Rapid identification of hemoplasma species by palindromic nucleotide substitutions at the GAAA tetraloop helix in the specificity domain of ribonuclease P RNA

We examined secondary structures of the ribonuclease P RNA sequences obtained from DNA databases, and identified a determinative prototype of the P12 helix peculiar to each species of hemoplasmas. This key structure will provide a rapid means for species identification of these uncultivable pathogens without making a phylogenetic tree based on alignments of nucleotide sequences. This procedure based on palindromic nucleotide substitutions at the stem portion of the P12 helix provide clear information such as the level of heterogeneity within a species, the relatedness between species, or facilitating the characterization and clustering of specific strains. In conclusion, the PNS analysis is based on the evaluation of only the strategic and highly conserved genomic region in the specificity domain of RNase P RNA.     

Therapeutic effect of anti-TNF-α antibody and levofloxacin (LVFX) in a mouse model of enterohemorrhagic Escherichia coli O157 infection

The ability of an anti-TNF-α antibody to confer protection against enterohaemorrhagic Escherichia coli (EHEC) O157 was investigated in germfree IQI mice. The use of an antibiotic levofloxacin (LVFX) alone or with the antibody was also studied. Protection included an increase in survival rate. Treatment with the anti-TNF-α antibody inhibited the histological signs associated with EHEC infection but did not prevent the colonization of EHEC or production of Shiga toxin (Stx). No clinical signs were observed and EHEC was completely eliminated in the mouse model receiving both anti-TNF-α antibody and LVFX. Anti-TNF-α antibody suppressed inflammatory cytokine response in the mouse kidney and brain by EHEC infection.     

Reproductive phenotypes in mice with targeted disruption of the 20alpha-hydroxysteroid dehydrogenase gene

In the corpus luteum of rats and mice, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) catalyzes the conversion of progesterone to a biologically inactive metabolite, 20alpha-dihydroprogesterone (20alpha-OHP). The reduction of progesterone by 20alpha-HSD is believed to be important for functional luteolysis in these rodent species. In addition to the corpus luteum, expression of 20alpha-HSD has been demonstrated in tissues such as the placenta, endometrial epithelia, and fetal skin, although the roles it plays in the latter tissues remain to be determined. To determine the contribution of 20alpha-HSD to functional luteolysis and to the rodent reproductive system more generally, we generated a strain of mice with targeted disruption of the 20alpha-HSD gene. In the 20alpha-HSD-/- mice we obtained, which lacked the genomic region essential for catalytic reaction, neither 20alpha-HSD activity in the corpus luteum nor an increase in the serum concentrations of 20alpha-OHP during pseudopregnancy or pregnancy was detected. The durations of the estrous cycle, pseudopregnancy, and pregnancy were significantly prolonged in the 20alpha-HSD-/- mice, although the serum progesterone levels decreased to levels low enough for delivery of pups at term of pregnancy. In addition, the number of pups, especially live pups, was markedly decreased in the 20alpha-HSD-/- mice. These findings suggest that the role of 20alpha-HSD in functional luteolysis is relatively minor but that it is involved in the survival of newborn mice.     

Peripartum changes in plasma estrone sulfate and estradiol-17beta profiles associated with and without the retention of fetal membranes in holstein-friesian cattle

