Identification and activity of a series of azole-based compounds with lactate dehydrogenase-directed anti-malarial activity

Plasmodium falciparum, the causative agent of malaria, relies extensively on glycolysis coupled with homolactic fermentation during its blood-borne stages for energy production. Selective inhibitors of the parasite lactate dehydrogenase (LDH), central to NAD(+) regeneration, therefore potentially provide a route to new antimalarial drugs directed against a novel molecular target. A series of heterocyclic, azole-based compounds are described that preferentially inhibit P. falciparum LDH at sub-micromolar concentrations, typically at concentrations about 100-fold lower than required for human lactate dehydrogenase inhibition. Crystal structures show these competitive inhibitors form a network of interactions with amino acids within the active site of the enzyme, stacking alongside the nicotinamide ring of the NAD(+) cofactor. These compounds display modest activity against parasitized erythrocytes, including parasite strains with known resistance to existing anti-malarials and against Plasmodium berghei in BALB/c mice. Initial toxicity data suggest the azole derivatives have generally low cytotoxicity, and preliminary pharmoco-kinetic data show favorable bioavailability and circulation times. These encouraging results suggest that further enhancement of these structures may yield candidates suitable for consideration as new therapeutics for the treatment of malaria. In combination these studies also provide strong support for the validity of targeting the Plasmodium glycolytic pathway and, in particular, LDH in the search for novel anti-malarials.     

Effects of grapefruit juice on the multidrug transporter P-glycoprotein in the human proximal tubular cell line HK-2

The multidrug transporter MDR-1 P-glycoprotein (Pgp) has been recently pointed out as an important mechanism underlying chemical interaction between drugs and many commonly ingested substances, including grapefruit juice (GFJ). Modulation of intestinal Pgp dependent transport by GFJ may lead to changes in bioavailability of drugs that are substrates of Pgp itself, by affecting their presystemic clearance. Since other cellular sites expressing Pgp and devoted to drug disposition, like kidney proximal tubules, could be involved in these pharmacokinetic interactions, we investigated the effect of GFJ on the expression and activity of Pgp in the human immortalized tubular cell line HK-2. Two flavonoid compounds related to GFJ, kaempferol and naringenin, were also tested for their effects on HK-2 Pgp. HK-2 cells cultured for 4 days in the presence of GFJ, showed a dose-dependent decrease in Pgp immunoblottable amount as well as a decrease in MDR-1 mRNA level, as shown by western blot analysis and RT-PCR, respectively. Both kaempferol and naringenin were also able to significantly decrease Pgp immunoblottable amount. To test whether the downregulation of HK-2 Pgp due to GFJ exposition could influence the cell sensitivity to drugs that are transported by Pgp itself, HK-2 cells precultured with GFJ were exposed to scalar concentrations of Cyclosporin A or Vinblastine and cell viability examined 36 hours later. The cytotoxicity of both drugs was increased. The calcein-AM test in untreated cells showed that GFJ, kaempferol or naringenin inhibited Pgp activity. Downregulation of Pgp as well inhibition of its function by GFJ or its related components in tubular cells could have a role in changing disposition kinetics of some important therapeutic agents.     

Caulimoviridae Tubule-Guided Transport Is Dictated by Movement Protein Properties

Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway.Following virus entry and replication, successful infection of a host requires viral spread to distal parts of the organism through the vascular tissue. In plants, virus movement involves mostly symplastic trafficking of different viral components through the connections of plasmodesmata (PD) (13). With this aim, plant viruses encode one or more movement proteins (MPs), which allow viral genomes to cross the host cell wall by altering the size exclusion limit (SEL) or the structure of PD (6, 11). Plant viruses have evolved distinct mechanisms to move their genomes within the host. These mechanisms can be grouped into two general strategies: one in which the genome is transported in the form of a nucleoprotein complex (NPC) and another in which nucleic acids are encapsidated and move as virus particles. In both cases, besides altering PD SEL, MPs are involved either in NPC assembly or in forming tubules traversing modified PD and helping transport of either NPC or virions to the neighboring cell. Within these two major strategies, there exists a wide range of variability in terms of the number and type of viral and host proteins helping MPs to mediate virus spread within the host (11).In spite of such variability, several different MPs have been classified into a 30K superfamily; these MPs, from 20 genera including both RNA and DNA genome viruses, are structurally related to the 30-kDa MP of Tobacco mosaic virus (TMV), independent of the movement strategy followed (14). Members of this family have a common core of predicted secondary structure elements (α-helices and β-elements) containing a nucleic acid binding domain. Distinct MPs belong to this family, including several tubule-forming MPs, although these are phylogenetically separated from the other members (14). Thus, 30K superfamily MPs are closely related, and some of them are functionally interchangeable in the viral context (2, 20). In particular, MPs from five distinct genera with an RNA genome can successfully replace the corresponding gene of Alfalfa mosaic virus (AMV) (19), indicating that one or more basic and fundamental movement properties might be associated with the common 30K structural core.Among all known plant viruses, only three viral families have evolved a DNA genome: Geminiviridae, Caulimoviridae, and Nanoviridae (6). One possible explanation for this restriction is that endogenous cell-to-cell transport via PD is specialized to use RNA as the communication and signaling molecule (12). To circumvent this restriction, and to allow the efficient exploitation of endogenous transport machineries, DNA genome viruses have evolved appropriate mechanisms involving their MPs. Interestingly, Begomovirus and Caulimovirus MPs also belong to the 30K superfamily discussed above (14). The MP encoded by Cauliflower mosaic virus (CaMV), the type member of Caulimoviridae, forms tubules that guide the movement of encapsidated virus via an indirect MP-virion interaction (16, 21), whereas geminivirus MPs selectively bind their genomes and transport them as NPCs (6, 9, 17). In this study, we investigated the evolutionary convergence of MPs encoded by DNA and RNA viruses by testing their exchangeability in the viral context.     

Evolutionary movement of centromeres in horse, donkey, and zebra

Centromere repositioning (CR) is a recently discovered biological phenomenon consisting of the emergence of a new centromere along a chromosome and the inactivation of the old one. After a CR, the primary constriction and the centromeric function are localized in a new position while the order of physical markers on the chromosome remains unchanged. These events profoundly affect chromosomal architecture. Since horses, asses, and zebras, whose evolutionary divergence is relatively recent, show remarkable morphological similarity and capacity to interbreed despite their chromosomes differing considerably, we investigated the role of CR in the karyotype evolution of the genus Equus. Using appropriate panels of BAC clones in FISH experiments, we compared the centromere position and marker order arrangement among orthologous chromosomes of Burchelli's zebra (Equus burchelli), donkey (Equus asinus), and horse (Equus caballus). Surprisingly, at least eight CRs took place during the evolution of this genus. Even more surprisingly, five cases of CR have occurred in the donkey after its divergence from zebra, that is, in a very short evolutionary time (approximately 1 million years).These findings suggest that in some species the CR phenomenon could have played an important role in karyotype shaping, with potential consequences on population dynamics and speciation.     

Comparative PCR-based restriction fragment length polymorphism analysis of the plasmid gene orf3 of Chlamydia trachomatis and Chlamydia psittaci

The BfaI digestion of PCR-based restriction fragment length polymorphism analysis of the plasmid orf3 of Chlamydia trachomatis and Chlamydia psittaci provided evidence for two distinct restriction patterns, respectively. The nucleotide sequences of orf3 genes confirmed these differences. Serum antibodies against recombinant C. psittaci protein (pgp3) encoded by orf3 were detected both in pigeons with C. psittaci infection and in a human patient with psittacosis.     

