Protein kinase C recognizes the protein kinase A-binding motif of nonstructural protein 3 of hepatitis C virus.

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) inhibits the nuclear transport and the enzymatic activity of the catalytic subunit of protein kinase A. This inhibition is mediated by an arginine-rich domain localized between amino acids 1487-1500 of the HCV polyprotein. The data presented here indicate that the arginine-rich domain, when embedded in recombinant fragments of NS3, interacts with the catalytic site of protein kinase C (PKC) and inhibits the phosphorylation mediated by this enzyme in vitro and in vivo. Furthermore, a direct binding of PKC to the NS3 fragments leads to an inhibition of the free shuttling of the kinase between the cytoplasm and the particulate fraction. In contrast, a peptide corresponding to the arginine-rich domain (HCV (1487-1500)), despite also being a PKC inhibitor, did not influence the PKC shuttling process and was transported to the particulate fraction by the translocating kinase upon activation with tetradecanoylphorbol-13-acetate. Using the tetradecanoylphorbol-13-acetate -stimulated respiratory burst of NS3-introduced neutrophils as a model system, we could demonstrate that NS3 is able to block PKC-mediated functions within intact cells. Our data support the possibility that NS3 disrupts the PKC-mediated signal transduction.     

Signal transduction around thymic stromal lymphopoietin (TSLP) in atopic asthma

T cells play a central role in many inflammatory diseases, hence the identification and validation of T cell-specific target genes will increase the understanding of T cell function in pathologic inflammatory situations. RNA interference (RNAi), with its ability to induce specific gene silencing in mammalian cells, represents a powerful technology to investigate and validate the function of pharmaceutical target genes in vitro and in vivo. The aim of the present study was to systematically explore RNAi-mediated gene-silencing of known T cell-specific model signaling molecules in primary murine T cells in vitro and in vivo. We demonstrate that siRNA delivery and subsequent silencing of T cell specific genes is substantially increased, if murine T cells were activated prior siRNA transfection. Silencing of ZAP70, p56Lck as well as PLC-γ1 protein expression resulted in impaired function of T cells in vitro. Furthermore, delayed type hypersensitivity (DTH) was ameliorated in vivo after adoptive transfer of ZAP70-silenced T cells. The combination of RNAi-mediated gene silencing and adoptive transfer of gene-silenced T cells, thus, may allow the identification and analysis of T cell-specific targets for therapeutic intervention. Additionally, this model system may represent an alternative to conventional time consuming and cost intensive gene targeting approaches.     

Copper deficiency inhibits Ca2+-induced swelling in rat cardiac mitochondria

Cu deficiency disrupts the architecture of mitochondria, impairs respiration, and inhibits the activity of cytochrome c oxidase - the terminal, Cu-dependent respiratory complex (Complex IV) of the electron transport chain. This suggests that perturbations in the respiratory chain may contribute to the changes in mitochondrial structure caused by Cu deficiency. This study investigates the effect of Cu deficiency on Ca2+-induced mitochondrial swelling as it relates to changes in respiratory complex activities in cardiac mitochondria of rats. Male weanling rats were fed diets containing either no added Cu (Cu0), 1.5 mg Cu/kg (Cu1.5), 3 mg Cu/kg (Cu3) or 6 mg Cu/kg (Cu6). The rate of Ca2+-induced mitochondrial swelling in the presence of succinate and oligomycin was reduced, and the time to reach maximal swelling was increased only in the rats consuming Cu0 diet. Cytochrome c oxidase activity was reduced 60% and 30% in rats fed Cu0 and Cu1.5, respectively, while NADH:cytochrome c reductase (Complex I+ComplexIII) activity was reduced 30% in rats consuming both Cu0 and Cu1.5. Mitochondrial swelling is representative of mitochondrial permeability transition pore (MPTP) formation and the results suggest that Ca2+-induced MPTP formation occurs in cardiac mitochondria of Cu-deficient rats only when cytochrome c oxidase activity falls below 30% of normal. Decreased respiratory complex activities caused by severe Cu deficiency may inhibit MPTP formation by increasing matrix ADP concentration or promoting oxidative modifications that reduce the sensitivity of the calcium trigger for MPTP formation.     

Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses

The process of selection and validation of reference genes is the first step in studies of gene expression by real-time quantitative polymerase chain reaction (RT-qPCR). The genome of lettuce, the most popular leaf vegetable cultivated worldwide, has recently been sequenced; therefore, suitable reference genes for reliable results in RT-qPCR analyses are required. In the present study, 17 candidate reference genes were selected, and their expression stability in lettuce leaves under drought, salt, heavy metal, and UV-C irradiation conditions and under the application of abscisic acid (ABA) was evaluated using geNorm and NormFinder software. The candidate reference genes included protein-coding traditional and novel reference genes and microRNAs (miRNAs). The results indicate that the expression stability is dependent on the experimental conditions. The novel protein-coding reference genes were more suitable than the traditional reference genes under drought, UV-C irradiation, and heavy metal conditions and under the application of ABA. Only under salinity conditions were the traditional protein-coding reference genes more stable than the novel genes. In addition, the miRNAs, mainly MIR169, MIR171/170 and MIR172, were stably expressed under the abiotic stresses evaluated, representing a suitable alternative approach for gene expression data normalization. The expression of phenylalanine ammonia lyase (PAL) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) was used to further confirm the validated protein-coding reference genes, and the expression of MIR172 and MIR398 was used to confirm the validated miRNA genes, showing that the use of an inappropriate reference gene induces erroneous results. This work is the first survey of the stability of reference genes in lettuce and provides guidelines to obtain more accurate RT-qPCR results in lettuce studies.     

Baseline Natural Killer and T Cell Populations Correlation with Virologic Outcome after Regimen Simplification to Atazanavir/Ritonavir Alone (ACTG 5201)

Objectives

Simplified maintenance therapy with ritonavir-boosted atazanavir (ATV/r) provides an alternative treatment option for HIV-1 infection that spares nucleoside analogs (NRTI) for future use and decreased toxicity. We hypothesized that the level of immune activation (IA) and recovery of lymphocyte populations could influence virologic outcomes after regimen simplification.

Methods

Thirty-four participants with virologic suppression ≥48 weeks on antiretroviral therapy (2 NRTI plus protease inhibitor) were switched to ATV/r alone in the context of the ACTG 5201 clinical trial. Flow cytometric analyses were performed on PBMC isolated from 25 patients with available samples, of which 24 had lymphocyte recovery sufficient for this study. Assessments included enumeration of T-cells (CD4/CD8), natural killer (NK) (CD3⁺CD56⁺CD16⁺) cells and cell-associated markers (HLA-DR, CD''s 38/69/94/95/158/279).

Results

Eight of the 24 patients had at least one plasma HIV-1 RNA level (VL) >50 copies/mL during the study. NK cell levels below the group median of 7.1% at study entry were associated with development of VL >50 copies/mL following simplification by regression and survival analyses (p = 0.043 and 0.023), with an odds ratio of 10.3 (95% CI: 1.92–55.3). Simplification was associated with transient increases in naïve and CD25⁺ CD4⁺ T-cells, and had no impact on IA levels.

Conclusions

Lower NK cell levels prior to regimen simplification were predictive of virologic rebound after discontinuation of nucleoside analogs. Regimen simplification did not have a sustained impact on markers of IA or T lymphocyte populations in 48 weeks of clinical monitoring.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00084019","term_id":"NCT00084019"}}NCT00084019     

Tn7-encoded proteins

Summary Proteins encoded by Tn7 have been studied in Escherichia coli maxicells harbouring either various deleted ColE1:: Tn7 plasmids or Tn7 fragments cloned in pBR322. Six Tn7-encoded proteins were detected and named p18, p32, p40, p54, p85-a and p85-b according to their apparent molecular weight. Protein p18 is dihydrofolate reductase type I and p32 is probably the protein conferring resistance to streptomycin/spectinomycin. Both genes map on the lefthand part of Tn7. The genes for the four other proteins are located on the right-hand part of Tn7. We propose that they fully cover a 6.9 kb DNA fragment without any overlapping. Starting from the right-hand end towards the middle of the transposon, these four genes are in the following order: p85-a, p54, p40 and p85-b. Transposition of Tn7 onto E. coli plasmids requires the proteins p85-a, p85-b, p54 and p40. However, transposition onto the chromosome does not require the p85-b and p40 products.     

