaling The stress-activated MAPK pathways, surrounding the Jun NHDrosophila, the single JNK homolog, Basket (bsk), is activated by the MAP2Ks Hemipterous (hep) and Mkk4 wallenda), Tak1, Takl2, and MLK (slpr) Drosophila homolog of mammalian mixed lineage kinases (MLKs), as the JNK kinase kinase required for embryonic dorsal closure Drosophila Slpr has been implicated in immune responsive p38 signaling in cultured cells, but this link has not been investigated in vivoThe JNK pathway has also been implicated in diverse cellular responses including apoptosis, wound healing, inflammation, and cell proliferation in many multicellular organisms. In in vitro and in vivo assays, we characterize this conserved phosphorylation site in Drosophila MLK/Slpr and demonstrate that it is required in the context of stress signaling but not necessarily in development. Namely, phosphorylation of a PXSP regulatory site in Slpr is enriched under specific stress conditions and mutation of this site alters stress responsive behavior of flies. Our results implicate Slpr and JNK signaling along with the p38 pathway in heat shock response and we argue that p38 MAPK-dependent modulation of Slpr phosphorylation might function in crosstalk to maintain Slpr-dependent signaling during stress response.Like many kinases, MLK activation is regulated by multiple molecular mechanisms and inputs. These mechanisms include relief of inhibition, dimerization, posttranslational modification, scaffolding, proteolysis, and changes in subcellular localization K53RUAS-bsk BL#9311 DNUAS-bsk BL#6409 WTUAS-bsk BL#9310 actUAS-hep BL#9306 UAS-hep.CA BL#6406 GG01785lic BL#19989, UAS-Dcr-2.D BL#24651 DNUAS-p38b1Mpk2 (1p38a null) 1hepE69puc (puc-lacZ) BS06slpr has been described 5475R-1Mpk2, 7393R-1p38b, 5680R-1bsk. 1118w was used as a control genotype. For constructs under the control of UAS sequences, expression is regulated by the presence of the Gal4 transcription factor +mW.hs = GAL4-arm.S}11)arm-Gal4 pnr-Gal4 69B-Gal4  according to manufacturer's instructions. Labeled proteins were analyzed by SDS-PAGE and autoradiography.In vitro translated protein was incubated with 1× λ-phosphatase at 30°C for 90 minutes in PMP buffer  supplemented with 1 mM MnCl2 (New England Biolabs). Proteins were subsequently visualized using SDS-PAGE and autoradiography, and dephosphorylation was detected by shifts in electrophoretic mobility. Aliquots of embryonic protein lysates in NP-40 buffer  were added to PMP buffer and treated similarly.Analyze>Gel function and represent grey values associated with the bands in each lane plotted versus distance migrated on the gel. Fold changes in relative levels of endogenous protein upon heat shock were determined from a minimum of four blots. Student's t-test was used for statistical analysis.Overnight collections of embryos expressing the transgenes were dechorionated and homogenized in NP-40 buffer with added protease inhibitors to generate a lysate for Western immunoblot. After separation by 8% SDS-PAGE, proteins were transferred to PVDF membrane and immunoblots were performed using the SNAP i.d.® system (Millipore). Mouse anti-HA  and mouse anti-β-tubulin  were diluted to 1∶300 and 1∶90, respectively, in 3 ml of 0.2% milk/TBST block. Rabbit anti-SlprSH3 was used at 1∶200 Immunofluorescence of embryos was performed as described For testing heat susceptibility of adult flies, stocks or crosses were raised at 25°C. Newly eclosed adults were collected in fresh vials and aged for 3–5 days. At this point, the experimental details for the mutant stocks versus the transgenic crosses were different and noted here.unaffected  or as affected (if sluggish or immobile on the surface of the food). Data from 3–10 separate experiments were combined, plotted as survival curves using Prism GraphPad software, and analyzed by the log-rank test.For the analysis of mutants, adults (gender combined) were transferred to fresh vials and placed at 37.5±1.0°C in a circulating water bath. Every 30 minutes, flies were scored as For experiments with transgenic flies, 3–5 day old adults from crosses were separated by gender in empty vials and heat-shocked at 37.5±1.0°C. After an initial 20 minutes, surviving adults were scored every half hour. Data from three separate experiments were combined, plotted, and analyzed in Prism GraphPad. Total numbers of adults tested per genotype is noted in the figure legends.For heat shock experiments, 14-hour egg collections on grape-juice agar plates were performed at 25°C. Up to 100 embryos were then handpicked to duplicate plates and aged for a total of three hours (3–17 hour-old population). Following this, control plates (−HS) were kept at 25°C while experimental plates (+HS) were transferred to a 37°C incubator for three hours (6–20 hour-old population) and then returned to 25°C. After an additional 24 hours, the number of dead embryos was recorded. Three trials per genotype were performed and the total numbers of embryos varied between 145 and 214. Data was displayed as mean percent lethality and the effect of heat shock on individual genotypes was