Effect of mimosine on DNA synthesis in mammalian cells

We have designed a general protocol to assess the rate of replicon initiation in mammalian cells in the presence of inhibitors of DNA synthesis. It is based on cross-linking DNA in vivo with trioxsalen, which effectively blocks the movement of the replication forks along DNA, while having little effect on initiation of replication. We applied this protocol to study the effect of the plant amino acid mimosine on the rate of replicon initiation in exponentially growing murine erythroleukemia F4N cells. We found out that during the first 2 h after application of 25-400 microM mimosine, the initiation step was inhibited more efficiently than the overall DNA synthesis. In this respect, the effect of mimosine was similar to that of gamma-ray irradiation and differed from that of hydroxyurea and aphidicolin. The results suggest that in addition to inhibiting the elongation step of DNA synthesis, mimosine inhibits the initiation of DNA replication as well.     

Comparison of nested PCR with immunofluorescent-antibody assay for detection of Ehrlichia canis infection in dogs treated with doxycycline

A partial 16S rRNA gene was amplified in Ehrlichia canis-infected cells by nested PCR. The assay was specific and did not amplify the closely related Ehrlichia chaffeensis, Ehrlichia muris, Neorickettsia helminthoeca, and SF agent 16S rRNA genes. The assay was as sensitive as Southern hybridization, detecting as little as 0.2 pg of E. canis DNA. By this method, all blood samples from four dogs experimentally infected with E. canis were positive as early as day 4 postinoculation, which was before or at the time of seroconversion. One hundred five blood samples from dogs from Arizona and Texas (areas of E. canis endemicity) and 30 blood samples from dogs from Ohio (area of E. canis nonendemicity) were examined by nested PCR and immunofluorescent-antibody (IFA) test. Approximately 84% of dogs from Arizona and Texas had been treated with doxycycline before submission of blood specimens. Among Arizona and Texas specimens, 46 samples were PCR positive (44%) and 80 were IFA positive (76%). Forty-three of 80 IFA-positive samples (54%) were PCR positive, and 22 of 25 IFA-negative samples (88%) were negative in the nested PCR. None of the Ohio specimens were IFA positive, but 5 specimens were PCR positive (17%). Our results indicate that the nested PCR is highly sensitive and specific for detection of E. canis and may be more useful in assessing the clearance of the organisms after antibiotic therapy than IFA, especially in areas in which E. canis is endemic.     

Point mutation flanking a CTL epitope ablates in vitro and in vivo recognition of a full-length viral protein

CD8+ T cells (T(CD8+)) recognize viral Ags as short peptides (epitopes) displayed at the cell surface by MHC class I molecules. Using a panel of recombinant vaccinia viruses, we show that single-point mutations flanking either side of an H-2Kd-restricted epitope, residues 147-155, within full-length influenza nucleoprotein (NP) can impact, even ablate, presentation of that epitope, while having no effect on presentation of distal epitopes. The most severe blocking mutation (Ala to Pro at position 146) did not inhibit NP(147-155) presentation in the context of a truncated minigene, implying that this peptide is not a functional processing intermediate. An amino-terminal proline replacement also significantly reduced presentation of NP(50-57) (H-2Kk restricted), while the same mutation did not affect a third NP epitope. Thus, while trends in processing specificity may exist, the epitope itself contributes to flanking sequence effects. These findings were paralleled by in vivo priming experiments in which, depending on viral dose, subtle in vitro blocking effects were absolute. Proteasome/synthetic peptide coincubation studies support a role for enhanced epitope destruction in preventing presentation, as did the effect of the peptide aldehyde, LLnL, which restored presentation of NP(147-155) from the mutated constructs. This reagent did not inhibit epitope presentation, even from wild-type NP, suggesting that its production may be proteasome independent. These results support the notion that point mutation of epitope flanking sequence can serve as a mechanism for viral immune evasion, shed light on the mechanisms involved, and suggest that in vitro assays may not be sensitive indicators of flanking sequence effects.     

