Transgenic mice which expressed human IGF-binding protein-3 (hIGFBP-3) were generated by pronuclear injection of an hIGFBP-3 cDNA driven by the mouse metallothionein 1 promoter. Two of the seven founder mice had measurable levels of hIGFBP-3 in the circulation. The serum levels of hIGFBP-3 increased as the mice were bred to homozygosity and were further induced by supplementing the drinking water with 25 mM ZnCl2. While the birth weight, litter size and body weight of transgenic mice were not significantly different from non-transgenic litter mates or wild-type mice derived from the same genetic background, the transgenic mice demonstrated selective organomegaly. The spleen, liver and heart of mice derived from both founders were significantly heavier compared with organs from non-transgenic mice (P < 0.05, P < 0.005 and P < 0.01 respectively). The weights of the brain and kidney were similar in transgenic and non-transgenic mice. Expression of the transgene was detected in the kidney, small intestine and colon by Northern blot analysis. Western ligand blotting of serum from transgenic mice did not demonstrate any change in the abundance of the IGFBPs detected by this method. When serum from transgenic mice was incubated with 125I-labeled IGF-I and analyzed by Sephacryl S-200 chromatography under neutral conditions a significantly (P < 0.05) increased amount of the radioactivity was found in the 140 kDa ternary complex compared with serum from wild-type mice. Immunoreactive hIGFBP-3 was detected in the 140 kDa ternary complex but the majority of immunoreactive hIGFBP-3 present in transgenic mouse serum eluted in later fractions indicating that it was not associated with the acid-labile subunit. These data demonstrate that modest constitutive expression of hIGFBP-3 has a selective effect on organ growth and development. The establishment of these IGFBP-3 transgenic mouse strains may provide useful models to investigate further the physiological role of IGFBP-3.

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The cAMP response element-binding protein (CREB) is a transcription factor that mediates the cellular response to metabolic and mitogenic signals. Whether CREB contributes to vascular function has received little attention, especially in relation to the processes associated with atherosclerotic disease progression and restenosis. This study examined the involvement of CREB in the mitogenic actions of angiotensin II (AngII), a growth factor that promotes neointimal hyperplasia in response to vascular injury. Treatments were performed on quiescent vascular smooth muscle cells (VSMCs) obtained from a porcine explant model. Organ culture was performed on porcine hearts subjected to angioplasty ex vivo. Stimulation of VSMCs with AngII resulted in transient CREB phosphorylation. Proliferation of smooth muscle cells in response to AngII was reduced by 90 % after infection with adenovirus expressing dominant-negative killer CREB (kCREB) mutant. Likewise, expression of kCREB prevented angioplasty-induced neointimal hyperplasia. AngII-induced CREB phosphorylation was independent of cAMP activation. Examination of putative CREB kinases revealed that MSK was responsible for phosphorylating CREB. In addition, inhibition of PKC revealed that this kinase operates upstream and activates MSK. These results indicate that activation of CREB via PKC and MSK is essential for SMC proliferation in response to AngII.

The resolution of racemic ibuprofen was studied by partial diastereomer salt formation. The resolution was performed via two methods: resolution with (+)-(R)-phenylethylamine as chiral agent and resolution with a mixture of (+)-(R)-phenylethylamine and benzylamine. The diastereomers and unreacted enantiomers were separated by supercritical fluid extraction with carbon dioxide at 15 MPa and 33 degrees C. The influence of the achiral benzylamine on the resolution efficiency was studied by varying the concentrations of the structurally related amines in their mixtures, keeping the sum molar ratio of the amines to racemic ibuprofen constant at 0.55 +/- 0.02. The presence of benzylamine positively influenced the resolution efficiency at certain concentrations. The crystal structure of the salts of (+)-(R)-phenylethylamine with (-)-(R)-ibuprofen and (+)-(S)-ibuprofen, respectively, as well as the cocrystal of the benzylamine-ibuprofen salt with neutral ibuprofen molecules are presented. These structures were determined by single crystal X-ray diffraction, proving the significantly different stoichiometry of the related amines with the chiral acid, in accordance with mass balance calculations.