analyzed using Student's t-test with Welches correction for unequal variance when appropriate.Embryonic protein lysates for Western immunoblots were prepared in NP-40 buffer from similar collection plates shocked at either 37°C (heat shock) or 4°C (cold shock) for three hours (6–20 hour-old population).2 254-nm light in a Stratalinker® 1800 UV Crosslinker (Stratagene). Embryos were allowed to recover in the absence or presence of light for 1 or 3 hours before protein lysates were prepared. The efficacy of irradiation was determined by removing 10–20 embryos from the irradiated sample to a new plate and scoring for death after an additional 24 hours of incubation at 25°C.UV irradiation was performed by exposing an overnight collection of dechorionated embryos spread on nytex membrane to 120 mJ/cmWith no more than 30 adults in a single vial, flies were maintained at 25°C and scored daily for survival. Flies were flipped to new vials with yeasted food every 3 or 4 days. Data from multiple vials were combined, plotted as survival curves, and analyzed using the log-rank test in Prism GraphPad. Total numbers are given in the legend.in vivo through the use of mutant transgenic constructs.Kinase activation often requires phosphorylation within the catalytic domain, but many kinases have additional regulatory sites. Given that mammalian MLK family members are extensively phosphorylated in vitro translated form of Slpr, called SKLC, comprising the N-terminal SH3, Kinase, Leucine Zipper, and CRIB domains . In the context of the full length Slpr protein, the D314Y mutation renders the protein nonfunctional and is presumed to inactivate the kinase In vitro translated SK migrated as a single band  and phosphomimetic  transgenes in the context of the full length Slpr protein , JNK and p38. To begin, we first used the mobility of the SKLC form of Slpr by SDS-PAGE as a way to ask whether Slpr modification might be influenced by a stress such as temperature shift. Western immunoblot analysis of lysates from embryos expressing the  of SKLC . Further of SKLC , providi lysates . Indeed, lysates . If embr lysates . Similar lysates ,C. Thus, lysates , suggestGiven that heat shock can induce apoptosis Drosophila, as depicted in Drosophila p38 and Bsk/JNK proteins are phosphorylated after heat treatment using phospho-specific antibodies directed against the active form of the kinases and second, whether animals with mutations in these proteins or their activators are compromised in their response to heat shock. Regarding the first question, immunoblots of lysates prepared from adult Drosophila revealed robust induction of p38 phosphorylation upon heat shock p38a mutant and wildtype flies for comparison 1118w flies were gradually affected by the thermal stress such that by 4 hr, more than 60% of the population was sluggish or unresponsive. In comparison, p38a null flies became catatonic and unresponsive more rapidly, with nearly 100% of the animals affected after 4 hours. To test whether Slpr was required for this stress response, BS06slpr null mutants were also subjected to the heat stress and showed sensitivity intermediate between control and p38a mutant flies. Given that the JNK pathway has not been implicated previously in heat shock response in flies, we tested another member of the JNK pathway, the JNK kinase, Hep  do not support that interpretation, and alternative explanations are conceivable. For example, other kinases are activated by p38 MAPKs, including the MAPKAP/MK, MSK, and MNK family members Drosophila has not yet been investigated; however, a recent study revealed that the MAPK phosphatase, Puckered, was phosphorylated by JNK and p38 in response to oxidative stress, though the consequences of this modification are still poorly understood in vivo. Also, the observation that phospho-SKLC was not enriched in lysates from UV-treated embryos, in which p38 is expected to be active, argues against a general p38 requirement and perhaps in favor of a temperature-dependent mechanism.Based on the appearance of two forms of SKLC in lysates prepared from ctivated  and the Drosophila heat shock response is clear . Taken together, it seems unlikely that feedback regulation of Slpr by Bsk/JNK could account for the temperature dependent enrichment of PXpSP even though the precedent of mammalian MLK3 phosphorylation by JNK as a positive feedback mechanism has been described Though the details of how p38 MAPKs are involved in regulating Slpr phosphorylation under stress conditions remain to be elucidated, the requirement for p38 MAPK signaling in is clear . Our resin vivo? In developmental processes that require Slpr-dependent JNK signaling, we found that mutants in the PXSP site behaved more or less like wildtype Slpr. When overexpressed, the PXAP and PXEP proteins were competent to upregulate JNK signaling in the embryo and to complement a slpr mutant to viability to variable extents. However, when expressed in animals that were also subjected to heat stress conditions, there was a deficit in the response of animals expressing the non-phosphorylatable