In one of the earliest events in human cytomegalovirus (HCMV)-infected cells, the major immediate-early (IE) protein IE1 initially targets to and then disrupts the nuclear structures known as PML oncogenic domains (PODs) or nuclear domain 10. Recent studies have suggested that modification of PML by SUMO is essential to form PODs and that IE1 both binds to PML and may disrupt PODs by preventing or removing SUMO adducts on PML. In this study, we showed that in contrast to herpes simplex virus type 1 (HSV-1) IE110 (ICP0), the loss of sumoylated forms of PML by cotransfected IE1 was resistant to the proteasome inhibitor MG132 and that IE1 did not reduce the level of unmodified PML. Reduced sumoylation of PML was also observed in U373 cells after infection with wild-type HCMV and proved to require IE1 protein expression. Mutational analysis revealed that the central hydrophobic domain of IE1, including Leu174, is required for both PML binding and loss of PML sumoylation and confirmed that all IE1 mutants tested that were deficient in these functions also failed both to target to PODs and to disrupt PODs. These same mutants were also inactive in several reporter gene transactivation assays and in inhibition of PML-mediated repression. Importantly, a viral DNA genome containing an IE1 gene with a deletion [IE1(⌬290-320)] that was defective in these activities was not infectious when transfected into permissive fibroblast cells, but the mutant IE1(K450R), which is defective in IE1 sumoylation, remained infectious. Our mutational analysis strengthens the idea that interference by IE1 with both the sumoylation of PML and its repressor activity requires a physical interaction with PML that also leads to disruption of PODs. These activities of IE1 also correlate with several unusual transcriptional transactivation functions of IE1 and may be requirements for efficient initiation of the lytic cycle in vivo.Human cytomegalovirus (HCMV), a member of the betaherpesvirus subfamily, typically causes nearly ubiquitous asymptomatic latent or persistent infections. However, primary infections of newborns and reactivation from latent infection in immunocompromised individuals, including recipients of organ transplantation and patients with AIDS, can lead to lifethreatening problems with overt systematic and chronic disease (56, 65). During lytic cycle infection, HCMV gene expression occurs in a three-step sequential fashion with immediate-early (IE or ␣), delayed-early (DE or ␤), and late (L or ␥) kinetics. Among the IE proteins, two nuclear regulatory phosphoproteins, IE1 (or IE72) and IE2 (or IE86), are the first and most abundantly expressed proteins and are synthesized by differential splicing from the same complex overlapping transcription unit within the major IE (MIE) locus (56).The 72-kDa IE1 protein (491 amino acids) is encoded by exons 1, 2, 3, and 4 (UL123), whereas the 86-kDa IE2 protein (579 amino acids) is encoded by exons 1, 2, 3, and 5 (UL122), and the two proteins share 85 amino acids at the N terminus. IE2 is a spe...

Donepezil is used for the palliative treatment of mild to moderate dementia of the Alzheimer’s disease. Donepezil crystallized as four solvent-free polymorphs including forms I, II, C, and F which differ in molecular conformations and packing. Conformational difference comes from the torsion of the 2, 3-dihydroinden-1-one moiety and methyl-benzyl ring with respect to piperidyl ring. Similar melting temperatures and heats of fusion were observed for four solvent-free polymorphic forms and made polymorph selection by solvent crystallization method poor. The relative thermodynamic stability relationships of each polymorph with respect to the amorphous form were determined using direct heat capacity (C p ) measurement and then used to evaluate the relative thermodynamic stability between polymorphs. Form F was the stable form over the temperature ranges we studied. Other than form F, form C was the stable form among three polymorphic forms including I, II, and C, below 53 °C, and is enantiotropically related to both forms I and II. Form II is the stable form above 53 °C and monotropically related to form I. The thermodynamic relationships between polymorphs were further confirmed by measuring the solubility over temperatures ranging from 35 to 60 °C in ethanol. Slurry conversion in ethanol, isopropyl alcohol, and cyclohexane was conducted to provide a guideline to obtain pure and desired polymorphic forms. The establishment of the thermodynamic relationships among four polymorphic forms greatly facilitated polymorph selections of donepezil.

The effect of mechanical impact on the polymorphic transformation of mefenamic acid (MFA) and the formation of a solid dispersion of mefenamic acid, a poor glass forming/poorly-water soluble compound, with polyvinylpyrrolidone (PVP) K12 was investigated. The implication of solid dispersion formation on solubility enhancement of MFA, prepared by cryomilling, was investigated. Solid state characterization was conducted using powder X-ray diffraction (PXRD) and Fourier-transform infrared (FTIR) spectroscopy combined with crystal structure analysis. Apparent solubility of the mixtures in pH 7.4 buffer was measured. A calculation to compare the powder patterns and FTIR spectra of solid dispersions with the corresponding physical mixtures was conducted. Solid state characterization showed that (1) MFA I transformed to MFA II when pure MFA I was cryogenically milled (CM); and (2) MFA forms a solid dispersion when MFA was cryogenically milled with PVP K12. FTIR spectral analysis showed that hydrogen bonding facilitated by mechanical impact played a major role in forming solid dispersions. The apparent solubility of MFA was significantly improved by making a solid dispersion with PVP K12 via cryomilling. This study highlights the importance of cryomilling with a good hydrogen bond forming excipient as a technique to prepare solid dispersion, especially when a compound shows a poor glass forming ability and therefore, is not easy to form amorphous forms by conventional method.

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