Overexpression of the rabies virus glycoprotein results in enhancement of apoptosis and antiviral immune response

A recombinant rabies virus (RV) carrying two identical glycoprotein (G) genes (SPBNGA-GA) was constructed and used to determine the effect of RV G overexpression on cell viability and immunity. Immunoprecipitation analysis and flow cytometry showed that tissue culture cells infected with SPBNGA-GA produced, on average, twice as much RV G as cells infected with RV carrying only a single RV G gene (SPBNGA). The overexpression of RV G in SPBNGA-GA-infected NA cells was paralleled by a significant increase in caspase 3 activity followed by a marked decrease in mitochondrial respiration, neither of which was observed in SPBNGA-infected cells. Furthermore, fluorescence staining and confocal microscopy revealed an increased extent of apoptosis and markedly reduced neurofilament and F actin in SPBNGA-GA-infected primary neuron cultures compared with neuronal cells infected with SPBNGA, supporting the concept that RV G or motifs of the RV G gene trigger the apoptosis cascade. Mice immunized with SPBNGA-GA showed substantially higher antibody titers against the RV G and against the nucleoprotein than SPBNGA-immunized mice, suggesting that the speed or extent of apoptosis directly determines the magnitude of the antibody response.     

Risk of all-cause mortality in HIV infected patients is associated with clinical, immunologic predictors and the CCR5 Δ32 deletion

Objective

Investigation of the interplay between the CCR5 Δ32/wt genotype and demographic, epidemiological, clinical and immunological factors associated with mortality in the cART era.

Design

Longitudinal data from 507 HIV-infected patients following the Δ32 allele detection were analyzed.

Methods

Cumulative 15 years mortality was calculated using Kaplan-Meyer methodology. Hazard ratios were estimated using univariate Cox models. Basing on Akakie information criteria and statistical significance multivariate Cox model was constructed and effect plots presenting adjusted hazard ratio time-dependency were drawn. Analysis of the association of all-cause mortality and CCR5 Δ32/wt genotype prior to the antiretroviral treatment (cART) initiation (n = 507) and on the therapy (n = 422) was also performed.

Results

A mortality rate of 2.66 (CI 2.57–3.19) per 100 person-years was observed. Univariate analysis factors modifying the risk of death included the CCR5 genotype, gender, history of cART, AIDS diagnosis and also CD4 lymphocyte nadir, zenith, the latest CD4 count and stable levels >500 cells/µl. For multivariate analysis the following predictors were selected: CCR5 genotype (HR for wt/wt 2.53, CI 1.16–5.53, p = 0.02), gender (HR for males 1.91, 95%CI 1.1–3.36, p = 0.023), introduction of combined antiretroviral treatment (HR 4.85, CI 3.0–7.89, if untreated or treated <1 month, p<0.0001) CD4 count of 500 cells/µl for six months or more (HR 4.16, CI 1.95–8.88 if not achieved, p = 0.028), the latest CD4 count (HR 5.44, CI 3.39–8.74 for <100 cells/µl, p<0.0001) and history of AIDS (HR 1.69, CI 1.03–2.79, p = 0.039). Among untreated individuals the Δ32/wt genotype was associated with notably better survival (p = 0.026), while among cART treated individuals the Δ32 mutation did not correlate significantly with higher survival rates (p = 0.23).

Conclusions

The Δ32 CCR5 allele is associated with a reduction of the risk of all-cause mortality in HIV (+) patients alongside clinical and immunologic predictors such as AIDS, history of cART, lymphocyte CD4 cell count and gender.     

A single amino acid change in rabies virus glycoprotein increases virus spread and enhances virus pathogenicity

Several rabies virus (RV) vaccine strains containing an aspartic acid (Asp) or glutamic acid (Glu) instead of an arginine (Arg) at position 333 of the RV glycoprotein (G) are apathogenic for immunocompetent mice even after intracranial inoculation. However, we previously showed that the nonpathogenic phenotype of the highly attenuated RV strain SPBNGA, which contains a Glu at position 333 of G, is unstable when this virus is passaged in newborn mice. While the Glu(333) remained unchanged after five mouse passages, an Asn(194)-->Lys(194) mutation occurred in RV G. This mutation was associated with increased pathogenicity for adult mice. Using site-directed mutagenesis to exchange Asn(194) with Lys(194) in the G protein of SPBNGA, resulting in SPBNGA-K, we show here that this mutation is solely responsible for the increase in pathogenicity and that the Asn(194)-->Lys(194) mutation does not arise when Asn(194) is exchanged with Ser(194) (SPBNGA-S). Our data presented indicate that the increased pathogenicity of SPBNGA-K is due to increased viral spread in vivo and in vitro, faster internalization of the pathogenic virus into cells, and a shift in the pH threshold for membrane fusion. These results are consistent with the notion that the RV G protein is a major contributor to RV pathogenesis and that the more pathogenic RVs escape the host responses by a faster spread than that of less pathogenic RVs.     