form, PXAP, and a measurable protection in animals expressing the phospho-mimetic PXEP protein. Moreover, flies expressing the PXAP form had a shorter median survival value at 41 days relative to controls with 51-day median survival in longevity experiments. So flies having this modification (PXEP), or being able to dynamically regulate it (PXSP), were at an advantage relative to flies expressing a form that could not be modified (PXAP), suggesting that phosphorylation at this site is functional and not just a byproduct of heat shock.What then is the physiological purpose of the conserved PXSP site? And does the enrichment of the phosphorylated form upon high or low temperature shock have any consequences The lower overall recovery of flies expressing PXAP transgenes might indicate that they are generally less fit than other genotypes used here. While this is a concern, the results we obtained using several independent transgenic lines provides evidence against a spurious effect on fitness due to the insertion site interfering with an unknown gene. Moreover, the results that PXAP expression in the embryo had a minimal impact on embryonic lethality or signaling under nonstress conditions, coupled with the evidence that UV radiation does not appear to elicit changes in the phosphorylation status at PXSP, as does heat shock, suggests a distinct, physiological response.While our data suggest a role for PXSP phosphorylation in thermostress signaling, it remains unclear by what mechanism this modification modulates Slpr activity. To probe the effect on Slpr when PXSP phosphorylation is lost, we performed several biochemical experiments (not shown). Proteolytic analysis of PXAP and SlprWT revealed similar proteolysis patterns with or without heat shock, indicating analogous protein folding between the two forms, thus ruling out the notion that the PXAP protein is grossly misfolded. Consistent with that observation, the unfolded protein response pathway was not induced in embryos overexpressing PXAP under non-stress conditions. Furthermore, we were unable to detect a biochemical interaction between Slpr and HSP70 or HSP90 under normal or stress conditions. Thus, while phosphorylation within the PXSP motif might not be required for proper protein folding, other explanations are credible, including regulation of protein turnover, spatial distribution, or activity. JNK phosphorylation of mammalian MLK3 at the serine-proline site regulates its distribution in the cell to modulate signaling intensity Drosophila and mammals arm-Gal4 directs transgenic Slpr protein expression weakly in larval imaginal discs and nonneural tissue, but strongly in the larval and adult nervous system, primarily in the mushroom bodies of the brain arm-Gal4 directed expression of Slpr transgenes in the insulin-producing cells of the adult brain or in other structures including the mushroom body is responsible for the phenotypic differences that we observe in adult stress response will be a topic for future investigations.Another question raised by our results is which tissue requires active JNK or p38 signaling during adult heat stress? Accumulating evidence suggests that the nervous system, in particular insulin producing neurosecretory cells, upregulate JNK signaling during oxidative stress response, which counteracts insulin/IGF signaling allowing an adaptive systemic response The results presented in this report demonstrate that the JNK pathway, including Slpr, is required for heat shock response. Modification of the PXSP site in Slpr is enriched in a temperature dependent fashion and correlates with the degree of susceptibility to heat stress. We argue that phosphorylation of the PXSP motif is important to sustain JNK signaling in attempt to reestablish homeostasis. The presence of phosphorylated Slpr protein at steady state under nonstress conditions might buffer signaling activity from complete failure in rapidly changing conditions.Figure S1Expression and localization of transgenes in embryonic epidermis. (A) Western immunoblot for the HA tag shows expression of the transgenic proteins in embryonic lysates. Specific transgenic lines are indicated in parentheses. (B) HA-directed immunofluorescence detecting the tagged transgenic proteins expressed under the control of pnr-Gal4. Dapi was used as a counterstain. Images are lateral views of stage 14 embryos.(TIF)Click here for additional data file.Figure S2Enrichment of endogenous Slpr, phosphorylated at the PXSP motif, upon heat shock. (A) Western immunoblots of embryonic lysates without or with λ-phosphatase treatment. Blot was first probed with anti-Slpr PXpSP antibody (P-Slpr), then stripped and reprobed with anti-Slpr SH3 antibody . (B) Western blot with the indicated antibodies probing amounts of protein before and after heatshock . (C) Quantification of phospho-Slpr versus total Slpr levels upon heat shock, normalized to β-tubulin as a loading control. The upregulation of the phosphorylated form of Slpr upon heat shock is significantly different than total Slpr (p = 5.6E-06).(TIF)Click here for additional data file.