The effects of diet, ad libitum overfeeding, and moderate dietary restriction on the rodent bioassay: the uncontrolled variable in safety assessment

Ad libitum (AL) overfeeding is the most significant, uncontrolled variable affecting the outcome of the current rodent bioassay. There is a highly significant correlation between AL food consumption, the resultant obesity and body weight, and low 2-yr survival in rodents. AL feeding of diets with lowered protein, metabolizable energy (ME), and increased fiber does not improve survival. Only dietary restriction (DR) of all diets tested significantly improves survival and delays the onset of spontaneous degenerative disease (i.e., nephropathy and cardiomyopathy) and diet-related tumors. Moderate DR results in an incidence of spontaneous tumors similar to AL-fed rats, but the tumors are found incidentally and do not cause early mortality. There is a decreased age-adjusted incidence of pituitary and mammary gland tumors in moderate DR-fed rats, but tumor growth time is similar between AL and DR rats with only a delay in tumor onset time seen in DR-fed groups. Moderate DR does not significantly alter drug-metabolizing enzyme activities nor the toxicologic response to 5 pharmaceuticals tested at maximum tolerated doses (MTDs). However, moderate DR-fed rats did require much higher doses of 4 additional pharmaceutical compounds before classical MTDs were produced. Toxicokinetic studies of 2 of these compounds demonstrated equal or higher steady-state systemic exposures to parent drug and metabolites in moderate DR-fed rats. Markers of oxidative stress (lipid peroxidation, protein oxidation) are decreased and cytoprotective anti-oxidant markers are preserved in moderate DR-fed rats. But moderate DR does not delay reproductive senescence in female rats. Only marked DR delays reproductive senescence compared to AL and moderate DR-fed female rats. These and other data indicate that moderate DR is the most appropriate method of dietary control for the rodent bioassay when used to assess pharmaceuticals for human safety and compounds for risk assessment.     

A method for approximating fractional power average relaxation times without inversion of multiexponential relaxation data

A method is presented for approximating fractional power averages of relaxation times for data equispaced in log time, without the need to invert multiexponential relaxation data. This form of average permits giving emphasis to short or long times depending on the choice of the p value, thus giving the possibility of representing different specific properties of porous media. This method has been tested on a large number of nuclear magnetic resonance (NMR) relaxation measurements in porous samples. This new algorithm appears to be robust with respect to both measurement and computation, and its major advantage is that it does not depend on a particular inversion method. Moreover, it permits a very fast computation.     

The role of supplemental translaminar screws in anterior lumbar interbody fixation: a biomechanical study

The immediate stabilization provided by anterior interbody cage fixation is often questioned. Therefore, the role of supplementary posterior fixation, particularly minimally invasive techniques such as translaminar screws, is relevant. The purpose of this biomechanical study was to determine the immediate three-dimensional flexibility of the lumbar spine, using six human cadaveric functional spinal units, in four different conditions: (1) intact, (2) fixed with translaminar screws (TLS), (3) instrumented with anterior interbody cage insertion with the BAK system and (4) instrumented with BAK cage with additional TLS fixation. Flexibility was determined in each testing condition by measuring the vertebral motions under applied pure moments (i.e. flexion-extension, bilateral axial rotation, bilateral lateral bending) in an unconstrained manner. Anterior fixation with the BAK alone provided significant stability in flexion and lateral bending. Additional posterior TLS significantly reduced the motion in extension and axial rotation. TLS fixation alone resulted in smaller rotations than BAK fixation in all loading directions. Based on these results, it seems that interbody cage fixation with the BAK system stabilizes the spine in some, but not all, loading directions. The problematic loading directions of extension and axial rotation can be substantially stabilized by using translaminar screw fixation. However, one should emphasize that the degree of stability needed to achieve solid fusion is not known.