We have generated transgenic mice that express green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter (MIP). The MIP-GFP mice develop normally and are indistinguishable from control animals with respect to glucose tolerance and pancreatic insulin content. Histological studies showed that the MIP-GFP mice had normal islet architecture with coexpression of insulin and GFP in the β-cells of all islets. We observed GFP expression in islets from embryonic day E13.5 through adulthood. Studies of β-cell function revealed no difference in glucose-induced intracellular calcium mobilization between islets from transgenic and control animals. We prepared single-cell suspensions from both isolated islets and whole pancreas from MIP-GFP-transgenic mice and sorted the β-cells by fluorescence-activated cell sorting based on their green fluorescence. These studies showed that 2.4 ± 0.2% ( n = 6) of the cells in the pancreas of newborn (P1) and 0.9 ± 0.1% ( n = 5) of 8-wk-old mice were β-cells. The MIP-GFP-transgenic mouse may be a useful tool for studying β-cell biology in normal and diabetic animals.
More than half of all pharmaceuticals are chiral compounds. Although the enantiomers of chiral compounds have the same chemical structure, they can exhibit marked differences in physiological activity; therefore, it is important to remove the undesirable enantiomer. Chromatographic separation of chiral enantiomers is one of the best available methods to get enantio-pure substances, but the optimization of the experimental conditions can be very time-consuming. One of the most widely used chiral stationary phases, amylose tris(3,5-dimethylphenyl carbamate) (ADMPC), has been extensively investigated using both experimental and computational methods; however, the dynamic nature of the interaction between enantiomers and ADMPC, as well as the solvent effects on the ADMPC-enantiomer interaction, are currently absent from models of the chiral recognition mechanism. Here we use QM/MM and molecular dynamics (MD) simulations to model the enantiomers of flavanone on ADMPC in either methanol or heptane/2-propanol (IPA) (90/10) to elucidate the chiral recognition mechanism from a new dynamic perspective. In atomistic MD simulations, the 12-mer model of ADMPC is found to hold the 4/3 left-handed helical structure in both methanol and heptane/IPA (90/10); however, the ADMPC polymer is found to have a more extended average structure in heptane/IPA (90/10) than in methanol. This results from the differences in the distribution of solvent molecules close to the backbone of ADMPC leads to changes in the distribution of the (φ, ψ) dihedral angles of the glycoside bond (between adjacent monomers) that define the structure of the polymer. Our simulations have shown that the lifetime of hydrogen bonds formed between ADMPC and flavanone enantiomers in the MD simulations are able to reproduce the elution order observed in experiments for both the methanol and the heptane/IPA solvent systems. Furthermore, the ratios of hydrogen-bonding-lifetime-related properties also capture the solvent effects, in that heptane/IPA (90/10) is found to make the separation between the two enantiomers of flavanone less effective than methanol, which agrees with the experimental separation factors of 0.9 versus 0.4 for R/S, respectively.
Chiral
high-performance liquid chromatography (HPLC) is commonly
performed to isolate the biologically active enantiomer of a drug
from the ineffective or even harmful ones. Understanding the molecular-level
recognition that underlies this process is necessary for trimming
down the very large number of possible combinations of chiral stationary
phases, solvent systems, and other experimental HPLC conditions, a
particularly important consideration when only small quantities of
the racemate are available. Fully atomistic molecular dynamics (MD)
simulation is a useful tool to provide this molecular-level understanding
and predict experimental separation factors under a given set of conditions.
To predict the chiral separation results for drug enantiomers by amylose
tris(3,5-dimethylphenyl carbamate) (ADMPC) chiral stationary phase,
we design a model of multiple ADMPC polymer strands coated on an amorphous
silica slab. Using various MD techniques, we successfully coat ADMPCs
onto the surface without losing the structural character of the backbone
in the presence of the solvent system. Not only is this model more
representative of the polymer surface on a solid support that is encountered
by the enantiomers, but it also provides more opportunities for chiral
molecules interacting with ADMPC, provides the possibility for large
drug molecules to interact with two polymer strands at the same instant,
and provides better agreement with experiment when we use the overall
average quantities as the predictive metric. For a better understanding
of why some metrics are better predictors than others, we use charts
of the distribution of hydrogen-bonding lifetimes for various donor–acceptor
pairs that contribute to the interaction events determining the relative
retention times for the enantiomers. We also examine the contribution
of ring–ring interactions to the molecular recognition process
and ultimately to the differential retention of enantiomers. The results
are more consistent than previous models and resolve the problematic
case of two drugs, thalidomide and valsartan.
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