Assays for analyzing exonucleases in vitro

Ribonucleases play essential roles in cell growth, differentiation, and the response to stress. This article deals with exoribonucleases, enzymes that degrade RNAs beginning at either the 5' or 3' end and proceed down the length of the RNA. The preparation of a crude extract of a mammalian 3'-to-5' exonuclease is described. Assay conditions for both 5'-to-3' and 3'-to-5' exonucleases are given. One of these is a yeast enzyme that is known to be involved in mRNA decay. Others are vertebrate exonucleases that are presumed to have a role in mRNA stability but have not yet been proven to do so.     

In vivo and in vitro activation of alveolar macrophages by recombinant interferon-gamma

In vivo administration of recombinant interferon-gamma (rIFN-gamma) was previously shown to result in activation of the microbicidal activities of peritoneal macrophages (PM phi). Because macrophages at different anatomical sites vary in their functional capacities, we considered it of interest to determine whether administration of murine rIFN-gamma, either in vitro or in vivo, can enhance the microbicidal activity of resident alveolar macrophages (AM phi) and to compare the effects of rIFN-gamma on AM phi and PM phi. After incubation in vitro with rIFN-gamma, the antimicrobial activities of both murine AM phi and PM phi were enhanced, as assessed by their ability to inhibit replication of the intracellular parasite, Toxoplasma gondii. This effect was dose dependent for AM phi over a range of 0.1 to 1 U/ml and for PM phi over a range of 0.5 to 1000 U/ml. In this assay, the minimum dosage required for in vitro activation of AM phi was one-half that required for activation of PM phi, suggesting a greater sensitivity of AM phi to the in vitro activity of rIFN-gamma. Macrophages from both anatomical sites were also activated when rIFN-gamma was administered in vivo. This effect was dose dependent over a range of 10(3) to 10(5) U/mouse. Freshly harvested AM phi and PM phi from mice injected 24 hr earlier with 10(4) U rIFN-gamma by either the i.v. or i.p. routes markedly inhibited intracellular multiplication of Toxoplasma. In contrast, AM phi and PM phi from control mice permitted fourfold to ninefold increases in numbers of intracellular Toxoplasma. The anti-toxoplasma activity of AM phi and PM phi gradually diminished over a period of 3 days when assayed at successive 24 hr periods after a single i.v. injection of rIFN-gamma. At 3 days after injection, a substantial loss of anti-toxoplasma activity was observed with PM phi as compared with controls; residual anti-toxoplasma activity was still demonstrable in AM phi at 3 days. These results demonstrate that in vitro as well as in vivo treatment with rIFN-gamma confers on AM phi an enhanced antimicrobial activity. These findings provide a rationale for evaluating rIFN-gamma in the treatment of pulmonary infections, especially those due to opportunistic pathogens against which AM phi play a major role in host defense.     

Lysosomal activation in mouse skeletal muscle induced by protamine in vitro

Summary Incubation of mouse skeletal muscle in a physiological Ringer solution containing protamine (60 g/ml) at +37° C for 1 h induced ultrastructural changes including proliferation of tubular profiles and vesicles at the I-band level close to the A-I junction, formation of numerous acid phosphatase positive lysosomes in the longitudinal sarcoplasmic reticulum and autophagic vacuolation starting at the level of the A-I junction.Biochemical determination of acid phosphatase in the incubated muscles showed that protamine caused an increase in acid phosphatase activity of about 25 % compared to enzyme activities obtained from muscles incubated without protamine at +37°C or with protamine at +4°C.The morphological findings suggest that the vesicles arising adjacent to the A-I junction originate from transverse tubules. Such vesicles, designated as endocytic, may acquire acid phosphatase activity in the longitudinal SR and be active in an autophagic process resulting in large vacuoles. A causal relationship between endocytosis and lysosomal activation is suggested.We wish to thank Miss Britt-Marie Svensson, Miss Ann-Christin Nilsson and Mrs. Lena Kvist for their able technical assistance. This work was supported by grants from the Medical Faculty, University of Lund, Sweden, and the Swedish Medical Research Council, Stockholm, Sweden (04P-4289, 14X-4286, 14X-3112) and from Muscular Dystrophy Association of America, Inc.     

