The RuvABC resolvase is indispensable for recombinational repair in sbcB15 mutants of Escherichia coli

The RuvABC proteins of Escherichia coli play an important role in the processing of Holliday junctions during homologous recombination and recombinational repair. Mutations in the ruv genes have a moderate effect on recombination and repair in wild-type strains but confer pronounced recombination deficiency and extreme sensitivity to DNA-damaging agents in a recBC sbcBC background. Genetic analysis presented in this work revealed that the (Delta)ruvABC mutation causes an identical DNA repair defect in UV-irradiated recBC sbcBC, sbcBC, and sbcB strains, indicating that the sbcB mutation alone is responsible for the extreme UV sensitivity of recBC sbcBC ruv derivatives. In experiments with gamma irradiation and in conjugational crosses, however, sbcBC (Delta)ruvABC and sbcB (Delta)ruvABC mutants displayed higher recombination proficiency than the recBC sbcBC (Delta)ruvABC strain. The frequency of conjugational recombination observed with the sbcB (Delta)ruvABC strain was quite similar to that of the (Delta)ruvABC single mutant, indicating that the sbcB mutation does not increase the requirement for RuvABC in a recombinational process starting from preexisting DNA ends. The differences between the results obtained in three experimental systems used suggest that in UV-irradiated cells, the RuvABC complex might act in an early stage of recombinational repair. The results of this work are discussed in the context of recent recombination models which propose the participation of RuvABC proteins in the processing of Holliday junctions made from stalled replication forks. We suggest that the mutant SbcB protein stabilizes these junctions and makes their processing highly dependent on RuvABC resolvase.     

Editing autoreactive TCR enables efficient positive selection

Allelic exclusion is inefficient at the TCRalpha locus, allowing a sizeable portion of T cells to carry two functional TCRs. The potential danger of dual TCR expression is a rescue of autoreactive TCRs during selection in the thymus and subsequent development of autoimmunity. In this study, we examine the reason(s) for replacing an autoreactive TCR and for allowing the survival of cells carrying two TCRs. We compared development of TCR transgenic CD4(+)CD8(-) thymocytes in the presence or absence of MHC class II autoantigen that does not induce deletion of thymocytes. Contrary to the expected negative effect of the presence of autoantigen, approximately 100% more CD4(+)CD8(-) thymocytes were found in the presence of MHC class II autoantigen than in the neutral background. A further increase in the strength of autoantigenic signal via expression of a human CD4 transgene led to an additional increase in the numbers of CD4(+)CD8(-) thymocytes. Thus, editing autoreactive TCR results in more efficient positive selection, and this may be both a reason and a reward for risking autoimmunity.     

Smoking habits, signs of chronic diseases and survival in inland and coastal regions of Croatia: a follow-up study

Aim of the study was to estimate, the relationship between survival, smoking habits, and the results of medical examinations in inland and coastal regions of Croatia. Age and sex stratified sample of general population (1,571 men and 1,793 women, 37-56 years old in 1972; followed in 1982: N = 1,090 men; 1,325 women and/or 1972-1999 controlling vital status). Relative risks and 95% confidence limits were estimated using Cox regression in the model with time dependent covariates, separately by sex. In all regions, the proportion of male smokers decreased between 1972 and 1982. The proportion of female smokers increased, differently in urban and rural regions. During the follow-up between 1972 and 1999, 568 deaths were recorded among men and 382 among women. In men, in addition to age, significant hazards of death were the number of smoked cigarettes per day, body mass index, sedative intake, vital lung capacity (FVC), 100FEV1/VC, systolic blood pressure, electrocardiogram, history of heart attack, and region. In women, in addition to age, significant predictors were the number of smoked cigarettes per day, systolic blood pressure, electrocardiogram, history of heart disease (excluding coronary diseases), and region. Survival relative risk increased with each additional smoked pack of cigarettes by 2.4% in women and 1.3% in men. Regional differences vs. smoking habit were observed. These data emphasize the need for prevention of smoking.     

Renal vascular resistance in glomerular diseases--correlation of resistance index with biopsy findings

Duplex Doppler sonography has been recognized as a noninvasive method to evaluate hemodynamic features of renal blood in renal and intrarenal arteries in patients with various renal diseases. The significance of duplex Doppler sonography in the evaluation of renal vascular resistance in glomerular diseases has not yet been clearly determined. The aim of the present study was to evaluate renal vascular resistance in patients with glomerular diseases by measuring intrarenal arterial resistance (RI) and to correlate RI with renal functional tests and other clinical and laboratory data. The Doppler parameters were also correlated with histopathological findings in the kidney which underwent the percutaneous biopsy. Duplex Doppler sonography was used to measure RIs in intrarenal arteries in 50 patients with glomerular diseases and 60 age-matched control subjects. The renal vascular resistance index (RI) was determined by the use of Doppler sonography. The mean RI in 50 patients with glomerular diseases was 0.68 +/- 0.09, which was statistically significantly higher than in 60 control subjects (the mean RI was 0.596 +/- 0.035). In a group of patients with membranoproliferative glomerulonephritis the mean RI was 0.817 +/- 0.624 which was statistically significantly higher than in other groups of glomerulonephritis. The renal vascular (resistance) RI significantly correlated with serum creatinine, creatinine clearance and beta2 microglobulin. Qualitative duplex sonography measure of renal arterial resistance-resistive index does not appear to be reliable in distinguishing different types of glomerulonephritis.     

