Synthesis, cloning, and expression of artificial genes, coding antigenic determinants of the foot-and-mouth virus substrain A22]

Chemical-enzymatic synthesis and cloning in Escherichia coli of double-stranded DNAs, coding for simple and complex antigenic determinants of foot-and-mouth disease virus (FMDV) strain A22, have been carried out. The simple antigenic determinants are a part of the viral coat protein VP1 (amino acid sequence 131-152 or 131-160) whereas the complex antigenic determinants comprise additionally the amino acid sequence 200-213 of VP1 linked to N-terminus of simple antigenic determinants through a tetrapeptide spacer Pro-Pro-Ser-Pro. Recombinant DNAs containing genes for antigenic determinants of FMDV fused with C-terminus of gene for human tumor necrosis factor (hrTNF) have been constructed. Expression of the hybrid genes and properties of the proteins coded were studied. All recombinant proteins were shown to interact specifically with polyclonal antibodies both against hrTNF and FMDV strain A22. The recombinant proteins produced by bacteria are perspective for study as a vaccine against FMDV.     

Solid phase ligation of synthetic DNA fragments

A synthetic oligodeoxyribonucleotide (oligo) covalently bound by an internucleotide linkage to the succinylated Sephacryl S-500 support through 1.9-diaminononane spacer was used as starting compound to assemble the E. coli rec A promoter DNA fragment from synthetic oligos by means of T4 DNA ligase. The solid-phase assembly of the designed DNA was performed by two ways: stepwise ligation of two pairs of oligos (2 dyads) or simultaneous ligation of four oligos (tetrad). Both ways gave equal results with some preference in the tetrad case. The reliability of E. coli promoter DNA fragment assembly was demonstrated by cloning it in a plasmid vector and sequencing the cloned DNA by the solid-phase Maxam--Gilbert technique.     

Heterologous expression of murine lymphotoxins in Escherichia coli and preparation of antibodies]

Genes encoding fragments of polypeptide chains of murine lymphotoxins (LT), namely, LT-alpha truncated from the N-terminus and the LT-beta extracellular domain, containing N-terminal hepta- and hexahistidine epitopes, respectively, were expressed in E. coli cells. The recombinant proteins purified by metallochelate chromatography were used to obtain polyclonal antibodies that specifically recognize murine LT.     

Immunoenzyme determination of human granulocyte-macrophage colony-stimulating factor using monoclonal antibodies

A set of seven hybridomas producing monoclonal antibodies (MAbs) to the human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was obtained. The properties of the monoclonal antibodies were characterized, and pairs of MAbs specific to different non-overlapping epitopes of GM-CSF were identified. A sensitive and simple method of two-site ELISA for GM-CSF was developed on the basis of two MAbs. According to this method, one MAb is absorbed onto a microtiter plate and another is labeled with biotin and used for the detection of GM-CSF bound to the first MAb. MAb labeled with biotin, in its turn, was visualized with the streptavidin-horseradish peroxidase conjugate. The sensitivity of this test was no less than 0.5 ng/ml, and a linear dose-response relationship was observed within a concentration interval from 0.5 to 32 ng/ml. No cross-reactivity was found with human tumor necrosis factor-alpha, granulocyte colony-stimulating factor, interleukin-2, or interleukin-3 in this test system.     

Immunotherapy with autologous tumor cells engineered to secrete Tag7/PGRP, an innate immunity recognition molecule

BACKGROUND: Recent studies indicate that the innate component of immune defense plays an important role in the establishment of antigen-specific immune response. We have previously isolated a novel mouse gene tag7/PGRP that was shown to be involved in the innate component of the immune system, and its insect homologue is an upstream mediator of Toll signaling in Drosophila. METHODS: Transiently or stably genetically modified mouse tumor cell lines expressing Tag7 were used. Tumor growth rate and animal survival were analyzed. Possible effector cells involved in tumor suppression were detected immunohistochemically. RESULTS: Transfection of mammary gland adenocarcinoma cells with the tag7 cDNA did not alter their growth rate in vitro but diminished their tumorogenicity in vivo in syngeneic and immunodeficient animals. Increased incidence of apoptosis was registered in the modified tumors. Transient expression of Tag7 by mouse melanoma M3 cells elicited protective immunity against parental tumor cells. Immunohistochemical analysis revealed that tumors after immunization with the genetically modified cells were infiltrated with Mac1(+) cells, B220(+) cells, and NK cells. Using nude mice we observed rejection of modified cells, but did not detect memory formation. CONCLUSIONS: We can conclude that secretion of the Tag7 protein by genetically modified cells can induce mobilization of antigen-presenting cells and innate effectors. Memory mechanisms are mediated by T cell response. For the first time our results demonstrate that local secretion of Tag7-the molecule involved in innate immunity-may play an important role in the induction of effective antitumor response in mice.     

Role of tumor necrosis factor alpha and sphingomyelin cycle activation in the induction of apoptosis by ischemia/reperfusion of the liver

The signal transduction pathways triggering apoptotic mechanisms after ischemia/reperfusion may involve TNF- secretion, ceramide generation, and initiation of lipid peroxidation. In the present study involvement of the TNF-, sphingomyelin cycle, and lipid peroxidation in the initiation of apoptosis induced in liver cells by ischemia and reperfusion was investigated. Wistar rats were subjected to total liver ischemia (for 15, 30 min, and 1 h) followed by subsequent reperfusion. Ischemia caused sharp decrease of neutral sphingomyelinase activity. Activity of acidic sphingomyelinase initially decreased (during 15-30 min ischemia) but then increased (after 1 h of ischemic injury). Reperfusion of the ischemic lobe of the liver caused increase in neutral sphingomyelinase activity and decrease in acidic sphingomyelinase activity. A small amount of TNF- detected by immunoblotting analysis was accumulated in the ischemic area of liver rapidly and the content of this cytokine dramatically increased after the reperfusion. TNF- is known to induce free radical production. We found that the accumulation of TNF and increase of sphingomyelinase activity during the development of ischemic/reperfusion injury coincided with increase in content of lipid peroxidation products (conjugated dienes) and DNA degradation detected by gel electrophoresis. Recently it was shown that superoxide radicals are used as signaling molecules within the sphingomyelin pathway. This suggests the existence of cross-talk between the oxidation system and the sphingomyelin cycle in cells, which may have important implications for the initial phase and subsequent development of post-ischemic injury.     

Multiadaptor 4.1 and RanBP9 protein family members as putative interaction partners for VARP, a Rab21 GTPase guanine nucleotide exchange factor

VARP is a novel VPS9 domain-containing protein which acts as a guanine nucleotide exchange factor for small GTPases Rab21 and Rab5, regulators of early endocytosis. However, the molecular mechanisms underlying VARP activity regulation and intracellular localization remain unknown. Using protein interaction cloning in yeast we isolated multiadaptor proteins of 4.1 protein family and RanBP9 as putative VARP interaction partners. The interactions revealed might be important for proper intracellular localization of VARP and its functions in early endocytosis.     

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.