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Statement of Problem: Dentists strive to create natural looking teeth for their patients. However, much of the research on esthetic dentistry has used artificial and computer-generated images to establish esthetic norms. Altered images are helpful in determining personal preferences but may be misleading for natural norms., Purpose: The purpose of this report from the Committee on Research in Fixed Prosthodontics of the American Academy of Fixed Prosthodontics was to establish an evidence base for clinical guidelines on lip mobility and the visibility of oral tissues., Material and Methods: A systematic search was conducted to collect data on the lip border movements and the visibility of teeth and gingiva of human participants., Results: One thousand and ninety-six articles were processed, and, based on inclusion criteria, 48 articles were reviewed. The findings determined that the upper lip lengthens with age resulting, at rest, in less display of the maxillary teeth and more of the mandibular arch. When evaluating the maxillary incisal edge position at rest, the canines are the most reproducible. Women tend to display more gingiva in a social smile than men, but this may not be so for a true smile of joy. Both the middle and lower facial thirds should be evaluated to detect a maximal smile. Care must be taken not to classify a person incorrectly as having a low smile line because they could still have a potential to show gingiva., Conclusions: Evidence-based clinical guidelines are offered to reduce the risk of esthetic failures., (Copyright © 2021 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.)

Objective: When multiple visible teeth are prepared, the prolonged treatment time may lead to patients needing a break that requires them to leave the operatory. Such a situation allows the patient to view their prepared teeth, a process that can be disconcerting to some patients., Clinical Considerations: An intraoperative provisional restoration can be made by using a thermoplastic vacuum-formed matrix of the patient's teeth that is filled with white-colored impression material and then placed over the prepared teeth to form a provisional restoration., Conclusions: The use of an intraoperative provisional restoration can be effectively used to cover prepared teeth while providing normal tooth morphology and facial appearance after preparation of visible teeth., Clinical Significance: When visible teeth are prepared, an intraoperative provisional restoration can be used to cover the prepared teeth and prevent concerned patients from viewing their prepared teeth. (J Esthet Restor Dent 29:189-192, 2017)., (© 2017 The Authors Journal of Esthetic and Restorative Dentistry Published by Wiley Periodicals, Inc.)

Statement of Problem: A patient's smile may not elicit the maximum amount of maxillary gingiva., Purpose: The purpose of this study was to measure the amount of gingival display with 4 different facial expressions., Material and Methods: Video images of 91 randomly selected adults were evaluated to measure the height of gingival display at the maxillary anterior teeth and first premolars when participants were asked to give their biggest smile (requested smile), make a Duchenne smile, mimic an intense grimace of disgust (grimace), and produce a funnel-shaped expression (funnel). Measurements were compared with the Friedman Test with post hoc comparisons (α=.05 for all tests)., Results: The intraclass correlation coefficient was (95% CI)=0.913(0.623, 0.984). At the central and lateral incisors, the grimace and funnel expressions produced the greatest amounts of gingival display. At the canines and the first premolars, both smiles (requested and Duchenne) exhibited the largest amount of gingival display of the 4 facial expressions., Conclusions: Neither smile type revealed a significantly greater amount of gingival display above the maxillary central or lateral incisors but the grimace and funnel facial expressions did (P ≤.001). Above the maxillary canines, both smiles displayed a significantly greater amount of gingiva than did the funnel expression (P<.001), but only the Duchenne smile displayed a greater amount than did the grimace expression (P=.05). Superiorly to the maxillary first premolars, both smile types revealed significantly greater amounts of soft tissue when compared with the other 2 facial expressions (P<.001)., (Copyright © 2014 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.)

The malaria parasite Plasmodium falciparum is able to synthesize de novo PLP (pyridoxal 5'-phosphate), the active form of vitamin B6. In the present study, we have shown that the de novo synthesized PLP is used by the parasite to detoxify 1O2 (singlet molecular oxygen), a highly destructive reactive oxygen species arising from haemoglobin digestion. The formation of 1O2 and the response of the parasite were monitored by live-cell fluorescence microscopy, by transcription analysis and by determination of PLP levels in the parasite. Pull-down experiments of transgenic parasites overexpressing the vitamin B6-biosynthetic enzymes PfPdx1 and PfPdx2 clearly demonstrated an interaction of the two proteins in vivo which results in an elevated PLP level from 12.5 μM in wild-type parasites to 36.6 μM in the PfPdx1/PfPdx2-overexpressing cells and thus to a higher tolerance towards 1O2. In contrast, by applying the dominant-negative effect on the cellular level using inactive mutants of PfPdx1 and PfPdx2, P. falciparum becomes susceptible to 1O2. Our results demonstrate clearly the crucial role of vitamin B6 biosynthesis in the detoxification of 1O2 in P. falciparum. Besides the known role of PLP as a cofactor of many essential enzymes, this second important task of the vitamin B6 de novo synthesis as antioxidant emphasizes the high potential of this pathway as a target of new anti-malarial drugs.

