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Plasmodium parasite resistance to antimalarial drugs is a serious threat to public health in malaria-endemic areas. Compounds that target core cellular processes like translation are highly desirable, as they should be capable of killing parasites in their liver and blood stage forms, regardless of molecular target or mechanism. Assays that can identify these compounds are thus needed. Recently, specific quantification of native Plasmodium berghei liver stage protein synthesis, as well as that of the hepatoma cells supporting parasite growth, was achieved via automated confocal feedback microscopy of the o-propargyl puromycin (OPP)-labeled nascent proteome, but this imaging modality is limited in throughput. Here, we developed and validated a miniaturized high content imaging (HCI) version of the OPP assay that increases throughput, before deploying this approach to screen the Pathogen Box. We identified only two hits; both of which are parasite-specific quinoline-4-carboxamides, and analogs of the clinical candidate and known inhibitor of blood and liver stage protein synthesis, DDD107498/cabamiquine. We further show that these compounds have strikingly distinct relationships between their antiplasmodial and translation inhibition efficacies. These results demonstrate the utility and reliability of the P. berghei liver stage OPP HCI assay for the specific, single-well quantification of Plasmodium and human protein synthesis in the native cellular context, allowing the identification of selective Plasmodium translation inhibitors with the highest potential for multistage activity., Competing Interests: The authors declare no conflict of interest.

Increasing numbers of antimalarial compounds are being identified that converge mechanistically at inhibition of cytoplasmic translation, regardless of the molecular target or mechanism. A deeper understanding of how their effectiveness as liver stage translation inhibitors relates to their chemoprotective potential could prove useful. Here, we probed that relationship using the Plasmodium berghei -HepG2 liver stage infection model. After determining translation inhibition EC 50 s for five compounds, we tested them at equivalent effective concentrations to compare the parasite response to, and recovery from, a brief period of translation inhibition in early schizogony, followed by parasites to 120 h post-infection to assess antiplasmodial effects of the treatment. We show compound-specific heterogeneity in single parasite and population responses to translation inhibitor treatment, with no single metric strongly correlated to the release of hepatic merozoites for all compounds. We also demonstrate that DDD107498 is capable of exerting antiplasmodial effects on translationally arrested liver stage parasites and uncover unexpected growth dynamics during the liver stage. Our results demonstrate that translation inhibition efficacy does not determine antiplasmodial efficacy for these compounds., (© 2024 McLellan and Hanson.)

The Plasmodium parasites that cause malaria undergo an obligate, asymptomatic developmental stage in the host liver before initiating the symptomatic blood-stage infection. The parasite liver stage is a key intervention point for antimalarial chemoprophylaxis: successful targeting of liver-stage parasites prevents disease development in individuals and can help to reduce parasite transmission in populations, as the gametocyte forms that transmit infection to mosquitos are exclusively found in the blood stage. Antimalarial drugs that can target multiple parasite stages are thus highly desirable, and one emerging cellular target for such multistage active compounds is the process of protein synthesis or translation. Quantitative study of liver stage translation, and thus mechanistic evaluation of translation inhibitors against liver stage parasites, is not amenable to the methods allowing quantification of asexual blood stage translation, such as radiolabeled amino acid incorporation or lysate-based translation of reporter transcripts. Here, we present a method using o-propargyl puromycin (OPP) labeling of host and parasite nascent proteomes in the P. berghei -HepG2 infection model, followed by automated confocal image acquisition and computational separation of P. berghei vs. H. sapiens nascent proteome signals to allow simultaneous readout of the effects of translation inhibitors on both host and parasite. This protocol details our HepG2 cell culture and infected monolayer handling optimized for microscopy, our OPP labeling workflow, and our approach to automated confocal imaging, image processing, and data analysis. Key features • Uses the o-propargyl puromycin labeling technique developed by Liu et al. to quantitatively analyze protein synthesis in Plasmodium berghei liver-stage parasites in actively translating hepatoma cells. • This quantitative approach should be adaptable for other puromycin-sensitive intracellular pathogens residing in actively translating host cells. • The P. berghei -infected HepG2 recovery and reseeding protocol presented here is of use in applications beyond nascent proteome labeling and quantification., Competing Interests: Competing interestsThe authors declare no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