Production of recombinant allergens in plants

A large percentage of allergenic proteins are of plant origin. Hence, plant-based expression systems are considered ideal for the recombinant production of certain allergens. First attempts to establish production of plant-derived allergens in plants focused on transient expression in Nicotiana benthamiana infected with recombinant viral vectors. Accordingly, allergens from birch and mugwort pollen, as well as from apple have been expressed in plants. Production of house dust mite allergens has been achieved by Agrobacterium-mediated transformation of tobacco plants. Beside the use of plants as production systems, other approaches have focused on the development of edible vaccines expressing allergens or epitopes thereof, which bypasses the need of allergen purification. The potential of this approach has been convincingly demonstrated for transgenic rice seeds expressing seven dominant human T cell epitopes derived from Japanese cedar pollen allergens. Parallel to efforts in developing recombinant-based diagnostic and therapeutic reagents, different gene-silencing approaches have been used to decrease the expression of allergenic proteins in allergen sources. In this way hypoallergenic ryegrass, soybean, rice, apple, and tomato were developed.     

Plasminogen activation and lysis of blood clots induced by cells in vitro

When human diploid fibroblasts were seeded onto the surface of blood clots, lysis of the clot occurred as a result of the release of cellular plasminogen activator. A number of aspects of this lysis were studied. 1. There was no significant difference in rates of lysis of whole blood clots, platelet-rich plasma clots, and platelet-poor plasma clots brought about by the same number of fibroblasts. 2. Clot lysis was promoted by nondividing cells and by proliferating cells. 3. Using cycloheximide to block protein synthesis it was found that the plasminogen activator released by fibroblasts had an active half-life of less than an hour. 4. When clots were washed prior to the addition of cells then lysis occurred at an increased rate. This was probably due to the removal of alpha 2-antiplasmin from the clots, since when antisera to alpha 2-antiplasmin was added to clots, lysis also proceeded at an increased rate. 5. Medium conditioned by fibroblasts did not promote clot lysis even when antiplasmin was removed by washing or by addition of antisera. 6. Cells had to be in direct contact with the clot in order to bring about lysis; when cells were separated from clots by permeable membranes there was no lysis. 7. When cross-linking of fibrin was reduced by the inhibition of transglutaminase, the rate of clot lysis was increased.     

Improving diagnostic tests for food allergy with recombinant allergens

Food allergy is one important manifestation of atopic allergy. Primary food allergy mainly affects young children (class I), whereas adults frequently develop food allergy as a consequence of an inhalant sensitization (class II). At present, the diagnostic instrument for proving class II food allergy is not satisfactory. Skin tests as well as serological tests are in general neither very specific nor highly sensitive because they depend on food extracts, which differ in their content of individual allergens, vary between manufacturers, and even between different batches. Since the presence of food allergen-specific IgE antibodies does not always correlate with clinical symptoms against the respective food, oral provocation tests (ideally double-blind placebo-controlled food challenges) have to be performed to validate serological diagnosis or skin tests. However, oral provocation tests are connected to several practical problems and include a specific risk for the allergic patient. Applying DNA technology, up to 40 food allergens have been produced in recombinant form, which implies standardized quality and unlimited quantity of the respective proteins. Hence, such molecules might be used to solve problems of clinical and molecular allergology in diagnosis, research, and therapy of class II food allergies. First experiments with recombinant food allergens in this respect appear very promising.     

In vitro activation of human sperm induced by amphibian egg extract

In this paper we characterize a system for the activation of human sperm using cell-free extracts from Xenopus laevis eggs. We characterize the kinetics of sperm activation in terms of morphological and DNA synthetic parameters. We have optimized some of the components which are necessary for chromatin decondensation and present data to demonstrate that the in vitro process is efficient over a wide range of salt concentrations and amounts of supplementary reducing agents.     

Plant virus expression systems for transient production of recombinant allergens in Nicotiana benthamiana

In recent years, several studies have demonstrated the use of autonomously replicating plant viruses as vehicles to express a variety of therapeutic molecules of pharmaceutical interest. Plant virus vectors for expression of heterologous proteins in plants represent an attractive biotechnological tool to complement the conventional production of recombinant proteins in bacterial, fungal, or mammalian cells. Virus vectors are advantageous when high levels of gene expression are desired within a short time, although the instability of the foreign genes in the viral genome may present problems. Similar levels of foreign protein production in transgenic plants often are unattainable, in some cases because of the toxicity of the foreign protein. Now virus-based vectors are for the first time investigated as a means of producing recombinant allergens in plants. Several plant virus vectors have been developed for the expression of foreign proteins. Here, we describe the utilization of tobacco mosaic virus- and potato virus X-based vectors for the transient expression of plant allergens in Nicotiana benthamiana plants. One approach involves the inoculation of tobacco plants with infectious RNA transcribed in vitro from a cDNA copy of the recombinant viral genome. Another approach utilizes the transfection of whole plants from wounds inoculated with Agrobacterium tumefaciens containing cDNA copies of recombinant plus-sense RNA viruses.     