Madurella mycetomatis. Thus, eumycetoma can precisely be diagnosed and confidently differentiated from similar conditions such as actinomycetoma by simple and inexpensive cytological techniques such as FNAC and imprint smears, employing routine May-Grünwald-Giemsa, Papanicolaou and simple PAS stains on cytological specimen, thus leading to rapid diagnosis for institution of correct treatment.Eumycetoma is a chronic cutaneous and subcutaneous infection caused by various genera of fungi producing specific colored granules known as grains. A 45-year-old farmer presented clinically with a left foot mass with multiple discharging sinuses existing for last 3 years. Clinical and radiological findings suggested a diagnosis of chronic osteomyelitis with suspicion of tuberculosis. Imprints plus fine needle aspiration cytology (FNAC) smears exhibited distinct brown-black colonies of a fungus having branching and septate hyphae embedded in matrix like material against a mixed inflammatory background. Periodic acid Schiff (PAS) stain gave positive staining and subsequent fungal culture confirmed the cytological diagnosis and aided in species identification as  It is mainly a disease of the tropical and subtropical zones, especially between the Tropic of Cancer and the Tropic of Capricorn, that is, between latitudes 15°S and 30°N. Mycetoma was first described in the mid-19ntified.1 It is enntified.15A 45-year-old man, farmer by occupation, presented clinically with a slow growing, slightly tender, firm mass on the plantar aspect of left foot for 3 years, measuring 5×4×3 cm. There were multiple discharging sinuses covered with blood mixed purulent exudates. Radiograph showed features of osteomyelitis and soft tissue shadow. A tentative clinical diagnosis of tuberculous osteomyelitis was proposed. Imprint smears were made from the discharge and fine needle aspiration cytology (FNAC) was done using a 23-gauge needle, attached to a 10-ml syringe. Aspirate consisted of sero-sanguinous, pus like material. Alcohol fixed smears were stained with Papanicolaou (Pap stain), while air-dried smears were stained with May-Grünwald-Giemsa (MGG) stain. Simultaneously, Periodic acid Schiff (PAS), Gram’s stain and acid fast bacilli (AFB) stains were performed. Additionally, fungal and bacterial cultures were carried out.Madurella mycetomatis on subsequent fungal culture.Both FNAC smears and imprint smears comprised plenty of pus cells, some lymphocytes, histiocytes and foreign body giant cells in a necrotic background. Several brown to black colonies were seen. On higher magnification, these consisted of septate, branching fungal hyphae embedded in a cement-like matrix Figures  and 2. PAgents that cause eumycetoma are primarily saprophytic micro-organisms that are found in the soil and on plant matter. Healthy persons become inoculated with these agents as a result of the traumatic implantation of thorns, splinters and other plant matter. Therefore, the incidence of mycetoma is more particularly occupation dependent as people like farmers, herdsmen and field workers are more likely to come in contact with the causative agents. The diseM. mycetomatis accounts for most cases worldwide. Pseudoallescheria boydii is the common aetiologic agent in the United States, while Madurella grisea is a common aetiologic agent in South America.[Eumycetoma may present as a small localised tumor like mass, with or without sinuses, or can be associated with significant morbidity in terms of gradual enlargement, destruction and deformity of the affected site. The diagnosis of eumycetoma is made tentatively clinically when discharging grains are visible to the naked eye. The grai America. In generet al.[et al.[The smears from eumycetoma lesions have a distinct cytological appearance, characterised by (brown to black) colonies of branching, septate (distinct) hyphae embedded in a matrix which stain positively with PAS or Gomori’s methenamine silver stains, both demonstrating large sized hyphae of eumycetoma.8 The dif4l.[et al. in their


The Peiligang Culture  in the Middle Yellow River region, North China, has long been considered representative of millet farming. It is still unclear, however, if broomcorn millet or foxtail millet was the first species domesticated during the Peiligang Culture. Furthermore, it is also unknown whether millet was cultivated singly or together with rice at the same period. In this study, phytolith analysis of samples from the Tanghu archaeological site reveals early crop information in the Middle Yellow River region, China. Our results show that broomcorn millet was the early dry farming species in the Peiligang Culture at 7800 cal. yr BP, while rice cultivation took place from 7800 to 4500 cal. yr BP. Our data provide new evidence of