Our previous study identified 52 antiplasmodial peptaibols isolated from fungi. To understand their antiplasmodial mechanism of action, we conducted phenotypic assays, assessed the in vitro evolution of resistance, and performed a transcriptome analysis of the most potent peptaibol, HZ NPDG-I. HZ NPDG-I and 2 additional peptaibols were compared for their killing action and stage dependency, each showing a loss of digestive vacuole (DV) content via ultrastructural analysis. HZ NPDG-I demonstrated a stepwise increase in DV pH, impaired DV membrane permeability, and the ability to form ion channels upon reconstitution in planar membranes. This compound showed no signs of cross resistance to targets of current clinical candidates, and 3 independent lines evolved to resist HZ NPDG-I acquired nonsynonymous changes in the P. falciparum multidrug resistance transporter, pfmdr1. Conditional knockdown of PfMDR1 showed varying effects to other peptaibol analogs, suggesting differing sensitivity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Importance: Plasmodium parasites cause malaria in humans. New multistage active antimalarial drugs are needed, and a promising class of drugs targets the core cellular process of translation, which has many potential molecular targets. During the obligate liver stage, Plasmodium parasites grow in metabolically active hepatocytes, making it challenging to study core cellular processes common to both host cells and parasites, as the signal from the host typically overwhelms that of the parasite. Here, we present and validate a flexible assay to quantify Plasmodium liver stage translation using a technique to fluorescently label the newly synthesized proteins of both host and parasite followed by computational separation of their respective nascent proteomes in confocal image sets. We use the assay to determine whether a test set of known compounds are direct or indirect liver stage translation inhibitors and show that the assay can also predict the mode of action for novel antimalarial compounds., Competing Interests: The authors declare no conflict of interest.

Protein synthesis is a core cellular process, necessary throughout the complex lifecycle of Plasmodium parasites, thus specific translation inhibitors would be a valuable class of antimalarial drugs, capable of both treating symptomatic infections in the blood and providing chemoprotection by targeting the initial parasite population in the liver, preventing both human disease and parasite transmission back to the mosquito host. As increasing numbers of antiplasmodial compounds are identified that converge mechanistically at inhibition of cytoplasmic translation, regardless of molecular target or mechanism, it would be useful to gain deeper understanding of how their effectiveness as liver stage translation inhibitors relates to their chemoprotective potential. Here, we probed that relationship using the P. berghei -HepG2 liver stage infection model. Using o-propargyl puromycin-based labeling of the nascent proteome in P. berghei- infected HepG2 monolayers coupled with automated confocal feedback microscopy to generate unbiased, single parasite image sets of P. berghei liver stage translation, we determined translation inhibition EC 50s for five compounds, encompassing parasite-specific aminoacyl tRNA synthetase inhibitors, compounds targeting the ribosome in both host and parasite, as well as DDD107498, which targets Plasmodium eEF2, and is a leading antimalarial candidate compound being clinically developed as cabamiquine. Compounds were then tested at equivalent effective concentrations to compare the parasite response to, and recovery from, a brief period of translation inhibition in early schizogony, with parasites followed up to 120 hours post-infection to assess liver stage antiplasmodial effects of the treatment. Our data conclusively show that translation inhibition efficacy per se does not determine a translation inhibitor's antiplasmodial efficacy. DDD107498 was the least effective translation inhibitor, yet exerted the strongest antimalarial effects at both 5x- and 10x EC 50 concentrations. We show compound-specific heterogeneity in single parasite and population responses to translation inhibitor treatment, with no single metric strongly correlated to release of hepatic merozoites for all compound, demonstrate that DDD107498 is capable of exerting antiplasmodial effects on translationally arrested liver stage parasites, and uncover unexpected growth dynamics during the liver stage. Our results demonstrate that translation inhibition efficacy cannot function as a proxy for antiplasmodial effectiveness, and highlight the importance of exploring the ultimate, as well as proximate, mechanisms of action of these compounds on liver stage parasites.