A series of previously unreported α-hydroxy hydrazonates were synthesized and tested for their antimalarial properties. Structure optimization of the antiplasmodially active α-hydroxy hydrazonate III furnished derivatives with strong in-vitro antimalarial activity against 3D7 strains of Plasmodium falciparum with IC(50) values lower than 2.0 µM., (2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Statement of Problem: The rigidity of the major connector for partial removable dental prostheses (PRDP) has not been well defined. There is little scientific evidence to support specific standards relative to the dimensions of a casting and their effect on long-term function of the PRDP or the supporting tissues., Purpose: The purpose of this study was to compare in vitro the effects of load deflection of 5 clinically used mandibular major connector forms., Material and Methods: A partially edentulous dental cast of a patient with the mandibular anterior teeth remaining was used as the test model. Lingual plate (LP), continuous bar (CB), sublingual bar (SB), lingual bar (LB), and dental lingual bar (DLB) specimens (n=5) were waxed on identical refractory casts, then cast in cobalt base alloy and finished. Each specimen was placed on a custom test table and statically loaded in the contralateral second molar region with 500 g in the horizontal and vertical planes. Deflections were measured at the terminal abutment rest seat, anterior to the load. Comparisons between group means were evaluated using the Dunnett T3 method, assuming unequal variances, for all pairwise tests at a .05 significance level and for 95% confidence intervals for group mean differences. Differences within the groups for vertical versus horizontal deflections were compared using a paired t test and Bonferroni correction (alpha=.05 for all tests)., Results: Horizontal deflection between LP and LB, CB and LB, and SB and LB were significant, with the first in each comparison having the least amount of deflection. A significant difference was also seen in the vertical deflection, with the first in each comparison observed to have deflected the least for LP versus CB, LP versus LB, LP versus DLB, CB versus DLB, LB versus DLB, and SB versus DLB. The amount of vertical deflection was significantly greater than the horizontal deflection for all 5 mandibular major connectors (P< or =.033)., Conclusions: In the horizontal plane, a significant difference in the amount of deflection was seen between the LP and LB, CB and LB, and LB and SB. In the vertical plane, the LP deflected significantly less than the CB, LB, and DLB. In addition, the DLB deflected significantly more in the vertical plane when compared to the CB, LB, and SB. All 5 mandibular major connectors had a significantly greater amount of vertical deflection as compared to horizontal deflection., (Copyright © 2010 The Editorial Council of the Journal of Prosthetic Dentistry. Published by Mosby, Inc. All rights reserved.)

As an intracellular proliferating parasite, Plasmodium falciparum exploits the human host to acquire nutrients. However, nutrients such as nucleotides and cofactors are mostly phosphorylated in the host cell cytosol and thus have to be dephosphorylated in order to be taken up by the parasite. Here we report the functional characterization of a unique secreted phosphatase in P. falciparum, which is expressed throughout the developmental stages in the red blood cell. We show that this enzyme, formerly described as anchoring glideosome-associated protein 50 (GAP50), reveals a broad substrate profile with preference for di- and triphosphates at pH 5-7. Bioinformatic studies of the protein sequence identified an N-terminal signal anchor (SA) as well as a C-terminal transmembrane domain. By means of live microscopy of parasites transfected with GFP-fusions of this secreted acid phosphatase (PfSAP), we demonstrate that PfSAP enters the secretory pathway en route to the parasite periphery - mediated by SA - and is subsequently engulfed into the food vacuole. We corroborate this with independent data where acid phosphatase activity is visualized in close proximity to hemozoin. The biochemical as well as the trafficking results support the proposed role of PfSAP in the acquisition of host nutrients by dephosphorylation.

Vitamin B1 is in its active form thiamine pyrophosphate (TPP), an essential cofactor for several key enzymes in the carbohydrate metabolism. Mammals must salvage this crucial nutrient from their diet in order to complement the deficiency of de novo synthesis. In the human pathogenic bacterium Staphylococcus aureus, two operons were identified which are involved in vitamin B1 metabolism. The first operon encodes for the thiaminase type II (TenA), 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase (ThiD), 5-(2-hydroxyethyl)-4-methylthiazole kinase (ThiM) and thiamine phosphate synthase (ThiE). The second operon encodes a phosphatase, an epimerase and the thiamine pyrophosphokinase (TPK). The open reading frames of the individual operons were cloned, their corresponding proteins were recombinantly expressed and biochemically analysed. The kinetic properties of the enzymes as well as the binding of TPP to the in vitro transcribed RNA of the proposed operons suggest that the vitamin B1 homeostasis in S. aureus is strongly regulated at transcriptional as well as enzymatic levels.

The human malaria parasite Plasmodium falciparum is able to synthesize de novo pyridoxal 5-phosphate (PLP), a crucial cofactor, during erythrocytic schizogony. However, the parasite possesses additionally a pyridoxine/pyridoxal kinase (PdxK) to activate B6 vitamers salvaged from the host. We describe a strategy whereby synthetic pyridoxyl-amino acid adducts are channelled into the parasite. Trapped upon phosphorylation by the plasmodial PdxK, these compounds block PLP-dependent enzymes and thus impair the growth of P. falciparum. The novel compound PT3, a cyclic pyridoxyl-tryptophan methyl ester, inhibited the proliferation of Plasmodium very efficiently (IC(50)-value of 14 microM) without harming human cells. The non-cyclic pyridoxyl-tryptophan methyl ester PT5 and the pyridoxyl-histidine methyl ester PHME were at least one order of magnitude less effective or completely ineffective in the case of the latter. Modeling in silico indicates that the phosphorylated forms of PT3 and PT5 fit well into the PLP-binding site of plasmodial ornithine decarboxylase (PfODC), the key enzyme of polyamine synthesis, consistent with the ability to abolish ODC activity in vitro. Furthermore, the antiplasmodial effect of PT3 is directly linked to the capability of Plasmodium to trap this pyridoxyl analog, as shown by an increased sensitivity of parasites overexpressing PfPdxK in their cytosol, as visualized by GFP fluorescence.