broomcorn millet and rice mixed farming at 7800 cal. yr BP in the Middle Yellow River region, which has implications for understanding the domestication process of the two crops, and the formation and continuance of the Ancient Yellow River Civilization. A prehiThe deposits from profile (130 cm in thickness) can be divided into five layers from top to bottom according to the structure of the stratigraphy, soil color, and the archaeological remains: 1) 0–22 cm, modern cultivated layer; 2) 22–66 cm, light yellow loess and loose deposits containing ceramic fragments; 3) 66–85 cm, brown soil, dense deposits containing charcoal and ceramic fragments; 4) 85–98 cm, red-brown soil, very dense deposits of red burned soil and charcoal; and 5) below 98 cm, pit deposit (pit H192). We successively collected the samples at 5-cm intervals from the eastern wall of excavation Unit 0316. In total 14 samples were taken from the 60 to 130 cm section, including five AMS dating samples .Another nine samples were collected from other pits  and houses  at the south part of the Tanghu site. These samples were dense deposits of red-brown, and rich in charcoal. Samples H110, H92, F39, and F34 were also used for AMS dating .c. 2 g) combining the techniques described by Piperno et al. 4P2O7), then treatment with 30% hydrogen peroxide (H2O2) and cold 15% hydrochloric acid (HCl), separation using zinc bromide  heavy liquid, and mounting on a slide using Canada Balsam. Phytolith counting and identification were performed using a Leica microscope with phase-contrast at 400 magnification. In most samples, more than 300 phytoliths were counted. Identification was aided by the use of reference materials Phytolith analysis was conducted on all 23 samples  , 100–105c. 370 phytoliths/g and c. 2230 phytoliths/g,.Sample H92  was from a typical storage pit according to the archaeological excavation et alMorphological characteristics  of each individual bulliform were measured following the definition of Zheng c. 0.3%). According to the archaeological stratigraphy and artifacts Rice bulliform and broomcorn millet phytoliths were found in samples H50 and F21-4 in small quantities , and tested the same age, 7800 cal. yr BP.Our phytolith results indicated that the millet remains in samples F21-4 and H92 from houses and pits at the Tanghu site were broomcorn millet. These results provided reliable evidence of broomcorn millet cultivation in the Peiligang Culture at 7800 cal. yr BP. Previous studies from Xinglonggou  c. 9000-8000 cal. yr BP c. 8000 cal. yr BP, is found at the Yuezhuang site north to the Lower Yellow River region c. 7800 to 4500 cal. yr BP in this region.The occurrence of rice at the Jiahu site in southern North China is the earliest presence of rice in North China and can be dated to as early as The morphology of rice bulliform phytoliths found at the Tanghu site has typical fish-scale-like features on the top and the two lateral protrusions, and more than nine scales arranged along the edge. The latter is considered the distinct morphological feature of cultivated rice et al.et al.et al.c.7800 to 4500 cal. yr BP.Additionally, the morphological comparisons of double-peaked rice husk phytoliths found from the Tanghu site with those in Zhao c. 5900 cal. yr BP, and then reached the Guanzhong Basin at c. 5600 cal. yr BP c. 5000 cal. yr BP The rice phytoliths found in the Tanghu site can also be considered evidence of a northward expansion of rice agriculture from the Middle-Lower reaches of the Yangtze River. Step by step, rice arrived at Nanjiaokou and nearby sites in the north part of Henan Province mo-pan), mullers (mo-bang), and others, recovered from the Tanghu site suggest that millet and rice farming could be traced back to 7800 cal. yr BP in the Middle Yellow River region Phytolith results together with numerous agricultural tools, i.e., denticulate sickles, adzes, slabs (metate, Our evidence indicates that broomcorn millet was cultivated in this region 7800 cal. yr BP during the Peiligang Culture. Rice exploitation took place in the period from 7800 to 4500 cal. yr BP. Our data provide new evidence of mixed broomcorn millet and rice farming record in the Peiligang Culture in the Middle Yellow River region. The findings have implications for understanding broomcorn millet domestication, northward spread of rice cultivation, and the formation of mixed millet-rice farming, thus the formation and continuance of the Ancient Yellow River Civilization.It should be pointed out that more work is needed to complete the picture of primitive agriculture at the Tanghu site in the Peiligang Culture due to the limited crop phytoliths found for this study. Well-dated multi-proxy data on pollen, starch analysis, and plant macrofossils are needed in future studies.