Molecular basis for the binding polyspecificity of an anti-cholera toxin peptide 3 monoclonal antibody

The onset of autoimmune diseases is proposed to involve binding promiscuity of antibodies (Abs) and T‐cells, an often reported yet poorly understood phenomenon. Here, we attempt to approach two questions: first, is binding promiscuity a general feature of monoclonal antibodies (mAbs) and second, what is the molecular basis for polyspecificity? To this end, the anti‐cholera toxin peptide 3 (CTP3) mAb TE33 was investigated for polyspecific binding properties. Screening of phage display libraries identified two epitope‐unrelated peptides that specifically bound TE33 with affinities similar to or 100‐fold higher than the wild‐type epitope. Substitutional analyses revealed distinct key residue patterns recognized by the antibody suggesting a unique binding mode for each peptide. A database query with one of the consensus motifs and a subsequent binding study uncovered 45 peptides (derived from heterologous proteins) that bound TE33. To better understand the structural basis of the observed polyspecificity we modeled the new cyclic epitope in complex with TE33. The interactions between this peptide and TE33 suggested by our model are substantially different from the interactions observed in the X‐ray structure of the wild‐type epitope complex. However, the overall binding conformation of the peptides is similar. Together, our results support the theory of a general polyspecific potential of mAbs. Copyright © 2005 John Wiley & Sons, Ltd.     

Microbial genomes and vaccine design: refinements to the classical reverse vaccinology approach

The advent of whole-genome sequencing of bacteria and advances in bioinformatics have revolutionized the study of bacterial pathogenesis, enabling the targeting of possible vaccine candidates starting from genomic information. Nowadays, the availability of hundreds of bacterial genomes enables identification of the genetic differences across several genomes from the same species. The unexpected degree of intra-species diversity suggests that a single genome sequence is not entirely representative and does not offer a complete picture of the genetic variability of a species. The practical consequence is that, in many cases, a universal vaccine is possible only by including a combination of antigens and this combination must take into account the pathogen population structure.     

Sphingosine 1-phosphate inhibits cell migration in C2C12 myoblasts

This study shows that sphingosine 1-phosphate (S1P) exerts an anti-migratory action in C2C12 myoblasts by reducing directional cell motility and fully abrogating the chemotactic response to insulin-like growth factor-1. The anti-migratory response to S1P required ligation to S1P(2), being attenuated in myoblasts where the receptor was down-regulated by specific antisense oligodeoxyribonucleotides or small interfering RNA (siRNA) and conversely potentiated in S1P(2)-overexpressing myoblasts. The investigation of RhoA and Rac GTPases, critically implicated in cell motility regulation, demonstrated that RhoA was rapidly activated by S1P, while Rac1 was unaffected within the first 5 min but stimulated thereafter. RhoA, but not Rac activation, was identified as a S1P(2)-dependent pathway in experiments in which receptor expression was attenuated by siRNA treatment or up-regulated by S1P(2)-encoding plasmid transfection. Finally, by expression of the dominant negative mutant of RhoA, the GTPase was found implicated in the anti-migratory action of S1P, whereas modulation of Rac1 functionality unaffected the anti-chemotactic effect of S1P, ruling out a role for this protein in the biological response. Since S1P was previously shown to inhibit myoblast proliferation and stimulate myogenesis, the here identified novel biological activity is in favour of a complex physiological role of the sphingolipid in the process of muscle repair.     

Cytokines and osteolysis around total hip prostheses

The aim of this work is to assess the correlation between the osteolysis around the prosthesis and the presence of cytokines favouring inflammation in the tissues at the interface between loosened prosthesis and bone. In this study, twenty-nine patients that underwent revision surgery were examined. Bioptic samples were collected at the interface between bone and implant both at the stem and socket level. Semiquantitative immunohistochemistry was performed to detect interleukin 1 alpha, interleukin 1 beta, interleukin 6 and tumour necrosis factor, cytokines that directly cause bone resorption and indirectly induce synthesis of other bone resorbing cytokines. Wear particles were identified and quantified by light microscopy. Radiographic evidence for osteolysis was scored by the Engh and Bobyn score. In tissues collected at the interface, the percentage of cells positive to IL1, IL6 and particularly to TNF increased in relation to the tissues collected at the interface with stable components. The cells occurring in the new capsule do not secrete cytokines in quantities that can be related to severity of wear. Cemented prostheses showed higher incidence of severe osteolysis, and higher levels of cytokines. It can be concluded that TNF, and to a lesser extent IL1 and IL6, are positively related to the severity of osteolysis around the prosthesis and therefore a pharmacological treatment can be hypothesized with anti-inflammatory or anti-cytokine drugs in order to limit or to avoid prosthesis loosening.