The present study reports the purification and characterization of GST from cytosolic fraction of Setaria cervi. GST activity was determined in various subcellular fractions of bovine filarial worms S. cervi (Bubalus bubalis Linn.) and was found to be localized mainly in the cytosolic and microsomal fractions. The soluble enzyme from S. cervi was purified to homogeneity using a combination of salt precipitation, centrifugation, cation exchange and GSH-Sepharose affinity chromatography followed by ultrafiltration. SDS-PAGE analysis revealed a single band and activity staining was also detected on PAGE gels. Gel filtration and MALDI-TOF studies revealed that the native enzyme is a homodimer with a subunit molecular mass of 24.6 kDa. Comparison of kinetic properties of the parasitic and mammalian enzymes revealed significant differences between them. The substrate specificity and inhibitor profile of cytosolic GST from S. cervi appeared to be different from GST from mammalian sources.

More than 30 years ago the potent ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) was designed as new anticancer drug. Its efficacy was not as expected since the polyamine metabolism in mammalian cells seemed to be far more complex. However when DFMO was applied to African trypanosomes its effect on this protozoan parasite was highly convincing. Thenceforward many researchers tested DFMO and also other polyamine synthesis inhibitors against different parasites among them the causative agent of malaria Plasmodium. This review recapitulates the different attempts to interfere chemically with the plasmodial polyamine metabolism, the impact on the disease as well as its biochemical and molecular background. It will show that this fast proliferating organism depends for growth on high amounts of polyamines and that Plasmodium has its own and unique polyamine synthesis, differing highly from the mammalian one mainly in the arrangement of the key enzymes, S-adenosylmethionine decarboxylase and ornithine decarboxylase (AdoMetDC/ODC), on a bifunctional protein.

Background: Plants, fungi, bacteria and the apicomplexan parasite Plasmodium falciparum are able to synthesize vitamin B6 de novo, whereas mammals depend upon the uptake of this essential nutrient from their diet. The active form of vitamin B6 is pyridoxal 5-phosphate (PLP). For its synthesis two enzymes, Pdx1 and Pdx2, act together, forming a multimeric complex consisting of 12 Pdx1 and 12 Pdx2 protomers., Methodology/principal Findings: Here we report amino acid residues responsible for stabilization of the structural and enzymatic integrity of the plasmodial PLP synthase, identified by using distinct mutational analysis and biochemical approaches. Residues R85, H88 and E91 (RHE) are located at the Pdx1:Pdx1 interface and play an important role in Pdx1 complex assembly. Mutation of these residues to alanine impedes both Pdx1 activity and Pdx2 binding. Furthermore, changing D26, K83 and K151 (DKK), amino acids from the active site of Pdx1, to alanine obstructs not only enzyme activity but also formation of the complex. In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK residues results in a hexameric assembly, and does not affect Pdx2 binding or its activity. While the modelled position of K151 is distal to the Pdx1:Pdx1 interface, it affects the assembly of hexameric Pdx1 into a functional dodecamer, which is crucial for PLP synthesis., Conclusions/significance: Taken together, our data suggest that the assembly of a functional Pdx1:Pdx2 complex follows a defined pathway and that inhibition of this assembly results in an inactive holoenzyme.

Vitamins are essential compounds mainly involved in acting as enzyme co-factors or in response to oxidative stress. In the last two years it became apparent that apicomplexan parasites are able to generate B vitamers such as vitamin B1 and B6 de novo. The biosynthesis pathways responsible for vitamin generation are considered as drug targets, since both provide a high degree of selectivity due to their absence in the human host. This report updates the current knowledge about vitamin B1 and B6 biosynthesis in malaria and other apicomplexan parasites. Owing to the urgent need for novel antimalarials, the significance of the biosynthesis and salvage of these vitamins is critically discussed in terms of parasite survival and their exploitation for drug development.

To elucidate the function of Omega class glutathione transferases (GSTs) (EC 2.5.1.18) in multicellular organisms, the GSTO-1 from Caenorhabditis elegans (GSTO-1; C29E4.7) was investigated. Disc diffusion assays using Escherichia coli overexpressing GSTO-1 provided a test of resistance to long-term exposure under oxidative stress. After affinity purification, the recombinant GSTO-1 had minimal catalytic activity toward classic GST substrates but displayed significant thiol oxidoreductase and dehydroascorbate reductase activity. Microinjection of the GSTO-1-promoter green fluorescent protein construct and immunolocalization by electron microscopy localized the protein exclusively in the intestine of all postembryonic stages of C. elegans. Deletion analysis identified an approximately 300-nucleotide sequence upstream of the ATG start site necessary for GSTO-1 expression. Site-specific mutagenesis of a GATA transcription factor binding motif in the minimal promoter led to the loss of reporter expression. Similarly, RNA interference (RNAi) of Elt-2 indicated the involvement of this gut-specific transcription factor in GSTO-1 expression. Transcriptional up-regulation under stress conditions of GSTO-1 was confirmed by analyzing promoter-reporter constructs in transgenic C. elegans strains. To investigate the function of GSTO-1 in vivo, transgenic animals overexpressing GSTO-1 were generated exhibiting an increased resistance to juglone-, paraquat-, and cumene hydroperoxide-induced oxidative stress. Specific silencing of the GSTO-1 by RNAi created worms with an increased sensitivity to several prooxidants, arsenite, and heat shock. We conclude that the stress-responsive GSTO-1 plays a key role in counteracting environmental stress.

Thiamine pyrophosphate (TPP), the active form of vitamin B1, is an essential cofactor for several enzymes. Humans depend exclusively on the uptake of vitamin B1, whereas bacteria, plants, fungi and the malaria parasite Plasmodium falciparum are able to synthesise thiamine monophosphate (TMP) de novo. TMP has to be dephosphorylated prior to pyrophosphorylation in order to obtain TPP. In P. falciparum the phosphatase capable to catalyse this reaction has been identified by analysis of the substrate specificity. The recombinant enzyme accepts beside vitamin B1 also nucleotides, phosphorylated sugars and the B6 vitamer pyridoxal 5'-phosphate. Vitamin B1 biosynthesis is known to occur in the cytosol. The cytosolic localisation of this phosphatase was verified by transfection of a GFP chimera construct. Stage specific Northern blot analysis of the phosphatase clearly identified an expression profile throughout the entire erythrocytic life cycle of P. falciparum and thereby emphasises the importance of dephosphorylation reactions within the malaria parasite.

