Design, synthesis and antibacterial evaluation of a polycationic calix[4]arene derivative alone and in combination with antibiotics.

The growing antibiotic resistance phenomenon continues to stimulate the search for new compounds and strategies to combat bacterial infections. In this study, we designed and synthesized a new polycationic macrocyclic compound (2) bearing four N-methyldiethanol ammonium groups clustered and circularly organized by a calix[4]arene scaffold. The in vitro activity of compound 2, alone and in combination with known antibiotics (ofloxacin, chloramphenicol or tetracycline), was assessed against strains of Staphylococcus aureus (ATCC 6538 and methicillin-resistant isolate 15), S. epidermidis (ATCC 35984 and methicillin-resistant isolate 57), and Pseudomonas aeruginosa (ATCC 9027 and antibiotic-resistant isolate 1). Calix[4]arene derivative 2 showed significant antibacterial activity against ATCC and methicillin-resistant Gram positive Staphylococci, improved the stability of tetracycline in water, and in combination with antibiotics enhanced the antibiotic efficacy against Gram negative P. aeruginosa by an additive effect.

Differential expression of erythroid genes in prion disease.

We previously reported reduced expression of erythroid-associated factor (ERAF) within haematopoietic tissues of rodent scrapie models, suggesting an unrecognized role for the erythroid lineage in prion disease. In the present study, we compared the expression of a panel of erythroid genes within four murine scrapie models and five virus infection models with parallels to prion disease pathogenesis. We report that differential expression of erythroid genes is not limited to ERAF, and is a common feature of murine scrapie, dependent on host expression of cellular prion protein. In contrast, erythroid gene expression was not altered following virus infection. Whilst these results further implicate cells of the erythroid lineage in the peripheral pathogenesis of prion disease, analysis of blood from BSE-infected cattle and scrapie-infected sheep reveals that the extent of differential expression of erythroid genes within peripheral blood is not sufficient to provide a discriminatory diagnostic test.

The biosynthesis and post-translational modification of Pbs21 an ookinete-surface protein of Plasmodium berghei.

Radiolabelled methionine incorporation into synchronised Plasmodium berghei gametocytes or ookinete cultures, showed that Pbs21 is not synthesised in bloodstage parasites; synthesis was detected within three hours of induction of gametogenesis; synthesis was triggered at gametogenesis, not by fertilisation. We show native Pbs21 to be a hydrophobic membrane protein that was insensitive to cleavage by phosphatidylinositol phospholipase C (PI-PLC), but sensitive to alkaline hydroxylamine, and partially sensitive to glycosylphosphatidylinositol-dependent phospholipase D (GPI-PLD) and HNO2. 3H-myristic and palmitic acid, 3H-glucosamine and mannose incorporation indicated Pbs21 was acylated and glycosylated. Linkage of the acyl group was sensitive to HNO2, which released an acyl-phosphatidylinositol more hydrophobic than that released from P3 of Trypanosoma brucei. All these properties are consistent with the presence of a malaria-specific glycosylphosphatidylinositol (GPI) anchor. In contrast recombinant Pbs21 (rPbs21), expressed in Spodoptera frugiperda cells, was sensitive to both PI-PLC and GPI-PLD, consistent with the protein being modified by a different (S. frugiperda) GPI anchor. Brefeldin A blocked secretion of rPbs21 within a cytoplasmic reticular compartment. Following deletion of the putative GPI anchor addition site (amino acids 189 213), the protein was transported to the cell surface and secreted directly into the aqueous phase of the culture medium. Deletion of amino acids 205-213 disrupted Pbs21 processing, transport through the ER and distribution onto the cell surface. Deletion of amino acids 1-28 prevented transport of Pbs21 into the ER. This suggests that correct processing of the GPI anchor in the ER-Golgi network is essential for the successful secretion of the recombinant protein, which is additionally dependent upon an N-terminal secretory signal sequence.

Embryonic activation and developmental expression of the murine prion protein gene.

While it is well established that cellular prion protein (PrP(C)) expression is required for the development of transmissible spongiform encephalopathies (TSEs), the physiological function of PrP(C) has yet to be determined. A number of studies have examined PrP expression in different tissues and in the later stages of embryonic development. However, the relative levels of expression of PrP RNA and protein in tissues outside the central nervous system (CNS) is not well documented and the exact point of transcriptional activation of PrP during embryogenesis is unknown. We have studied PrP mRNA expression in murine embryos and both mRNA and protein expression in a variety of adult tissues. PrP RNA was detected at different levels in all tissues tested while PrP(C) protein was detectable in all adult tissues tested with the exception of kidney and liver. RNA and protein levels were also assessed at four points during postnatal brain development and levels of both were seen to increase with development. We also established that, during embryogenesis, induction of PrP RNA expression occurs between E8.5 and E9, during the period of transition from anaerobic to aerobic metabolism. Preliminary experiments investigating the effects of superoxide radicals on PrP expression in cultured neuroblastoma and astrocyte cells support the suggestion that PrP(C) forms part of a cellular antioxidant defense mechanism.