Gene expression in malaria parasites is subject to various layers of regulation, including histone post-translational modifications (PTMs). Gene regulatory mechanisms have been extensively studied during the main developmental stages of Plasmodium parasites inside erythrocytes, from the ring stage following invasion to the schizont stage leading up to egress. However, gene regulation in merozoites that mediate the transition from one host cell to the next is an understudied area of parasite biology. Here, we sought to characterize gene expression and the corresponding histone PTM landscape during this stage of the parasite lifecycle through RNA-seq and ChIP-seq on P. falciparum blood stage schizonts, merozoites, and rings, as well as P. berghei liver stage merozoites. In both hepatic and erythrocytic merozoites, we identified a subset of genes with a unique histone PTM profile characterized by a region of H3K4me3 depletion in their promoter. These genes were upregulated in hepatic and erythrocytic merozoites and rings, had roles in protein export, translation, and host cell remodeling, and shared a DNA motif. These results indicate that similar regulatory mechanisms may underlie merozoite formation in the liver and blood stages. We also observed that H3K4me2 was deposited in gene bodies of gene families encoding variant surface antigens in erythrocytic merozoites, which may facilitate switching of gene expression between different members of these families. Finally, H3K18me and H2K27me were uncoupled from gene expression and were enriched around the centromeres in erythrocytic schizonts and merozoites, suggesting potential roles in the maintenance of chromosomal organization during schizogony. Together, our results demonstrate that extensive changes in gene expression and histone landscape occur during the schizont-to-ring transition to facilitate productive erythrocyte infection. The dynamic remodeling of the transcriptional program in hepatic and erythrocytic merozoites makes this stage attractive as a target for novel anti-malarial drugs that may have activity against both the liver and blood stages., (© 2023. The Author(s).)

The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome ( Pf 20S) β5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf 20S than to human constitutive 20S ( Hs 20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf 20S and Hs 20Sc in complex with MPI-5 and Pf 20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum , underpinning the design of potent and selective antimalarial proteasome inhibitors., Competing Interests: The authors declare no competing interest.

The adherens junctions (AJs) and tight junctions (TJs) provide critical adhesive contacts between neighboring epithelial cells and are crucial for epithelial adhesion, integrity, and barrier functions in a wide variety of tissues and organisms. The striatin protein family, which are part of the striatin interaction phosphatases and kinases complex, are multidomain scaffolding proteins that play important biologic roles. We have previously shown that striatin colocalizes with the tumor suppressor protein adenomatous polyposis coli in the TJs of epithelial cells. Here we show that striatin affects junction integrity and cell migration, probably through a mechanism that involves the adhesion molecule E-cadherin. Cells engaged in cell-cell adhesion expressed a high MW-modified form of striatin that forms stable associations with detergent-insoluble, membrane-bound cellular fractions. In addition, striatin has recently been suggested to be a target of the poly (ADP-ribose) polymerases Tankyrase 1, and we have found that striatin interacts with Tankyrase 1 and is subsequently poly-ADP-ribosylated. Taken together, our results suggest that striatin is a novel cell-cell junctional protein that functions to maintain correct cell adhesion and may have a role in establishing the relationship between AJs and TJs that is fundamental for epithelial cell-cell adhesion.-Lahav-Ariel, L., Caspi, M., Nadar-Ponniah, P. T., Zelikson, N., Hofmann, I., Hanson, K. K., Franke, W. W., Sklan, E. H., Avraham, K. B., Rosin-Arbesfeld, R. Striatin is a novel modulator of cell adhesion.

Transmission of the malaria parasite Plasmodium falciparum involves infection of Anopheles mosquitoes. Here we characterize SOPT, a protein expressed in P. falciparum ookinetes that facilitates infection of the mosquito midgut. SOPT was identified on the basis that it contains a signal peptide, a PEXEL-like sequence and is expressed in asexual, ookinete and sporozoite stages, suggesting it is involved in infecting the human or mosquito host. SOPT is predicted to contain a subtilisin-like fold with a non-canonical catalytic triad and is orthologous to P. berghei PIMMS2. Localization studies reveal that SOPT is not exported to the erythrocyte but is expressed in ookinetes at the parasite periphery. SOPT-deficient parasites develop normally through the asexual and sexual stages and produce equivalent numbers of ookinetes to NF54 controls, however, they form fewer oocysts and sporozoites in mosquitoes. SOPT-deficient parasites were also unable to activate the immune-responsive midgut invasion marker SRPN6 after mosquito ingestion, suggesting they are defective for entry into the midgut. Disruption of SOPT in P. berghei (PIMMS2) did not affect other lifecycle stages or ookinete development but again resulted in fewer oocysts and sporozoites in mosquitoes. Collectively, this study shows that SOPT/PIMMS2 plays a conserved role in ookinetes of different Plasmodium species., (© 2018 John Wiley & Sons Ltd.)

