An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Structure based virtual screening of ligands to identify cysteinyl leukotriene receptor 1 antagonist

Montelukast and Zafirlukast are known leukotriene receptor antagonists prescribed in asthma treatment. However, these fall short as mono therapy and are frequently used in combination with inhaled glucocorticosteroids with or without long acting beta 2 agonists. Therefore, it is of interest to apply ligand and structure based virtual screening strategies to identify compounds akin to lead compounds Montelukast and Zafirlukast. Hence, compounds with structures having 95% similarity to these compounds were retrieved from NCBI׳s PubChem database. Compounds similar to lead were grouped and docked at the antagonist binding site of cysteinyl leukotriene receptor 1. This exercise identified compounds UNII 70RV86E50Q (Pub Cid 71587778) and Sure CN 9587085 (Pub Cid 19793614) with higher predicted binding compared to Montelukast and Zafirlukast. It is shown that the compound Sure CN 9587085 showed appreciable ligand receptor interaction compared to UNII 70RV86E50Q. Thus, the compound Sure CN 9587085 is selected as a potent antagonist to cysteinyl leukotriene receptor 1 for further consideration in vitro and in vivo validation.     

Evidence that pinocytosis in lymphoid cells has a low capacity

In contrast to adherent cells, human B and T lymphoblasts, marmoset monkey T lymphoblasts, and mouse T lymphoblasts do not form monolayers and have a poor ability to pinocytose. After a 10-min incubation of lymphoblasts at 37 degrees C, the level of internalized medium reached a plateau. During this time, lymphoblasts pinocytosed 3-4 femtoliters (1 fl = 10(-15) l) of medium per cell as calculated by the quantity of the entrapped pinocytic marker 5(6)-carboxyfluorescein. The levels of pinocytosed liquid did not increase during a subsequent 90-min incubation of cells at 37 degrees C. Adherent HeLa cells took up 27 fl of medium per cell per hour. Other types of adherent cells were reported by others to pinocytose 20 to 90 fl of medium per cell per hour. The process of pinocytosis in lymphoblasts appeared to be reversible since cells which were pre-loaded with carboxyfluorescein and then incubated at 37 degrees C in fresh medium lost the marker almost completely within 40 min. Similar results were obtained with horseradish peroxidase as the pinocytic marker. Further evidence that lymphoblasts have a low capacity for pinocytic internalization relative to adherent cells was obtained from the observation that Namalwa lymphoblasts were approximately 100 times more resistant to the cytotoxic action of the protein toxin gelonin than the adherent HeLa cells. Gelonin is a ribosome-inactivating toxin which is not capable of binding to cells, and its only mode for internalization appears to be pinocytosis. Ribosomes in cell lysates of the two lines were equally sensitive to gelonin. It is speculated that the poor pinocytic ability of lymphoid cells may reflect a fundamental difference between adherent and non-adherent cells and that this may impede the targeting of drugs into lymphoid cells.     

Epstein Barr virus binding induces internalization of the C3d receptor: a novel immunotoxin delivery system

Epstein Barr virus (EBV) infection of human B lymphocytes is initiated by selective binding of the virus to the C3d receptor (EBV/C3d receptor) on the cell surface and results in polyclonal proliferation of infected cells. In these studies we examined the fate of the EBV/C3d receptor during viral infection by using an immunotoxin made from a monoclonal antibody (HB5) reactive with the receptor and the potent toxin, gelonin. Binding of the HB5-gelonin conjugate to the EBV/C3d receptor before EBV infection (at concentrations as low as 10(-11) M) significantly inhibited the subsequent polyclonal proliferation of virus-infected B lymphocytes. HB5 antibody and gelonin alone did not inhibit proliferation. Because internalization of gelonin-antibody conjugates is required to cause cytotoxicity, these results indicate that infection of B lymphocytes with EBV selectively induced endocytosis of the EBV/C3d receptor with concomitant internalization of the immunotoxin. Proliferation of B lymphocytes that were activated by prior infection with EBV, or activated by cross-linking of their surface immunoglobulin molecules, was not inhibited by the antibody-toxin conjugate even at concentrations as high as 10(-7) M. Also, the growth of B lymphoblastoid cell lines cultured in the presence or absence of infectious EBV was not inhibited by HB5-gelonin. Thus, our results suggest that the EBV/C3d receptor is internalized only during the infection of normal B lymphocytes by EBV, with co-internalization of immunotoxin, and indicate that internalization of the EBV/C3d receptor-immunotoxin complex does not occur simply as a consequence of activation and proliferation of B lymphocytes. The use of a ligand to induce endocytosis of its receptor offers a new strategy for the selective delivery of immunotoxins to cells and may be more generally applicable.     