Cyanobacteria produce metabolites with diverse bioactivities, structures and pharmacological properties. The effects of microcystins (MCYs), a family of peptide type protein-phosphatase inhibitors and cylindrospermopsin (CYN), an alkaloid type of protein synthesis blocker will be discussed in this review. We are focusing mainly on cyanotoxin-induced changes of chromatin organization and their possible cellular mechanisms. The particularities of plant cells explain the importance of such studies. Preprophase bands (PPBs) are premitotic cytoskeletal structures important in the determination of plant cell division plane. Phragmoplasts are cytoskeletal structures involved in plant cytokinesis. Both cyanotoxins induce the formation of multipolar spindles and disrupted phragmoplasts, leading to abnormal sister chromatid segregation during mitosis. Thus, MCY and CYN are probably inducing alterations of chromosome number. MCY induces programmed cell death: chromatin condensation, nucleus fragmentation, necrosis, alterations of nuclease and protease enzyme activities and patterns. The above effects may be related to elevated reactive oxygen species (ROS) and/or disfunctioning of microtubule associated proteins. Specific effects: MCY-LR induces histone H3 hyperphosphorylation leading to incomplete chromatid segregation and the formation of micronuclei. CYN induces the formation of split or double PPB directly related to protein synthesis inhibition. Cyanotoxins are powerful tools in the study of plant cell organization. The marine and freshwater habitats are considered to be a source of potential drugs. To date approximately 16,000 natural products have been isolated from marine, freshwater organisms so it is not surprising that these organisms are a wonderful source of biologically active natural products with diverse chemistry and pharmacology too .Marine biotoxins have drawn worldwide attention because of their involvement in human intoxication and the socio-economic impacts brought by those incidents. To understand the chemistry and the mechanism of action of the toxins is important because it helps in developing the adequate countermeasures, such as detection, determination and therapeutic methods. In addition many of the toxins have been found to be useful tools for probing biological or pharmacological phenomena, like use of okadaic acid in protein phosphatase studies .The unicellular life forms such as several algae and cyanobacteria, which can become abundant under favorable environmental conditions in waters, are readily collectable, culturable, and thus, have become good targets of many natural products researchers . In fresCyanobacterial toxins are classified as hepatotoxins (microcystins and nodularins), neurotoxins , cytotoxins (cylindrospermopsins) and dermotoxins . Their pWe are focusing in this review on the two main cyanotoxin-MCY and CYN induced changes of chromatin organization and the possible cellular mechanisms of those alterations in plant cells.d-amino acids and two unusual amino acids namely, N-methyldehydroalanine (Mdha) and a hydrophobic d-amino acid, 3-amino-9-methoxy-2-6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) [l-amino acids along with alterations to side chains [Microcystis, Anabaena, Planktothrix, Nostoc [50 of protein phosphatase inhibition by MCY-LR is (0.1–0.25 nM) in vitro. Liver damage is one of the most common phenomena in the intoxication of microcystins in animal organisms which starts with loss of membrane integrity, cytoskeletal disorganization, cellular disruption, lipid peroxidation, DNA damage, apoptosis, necrosis, and ultimately death by hemorrhagic shock [MCYs are cyclic heptapeptides consisting of seven amino acids, including , Nostoc . The tox, Nostoc . It is t, Nostoc . The Add, Nostoc . Microcy, Nostoc . The IC5ic shock .Umezakia natans from Japan and in Aphanizomenon ovalisporum from Israel samples have the same epimeric form, which is of opposite orientation to the toxin of C. raciborskii from Australia [ in vitro. Inhibition of protein synthesis occurs at the ribosome during the peptide chain elongation step [CYN is a low molecular weight (MW.415) tricyclic alkaloid with guanidino group linked at C7 to hydroxymethyl uracil. Because of the negatively charged sulfate group and the positively charged guanidino group, the molecule is a zwitterion and very soluble in