Proteases have been implicated in a variety of developmental processes during the malaria parasite lifecycle. In particular, invasion and egress of the parasite from the infected hepatocyte and erythrocyte, critically depend on protease activity. Although falcipain-1 was the first cysteine protease to be characterized in P. falciparum, its role in the lifecycle of the parasite has been the subject of some controversy. While an inhibitor of falcipain-1 blocked erythrocyte invasion by merozoites, two independent studies showed that falcipain-1 disruption did not affect growth of blood stage parasites. To shed light on the role of this protease over the entire Plasmodium lifecycle, we disrupted berghepain-1, its ortholog in the rodent parasite P. berghei. We found that this mutant parasite displays a pronounced delay in blood stage infection after inoculation of sporozoites. Experiments designed to pinpoint the defect of berghepain-1 knockout parasites found that it was not due to alterations in gliding motility, hepatocyte invasion or liver stage development and that injection of berghepain-1 knockout merosomes replicated the phenotype of delayed blood stage growth after sporozoite inoculation. We identified an additional role for berghepain-1 in preparing blood stage merozoites for infection of erythrocytes and observed that berghepain-1 knockout parasites exhibit a reticulocyte restriction, suggesting that berghepain-1 activity broadens the erythrocyte repertoire of the parasite. The lack of berghepain-1 expression resulted in a greater reduction in erythrocyte infectivity in hepatocyte-derived merozoites than it did in erythrocyte-derived merozoites. These observations indicate a role for berghepain-1 in processing ligands important for merozoite infectivity and provide evidence supporting the notion that hepatic and erythrocytic merozoites, though structurally similar, are not identical.

As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment., (Copyright © 2017, American Association for the Advancement of Science.)

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

Iron is an essential micronutrient but is also highly toxic. In yeast and plant cells, a key detoxifying mechanism involves iron sequestration into intracellular storage compartments, mediated by members of the vacuolar iron-transporter (VIT) family of proteins. Here we study the VIT homologue from the malaria parasites Plasmodium falciparum (PfVIT) and Plasmodium berghei (PbVIT). PfVIT-mediated iron transport in a yeast heterologous expression system is saturable (Km ∼ 14.7 μM), and selective for Fe(2+) over other divalent cations. PbVIT-deficient P. berghei lines (Pbvit(-)) show a reduction in parasite load in both liver and blood stages of infection in mice. Moreover, Pbvit(-) parasites have higher levels of labile iron in blood stages and are more sensitive to increased iron levels in liver stages, when compared with wild-type parasites. Our data are consistent with Plasmodium VITs playing a major role in iron detoxification and, thus, normal development of malaria parasites in their mammalian host.

Prior to invading nonreplicative erythrocytes, Plasmodium parasites undergo their first obligate step in the mammalian host inside hepatocytes, where each sporozoite replicates to generate thousands of merozoites. While normally quiescent, hepatocytes retain proliferative capacity and can readily reenter the cell cycle in response to diverse stimuli. Many intracellular pathogens, including protozoan parasites, manipulate the cell cycle progression of their host cells for their own benefit, but it is not known whether the hepatocyte cell cycle plays a role during Plasmodium liver stage infection. Here, we show that Plasmodium parasites can be observed in mitotic hepatoma cells throughout liver stage development, where they initially reduce the likelihood of mitosis and ultimately lead to significant acquisition of a binucleate phenotype. However, hepatoma cells pharmacologically arrested in S phase still support robust and complete Plasmodium liver stage development, which thus does not require cell cycle progression in the infected cell in vitro. Furthermore, murine hepatocytes remain quiescent throughout in vivo infection with either Plasmodium berghei or Plasmodium yoelii, as do Plasmodium falciparum-infected primary human hepatocytes, demonstrating that the rapid and prodigious growth of liver stage parasites is accomplished independent of host hepatocyte cell cycle progression during natural infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Organisms have evolved numerous strategies to control infection by an array of intracellular pathogens. One cell autonomous pathogen control strategy is global inhibition of protein synthesis via stress granule (SG) formation. SGs are induced by stressful stimuli such as oxidative stress and nutrient deprivation, and are known to counteract both viral and bacterial infections. Pathogens, in turn, may actively block an infected cell's ability to form SGs. In vitro and in vivo, many liver stage malaria parasites are eliminated during development. We show here that SG formation is not amongst the strategies used for elimination of parasites from hepatocytes. Neither cell traversal, sporozoite invasion, nor rapid parasite growth leads to the formation of SGs. Furthermore, Plasmodium berghei infection does not compromise the ability of infected cells to assemble SGs in response to oxidative or nutritional stress. Plasmodium infection is therefore not detected by hepatocytes as a strong stressor necessitating global translational repression in response, highlighting the idea that Plasmodium has evolved strategies to ensure its remarkable growth in the hepatocyte while maintaining host cell homeostasis.