Porcelain laminate veneers have been proven to be a successful treatment modality for maxillary incisors in clinical practice and in controlled clinical studies. However, the data in clinical studies on the success of veneers for restoring mandibular incisors are limited. Clinically, the successful restoration of mandibular incisors with porcelain laminate veneers is one of the more challenging procedures in all of esthetic restorative dentistry. Limited coronal dimensions, the small amount of enamel available for bonding (particularly in the cervical areas), materials and techniques for the bonding procedures, and the response of the tooth-veneer complex to forces generated during the incisal loading in both functional as well as parafunctional contacts must be considered as potential sources of success or failure. This Critical Appraisal reviews three recent scientific articles to shed some light on these issues and, as in all research endeavors, leads the reader to identify additional areas of concern that might stimulate further scientific inquiry. The first publication studied predictors for enamel thickness for mandibular incisors. The second examined bonding protocols for exposed dentin and suggested immediate dentin sealing. The third paper addressed fracture behavior of mandibular incisors restored with porcelain laminate veneers in vitro.

The synthesis and in-vitro antimalarial activity of gamma-substituted bis(pivaloyloxymethyl)ester analogues of the drug candidate fosmidomycin have been investigated. In contrast to the high antimalarial activity of alpha-aryl substituted fosmidomycin analogues like alpha-phenylfosmidomycin, gamma-substituted derivatives display only weak to moderate activity against the chloroquine-sensitive strain 3D7 of Plasmodium falciparum.

Plasmodium falciparum is the causative agent of the most severe type of malaria, a life-threatening disease affecting the lives of over three billion people. Factors like widespread resistance against available drugs and absence of an effective vaccine are seriously compounding control of the malaria parasite. Thus, there is an urgent need for the identification and validation of new drug targets. The enzymes of the polyamine biosynthesis pathway have been suggested as possible targets for the treatment of malaria. One of these enzymes is spermidine synthase (SPDS, putrescine aminopropyltransferase), which catalyzes the transfer of an aminopropyl moiety from decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine, leading to the formation of spermidine and 5'-methylthioadenosine. Here we present the three-dimensional structure of P. falciparum spermidine synthase (pfSPDS) in apo form, in complex with dcAdoMet and two inhibitors, S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and trans-4-methylcyclohexylamine (4MCHA). The results show that binding of dcAdoMet to pfSPDS stabilizes the conformation of the flexible gatekeeper loop of the enzyme and affects the conformation of the active-site amino acid residues, preparing the protein for binding of the second substrate. The complexes of AdoDATO and 4MCHA with pfSPDS reveal the mode of interactions of these compounds with the enzyme. While AdoDATO essentially fills the entire active-site pocket, 4MCHA only occupies part of it, which suggests that simple modifications of this compound may yield more potent inhibitors of pfSPDS.

The synthesis and in-vitro antimalarial activity of conformationally restrained bis(pivaloyloxymethyl) ester analogues of the natural product fosmidomycin is presented. In contrast to alpha-aryl-substituted analogues, conformationally restrained aromatic analogues exhibit only moderate in-vitro antimalarial activity against the chloroquine-sensitive strain 3D7 of Plasmodium falciparum. The most active derivative displays an IC(50) value of 47 microM.

Glutathione-S-transferase(s) (E.C.2.5.1.18, GSTs) have been investigated in parasitic protozoans with respect to their biochemistry and they have been identified as potential vaccine candidates in protozoan parasites and as a target in the synthesis of new antiparasitic agents. In a search towards the identification of novel biochemical targets for antimalarial drug design, the area of Plasmodium glutathione metabolism provides a number of promising chemotherapeutic targets. GST activity was determined in various subcellular fractions of malarial parasites Plasmodium yoelii and was found to be localized mainly in the cytosolic fraction (specific activity, c. 0.058 +/- 0.016 micromol/min/mg protein). Hemin, a known inhibitor of mammalian GST(s), maximally inhibited this enzyme from P. yoelii to nearly 86%. In a search towards synthetic modulators of malarial GST(s), 575 compounds belonging to various chemical classes were screened for their effect on crude GST from P. yoelii and 92 compounds belonging to various chemical classes were studied on recombinant GST from P. falciparum. Among all the compounds screened, 83 compounds inhibited/stimulated the enzyme from P. yoelii/P. falciparum to the extent of 40% or more.

Spermidine synthase is currently considered as a promising drug target in the malaria parasite, Plasmodium falciparum, due to the vital role of spermidine in the activation of the eukaryotic translation initiation factor (eIF5A) and cell proliferation. However, very limited information was available regarding the structure and mechanism of action of the protein at the start of this study. Structural and mechanistic insights of the P. falciparum spermidine synthase (PfSpdSyn) were obtained utilizing molecular dynamics simulations of a homology model based on the crystal structures of the Arabidopsis thaliana and Thermotoga maritima homologues. Our data are supported by in vitro site-directed mutagenesis of essential residues as well as by a crystal structure of the protein that became available recently. We provide, for the first time, dynamic evidence for the mechanism of the aminopropyltransferase action of PfSpdSyn. This characterization of the structural and mechanistic properties of the PfSpdSyn as well as the elucidation of the active site residues involved in substrate, product, and inhibitor interactions paves the way toward inhibitor selection or design of parasite-specific inhibitors.