polar solvents . CYN wasustralia . The uraustralia . The mecion step . Evaluation step . Recent ion step . Glutathion step . The maiion step ,23, althion step . Plant cells have particularities not found in other eukaryotic cells. These include plastids, the presence of cellulose cell wall, storage vacuoles and plasmodesmata, the latter connecting neighboring cells in tissues  ,31,32,33Concerning chromatin in non-mitotic plant cells, both the disturbance of protein phosphatase activities by MCYs and protein synthesis by CYN are likely to interfere with a wide range of intracellular events, leading to cell death. Programmed cell death (PCD) of plant cells bears several particularities, like: (i) It is not always accompanied by internucleosomal DNA cleavage (DNA laddering) . In contrast, at higher MCY-LR doses, mitosis is inhibited, that is correlated with the induction of PCD and necrosis ,104. ThiPhragmites australis and Sinapis alba. The inhibition of mitosis could be correlated to the inhibition of growth. At a close examination of distinct mitotic phases, the toxin stimulates early as well as late mitosis at different doses at relatively long-term exposures of roots [Ceratophyllum demersum, where MCY-LR clearly blocked cells in prophase/prometaphase [V. faba showed apparently opposite effects: At low doses (1 μg mL−1), MCY-LR did not increase significantly the rate of cell division, but increased late mitosis index and delayed metaphase-anaphase transition [Tradescantia stamen hair cells microinjected with the toxin [−1), there was a delayed entry into M phase, but the acceleration of cell cycle and overall stimulation of mitosis, with a relatively low rate of late mitosis in V. faba [P. australis roots exposed to MCY-LR for 10 days [Similar observations have been made in plants. Stimulation of mitosis was observed at low MCY-LR concentrations in several model systems, while inhibition of mitotic activity occurs in long-term treated, non-synchronized root tip meristematic cells of of roots ,80,105. etaphase . Furtherhe toxin . Since dhe toxin , this efArabidopsis thaliana and Nicotiana tabacum are not able of mitotic or non-mitotic MT binding, when they are phosphorylated, e.g., PP1 and PP2A do not dephosphorylate them [ple4 genotype that bears a mutation in the AtMAP65-3/PLEIADE gene is characterized by alteration of AtMAP65-1 binding to MTs and multinucleate cells in root meristems: this mutant shows a clear relationship between mitotic MT assembly and correct chromatid segregation [i.e., PP1 and PP2A inhibition) on mitotic and non-mitotic MT dynamics and consequent chromatin organization. However, such studies are still awaited. Several studies proved MCY-induced alterations of mitotic spindle formation and consequently, abnormal chromosome movement during animal and human cell division. Characteristic anomalies were monopolar, multipolar or disrupted spindles ,108,109.ate them ,112. AtMate them . Thereforegation . We can V. faba root tips the toxin induced high levels of phosphorylation not only at the pericentromeric regions, but in chromosome arms as well. This was accompanied by chromosome hypercondensation in metaphase and the formation of lagging chromosomes leading to the formation of micronuclei  or reorientation (Ceratophyllum demersum shoot tips). Besides cell death, these processes are correlated to changes of cell shape and alterations in the morphology of axial organs  Increases in several protease isoenzyme activities in the aquatic plants  arrhiza ; (ii) Ineedlings ,78; (iiiP. australis roots, but not in S. alba seedlings [It is worth mentioning that the cyanotoxin induces the formation of necrotic-like tissue in eedlings ,72. OtheThe effects of MCY and CYN on chromatin organization in plants are summarized on  in vitro grown P. australis plantlets only.Concerning CYN, it induces spindle-phragmoplast disruption and abnormal PPB formation, probably due to its protein synthesis inhibitory effects in mitotic plant cells. To date, there is no direct evidence of CYN induced PCD in plant cells, but there are several indirect proofs e.g., modulation of protease activities and increases in ROS and oxygen radical scavenging enzymes-that raise the possibility of inducing this phenomenon. CYN-induced necrosis has been proven inA generalized diagram for the effects of MCY on plant chromatin is shown on As proposed in