Before they infect red blood cells and cause malaria, Plasmodium parasites undergo an obligate and clinically silent expansion phase in the liver that is supposedly undetected by the host. Here, we demonstrate the engagement of a type I interferon (IFN) response during Plasmodium replication in the liver. We identified Plasmodium RNA as a previously unrecognized pathogen-associated molecular pattern (PAMP) capable of activating a type I IFN response via the cytosolic pattern recognition receptor Mda5. This response, initiated by liver-resident cells through the adaptor molecule for cytosolic RNA sensors, Mavs, and the transcription factors Irf3 and Irf7, is propagated by hepatocytes in an interferon-α/β receptor-dependent manner. This signaling pathway is critical for immune cell-mediated host resistance to liver-stage Plasmodium infection, which we find can be primed with other PAMPs, including hepatitis C virus RNA. Together, our results show that the liver has sensor mechanisms for Plasmodium that mediate a functional antiparasite response driven by type I IFN.

Residence within a customized vacuole is a highly successful strategy used by diverse intracellular microorganisms. The parasitophorous vacuole membrane (PVM) is the critical interface between Plasmodium parasites and their possibly hostile, yet ultimately sustaining, host cell environment. We show that torins, developed as ATP-competitive mammalian target of rapamycin (mTOR) kinase inhibitors, are fast-acting antiplasmodial compounds that unexpectedly target the parasite directly, blocking the dynamic trafficking of the Plasmodium proteins exported protein 1 (EXP1) and upregulated in sporozoites 4 (UIS4) to the liver stage PVM and leading to efficient parasite elimination by the hepatocyte. Torin2 has single-digit, or lower, nanomolar potency in both liver and blood stages of infection in vitro and is likewise effective against both stages in vivo, with a single oral dose sufficient to clear liver stage infection. Parasite elimination and perturbed trafficking of liver stage PVM-resident proteins are both specific aspects of torin-mediated Plasmodium liver stage inhibition, indicating that torins have a distinct mode of action compared with currently used antimalarials.

The chromosomal passenger complex (CPC) is a key regulator of mitosis in many organisms, including yeast and mammals. Its components co-localise at the equator of the mitotic spindle and function interdependently to control multiple mitotic events such as assembly and stability of bipolar spindles, and faithful chromosome segregation into daughter cells. Here, we report the first detailed characterisation of a CPC mutation in Drosophila, using a loss-of-function allele of borealin (borr). Like its mammalian counterpart, Borr colocalises with the CPC components Aurora B kinase and Incenp in mitotic Drosophila cells, and is required for their localisation to the mitotic spindle. borr mutant cells show multiple mitotic defects that are consistent with loss of CPC function. These include a drastic reduction of histone H3 phosphorylation at serine 10 (a target of Aurora B kinase), a pronounced attenuation at prometaphase and multipolar spindles. Our evidence suggests that borr mutant cells undergo multiple consecutive abnormal mitoses, producing large cells with giant nuclei and high ploidy that eventually apoptose. The delayed apoptosis of borr mutant cells in the developing wing disc appears to cause non-autonomous repair responses in the neighbouring wild-type epithelium that involve Wingless signalling, which ultimately perturb the tissue architecture of adult flies. Unexpectedly, during late larval development, cells survive loss of borr and develop giant bristles that may reflect their high degree of ploidy.

We identified Drosophila Smurf (DSmurf) as a negative regulator of signaling by the BMP2/4 ortholog DPP during embryonic dorsal-ventral patterning. DSmurf encodes a HECT domain ubiquitin-protein ligase, homologous to vertebrate Smurf1 and Smurf2, that binds the Smad1/5 ortholog MAD and likely promotes its proteolysis. The essential function of DSmurf is restricted to its action on the DPP pathway. DSmurf has two distinct, possibly mechanistically separate, functions in controlling DPP signaling. Prior to gastrulation, DSmurf mutations cause a spatial increase in the DPP gradient, as evidenced by ventrolateral expansion in expression domains of target genes representing all known signaling thresholds. After gastrulation, DSmurf mutations cause a temporal delay in downregulation of earlier DPP signals, resulting in a lethal defect in hindgut organogenesis.