The aim of this study was to evaluate the changes in plasma concentrations of estrone sulfate (E(1)S) and estradiol-17beta (E(2)beta) during the peripartum period (from day 10 prepartum to day 1 postpartum) associated with and without retention of fetal membranes (RFM) in Holstein-Friesian cattle (n=42). Plasma samples were analyzed for E(1)S and E(2)beta by ELISA. All parturitions were spontaneous and normal. Of 38 cattle delivering singletons, 29 had no RFM (singleton-normal group) and nine had RFM for more than 12 h (singleton-RFM group). Four cows gave birth to twins, and each twin had its own fetal membrane (FM). Two twinning cows expelled both FMs normally within 12 h (twin-normal group). In the remaining 2 twinning cows (twin-RFM group), the FM was expelled normally for one twin (first), while the FM of the other (second) was retained. There were no significant differences in the E(1)S concentrations or their increments from the concentrations on the preceding day between the normal and RFM groups of singleton cows on any peripartum day. The mean plasma E(2)beta concentrations on each day from day 10 to day 3 prepartum were significantly lower (P<0.05) in the singleton-RFM group compared with the singleton-normal group; however, on days 2 and 1 prepartum, the increments in the E(2)beta concentrations from the concentrations on the preceding days were significantly higher (P<0.05) in the singleton-RFM group than in the singleton-normal group. Thus, the plasma E(1)S concentrations just before parturition may not be associated with RFM. In the cows with RFM, the lower plasma E(2)beta concentrations that were found prior to day 2 prepartum may have been associated with immature placentomes, and the rapid rise in plasma E(2) beta within 1 to 2 days prior to calving may have produced asynchrony of placental and/or fetal maturation in relation to calving, thus resulting in RFM.     

Lack of inhibitory effects of several fluoroquinolones on cytochrome P-450 3A activities at clinical dosage in dogs

Inhibitory effects of several fluoroquinolones (FQs) on liver CYP3A activities were examined by in vitro and in vivo tests in dogs. Midazolam (MDZ) hydroxylation rate was used to determine the CYP3A activities in liver microsomes. Enrofloxacin (EFX), ofloxacin (OFX) orbifloxacin (OBFX) and ciprofloxacin (CFX) were tested. None of the FQs changed Vmax, Km or intrinsic clearance (Vmax/Km) of MDZ. For in vivo test, we examined the effects of oral administration of EFX and OFX on the pharmacokinetics of quinidine (QN), a CYP3A substrate. EFX or OFX (10 mg/kg) was administered once a day for 3 days. QN (2 mg/kg) was intravenously injected at 2 h after the final dose of FQs administration. The same dose of QN was intravenously injected 3 weeks before the start of FQs administration for control. Neither EFX nor OFX changed the pharmacokinetic parameters of QN. These in vitro and in vivo consisted results suggest that these FQs lack the inhibitory effects on CYP3A activities in dogs. Hence, given these results, the risk of drug-drug interaction is unlikely to occur between FQs and CYP3A substrates in clinical situation in dogs.     

Histopathological evaluation of the ocular-irritation potential of shampoos,make-up removers and cleansing foams in the bovine corneal opacity and permeability assay

The bovine corneal opacity and permeability (BCOP) assay is an alternative method to the in vivo Draize eye test in rabbits for evaluating eye irritation in vitro. Here, we compared the numerical results of the BCOP assay with the corresponding histopathology for three different corneas for each test substance, including commercially available shampoos, make-up removers and cleansing foams that contained surfactants and other ingredients. The histopathological score was defined based on the severity of lesions in the corneal epithelium. The histopathological findings and scores of the three sections for each test substance were comparable. The in vitro irritancy score (IVIS) generally corresponds to the corneal irritant potential of the test substances assigned on the basis of the histopathological findings in this study. In the present study, we characterized the histopathology of the corneal epithelium and stroma and especially showed that the corneal epithelial injury caused by test substances might be important in assessment of test substances that are mild eye irritants (category 2B) as classified by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS), as corneal lesions suggestive of classification into category 2B were localized on the border between the corneal epithelium and stroma, which contained cell elements related to assessment of prognosis of an in vivo eye injury. Histopathological assessment might be useful in predicting in vivo ocular irritation, particularly for test substances with an IVIS >3.1 but ≤25 that are classified as mild irritants (category 2B) according to the UN GHS.     

Isolation and structure determination of blepharismin, a conjugation initiating gamone in the ciliate blepharisma

One of the gamones (gamone II) which are effective for the induction of conjugation in Blepharisma intermedium has been isolated in a crystalline form and designated as blepharismin. From the result of chemical and spectroscopic investigations, in which x-ray crystallographic analysis was used as a definitive tool, blepharismin has been found to have the structure of calcium 3-(2'-formylamino-5'-hydroxybenzoyl)lactate.