Characterization of the modes of action of anti-Pbs21 malaria transmission-blocking immunity: ookinete to oocyst differentiation in vivo.

The impact of immune sera, and peripheral blood cells (PBC) from mice immunized with Plasmodium berghei ookinetes; and of purified immunoglobulin or Fab fragments from anti-Pbs21 monoclonal antibody 13.1, upon establishment of oocyst infections in the mosquito was studied. Infections were initiated either from gametocyte-infected mice, or membrane feeders which contained either gametocytes or mature ookinetes. PBC from ookinete-immunized mice presented with non-immune serum failed to show any transmission-blocking activity. Anti-ookinete serum, intact anti-Pbs21 monoclonal antibody 13.1 or its Fab fragments, all inhibited oocyst formation significantly. When gametocyte-infected mice or gametocytes in membrane feeds were used, inhibition did not directly correlate with antibody concentration. In membrane feeders that contained ookinetes and antibody, concentration-dependent inhibition usually occurred. The efficacy of purified 13.1 IgG was dependent upon the ookinete concentration. The ookinete plasmalemma and cytoplasm were significantly disturbed after 12 h in bloodmeals that contained antibody 13.1, but not in the isotype controls. These changes may have caused the observed failure of the ookinete to migrate as rapidly as the controls from the destructive environment of the bloodmeal.

Melanins in physiological conditions protect against lipoperoxidation. A study on albino and pigmented Xenopus.

The susceptibility to lipoperoxidation in liver of albino and pigmented Xenopus laevis Daudin, has been studied. Albino Xenopus liver was richer in polyunsaturated fatty acids (PUFAs) than the pigmented one; moreover, it was also richer in mitochondrial superoxide dismutase (MnSOD) and in reduced glutathione (GSH). The thiobarbituric acid-reactive substances (TBARS) values were more abundant in the albino tissue compared to the pigmented tissue both during spontaneous and Fe++ induced lipoperoxidation. Therefore, when isolated and purified melanin, in physiological quantities, was added to albino tissue, the TBARS values drastically decreased. Thus, melanin, in our experimental conditions, protects the albino tissue even more than SOD and GSH do. Melanin, in our opinion, acts as an antioxidant, because it is able to scavenge O2-.

Studies on the immunogenicity of a recombinant ookinete surface antigen Pbs21 from Plasmodium berghei expressed in Escherichia coli.

Plasmodium berghei ookinete surface antigen (Pbs21), was produced as a fusion product with maltose binding protein (MBP) in Escherichia coli and used to induce transmission-blocking immunity in mice. Specificity of induced antibody was confirmed by Western blotting with native ookinete Pbs21, and by the indirect immunofluorescent antibody test on ookinete bloodfilms. Immunized mice were infected with P. berghei and transmission to Anopheles stephensi mosquitoes determined by both the intensity and prevalence of oocyst infections. Compared with a control group immunized with MBP alone the maximum blockade of oocyst intensity was 66% in the mice immunized with recombinant MBP-Pbs21. Over nine experiments blockade averaged only 33%. By comparison with native Pbs21 protein, which usually induces > or = 90% blockade, our data suggests the recombinant protein produced in this bacterial system is a less effective immunogen despite expressing epitopes recognized by known transmission-blocking monoclonal antibodies.

Characterization of the effector mechanisms of a transmission-blocking antibody upon differentiation of Plasmodium berghei gametocytes into ookinetes in vitro.

The transmission-blocking monoclonal antibody 13.1, which recognizes the ookinete surface antigen Pbs21 of Plasmodium berghei, and an IgG2a isotype control antibody 26.37 were purified by caprylic acid and ammonium sulphate precipitation. Fab fragments were prepared by papain digestion. IgG but not Fab from antibody 13.1 reduced ookinete formation by P. berghei in culture by as much as 94% at a concentration of 100 micrograms/ml. There was little difference in antibody efficacy in the range 6.25-400 micrograms/ml in this assay. The parasite was most sensitive to antibody activity in the first 6-9 h of culture, i.e. the gamete/zygote and early retort stages. Peripheral blood leucocytes (PBL) were essential to achieve maximal inhibition by mAb 13.1 (activity was abrogated totally if PBL were removed). Together the data suggest that one of the mechanisms of action of this antibody is antibody-mediated PBL killing. Phagocytosis of parasites was noted in these experiments in all cultures. We have not attempted in this study to distinguish between Fc-mediated opsonization, as opposed to antibody-dependent cellular cytotoxicity.