An Investigation into the Influence of Experimental Conditions on <Emphasis Type="Italic">In Vitro</Emphasis> Drug Release from Immediate-Release Tablets of Levothyroxine Sodium and Its Relation to Oral Bioavailability

The aim of this study was to investigate the influence of experimental conditions on levothyroxine sodium release from two immediate-release tablet formulations which narrowly passed the standard requirements for bioequivalence studies. The in vivo study was conducted as randomised, single-dose, two-way cross-over pharmacokinetic study in 24 healthy subjects. The in vitro study was performed using various dissolution media, and obtained dissolution profiles were compared using the similarity factor value. Drug solubility in different media was also determined. The in vivo results showed narrowly passing bioequivalence. Considering that levothyroxine sodium is classified as Class III drug according to the Biopharmaceutics Classification System, drug bioavailability will be less sensitive to the variation in its dissolution characteristics and it can be assumed that the differences observed in vitro in some of investigated media probably do not have significant influence on the absorption process, as long as rapid and complete dissolution exists. The study results indicate that the current regulatory criteria for the value of similarity factor in comparative dissolution testing, as well as request for very rapid dissolution (more than 85% of drug dissolved in 15 min), are very restricted for immediate-release dosage forms containing highly soluble drug substance and need further investigation. The obtained results also add to the existing debate on the appropriateness of the current bioequivalence standards for levothyroxine sodium products.     

Disease intensity of some tomato viroses in Serbia

In this paper we present the data on the disease intensity of the tomato plants grown in glass and plastic-houses, and in the open field. The infection was caused by the following viruses: Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Alfalfa mosaic virus (AMV), Potato virus X (PVX), Potato virus Y (PVY), Tomato black ring virus (TBRV), Tomato ringspot virus (ToRSV), Tomato aspermy virus (TAV), and Cucumber mosaic virus (CMV). These viruses represented most frequent tomato pathogens in Serbia. According to the obtained results, it could be concluded that 92.94% of the tested tomato plants grown in glass and plastic-houses, and 89.82% grown in the open field were infected by one of the above viruses. Most of the plant samples were infected by two or more viruses. The most frequent viruses — tomato pathogens in Serbia were ToMV, PVY and TMV.     

Sex Bias in Pathogenesis of Autoimmune Neuroinflammation: Relevance for Dimethyl Fumarate Immunomodulatory/Anti-oxidant Action

In the present study, upon showing sexual dimorphism in dimethyl fumarate (DMF) efficacy to moderate the clinical severity of experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats, cellular and molecular substrate of this dimorphism was explored. In rats of both sexes, DMF administration from the day of immunization attenuated EAE severity, but this effect was more prominent in males leading to loss of the sexual dimorphism observed in vehicle-administered controls. Consistently, in male rats, DMF was more efficient in diminishing the number of CD4+ T lymphocytes infiltrating spinal cord (SC) and their reactivation, the number of IL-17+ T lymphocytes and particularly cellularity of their highly pathogenic IFN-γ+GM-CSF+IL-17+ subset. This was linked with changes in SC CD11b+CD45+TCRαβ? microglia/proinflammatory monocyte progeny, substantiated in a more prominent increase in the frequency of anti-inflammatory phygocyting CD163+ cells and the cells expressing high surface levels of immunoregulatory CD83 molecule (associated with apoptotic cells phagocytosis and implicated in downregulation of CD4+ T lymphocyte reactivation) among CD11b+CD45+TCRαβ– cells in male rat SC. These changes were associated with greater increase in the nuclear factor (erythroid-derived 2)-like 2 expression in male rats administered with DMF. In accordance with the previous findings, DMF diminished reactive nitrogen and oxygen species generation and consistently, SC level of advanced oxidation protein products, to the greater extent in male rats. Overall, our study indicates sex-specificity in the sensitivity of DMF cellular and molecular targets and encourages sex-based clinical research to define significance of sex for action of therapeutic agents moderating autoimmune neuroinflammation-/oxidative stress-related nervous tissue damage.     

Cell wall-associated malate dehydrogenase activity from maize roots

Isolated cell walls from maize (Zea mays L.) roots exhibited ionically and covalently bound NAD-specific malate dehydrogenase activity. The enzyme catalyses a rapid reduction of oxaloacetate and much slower oxidation of malate. The kinetic and regulatory properties of the cell wall enzyme solubilized with 1 M NaCl were different from those published for soluble, mitochondrial or plasma membrane malate dehydrogenase with respect to their ATP, Pi, and pH dependence. Isoelectric focusing of ionically-bound proteins and specific staining for malate dehydrogenase revealed characteristic isoforms present in cell wall isolate, different from those present in plasma membranes and crude homogenate. Much greater activity of cell wall-associated malate dehydrogenase was detected in the intensively growing lateral roots compared to primary root with decreased growth rates. Presence of Zn²⁺ and Cu²⁺ in the assay medium inhibited the activity of the wall-associated malate dehydrogenase. Exposure of maize plants to excess concentrations of Zn²⁺ and Cu²⁺ in the hydroponic solution inhibited lateral root growth, decreased malate dehydrogenase activity and changed isoform profiles. The results presented show that cell wall malate dehydrogenase is truly a wall-bound enzyme, and not an artefact of cytoplasmic contamination, involved in the developmental processes, and detoxification of heavy metals.