Biosynthesis of oleanolic acid glycosides in protoplasts isolated from Calendula officinalis L. roots

Many medicinal plants contain oleanane saponins in roots, however, only scarce data on their biosynthesis in this organ are available so far, including our previous results concerning Calendula officinalis plant. Thus, the purpose of the present work was to confirm the presumable biosynthetic pathway of oleanolic acid glycosides in roots of young C. officinalis plants. First of all, the effective method of isolation of protoplasts from C. officinalis roots was established. Then, isolated root protoplasts were supplied with radioactive precursors, [2-¹⁴C] mevalonate (MVA) and [3-³H] oleanolic acid (OL) and their transformations were studied with comparison to results obtained with excised roots. The penetration of both precursors into protoplasts was more rapid and effective than in the case of excised roots. The labeling of sterols and OL during the incubation with MVA showed that the isoprenoid pathway leading to triterpenoids was operative in excised roots as well as isolated root protoplasts. Moreover, the transformations of OL into two series of its glycosides, i.e. glucosides and glucuronides were investigated. It has been shown that both series of OL glycosides are synthesized in isolated root protoplasts in the same way as in excised roots of young marigold plants.     

SNF1-Related Protein Kinases Type 2 Are Involved in Plant Responses to Cadmium Stress

Cadmium ions are notorious environmental pollutants. To adapt to cadmium-induced deleterious effects plants have developed sophisticated defense mechanisms. However, the signaling pathways underlying the plant response to cadmium are still elusive. Our data demonstrate that SnRK2s (for SNF1-related protein kinase2) are transiently activated during cadmium exposure and are involved in the regulation of plant response to this stress. Analysis of tobacco (Nicotiana tabacum) Osmotic Stress-Activated Protein Kinase activity in tobacco Bright Yellow 2 cells indicates that reactive oxygen species (ROS) and nitric oxide, produced mainly via an l-arginine-dependent process, contribute to the kinase activation in response to cadmium. SnRK2.4 is the closest homolog of tobacco Osmotic Stress-Activated Protein Kinase in Arabidopsis (Arabidopsis thaliana). Comparative analysis of seedling growth of snrk2.4 knockout mutants versus wild-type Arabidopsis suggests that SnRK2.4 is involved in the inhibition of root growth triggered by cadmium; the mutants were more tolerant to the stress. Measurements of the level of three major species of phytochelatins (PCs) in roots of plants exposed to Cd²⁺ showed a similar (PC2, PC4) or lower (PC3) concentration in snrk2.4 mutants in comparison to wild-type plants. These results indicate that the enhanced tolerance of the mutants does not result from a difference in the PCs level. Additionally, we have analyzed ROS accumulation in roots subjected to Cd²⁺ treatment. Our data show significantly lower Cd²⁺-induced ROS accumulation in the mutants’ roots. Concluding, the obtained results indicate that SnRK2s play a role in the regulation of plant tolerance to cadmium, most probably by controlling ROS accumulation triggered by cadmium ions.Cadmium is one of the most toxic soil pollutants. Cadmium ions accumulate in plants and affect, via the food chain, animal and human health. In plants, cadmium is taken up by roots and is transported to aerial organs, leading to chromosomal aberrations, growth reduction, and inhibition of photosynthesis, transpiration, nitrogen metabolism, nutrient and water uptake, eventually causing plant death (for review, see DalCorso et al., 2008). Plants are challenged not only by cadmium ions themselves, but also by Cd²⁺-induced harmful effects including oxidative stress (Schützendübel et al., 2001; Olmos et al., 2003; Cho and Seo, 2005; Sharma and Dietz, 2009). The extent of the detrimental effects on plant growth and metabolism depends on the level of cadmium ions present in the surrounding environment and on the plant’s sensitivity to heavy metal stress.Tolerant plants avoid heavy metal uptake and/or induce the expression of genes encoding products involved, directly or indirectly, in heavy metal binding and removal from potentially sensitive sites, by sequestration or efflux (Clemens, 2006). The best-characterized heavy metal binding ligands in plants are thiol-containing compounds metallothioneins and phytochelatins (PCs), whose production is stimulated by Cd²⁺. PCs bind metal ions and transport them to the vacuole, thus reducing the toxicity of the metal in the cytosol (for review, see Cobbett, 2000; Cobbett and Goldsbrough, 2002). PCs are synthesized from reduced glutathione (GSH). Therefore, production of compounds involved in cadmium detoxification and, at the same time, in cadmium tolerance closely depends on sulfur metabolism. So far, our knowledge on the cellular processes induced by cadmium that lead to changes in sulfur metabolism in plants has been rather limited.Protein kinases and phosphatases are considered major signal transduction elements. However, until now only a few of them have been described to be involved in cadmium stress response or sulfur metabolism. For instance, excessive amounts of cadmium or copper activate mitogen-activated protein kinases (MAPKs) in Medicago sativa (Jonak et al., 2004), rice (Oryza sativa; Yeh et al., 2007), and Arabidopsis (Arabidopsis thaliana; Liu et al., 2010). Studies on rice MAPKs involved in heavy metal stress response indicate that the activity of these kinases depends on the oxidative stress induced by Cd²⁺. Moreover, Yeh et al. (2007) suggested that the activation of MAPKs in rice by cadmium or copper required the activity of calcium-dependent protein kinase (CDPK) and PI3 kinase, since the MAPK pathways involved in cadmium and copper stress response could be inhibited by a CDPK antagonist (W7) or a PI3 kinase inhibitor (wortmannin). However, so far the function of the identified kinases in plant adaptation to heavy metal pollution has not been established. There is some information concerning an involvement of CDPK in sulfur metabolism (Liu et al., 2006). Soybean (Glycine max) Ser acetyltransferase (GmSerat2;1), the enzyme that catalyzes the first reaction in the biosynthesis of Cys from Ser, is phosphorylated by CDPK. The phosphorylation has no effect on GmSerat2;1 activity, but it renders the enzyme insensitive to the feedback inhibition by Cys (Liu et al., 2006). There is growing evidence that SnRK2s (for SNF1-related protein kinase2) play a role in the regulation of sulfur metabolism. Most information showing a connection between SnRK2s and sulfur metabolism comes from experiments on the lower plant Chlamydomonas reinhardtii (Davies et al., 1999; Irihimovitch and Stern, 2006; González-Ballester et al., 2008, 2010). SNRK2.1 is considered a general regulator of S-responsive gene expression in C. reinhardtii (González-Ballester et al., 2008).In higher plants the SnRK2 family members are known to be involved in plant response to drought, salinity, and in abscisic acid (ABA)-dependent plant development (Boudsocq and Laurière, 2005; Fujii et al., 2007, 2011; Fujii and Zhu, 2009; Fujita et al., 2009; Nakashima et al., 2009; Kulik et al., 2011). Ten members of the SnRK2 family have been identified in Arabidopsis and in rice (Boudsocq et al., 2004; Kobayashi et al., 2004). All of them, except SnRK2.9 from Arabidopsis, are rapidly activated by treatment with different osmolytes, such as Suc, mannitol, sorbitol, and NaCl, and some of them also by ABA. Results presented by Kimura et al. (2006) suggest that in Arabidopsis, similarly to C. reinhardtii, some SnRK2s are involved in the regulation of S-responsive gene expression and O-acetyl-l-Ser accumulation under limited sulfur supply, indicating that also higher plants’ SnRK2s could be involved in sulfur metabolism.As it was mentioned before, oxidative stress induced by cadmium ions significantly contributes to the metal toxicity. Reactive oxygen species (ROS) can be produced in many different reactions in various compartments of the cell in response to cadmium (Romero-Puertas et al., 2004; Heyno et al., 2008; Tamás et al., 2009). The best-characterized ROS-generating enzymes that take part in the response to cadmium are the plasma-membrane-bound NADPH oxidases (Olmos et al., 2003; Romero-Puertas et al., 2004; Garnier et al., 2006). There are some indications that plant NADPH oxidases are phosphorylated by SnRK2s (Sirichandra et al., 2009), therefore it is highly plausible that SnRK2s play a role in the regulation of ROS accumulation in plants subjected to cadmium stress. Taking into consideration all facts mentioned above we hypothesized that SnRK2s could be involved in the plant response to stress induced by cadmium ions. To verify this conjecture, we analyzed the activity and potential role of selected SnRK2s, in tobacco (Nicotiana tabacum) cells and Arabidopsis plants, in the response to cadmium ions.     