Vitamin B(1) is an essential cofactor for key enzymes such as 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase. Plants, bacteria and fungi, as well as Plasmodium falciparum, are capable of synthesising vitamin B(1)de novo, whereas mammals have to take up this cofactor from their diet. Thiamine, a B(1) vitamer, has to be pyrophosphorylated by thiamine pyrophosphokinase (TPK) to the active form. The human malaria parasite P. falciparum expresses an N-terminally extended pyrophosphokinase throughout the entire erythrocytic life cycle, which was analysed by Northern and Western blotting. The recombinant enzyme shows a specific activity of 27 nmol min(-1) mg(-1) protein and specificity for thiamine with a K(m) value of 73 microM, while thiamine monophosphate is not accepted. Mutational analysis of the N-terminal extension of the plasmodial TPK showed that it influences thiamine binding as well as metal dependence, which suggests N-terminal participation in the conformation of the active site. Protein sequences of various plasmodial TPKs were analysed for their phylogeny, which classified the Plasmodium TPKs to a group distinct from the mammalian TPKs. To verify the location of the parasite TPK within the cell, immunofluorescence analyses were performed. Co-staining of PfTPK with a GFP marker visualised its cytosolic localisation.

The phosphonohydroxamic acid Fosmidomycin is a drug candidate for the treatment of Malaria, currently in phase II trials in combination with Clindamycin. In order to obtain compounds of higher lipophilicity, we recently synthesized alpha-phenyl substituted Fosmidomycin derivatives which display high antimalarial activity. We now report the synthesis and in vitro antimalarial activity of arylmethyl substituted bis(pivaloyloxymethyl) ester prodrugs of Fosmidomycin and its acetyl analogue FR900098. The 3,4-dichlorobenzyl substituted derivative of Fosmidomycin proved to be about twice as active as the respective Fosmidomycin prodrug, however, less active than the corresponding FR900098 prodrug. Electron donating substituents as well as voluminous substituents led to a significant reduction of activity.

Fosmidomycin is a promising antimalarial drug candidate with a unique chemical structure and a novel mode of action. Chain substituted pivaloyloxymethyl ester derivatives of Fosmidomycin and its acetyl analogue FR900098 have been synthesized and their in vitro antimalarial activity versus the Chloroquine sensitive strain 3D7 of Plasmodium falciparum has been determined.

In the malaria parasite, the two main regulatory activities of polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) occur in a single bifunctional protein. The AdoMetDC domain was modeled using the human and potato X-ray crystal structures as templates. Three parasite-specific inserts and the core active site region was identified using a structure-based alignment approach. The domain was modeled without the two largest inserts, to give a root mean square deviation of 1.85 angstroms from the human template. Contact with the rest of the bifunctional complex is predicted to occur on one face of the Plasmodium falciparum AdoMetDC (PfAdoMetDC) domain. In the active site there are four substitutions compared to the human template. One of these substitutions may be responsible for the lack of inhibition by Tris, compared to mammalian AdoMetDC. The model also provides an explanation for the lack of putrescine stimulation in PfAdoMetDC compared to mammalian AdoMetDC. A network of residues that connects the putrescine-binding site with the active site in human AdoMetDC is conserved in the malarial and plant cognates. Internal basic residues are found to assume the role of putrescine, based on the model and site-directed mutagenesis: Arg11 is absolutely required for normal activity, while disrupting Lys15 and Lys215 each cause 50% inhibition of AdoMetDC activity. These novel features of malarial AdoMetDC suggest possibilities for the discovery of parasite-specific inhibitors.

Vitamin B1 (thiamine) is an essential cofactor for several key enzymes of carbohydrate metabolism. Mammals have to salvage this crucial nutrient from their diet to complement their deficiency of de novo synthesis. In contrast, bacteria, fungi, plants and, as reported here, Plasmodium falciparum, possess a vitamin B1 biosynthesis pathway. The plasmodial pathway identified consists of the three vitamin B1 biosynthetic enzymes 5-(2-hydroxy-ethyl)-4-methylthiazole (THZ) kinase (ThiM), 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP)/HMP-P kinase (ThiD) and thiamine phosphate synthase (ThiE). Recombinant PfThiM and PfThiD proteins were biochemically characterised, revealing K(m)app values of 68 microM for THZ and 12 microM for HMP. Furthermore, the ability of PfThiE for generating vitamin B1 was analysed by a complementation assay with thiE-negative E. coli mutants. All three enzymes are expressed throughout the developmental blood stages, as shown by Northern blotting, which indicates the presence of the vitamin B1 biosynthesis enzymes. However, cultivation of the parasite in minimal medium showed a dependency on the provision of HMP or thiamine. These results demonstrate that the human malaria parasite P. falciparum possesses active vitamin B1 biosynthesis, which depends on external provision of thiamine precursors.

Lipid-binding proteins exhibit important functions in lipid transport, cellular signaling, gene transcription, and cytoprotection. Their functional analogues in nematodes are nematode polyprotein allergens/antigens and fatty acid and retinoid-binding proteins. This work describes a novel 55-kDa protein, Ag-lbp55, purified from the parasitic nematode Ascaridia galli. By direct N-terminal sequencing, a partial amino acid sequence was obtained that allowed the design of oligonucleotide primers to obtain the full-length cDNA sequence. Sequence analysis revealed the presence of an N-terminal signal peptide of 25 amino acid residues and a FAR domain at the C terminus. Data base searches showed almost no significant homologies to other described proteins. The secondary structure of Ag-lbp55 was predominantly alpha-helical (65%) as shown by CD spectroscopy. It was found to bind with high affinity fatty acids (caprylic, oleic, and palmitic acid) and their fluorescent analogue dansylaminoundecanic acid. Immunolocalization showed that Ag-lbp55 is a highly abundant protein, mainly distributed in the inner hypodermis and extracellularly in the pseudocoelomatic fluid. A similar staining pattern was observed in other pathogenic nematodes, indicating the existence of similar proteins in these species.