Role of the recombinant non-integrin platelet collagen receptor P65 on platelet activation induced by convulxin

Convulxin (Cvx) isolated from Crotalus durissus terrificus venom selectively binds with a high affinity to platelets and induces platelet aggregation by a mechanism that resembles that induced by collagen. Taking advantage that P65 has been recently cloned and expressed as a recombinant soluble protein (rec-P65), we examined the role of this non-integrin collagen receptor in platelet activation induced by Cvx. Rec-P65 blocked platelet adhesion to collagen-coated surfaces and inhibited platelet aggregation and ATP secretion induced by type I collagen. On the other hand, rec-P65 did not inhibit platelet aggregation and ATP secretion induced by Cvx, and it did not affect platelet adhesion to Cvx. In addition, ligand-blotting indicated that the Cvx binding to the collagen receptor GPVI was preserved in the presence of rec-P65. These observations indicate that P65 does not play a significant role in platelet activation by Cvx; in contrast, platelet response to collagen involves multiple receptors.     

In vitro anti-tumor immune response induced by dendritic cells transfected with EBV-LMP2 recombinant adenovirus

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is a high-incidence tumor in southern China. Latent membrane proteins 2 (LMP2) is a subdominant antigen of EBV. The present study was to develop a dendritic cells (DCs)-based cancer vaccine (rAd-LMP2-DC) and to study its biological characteristics and its immune functions. Our results showed that LMP2 gene transfer did not alter the typical morphology of mature DC, and the representative phenotypes of mature DC (CD80, CD83, and CD86) were highly expressed in rAd-LMP2-DCs. The expression of LMP2 in rAd-LPM2-DCs was about 84.54%, which suggested efficient gene transfer. Transfected DCs markedly increased antigen-specific T-cell proliferation. The specific cytotoxicity against NPC cell was significantly higher than that in controls (p < 0.05), and enhanced with increased stimulations by transfected DCs. In addition, phenotypic analysis demonstrated that the LMP2-specific CTLs consisted of both CD4(+) and CD8(+) T cells. These results showed that development of DC-based vaccine by transfection with malignancy-associated virus antigens could elicit potent CTL response and provide a potential strategy of immunotherapy for EBV-associated NPC.     

Prophenoloxidase activation and antimicrobial peptide expression induced by the recombinant microbe binding protein of Manduca sexta

Manduca sexta microbe binding protein (MBP) is a member of the β-1,3-glucanase-related protein superfamily that includes Gram-negative bacteria-binding proteins (GNBPs), β-1,3-glucan recognition proteins (βGRPs), and β-1,3-glucanases. Our previous and current studies showed that the purified MBP from baculovirus-infected insect cells had stimulated prophenoloxidase (proPO) activation in the hemolymph of naïve and immune challenged larvae and that supplementation of the exogenous MBP and peptidoglycans (PGs) had caused synergistic increases in PO activity. To explore the underlying mechanism, we separated by SDS-PAGE naïve and induced larval plasma treated with buffer or MBP and detected on immunoblots changes in intensity and/or mobility of hemolymph (serine) proteases [HP14, HP21, HP6, HP8, proPO-activating proteases (PAPs) 1–3] and their homologs (SPH1, SPH2). In a nickel pull-down assay, we observed association of MBP with proHP14 (slightly), βGRP2, PG recognition protein-1 (PGRP1, indirectly), SPH1, SPH2, and proPO2. Further experiments indicated that diaminopimelic acid (DAP) or Lys PG, MBP, PGRP1, and proHP14 together trigger the proPO activation system in a Ca²⁺-dependent manner. Injection of the recombinant MBP into the 5th instar naïve larvae significantly induced the expression of several antimicrobial peptide genes, revealing a possible link between HP14 and immune signal transduction. Together, these results suggest that the recognition of Gram-negative or -positive bacteria via their PGs induces the melanization and Toll pathways in M. sexta.     