Cytopathic effects of the cytomegalovirus-encoded apoptosis inhibitory protein vMIA

Replication of human cytomegalovirus (CMV) requires the expression of the viral mitochondria-localized inhibitor of apoptosis (vMIA). vMIA inhibits apoptosis by recruiting Bax to mitochondria, resulting in its neutralization. We show that vMIA decreases cell size, reduces actin polymerization, and induces cell rounding. As compared with vMIA-expressing CMV, vMIA-deficient CMV, which replicates in fibroblasts expressing the adenoviral apoptosis suppressor E1B19K, induces less cytopathic effects. These vMIA effects can be separated from its cell death-inhibitory function because vMIA modulates cellular morphology in Bax-deficient cells. Expression of vMIA coincided with a reduction in the cellular adenosine triphosphate (ATP) level. vMIA selectively inhibited one component of the ATP synthasome, namely, the mitochondrial phosphate carrier. Exposure of cells to inhibitors of oxidative phosphorylation produced similar effects, such as an ATP level reduced by 30%, smaller cell size, and deficient actin polymerization. Similarly, knockdown of the phosphate carrier reduced cell size. Our data suggest that the cytopathic effect of CMV can be explained by vMIA effects on mitochondrial bioenergetics.     

Synthesis and biological evaluation of novel taxoids designed for targeted delivery to tumors

The use of drug-antibody conjugates affords a method for the targeted delivery of anticancer drugs specifically to cancer cells. Monoclonal antibodies alone usually do not possess high therapeutic efficacy, however, they are capable of targeting tumor markers selectively. We have prepared taxoids with significantly higher cytotoxicity than paclitaxel and docetaxel. These taxoids now meet the high potency required for use in a targeted-delivery approach using monoclonal antibodies. The synthesis and biological evaluation of these taxoids are reported.     

Processing of human cytomegalovirus UL37 mutant glycoproteins in the endoplasmic reticulum lumen prior to mitochondrial importation

The human cytomegalovirus (HCMV) UL37 glycoprotein (gpUL37) is internally cleaved and its products divergently traffic to mitochondria or are retained in the secretory pathway. To define the requirements for gpUL37 cleavage, residues -1 and -3 of the consensus endoplasmic reticulum (ER) signal peptidase I site within exon 3 (UL37x3) were replaced by bulky tyrosines (gpUL37 cleavage site mutant I). Internal cleavage of this UL37x3 mutant was inhibited, verifying usage of the consensus site at amino acids (aa) 193/194. The full-length mitochondrial species of gpUL37 cleavage site mutant I was N glycosylated and endoglycosidase H sensitive, indicating that ER translocation and processing took place prior to its mitochondrial importation. Moreover, these results suggest that internal cleavage of gpUL37 is not necessary for its N glycosylation. Partial deletion or disruption of the UL37 hydrophobic core immediately upstream of the cleavage site resulted in decreased protein abundance, suggesting that the UL37x3 hydrophobic alpha-helix contributes to either correct folding or stability of gpUL37. Insertion of the UL37x3 hydrophobic core and cleavage site into pUL37(M), a splice variant of gpUL37 which lacks these sequences and is neither proteolytically cleaved nor N glycosylated, resulted in its internal cleavage and N glycosylation. Its NH(2)-terminal fragment, pUL37(M-NH2), was detected more abundantly in mitochondria, while its N-glycosylated C-terminal fragment, gpUL37(M-COOH), was detected predominantly in the ER in a manner analogous to that of gpUL37 cleavage products. These results indicate that UL37x3 aa 178 to 205 are prerequisite for gpUL37 internal cleavage and alter UL37 protein topology allowing N glycosylation of its C-terminal sequences. In contrast, the NH(2)-terminal UL37x1 hydrophobic leader, present in pUL37x1, pUL37(M), and gpUL37, is not cleaved from mature UL37 protein, retaining a membrane anchor for UL37 isoforms during trafficking. Taken together, these results suggest that HCMV gpUL37 undergoes sequential trafficking, during which it is ER translocated, processed, and then mitochondrially imported.     