The polyamine synthesis enzyme spermidine synthase (SPDS) has been cloned from the model nematode Caenorhabditis elegans. Biochemical characterisation of the recombinantly expressed protein revealed a high degree of similarity to other eukaryotic SPDS with the exception of a low affinity towards the substrate decarboxylated S-adenosylmethionine (Km = 110 microM) and a less pronounced feedback inhibition by the second reaction product 5'-methylthioadenosine (IC50 = 430 microM). The C. elegans protein that carries a nematode-specific insertion of 27 amino acids close to its N-terminus was crystallized, leading to the first X-ray structure of a dimeric eukaryotic SPDS.

The gene encoding spermidine synthase was cloned from the human malaria parasite Plasmodium falciparum. Northern and Western blot analyses revealed a stage specific expression during the erythrocytic schizogony with the maximal amount of transcript and protein in mature trophozoites. Immunofluorescence assays (IFAs) suggest a cytoplasmatic localisation of the spermidine synthase in P. falciparum. The spermidine synthase polypeptide of 321 amino acids has a molecular mass of 36.6kDa and contains an N-terminal extension of unknown function that, similarly, is also found in certain plants but not in animal or bacterial orthologues. Omitting the first 29 amino acids, a truncated form of P. falciparum spermidine synthase has been recombinantly expressed in Escherichia coli. The enzyme catalyses the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dcAdoMet) onto putrescine with Km values of 35 and 52microM, respectively. In contrast to mammalian spermidine synthases, spermidine can replace to some extent putrescine as the aminopropyl acceptor. Hence, P. falciparum spermidine synthase has the capacity to catalyse the formation of spermine that is found in small amounts in the erythrocytic stages of the parasite. Among the spermidine synthase inhibitors tested against P. falciparum spermidine synthase, trans-4-methylcyclohexylamine (4MCHA) was found to be most potent with a Ki value of 0.18microM. In contrast to the situation in mammals, where inhibition of spermidine synthase has no or only little effect on cell proliferation, 4MCHA was an efficient inhibitor of P. falciparum cell growth in vitro with an IC50 of 35microM, indicating that P. falciparum spermidine synthase represents a putative drug target.

The intraerythrocytic development of Plasmodium falciparum correlates with increasing levels of the polyamines putrescine, spermidine, and spermine in the infected red blood cells; and compartmental analyses revealed that the majority is associated with the parasite. Since depletion of cellular polyamines is a promising strategy for inhibition of parasite proliferation, new inhibitors of polyamine biosynthesis were tested for their antimalarial activities. The ornithine decarboxylase (ODC) inhibitor 3-aminooxy-1-aminopropane (APA) and its derivatives CGP 52622A and CGP 54169A as well as the S-adenosylmethionine decarboxlyase (AdoMetDC) inhibitors CGP 40215A and CGP 48664A potently affected the bifunctional P. falciparum ODC-AdoMetDC, with K(i) values in the low nanomolar and low micromolar ranges, respectively. Furthermore, the agents were examined for their in vitro plasmodicidal activities in 48-h incubation assays. APA, CGP 52622A, CGP 54169A, and CGP 40215A were the most effective, with 50% inhibitory concentrations below 3 microM. While the effects of the ODC inhibitors were completely abolished by the addition of putrescine, growth inhibition by the AdoMetDC inhibitor CGP 40215A could not be antagonized by putrescine or spermidine. Moreover, CGP 40215A did not affect the cellular polyamine levels, indicating a mechanism of action against P. falciparum independent of polyamine synthesis. In contrast, the ODC inhibitors led to decreased cellular putrescine and spermidine levels in P. falciparum, supporting the fact that they exert their antimalarial activities by inhibition of the bifunctional ODC-AdoMetDC.

Onchocerciasis is a debilitating parasitic disease caused by the filarial worm Onchocerca volvulus. Similar to other helminth parasites, O. volvulus is capable of evading the host's immune responses by a variety of defense mechanisms, including the detoxification activities of the glutathione S-transferases (GSTs). Additionally, in response to drug treatment, helminth GSTs are highly up-regulated, making them tempting targets both for chemotherapy and for vaccine development. We analyzed the three-dimensional x-ray structure of the major cytosolic GST from O. volvulus (Ov-GST2) in complex with its natural substrate glutathione and its competitive inhibitor S-hexylglutathione at 1.5 and 1.8 angstrom resolution, respectively. From the perspective of the biochemical classification, the Ov-GST2 seems to be related to pi-class GSTs. However, in comparison to other pi-class GSTs, in particular to the host's counterpart, the Ov-GST2 reveals significant and unusual differences in the sequence and overall structure. Major differences can be found in helix alpha-2, an important region for substrate recognition. Moreover, the binding site for the electrophilic co-substrate is spatially increased and more solvent-accessible. These structural alterations are responsible for different substrate specificities and will form the basis of parasite-specific structure-based drug design investigations.

