ATP-dependent remodeling of the spliceosome: intragenic suppressors of release-defective mutants of Saccharomyces cerevisiae Prp22

The essential splicing factor Prp22 is a DEAH-box helicase that catalyzes the release of mRNA from the spliceosome. ATP hydrolysis by Prp22 is necessary but not sufficient for spliceosome disassembly. Previous work showed that mutations in motif III (635SAT637) of Prp22 that uncouple ATP hydrolysis from spliceosome disassembly lead to severe cold-sensitive (cs) growth defects and to impaired RNA unwinding activity in vitro. The cs phenotype of S635A (635AAT) can be suppressed by intragenic mutations that restore RNA unwinding. We now report the isolation and characterization of new intragenic mutations that suppress the cold-sensitive growth phenotypes of the T637A motif III mutation (SAA), the H606A mutation in the DEAH-box (DEAA), and the R805A mutation in motif VI (804QAKGRAGR811). Whereas the T637A and H606A proteins are deficient in releasing mRNA from the spliceosome at nonpermissive temperature in vitro, the suppressor proteins have recovered mRNA release activity. To address the mechanisms of suppression, we tested ATPase and helicase activities of Prp22 suppressor mutant proteins and found that the ability to unwind a 25-bp RNA duplex was not restored in every case. This finding suggests that release of mRNA from the spliceosome is less demanding than unwinding of a 25-bp duplex RNA; the latter reaction presumably reflects the result of several successive cycles of ATP binding, hydrolysis, and unwinding. Increasing the reaction temperature allows H606A and T637A to effect mRNA release in vitro, but does not restore RNA unwinding by T637A.     

Crystal structure of the tetrameric cytidine deaminase from Bacillus subtilis at 2.0 A resolution

Cytidine deaminases (CDA, EC 3.5.4.5) are zinc-containing enzymes in the pyrimidine salvage pathway that catalyze the formation of uridine and deoxyuridine from cytidine and deoxycytidine, respectively. Two different classes have been identified in the CDA family, a homodimeric form (D-CDA) with two zinc ions per dimer and a homotetrameric form (T-CDA) with four zinc ions per tetramer. We have determined the first structure of a T-CDA from Bacillus subtilis. The active form of T-CDA is assembled of four identical subunits with one active site apiece. The subunit of D-CDA is composed of two domains each exhibiting the same fold as the T-CDA subunits, but only one of them contains zinc in the active site. The similarity results in a conserved structural core in the two CDA forms. An intriguing difference between the two CDA structures is the zinc coordinating residues found at the N-terminal of two alpha-helices: three cysteine residues in the tetrameric form and two cysteine residues and one histidine residue in the dimeric form. The role of the zinc ion is to activate a water molecule and thereby generate a hydroxide ion. How the zinc ion in T-CDA surrounded with three negatively charged residues can create a similar activity of T-CDA compared to D-CDA has been an enigma. However, the structure of T-CDA reveals that the negative charge caused by the three ligands is partly neutralized by (1) an arginine residue hydrogen-bonded to two of the cysteine residues and (2) the dipoles of two alpha-helices.     

Impaired Functionality of Antiviral T Cells in G-CSF Mobilized Stem Cell Donors: Implications for the Selection of CTL Donor

Adoptive transfer of antiviral T cells enhances immune reconstitution and decreases infectious complications after stem cell transplantation. Information on number and function of antiviral T cells in stem cell grafts is scarce. We investigated (1) immunomodulatory effects of G-CSF on antiviral T cells, (2) the influence of apheresis, and (3) the optimal time point to collect antiviral cells.CMV-, EBV- and ADV-specific T cells were enumerated in 170 G-CSF-mobilized stem cell and 24 non-mobilized platelet donors using 14 HLA-matched multimers. T-cell function was evaluated by IFN-γ ELISpot and granzyme B secretion. Immunophenotyping was performed by multicolor flow cytometry.G-CSF treatment did not significantly influence frequency of antiviral T cells nor their in vitro expansion rate upon antigen restimulation. However, T-cell function was significantly impaired, as expressed by a mean reduction in secretion of IFN-γ (75% in vivo, 40% in vitro) and granzyme B (32% target-independent, 76% target-dependent) as well as CD107a expression (27%). Clinical follow up data indicate that the first CMV-reactivation in patients and with it the need for T-cell transfer occurs while the donor is still under the influence of G-CSF.To overcome these limitations, T-cell banking before mobilization or recruitment of third party donors might be an option to optimize T-cell production.     

