A single tube RT-PCR assay for the detection of mosquito-borne flaviviruses.

Mosquito-borne flaviviruses include several important agents of human disease and have provided striking examples of emerging infections. In this study we present the design and validation of a single tube RT-PCR assay using a pair of consensus primers for the detection of mosquito-borne flaviviruses. Sequencing of the amplicons permits the species identification. The assay was validated using RNA from the yellow fever virus vaccine strain and from representative strains of dengue viruses 1, 2, 3 and 4, West Nile virus, Kunjin virus (a clade of West Nile virus), and St. Louis encephalitis virus.

Changes in some factors of the innate immunity and serum zinc and iron concentrations in pigs following intravenous administration of Escherichia coli lipopolysaccharide.

The changes in some factors of the innate immunity (phagocytosis, complement, lysozyme); haematological parameters-leukocytes, erythrocytes, differential white blood cell counts, haemoglobin, haematocrit and the serum concentrations of the microelements zinc and iron in six 2- to 3-months-old female piglets after the intravenous administration of lipopolysaccharide from Escherichia coli 0111:B4 were determined. It was found out that 1 h after the administration of lipopolysaccharide at the dosage rate of 10 microg/kg body weight resulted in a decrease in the phagocytic parameters, i.e. the phagocytic number and the index of phagocytic activity, which was followed by an increase in their values between post treatment hours 2 and 4. The leukocyte counts had decreased by hour 2 after the injection, but thereafter increased, and at post treatment hour 72, a leukocytosis was observed. The differential white blood cell counts were characterized by a shift to the left between hours 2 and 4 and a statistically significant increase in lymphocyte counts at hour 48 of the experiment. The serum zinc concentrations were increased an hour after the lipopolysaccharide application; after which their average values were lower. The haemolytic activities (CH50) of the classical and the alternative pathways of complement activation decreased. The haemolytic activity (CH50) for the classical pathway began to increase at hour 48 following the treatment. Significant changes were not observed in lysozyme activity, serum iron concentrations or the related haematological parameters (erythrocytes and haemoglobin).

The binding of Ku antigen to homeodomain proteins promotes their phosphorylation by DNA-dependent protein kinase.

The Ku antigen (70- and 80-kDa subunits) is a regulatory subunit of DNA-dependent protein kinase (DNA-PK) that promotes the recruitment of the catalytic subunit of DNA-PK (DNA-PKcs) to DNA ends and to specific DNA sequences from which the kinase is activated. Ku and DNA-PKcs plays essential roles in double-stranded DNA break repair and V(D)J recombination and have been implicated in the regulation of specific gene transcription. In a yeast two-hybrid screen of a Jurkat T cell cDNA library, we have identified a specific interaction between the 70-kDa subunit of Ku heterodimer and the homeodomain of HOXC4, a homeodomain protein expressed in the hematopoietic system. Unexpectedly, a similar interaction with Ku was observed for several additional homeodomain proteins including octamer transcription factors 1 and 2 and Dlx2, suggesting that specific binding to Ku may be a property shared by many homeodomain proteins. Ku-homeodomain binding was mediated through the extreme C terminus of Ku70 and was abrogated by amino acid substitutions at Lys595/Lys596. Ku binding allowed the recruitment of the homeodomain to DNA ends and dramatically enhanced the phosphorylation of homeodomain-containing proteins by DNA-PK. These results suggest that Ku functions as a substrate docking protein for signaling by DNA-PK to homeodomain proteins from DNA ends.

Effects of indomethacin on lipopolysaccharide-induced plasma PGE2 concentrations and clinical pathological disorders in experimental endotoxemia.

The effects of indomethacin, a cyclooxygenase inhibitor, upon plasma concentrations of prostaglandin E2 (PGE2), the febrile response, and metabolic and hematological alterations induced by lipopolysaccharide (LPS) were studied. Experimental endotoxemia was provoked via i.p. injection of 1.0 mg E coli LPS/kg in rats (group A). Indomethacin was introduced/os (2.5 mg/kg) 30 min prior to LPS challenge (group B). Pretreatment with this medication completely inhibited the hyperthermic response to LPS and eliminated the LPS-induced non-specific symptoms of anorexia, adipsia, reduced locomotory activity and gastrointestinal troubles. Plasma PGE2 concentrations increased as early as the 2nd h after the LPS challenge but were blocked when endotoxin application was preceded by indomethacin treatment. Indomethacin did not significantly influence hematological parameters. The dynamics of hematocrit and erythrocyte counts were similar in both groups with a decrease up to the 2nd h followed by an increase to maximum at post-treatment day 3. Pretreatment with indomethacin did not influence the endotoxin-induced leukopenia observed at the 2nd h or the accompanying neutropenia and left shift. Cyclooxygenase inhibition affected total protein concentrations; they were decreased in the early hours of the study (hours 4-6) in both groups. The later tendency towards increase in total protein concentrations was more expressed in animals from group B. Changes in blood glucose were characterized by a permanent tendency towards decrease after hour 2 of LPS challenge up to day 6 (group A). In group B, a similar tendency was observed, but glucose concentrations decreased between hours 2-6 and then returned to initial values.