Volatile anaesthetics have historically been considered to act in a nonspecific manner on the central nervous system. More recent studies, however, have revealed that the receptors for inhibitory neurotransmitters such as gamma-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABA(A) and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA rho1 receptor is reduced. We have used this difference in pharmacology to investigate the molecular basis for modulation of these receptors by anaesthetics and alcohols. By using chimaeric receptor constructs, we have identified a region of 45 amino-acid residues that is both necessary and sufficient for the enhancement of receptor function. Within this region, two specific amino-acid residues in transmembrane domains 2 and 3 are critical for allosteric modulation of both GABA(A) and glycine receptors by alcohols and two volatile anaesthetics. These observations support the idea that anaesthetics exert a specific effect on these ion-channel proteins, and allow for the future testing of specific hypotheses of the action of anaesthetics.

Retinoic acid receptors (RAR) are members of the steroid/thyroid hormone receptor superfamily and serve as ligand-activated transcription factors. In order to facilitate studies of receptor protein, we have generated a monoclonal antibody to the human RAR gamma, and have developed a procedure to purify the full-length receptor expressed in insect cells. The monoclonal antibody (A10) was developed using as antigen a carboxy-terminal fragment of the human RAR gamma expressed as a bacterial fusion protein. The A10 monoclonal antibody binds to both native and denatured forms of the human RAR gamma. This antibody was immobilized on a resin and used to purify full-length, baculovirus-expressed human RAR gamma to near homogeneity. The immunoaffinity-purified receptor is > 90-95% pure as revealed by silver-stained gels. The identity of the single protein band as RAR gamma was verified by immunoblotting using a polyclonal antibody to an epitope distinct from that recognized by the A10 antibody. The pure human RAR gamma is functional with respect to both ligand and DNA binding. Scatchard analysis of 3H-labeled all-trans retinoic acid binding to purified human RAR gamma revealed a single, high-affinity binding site with a Kd of approximately 2 nM. Binding of the pure RAR gamma to a DR5-type retinoic acid response element was also studied. Response element binding by RAR gamma required the presence of the retinoid X receptor, but did not require the presence of additional proteins. Human RAR gamma protein purified in this fashion will be useful in future structural and functional studies.

The maize dwarf mosaic virus strain B (MDMV-B) coat protein (cp) gene was cloned into a monocot expression cassette and introduced into sweet corn cell suspension cultures via particle bombardment or electroporation. Transformed cells were selected on culture media containing 300 mg/l kanamycin, and plants were regenerated. Cells from all transformed lines expressed the cp gene; and one transgenic line synthesized approximately 100-200 micrograms MDMV-cp per gram fresh weight. Plants regenerated from this line were challenged with a virus inoculum concentration adjusted to produce symptoms in nontransgenic controls at six days post inoculation. In growth chamber studies, the presence of the MDMV-cp provided resistance to inoculations with MDMV-A or MDMV-B and to mixed inoculations of MDMV and maize chlorotic mottle virus.

Competition of all-trans-retinol and all-trans-retinaldehyde with 3H-labeled all-trans-retinoic acid (RA) for binding to retinoic acid receptors (RARs) was examined in human neuroblastoma cell nuclear extracts. All-trans-retinol was 35-fold less potent than all-trans-RA, whereas all-trans-retinaldehyde was 500-fold less active in binding to the nuclear receptors. To confirm that all-trans-retinol binds to RARs, experiments were carried out with RARs alpha, beta, and gamma expressed as bacterial fusion proteins. All-trans-retinol was only 4- to 7-fold less potent than all-trans-RA in binding to all three RAR subtypes. The all-trans-retinol binding observed was not the result of metabolism of retinol to RA or some other active compound during the binding experiment. Retinyl acetate was virtually inactive in competition binding experiments, while very slight activity was observed with 13-cis-RA and all-trans-retinaldehyde. Significant competition occurred with 4-hydroxy-RA and 4-keto-RA, which were 15- to 40-fold less potent than all-trans-RA. The 9-cis isomer of RA was equipotent with all-trans-retinol in these studies. These results suggest that all-trans-retinol cannot be excluded as a physiologically significant ligand for RAR-mediated gene expression.