Nonstructural protein 3 of hepatitis C virus blocks the distribution of the free catalytic subunit of cyclic AMP-dependent protein kinase.

Chronic hepatitis resulting from hepatitis C virus (HCV) infection develops into cirrhosis in at least half of infected patients and increases the risk of hepatocellular carcinoma. The pathogenic effects of a number of viruses result from the disturbance of intracellular signal cascades caused by viral antigens. Therefore, we investigated the interaction of nonstructural protein 3 (NS3) of HCV with the cyclic AMP-dependent signal pathway. We found a similarity between the HCV sequence Arg-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg localized in NS3 and the general consensus sequence of protein kinase A (PKA). Consequently, the catalytic (C) subunit of PKA bound to a bacterially expressed fragment of HCV polyprotein containing amino acid residues 1189 to 1525. When this fragment was introduced into cells, it inhibited the translocation of the C subunit into the nucleus after stimulation with forskolin. The result of this inhibition was significantly reduced histone phosphorylation. Therefore, the presence of NS3 in the cytoplasm of infected cells may affect a wide range of PKA functions and contribute to the pathogenesis of the diseases caused by HCV.     

Conformational analysis of Amphotericin B

Within a theoretical approach to the problem of antifungal action of Amphotericin B (AmB), a conformational analysis of the neutral and zwitterionic form of this antibiotic in vacuo was performed by the MM2P and AM1 methods. The analysis was carried out with regard to the mutual orientation of the macrolidic and glycosidic fragments of the molecule, which is defined by the phi and psi steric angles. This orientation defines the overall shape of the molecule and is postulated to be important for the antifungal action of the drug. As a result of the MM2P calculations, phi, psi steric energy and population maps were prepared. Several conformers were found on these maps but only two of them (one each for the zwitterionic and the neutral forms of the antibiotic) were previously observed experimentally for isolated molecules. Our other calculated conformers were not observed experimentally but we propose that they may also appear in the AmB channel structure. The results of our conformational analysis were compared with experimental NMR data (nuclear Overhauser effects between selected hydrogen atoms) obtained previously. New structural information obtained for AmB in the present work will be useful for building a molecular model of AmB-target interactions as well as for designing new derivatives of AmB.     

Usefulness of the diagnostic kit Enteroplast for identification of Enterobacteriaceae

Comparative evaluation of biochemical properties determined by ENTEROPLAST kit (P.P.Z. Plastomed) and biochemical set of tests (conventional method) of 140 strains representing 12 genera of Enterobacteriaceae family was undertaken. Consistent results positive or negative (in 12 biochemical tests) were obtained in 93%. The highest percentage of inconsistent results of biochemical reactions (positive in conventional method and negative in ENTEROPLAST kit) were observed in following tests: growth on Simmons medium--17.8%, MR--7.8%, fermentation of raffinose--7.2%. Significantly lower percentage of inconsistent results was found in the case of false positive reactions (negative in conventional method and positive in ENTEROPLAST kit). In summary, it seems that Enteroplast kit can be used for routine diagnostic examinations for identification of rods of Enterobacteriaceae family, basing on their biochemical properties.     

Isolation and identification of the products of Streptococcus faecalis inhibiting mycelial transformation of Candida albicans

An attempt was made to isolate and identify Streptococcus faecalis products responsible for the inhibition of mycelial transformation of Candida albicans. Five of streptococcal strains which 48 h broth culture supernatants run at 37 degrees C inhibited the most transformation of Candida albicans from yeast phase to mycelial phage. The strains were cultivated for 48 h in Tryptic Soy Broth at 37 degrees C, centrifuged and culture supernatants sterilised by means of filtration on millipore membranes of 0.4 micron diameter. After multistep purification of supernatants filtration on Diaflo PM 10 ultrafiltration membranes, Sephadex G 25, polyacrylamide gel electrophoresis) a homogenous, active fraction was obtained containing peptides of molecular weight around 6,000 Da. The peptides lost ability to induce mycelial transformation of C. albicans after heating at 100 degrees C for 10 min. Significant inhibition of morphological transformation of fungal cells was seen at the preparation concentration of 0.12 microgram/ml.