Sequence-specific DNA binding and transcription factor phosphorylation by Ku Autoantigen/DNA-dependent protein kinase. Phosphorylation of Ser-527 of the rat glucocorticoid receptor.

NRE1 is a DNA sequence element through which Ku antigen/DNA-dependent protein kinase (DNA-PK) catalytic subunit represses the induction of mouse mammary tumor virus transcription by glucocorticoids. Although Ku is an avid binder of DNA ends and has the ability to translocate along DNA, we report that direct sequence-specific Ku binding occurs with higher affinity (Kd = 0.84 +/- 0.24 nM) than DNA end binding. Comparison of Ku binding to several sequences over which Ku can accumulate revealed two classes of sequence. Sequences with similarity to NRE1 competed efficiently for NRE1 binding. Conversely, sequences lacking similarity to NRE1 competed poorly for Ku and were not recognized in the absence of DNA ends. Phosphorylation of glucocorticoid receptor (GR) fusion proteins by DNA-PK reflected Ku DNA-binding preferences and demonstrated that co-localization of GR with DNA-PK on DNA in cis was critical for efficient phosphorylation. Phosphorylation of the GR fusion protein by DNA-PK mapped to a single site, Ser-527. This site occurs adjacent the GR nuclear localization sequence between the DNA and ligand binding domains of GR, and thus its phosphorylation, if confirmed, has the potential to affect receptor function in vivo.

Changes in some parameters of the immune response in rats after cold stress.

The influence of stress upon the endocrine system and its relationship to the immune system have been studied. The simulation of chronic cold stress in rats caused an increase in ACTH, corticosterone, and T3 and T4 levels. The different hormonal background in stressed animals affected the characteristics and the intensity of the immune response, which was traced in dynamics and through the action of 10(9) ovine erythrocytes. A strong suppression of B-lymphocytes and of PFC count was observed. Increased Fc gamma-RFC-suppressing activity was also determined. The PFC suppression changed simultaneously with the rate and dynamics of Fc gamma-RFC-suppressing activity in animals under stress. In control rats (immunized with ovine erythrocytes only), the antigen caused insignificant changes in corticosterone level, which was not typical for stress and was not accompanied by suppressing activity in the immune response.

[Acid-base status and antibody production in pigs immunized with a vaccine against erysipelas]. / Kiselinno-alkalen status i antitialoproduktsiia pri praseta, imunizirani s vaksina protiv chervenka.

Studied was the correlation between the acid-alkaline status and the antibody production in pigs treated with the VR-2 vaccine against swine erysipelas. A progressive growth of the values of the B-system indices was established as early as the 24th hour in correlation with the higher HCO3 and CO2 values. The latter substantiated a new profile of the acid-alkaline state, which persisted up to the 10th day. The subsidiary increase in the concentration of HCO3 from the 10th to the 15th day as well as of pCO2 on the 15th day was associated with the highest percent and absolute number of B lymphocytes and 'AOK' at 10(6). This confirmed the presence of a gradual correlation between the indices of the acid-alkaline state and the B system.

[Changes in the white blood picture and the T- and B-lymphocyte counts in the peripheral blood of pigs after treatment with E. coli endotoxin]. / Promeni v bialata kruvna kartina i broia na T- i B-limfotsitite v perifernata kruv na praseta sled tretirane s endotoksin na E. coli.

Studied were the changes in the total count of the white blood cells, the leukocyte formula, and the T- and B-lymphocytes of young pigs (2-3 months old) at various intervals following treatment with an E. coli enterotoxin. It was found that there set in an essential drop of the total count of the white blood cells during the first 24 hours after treatment, with a following rising trend. There were also characteristic changes in the ratio between the various types of white blood cells, with a rise of the lymphocytes and monocytes. The level of T-POK and B-POK rose after the endotoxin was injected--for the T cells it reached peak values between the 3d and 5th day, while for the B cells it was highest between the 5th and 7th day. There was a following drop with both types of cells, and by the 15th day the initial level was reached.