The specific cytotoxicity of immunoconjugates containing blocked ricin is dependent on the residual binding capacity of blocked ricin: evidence that the membrane binding and A-chain translocation activities of ricin cannot be separated.

Recently we have developed blocked ricin, a derivative of native ricin in which the galactose-binding sites of the B-chain are blocked by covalent modification with affinity ligands. This modification impedes the binding function of the B-chain, while sparing its ability to facilitate the entry of the toxic subunit of ricin, the A-chain, into the cytoplasm. Immunotoxins prepared with blocked ricin approach the cytotoxic potency of native ricin with antibody-dependent specificity. Here we report that the high cytotoxic potency of these immunoconjugates, which is attributed to the preserved translocation function of the ricin B-chain, is dependent on the minimal residual lectin activity of blocked ricin. Our findings support the notion that two functions of ricin, membrane binding and translocation, cannot be separated.     

Antibody-complement-mediated cytotoxicity is enhanced by ribosome-inactivating proteins

The antibody-mediated cytotoxicity of complement can be increased by the ribosome-inactivating proteins gelonin and PAP-S. Treatment of human lymphoid cells that express CALLA with an anti-CALLA monoclonal antibody, J5, and then with rabbit complement, leaves about 6% of the cells alive. The same treatment in the presence of a sublethal concentration of gelonin or PAP-S leaves only about 0.02 and 0.3% of the cells alive respectively. This synergistic effect has potential implications for the in vitro elimination of malignant cells or of immunocompetent cells from bone marrow before its transplantation.     

vMIA,a viral inhibitor of apoptosis targeting mitochondria

Human cytomegalovirus encodes a powerful cell death suppressor vMIA (viral mitochondria-localized inhibitor of apoptosis), also known as pUL37x1. vMIA, a product of the immediate early gene UL37 exon 1, is predominantly localized in mitochondria, where it appears to form a complex with adenine nucleotide translocator, believed to be a component of the mitochondrial transition pore complex. vMIA suppresses apoptosis by blocking permeabilization of the mitochondrial outer membrane. Expression of vMIA protects cells against apoptosis triggered by diverse stimuli, including ligation of death receptors, exposure to certain cytotoxic drugs, and infection with an adenovirus mutant deficient in E1B19K. Deletion mutagenesis of vMIA revealed two domains that are necessary and, together, sufficient for its anti-apoptotic activity. The first domain contains a mitochondrial targeting signal. The function of the second domain is still unknown. vMIA does not share any significant amino acid sequence homology with Bcl-2, and, unlike Bcl-2 or Bcl-x(L), it does not bind BAX or VDAC. These structural and functional differences between vMIA and Bcl-2 suggest that vMIA represents a separate class of cell death suppressors. Experiments with vMIA-deficient CMV (human cytomegalovirus) mutants provide strong evidence that the anti-apoptotic function of vMIA is required to prevent CMV-induced apoptosis, and is necessary for viral replication. In addition to vMIA, UL37 encodes two longer splice-variant proteins, gpUL37 and GP37(M). Biological functions of these proteins have not yet been identified, and may be unrelated to their anti-apoptotic activity. The identification of vMIA and the finding that its anti-apoptotic function is required for CMV replication provides a rationale for the development of anti-CMV pharmaceuticals that would inactivate vMIA and thus restore apoptosis in cells infected with CMV.