Macrophage activation to kill Leishmania major: activation of macrophages for intracellular destruction of amastigotes can be induced by both recombinant interferon-gamma and non-interferon lymphokines

Macrophages treated with lymphokine (LK)-rich culture fluids from antigen- or mitogen-stimulated spleen cells or the hybridoma T cell 24/G1, or murine recombinant interferon-gamma (IFN-gamma) from either transfected monkey kidney cells (cos rIFN-gamma) or bacterial (E. coli) DNA (rIFN-gamma) developed the capacity to kill intracellular amastigotes of Leishmania major. Removal of IFN activity from LK by neutralizing fluid phase monoclonal anti-rIFN-gamma antibody, or by solid phase immunoadsorption, left residual macrophage activation factors that induced approximately 50% of the macrophage anti-leishmanial activity of untreated LK. In contrast, rIFN-gamma subjected to the same antibody treatments lost all capacity to induce this macrophage effector function. These results suggest that the intracellular destruction of amastigotes is regulated by several different factors. One of these factors is clearly IFN-gamma, which is pleiotropic in its effects on macrophage functions. The other non-IFN LK factors are immunochemically unrelated to IFN-gamma, and may regulate macrophage microbicidal activities in a more selective manner.     

Expression,refolding, and in vitro activation of a recombinant snake venom pro-metalloprotease

Metalloproteases comprise a family of Zn(2+)-endopeptidases that degrade most components of the extracellular matrix. Snake venoms are rich sources of metalloproteases, which also digest fibrinogen as well as fibrin, and in some cases, induce hemorrhage. A few low-molecular weight snake venom metalloproteases (svMPs) have been described as being devoid of hemorrhagic activity, but they have strong direct-acting fibrinolytic activity. This property could be very helpful in thrombosis therapy. ACLF is a fibrinolytic, non-hemorrhagic metalloprotease from the venom of the North American snake Agkistrodon contortrix laticinctus. We have developed an expression system for production of a recombinant pro-ACLF from a clone (ACLPREF) isolated from a venom gland cDNA library. The coding region including both the pro-enzyme domain and the mature protein domain was amplified by PCR and subcloned into the pET28a vector and the new plasmid was used to transform BL21(DE3) Escherichia coli cells. Culture of the transformants at 37 degrees C led to the overexpression of an insoluble 48kDa protein after induction with 1.0mM IPTG. The expressed protein was recovered from inclusion bodies with 6M buffered urea and purified by affinity chromatography under denaturing conditions. After dithiothreitol treatment, protein refolding was performed by gradual removal of the denaturing agent by dialysis. The pro-enzyme underwent auto-activation during refolding and it was active on fibrinogen and on a synthetic substrate. To control the activation step, the denaturing agent was rapidly removed to keep the protein in an unprocessed form, followed by later addition of Ca(2+) and Zn(2+) ions. This allowed controlling the enzyme activation, when it is needed.     

CD18 activation epitopes induced by leukocyte activation.

The cell surface adhesion molecule LFA-1 coordinates leukocyte trafficking and is a costimulatory molecule for T cell activation. We developed a panel of mAbs that recognize activation epitopes on the CD18 subunit, and show that stimulation of T lymphocytes appears to be accompanied by a conformational change in a subpopulation of LFA-1 that does not require ligand binding. Activation epitope up-regulation requires divalent cations, is sensitive to cellular signal transduction events, and correlates with cell adhesion. In addition, the stimulated appearance of these activation epitopes is absent in cell lines from patients with leukocyte adhesion deficiency-1/variant that has previously been shown to be defective in LFA-1 activation. Thus, these activation epitope Abs can be used to dissect signal transmission to CD18. Evidence suggests that these CD18 activation epitopes are induced early in cellular activation and are independent of actin rearrangement necessary for avid adhesion. We have also determined that function-blocking CD18 Abs inhibit the induction of activation epitopes. One activation epitope Ab binds to a site on CD18 distinct from that of the blocking Abs, indicating that the blocking Abs suppress a conformational change in LFA-1. We also find that these neoepitopes are present on rLFA-1 with high affinity for ICAM-1 and their binding is modulated in parallel with the affinity of LFA-1 for ICAM-1. Collectively, these neoepitope Abs identify a subpopulation of LFA-1 most likely with high affinity for ICAM-1 and necessary for LFA-1 function.