Methylmalonyl-CoA epimerase (MCE) is an enzyme involved in the propionyl-CoA metabolism that is responsible for the degradation of branched amino acids and odd-chain fatty acids. This pathway typically functions in the reversible conversion of propionyl-CoA to succinyl-CoA. The Caenorhabditis elegans genome contains a single gene encoding MCE (mce-1) corresponding to a 15 kDa protein. This was expressed in Escherichia coli and the enzymatic activity was determined. Analysis of the protein expression pattern at both the tissue and subcellular level by microinjection of green fluorescent protein constructs revealed expression in the pharynx, hypodermis and, most prominently in body wall muscles. The subcellular pattern agrees with predictions of mitochondrial localization. The sequence similarity to an MCE of known structure was high enough to permit a three-dimensional model to be built, suggesting conservation of ligand and metal binding sites. Comparison with corresponding sequences from a variety of organisms shows more than 1/6 of the sequence is completely conserved. Mutants allelic to mce-1 showed no obvious phenotypic alterations, demonstrating that the enzyme is not essential for normal worm development under laboratory conditions. However, survival of the knockout mutants was altered when exposed to stress conditions, with mutants surprisingly showing an increased resistance to oxidative stress.

Vitamin B6 is an essential cofactor for more than 100 enzymatic reactions. Mammalian cells are unable to synthesize vitamin B6 de novo, whereas bacteria, plants, fungi, and as shown here Plasmodium falciparum possess a functional vitamin B6 synthesis pathway. P. falciparum expresses the proteins Pdx1 and Pdx2, corresponding to the yeast enzymes Snz1-p and Sno1-p, which are essential for the vitamin B6 biosynthesis. An involvement of PfPdx1 and PfPdx2 in the de novo synthesis of vitamin B6 was shown by complementation of pyridoxine auxotroph yeast cells. Both plasmodial proteins act together in the glutaminase activity with a specific activity of 209 nmol min(-1) mg(-1) and a K(m) value for glutamine of 1.3 mm. Incubation of the parasites with methylene blue revealed by Northern blot analysis an elevated transcriptional level of pdx1 and pdx2, suggesting a participation of these proteins in the defenses against singlet oxygen. To be an active cofactor, vitamin B6 has to be phosphorylated by the pyridoxine kinase (PdxK). The recombinant plasmodial PdxK revealed K(m) values for the B6 vitamers pyridoxine and pyridoxal and for ATP of 212, 70, and 82 microM, respectively. All three enzymes expose a stage-specific transcription pattern within the trophozoite stage that guarantees the concurrent expression of Pdx1, Pdx2, and PdxK for the indispensable provision of vitamin B6. The occurrence of the vitamin B6 de novo synthesis pathway displays a potential new drug target, which can be exploited for the development of new chemotherapeutics against the human malaria parasite P. falciparum.

The human malaria parasite Plasmodium falciparum possesses a single gene with high similarity to the metalloproteins arginase and agmatinase. The recombinant protein reveals strict specificity for arginine, and it has been proposed that its function in ornithine production is as a precursor for polyamine biosynthesis. The specific activity of the plasmodial arginase was found to be 31 micromol min(-1) mg(-1) protein and the k(cat) was calculated as 96 (s-1) . The Km value for arginine and Ki value for ornithine were determined as 13 mM and 19 mM, respectively. The active arginase is a homotrimer of ca. 160 kDa. Dialysis of the arginase against EDTA results in monomers of approximately 48 kDa; however, the quaternary structure can be restored by addition of Mn 2+ . Mutagenic analyses of all the amino acid residues proposed to be involved in metal binding led to complex dissociation, except for the His-193-Ala mutant, which was also inactive but retained the trimeric structure. Substitution of His-233, which has been suggested to be in charge of proton shuttling within the active site, disrupted the trimeric structure and thereby the activity of the Pf arginase. Northern blot analysis identified a stage-specific expression pattern of the plasmodial arginase in the ring/young trophozoite stage, which guarantees the provision of ornithine for essential polyamine biosynthesis.

Ag-NPA-1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascaridia galli, is a member of the nematode polyprotein allergen/antigen (NPA) family. Spectroscopic analysis shows that Ag-NPA-1 is a highly ordered, alpha-helical protein and that ligand binding slightly increases the ordered secondary structure content. The conserved, single Trp residue (Trp17) and three Tyr residues determine the fluorescence properties of Ag-NPA-1. Analysis of the efficiency of the energy transfer between these chromophores shows a high degree of Tyr-Trp dipole-dipole coupling. Binding of fatty acids and retinol was accompanied by enhancement of the Trp emission, which allowed calculation of the affinity constants of the binary complexes. The distance between the single Trp of Ag-NPA-1 and the fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1- sulfonyl)amino]undecanoic acid (DAUDA) from the protein binding site is 1.41 nm as estimated by fluorescence resonance energy transfer. A chemical modification of the Cys residues of Ag-NPA-1 (Cys66 and Cys122) with the thiol reactive probes 5-({[(2-iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid (IAEDANS) and N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), followed by MALDI-TOF analysis showed that only Cys66 was labeled. The observed similar affinities for fatty acids of the modified and native Ag-NPA-1 suggest that Cys66 is not a part of the protein binding pocket but is located close to it. Ag-NPA-1 is one of the most abundant proteins in A. galli and it is distributed extracellularly mainly as shown by immunohistology and immunogold electron microscopy. This suggests that Ag-NPA-1 plays an important role in the transport of fatty acids and retinoids.