Synthesis and cytotoxic activity of novel acyclic nucleoside analogues with functionality in click chemistry

We describe synthesis of novel acyclic nucleoside analogues which are building blocks for CuAAC reaction and their activity against two types of human cancer cell lines (HeLa, KB). Three of chosen compounds show promising cytotoxic activity. Synthesis pathway starting from simple and easily accessible substrates employing DMT or TBDPS protective groups is described. Adenosine and thymidine analogues containing alkyne moiety and adenosine analogue containing azido group were synthesized. The obtained units showed ability of forming triazole motif under the CuAAC reaction conditions.     

On methylene‐bridged cysteine and lysine residues in proteins

Cysteine residues ubiquitously stabilize tertiary and quaternary protein structure by formation of disulfide bridges. Here we investigate another linking interaction that involves sulfhydryl groups of cysteines, namely intra‐ and intermolecular methylene‐bridges between cysteine and lysine residues. A number of crystal structures possessing such a linkage were identified in the Protein Data Bank. Inspection of the electron density maps and re‐refinement of the nominated structures unequivocally confirmed the presence of Lys‐CH₂‐Cys bonds in several cases.     

Immobilization of Zidovudine Derivatives on the SBA-15 Mesoporous Silica and Evaluation of Their Cytotoxic Activity

Novel zidovudine derivatives, able to be covalently conjugated to silica surface, have been obtained and grafted to SBA-15 mesoporous silica. Cytotoxic activity of the hybrid organic-inorganic (zidovudine derivatives-silica) systems against HeLa and KB cell lines has been analyzed. Addition of folic acid had a positive influence on the cytotoxicity. Up to 69% of HeLa and 65% of KB tumor cells growth inhibition has been achieved at low silica concentration used (10 μg/mL).