Defense Responses in Two Ecotypes of Lotus japonicus against Non-Pathogenic Pseudomonas syringae

Lotus japonicus is a model legume broadly used to study many important processes as nitrogen fixing nodule formation and adaptation to salt stress. However, no studies on the defense responses occurring in this species against invading microorganisms have been carried out at the present. Understanding how this model plant protects itself against pathogens will certainly help to develop more tolerant cultivars in economically important Lotus species as well as in other legumes. In order to uncover the most important defense mechanisms activated upon bacterial attack, we explored in this work the main responses occurring in the phenotypically contrasting ecotypes MG-20 and Gifu B-129 of L. japonicus after inoculation with Pseudomonas syringae DC3000 pv. tomato. Our analysis demonstrated that this bacterial strain is unable to cause disease in these accessions, even though the defense mechanisms triggered in these ecotypes might differ. Thus, disease tolerance in MG-20 was characterized by bacterial multiplication, chlorosis and desiccation at the infiltrated tissues. In turn, Gifu B-129 plants did not show any symptom at all and were completely successful in restricting bacterial growth. We performed a microarray based analysis of these responses and determined the regulation of several genes that could play important roles in plant defense. Interestingly, we were also able to identify a set of defense genes with a relative high expression in Gifu B-129 plants under non-stress conditions, what could explain its higher tolerance. The participation of these genes in plant defense is discussed. Our results position the L. japonicus-P. syringae interaction as a interesting model to study defense mechanisms in legume species.     

Immunization of Chlamydia pneumoniae (Cpn)-Infected Apobtm2SgyLdlrtm1Her/J Mice with a Combined Peptide of Cpn Significantly Reduces Atherosclerotic Lesion Development

Objective

To investigate the antigenic effect of a peptide containing two epitopes of Chlamydia pneumoniae (Cpn) on atherosclerotic lesion formation in mice infected with Cpn.

Materials and Methods

Six-week-old Apob^tm2SgyLdlr^tm1Her/J mice were immunized using a repetitive immunization multiple-sites strategy with KLH-conjugated peptides derived from the major outer membrane protein and the putative outer membrane protein 5 of Cpn. Mice were fed a high-fat diet and infected with Cpn twice during the 10-week diet period. Lesions were evaluated histologically; local and systemic immune responses were analyzed by immunohistochemistry of aorta samples and cytokine measurements in plasma samples and splenocyte supernatants.

Results

Mice immunized with the combined Cpn peptide showed a greater reduction in lesion size compared to mice immunized with either epitope alone [54.7% vs 39.8% or 41.72%] and was also associated with a significant decrease in lesion area in descending aortas compared with those in controls (88.9% for combined Cpn peptide, 81.9% for MOMP peptide and 75.7% for Omp5, respectively). This effect was associated with a shift in the cellular composition of plaques towards decreased inflammatory cell and increased regulatory T-cell content. Additionally, the effect was also connected with decreased secretion of proinflammatory cytokines and increased production of anti-inflammatory cytokines demonstrated in plasma and in supernatant on stimulated spleen cells.

Conclusions

Atherosclerotic lesion formation may be promoted by Cpn infection in the presence of a high-fat diet, and reduced by immunization with the combined Cpn peptide. The combined peptide has more potential than either epitope alone in reducing atherosclerotic lesion development through Treg expansion.     

Skin disease in a geriatric patients group in outpatient dermatologic clinic Karlovac, Croatia

To determine the characteristic pattern and frequency of dermatoses in dermatologic patients over 65 years we used electronic data base of General hospital Karlovac and conducted a retrospective cross sectional study evaluating age, sex and proportion of dermatoses. Total number of patients was 3200. There were 822 (25.69%) patients older than 65 years, and 2378 (74.31%) patients aged from 18-64 years. Ratio male/female in population over 65 years in our study was 0.76:1. Males had higher frequncy of verrucae vulgares 26 (7.3%) and dermatitis nummularis 47 (13.2%) then female and that difference was statistically significant. Female had higher frequency of keratosis acitinca 124 (26.61%) and fibroma 23 (4.94%), and that difference was statistically significant. The ten most common diagnosis in population over 65 years old were: keratosis actinica in 184 patients (22.38%) verrucae seborrhoicae in 156 (18.98%), dermatitits nummularis in 77 patients (9.37%), dermatitis allergica e contactu in 60 (7.30%) patients, mycosis in 56 (6.81%) patients, psoriasis in 51 (6.20%), verrucae vulgares in 39 (4.74%), fibromas in 27 (3.28%), naevi in 9 (1.09%) and acne in 1 (0.12%) patient. The number of patients with diagnosis of keratosis actinica, verrucae seborrhoicae and mycoses in population over 65 years old are greater then in younger subpopulation and that difference was statistically significant. There are specific pattern of frequency of dermatoses in elderly. Verrucae seborrhoica, keratosis actinica and mycoses are more common then in general population. In elderly risk for development of skin cancer is increased. Early detection of skin cancers and treatment of precanceroses is of utmost interest of health providers.     