Polyamines are essential cell constituents involved in growth processes. In Caenorhabditis elegans the polyamine synthetic pathway consists of three enzymes, ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine synthase. Their gene expression pattern was determined in C. elegans by microinjection of green fluorescent protein (GFP) reporter gene constructs. All transgenic animals exhibited GFP expression in their intestinal cells. For the AdoMetDC promoter, fluorescence was additionally observed in dopaminergic neurons, while the ODC promoter also drives a male-specific GFP expression in the distal part of the reproductive system. The minimal promoter regions for intestine-specific expression of the AdoMetDC and spermidine synthase genes were determined by deletion mutants. Using the Seqcomp and Family Relation programs, a similar arrangement of putative cis-regulatory elements within these regions and also within the respective regions of the orthologous Caenorhabditis briggsae genes were found. The functional conservation of the latter was confirmed by heterologous transformation experiments. Moreover, the involvement of putative GATA- and initiator-(Inr)-like-elements in gene expression was determined by mutagenesis studies. RNase protection assay revealed that the Inr-like-element does not represent the main transcriptional start site, at least of C. elegans spermidine synthase. In conclusion, a similar minimal promoter architecture was found for C. elegans as well as C. briggsae AdoMetDC and spermidine synthase, two genes that participate in the same metabolic pathway.

Glutathione S-transferases (GSTs) are a family of detoxification enzymes that catalyse the conjugation of glutathione to xenobiotic and endogenous electrophilic compounds, thus facilitating their elimination from cells. The recombinant Onchocerca volvulus GST2 has been expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion technique. Two different crystal forms were grown under identical conditions. They belong to space groups P2(1)2(1)2 and P2(1), respectively. The unit-cell parameters obtained are a = 112.6, b = 84.3, c = 45.1 A for the P2(1)2(1)2 crystal form and a = 51.6, b = 82.3, c = 56.7 A, beta = 95.89 degrees for the P2(1) form. Complete data sets to 2.6 and 1.5 A, respectively, have been collected at 100 K with synchrotron radiation.

The ethyl 4-chlorobenzothieno[3,2-b]pyridine-3-carboxylate (2) reacted with the hydrochlorides of the mono- and bis-phenol Mannich bases 6 to yield the amodiaquine and pyronaridine analogues 9. The chloroquine analogue 10 was formed by melting 2 with the novaldiamine base (7) in phenol. The stability of the 4-aminophenols 9 was investigated by anodic oxidation using the rotating platinum electrode by means of difference pulse voltammetry. The half wave potentials were measured giving E(1/2) approximately 1.05 V. Compound 9g displayed the highest activity against the growth of the malaria parasite Plasmodium falciparum. Testing against the chloroquine sensitive 3D7 and the chloroquine resistant Dd2 strain resulted in IC50 values of 150 nM and 210 nM, respectively. Surprisingly, the 3-carbinol 4 and the 3-chloromethyl derivative 5, synthesized from the 3-carboxylic acid ester 2, reacted with the phenol Mannich base 6a and the novaldiamine base (7), respectively, to yield the 4-pyridone 8.

Pyrido[3,2-b]indol-4-yl-amines--synthesis and investigation of activity against malaria Starting with 3-aminoindole-2-carboxylic acid ester 1 the annulated pyrido[3,2-b]indoles 6 and 8 were synthesized as key substances. The 4-chloropyridine derivative 8 reacted with the phenol Mannich bases 11 and the novaldiamine base 13, respectively, to yield the amodiaquine and cycloquine analogues 12 as well as the chloroquine analogue 14. The stability of the compounds 12 and 14 were proven by the half wave potentials measured by differential pulse voltammetry. Compounds 12 and 14 were tested for in vitro antimalarial activity using a chloroquine sensitive and a chloroquine resistant Plasmodium falciparum strain. The highest activity was shown by 12g with IC50 values of 50 nM and 38 nM, respectively. The in vivo activity of 12g was tested in Plasmodium vinckei infected mice resulting in ED50 values of 22 mg/kg and 26 mg/kg after intraperitoneal and oral administration, respectively.

Polyamine biosynthesis of the malaria parasite, Plasmodium falciparum, is regulated by a single, hinge-linked bifunctional PfAdoMetDC/ODC [ P. falciparum AdoMetDC (S-adenosylmethionine decarboxylase)/ODC (ornithine decarboxylase)] with a molecular mass of 330 kDa. The bifunctional nature of AdoMetDC/ODC is unique to Plasmodia and is shared by at least three species. The PfAdoMetDC/ODC contains four parasite-specific regions ranging in size from 39 to 274 residues. The significance of the parasite-specific inserts for activity and protein-protein interactions of the bifunctional protein was investigated by a single- and multiple-deletion strategy. Deletion of these inserts in the bifunctional protein diminished the corresponding enzyme activity and in some instances also decreased the activity of the neighbouring, non-mutated domain. Intermolecular interactions between AdoMetDC and ODC appear to be vital for optimal ODC activity. Similar results have been reported for the bifunctional P. falciparum dihydrofolate reductase-thymidylate synthase [Yuvaniyama, Chitnumsub, Kamchonwongpaisan, Vanichtanankul, Sirawaraporn, Taylor, Walkinshaw and Yuthavong (2003) Nat. Struct. Biol. 10, 357-365]. Co-incubation of the monofunctional, heterotetrameric approximately 150 kDa AdoMetDC domain with the monofunctional, homodimeric ODC domain (approximately 180 kDa) produced an active hybrid complex of 330 kDa. The hinge region is required for bifunctional complex formation and only indirectly for enzyme activities. Deletion of the smallest, most structured and conserved insert in the ODC domain had the biggest impact on the activities of both decarboxylases, homodimeric ODC arrangement and hybrid complex formation. The remaining large inserts are predicted to be non-globular regions located on the surface of these proteins. The large insert in AdoMetDC in contrast is not implicated in hybrid complex formation even though distinct interactions between this insert and the two domains are inferred from the effect of its removal on both catalytic activities. Interference with essential protein-protein interactions mediated by parasite-specific regions therefore appears to be a viable strategy to aid the design of selective inhibitors of polyamine metabolism of P. falciparum.