Shear-induced variability in the United States Pharmacopeia Apparatus 2: modifications to the existing system.

The hydrodynamics within the United States Pharmacopeia Apparatus 2 have been shown to be highly non-uniform with a potential to yield substantial variability in dissolution rate measurements. Through the use of readily available engineering tools, several geometric modifications to the device were evaluated in this study. Specifically, we examined the influence of impeller clearance, agitator type (radial and axial), and vessel geometry (PEAK vessel) on the fluid flow properties and their relation to measured dissolution rates. Increasing the impeller clearance was observed to exacerbate the heterogeneity in shear and would likely result in greater variability in dissolution measurements. Altering the impeller type was shown to yield changes in the hydrodynamic behavior; however, the overall properties and problems with the test remain the same. Use of the PEAK vessel was observed to reduce shear heterogeneity in the regions where tablets are most likely to visit during testing; however, higher shear rates may result in the inability to discriminate between true differences in dissolution rates.

Hydrodynamics-induced variability in the USP apparatus II dissolution test.

The USP tablet dissolution test is an analytical tool used for the verification of drug release processes and formulation selection within the pharmaceutical industry. Given the strong impact of this test, it is surprising that operating conditions and testing devices have been selected empirically. In fact, the flow phenomena in the USP test have received little attention in the past. An examination of the hydrodynamics in the USP apparatus II shows that the device is highly vulnerable to mixing problems that can affect testing performance and consistency. Experimental and computational techniques reveal that the flow field within the device is not uniform, and dissolution results can vary dramatically with the position of the tablet within the vessel. Specifically, computations predict sharp variations in the shear along the bottom of the vessel where the tablet is most likely to settle. Experiments in which the tablet location was carefully controlled reveal that the variation of shear within the testing device can affect the measured dissolution rate.

Delayed rRNA processing results in significant ribosome biogenesis and functional defects.

mof6-1 was originally isolated as a recessive mutation in Saccharomyces cerevisiae which promoted increased efficiencies of programmed -1 ribosomal frameshifting and rendered cells unable to maintain the killer virus. Here, we demonstrate that mof6-1 is a unique allele of the histone deacetylase RPD3, that the deacetylase function of Rpd3p is required for controlling wild-type levels of frameshifting and virus maintenance, and that the closest human homolog can fully complement these defects. Loss of the Rpd3p-associated histone deacetylase function, either by mutants of rpd3 or loss of the associated gene product Sin3p or Sap30p, results in a delay in rRNA processing rather than in an rRNA transcriptional defect. This results in production of ribosomes having lower affinities for aminoacyl-tRNA and diminished peptidyltransferase activities. We hypothesize that decreased rates of peptidyl transfer allow ribosomes with both A and P sites occupied by tRNAs to pause for longer periods of time at -1 frameshift signals, promoting increased programmed -1 ribosomal frameshifting efficiencies and subsequent loss of the killer virus. The frameshifting defect is accentuated when the demand for ribosomes is highest, suggesting that rRNA posttranscriptional modification is the bottleneck in ribosome biogenesis.

The 9-A solution: how mRNA pseudoknots promote efficient programmed -1 ribosomal frameshifting.

There is something special about mRNA pseudoknots that allows them to elicit efficient levels of programmed -1 ribosomal frameshifting. Here, we present a synthesis of recent crystallographic, molecular, biochemical, and genetic studies to explain this property. Movement of 9 A by the anticodon loop of the aminoacyl-tRNA at the accommodation step normally pulls the downstream mRNA a similar distance along with it. We suggest that the downstream mRNA pseudoknot provides resistance to this movement by becoming wedged into the entrance of the ribosomal mRNA tunnel. These two opposing forces result in the creation of a local region of tension in the mRNA between the A-site codon and the mRNA pseudoknot. This can be relieved by one of two mechanisms; unwinding the pseudoknot, allowing the downstream region to move forward, or by slippage of the proximal region of the mRNA backwards by one base. The observed result of the latter mechanism is a net shift of reading frame by one base in the 5' direction, that is, a -1 ribosomal frameshift.