Genotype-Dependent Effect of Exogenous Nitric Oxide on Cd-induced Changes in Antioxidative Metabolism, Ultrastructure, and Photosynthetic Performance in Barley Seedlings (Hordeum vulgare)

A greenhouse hydroponic experiment was performed using Cd-sensitive (cv. Dong 17) and Cd-tolerant (Weisuobuzhi) barley seedlings to evaluate how different genotypes responded to cadmium (Cd) toxicity in the presence of sodium nitroprusside (SNP), a nitric oxide (NO) donor. Results showed that 5 μM Cd increased the accumulation of O₂^•−, H₂O₂, and malondialdehyde (MDA) but reduced plant height, chlorophyll content, net photosynthetic rate (P _n), and biomass, with a much more severe response in the Cd-sensitive genotype. Antioxidant enzyme activities increased significantly under Cd stress in the roots of the tolerant genotype, whereas in leaves of the sensitive genotype, superoxide dismutase (SOD) and ascorbate peroxide (APX), especially cytosol ascorbate peroxidase (cAPX), decreased after 5–15 days Cd exposure. Moreover, Cd induces NO synthesis by stimulating nitrate reductase and nitric oxide synthetase-like enzymes in roots/leaves. A Cd-induced NO transient increase in roots of the Cd-tolerant genotype might partly contribute to its Cd tolerance. Exogenous NO dramatically alleviated Cd toxicity, markedly diminished Cd-induced reactive oxygen species (ROS) and MDA accumulation, ameliorated Cd-induced damage to leaf/root ultrastructure, and increased chlorophyll content and P _n. External NO counteracted the pattern of alterations in certain antioxidant enzymes induced by Cd; for example, it significantly elevated the depressed SOD, APX, and catalase (CAT) activities in the Cd-sensitive genotype after 10- and 15-day treatments. Furthermore, NO significantly increased stromal APX and Mn-SOD activities in both genotypes and upregulated Cd-induced decrease in cAPX activity and gene expression of root/leaf cAPX and leaf CAT1 in the Cd-sensitive genotype. These data suggest that under Cd stress, NO, as a potent antioxidant, protects barley seedlings against oxidative damage by directly and indirectly scavenging ROS and helps to maintain stability and integrity of the subcellular structure.     

Evaluation of a flat membrane hepatocyte bioreactor for pharmacotoxicological applications: evidence that inhibition of spontaneously produced nitric oxide improves cell functionality

A laboratory-scale bioreactor was re-evaluated, with the aim of improving its use for the perfused culture of rat hepatocytes. In contrast to conventional culture systems, the flat membrane bioreactor (FMB) showed good functionality and biochemical competence during 2-3 days. Hepatocytes cultured in the FMB, specifically in a "sandwich" configuration, were functionally stable, as shown by a high rate of urea biosynthesis after challenge with NH4Cl, a low alanine-aminotransferase leakage and suppressed spontaneous nitric oxide (NO) production. Moreover, the time-course of the disappearance of cyclosporin A (CsA) from the perfusate demonstrated the high biotransformation capacity of cells in the FMB. The effect of CsA on the modulation of urea and spontaneous NO production demonstrated flexibility, in that minor changes could be observed at diverse time intervals and in a non-destructive way. The monitoring of nitrite levels during various steps of isolation and culture suggested that spontaneously produced NO has a negative impact on hepatocyte metabolic and functional integrity. In spite of the sophisticated techniques that are being used for the preparation of bioreactors, with hepatocytes surviving for longer periods, our data have shed light on some factors that could be important for the successful use of similar models for pharmacotoxicological and other biomedical applications.