These findings indicate that a synthetic polymer scaffold can serve as a platform for islet transplantation and improves the function of extrahepatically transplanted islets compared to islets transplanted without a scaffold. The scaffold may also be useful to deliver bioactive molecules to modify the microenvironment surrounding the transplanted islets and, thus, enhance islet survival and function.

Neurons express adaptor (AP)-3 complexes assembled with either ubiquitous (␤3A) or neuronal-specific (␤3B) ␤3 isoforms. However, it is unknown whether these complexes indeed perform distinct functions in neuronal tissue. Here, we explore this hypothesis by using genetically engineered mouse models lacking either ␤3A-or ␤3B-containing AP-3 complexes. Somatic and neurological phenotypes were specifically associated with the ubiquitous and neuronal adaptor deficiencies, respectively. At the cellular level, AP-3 isoforms were localized to distinct neuronal domains. ␤3B-containing AP-3 complexes were preferentially targeted to neuronal processes. Consistently, ␤3B deficiency compromised synaptic zinc stores assessed by Timm's staining and the synaptic vesicle targeting of membrane proteins involved in zinc uptake (ZnT3 and ClC-3). Surprisingly, despite the lack of neurological symptoms, ␤3A-deficient mouse brain possessed significantly increased synaptic zinc stores and synaptic vesicle content of ZnT3 and ClC-3. These observations indicate that the functions of ␤3A-and ␤3B-containing complexes are distinct and divergent. Our results suggest that concerted nonredundant functions of neuronal and ubiquitous AP-3 provide a mechanism to control the levels of selected membrane proteins in synaptic vesicles.

β-Cell mass increases during pregnancy to accommodate for insulin resistance. This increase is mainly due to β-cell proliferation, a process that requires intact prolactin receptor (Prlr) signaling. Signaling molecules that are known to regulate β-cell proliferation include Jak2, Akt, the tumor suppressor menin, and cell cycle proteins. Whether these pathways are involved in prolactin-mediated β-cell proliferation is unknown. Using the heterozygous prolactin receptor-null (Prlr(+/-)) mice, we isolated pancreatic islets from both Prlr(+/+) and Prlr(+/-) mice on d 0 and 15 of pregnancy and examined the expression levels of these signaling molecules. In the wild-type mice (Prlr(+/+)), both phospho-Jak2 and phospho-Akt expression in pancreatic islets increased during pregnancy, which were attenuated in the pregnant Prlr(+/-) mice. During pregnancy, menin expression was reduced by 50 and 20% in the Prlr(+/+) and the Prlr(+/-) mice, respectively, and the pregnant Prlr(+/-) mice had higher islet p18 levels than the Prlr(+/+) mice. Interestingly, between d 0 and 15 of pregnancy, expression of cyclin inhibitory protein p21(cip) was increased in the Prlr(+/+) mice, but this increase was blunted in the Prlr(+/-) mice. Lastly, we did not find any difference in the expression levels of cyclins D1, D2, and inhibitory kinases between the pregnant Prlr(+/+) and Prlr(+/-) mice. Therefore, we conclude that during pregnancy, placental hormones act through the prolactin receptor to increase β-cell mass by up regulating β-cell proliferation by engaging Jak2, Akt, menin/p18, and p21. Future studies will determine the relative contribution of these molecules in maintaining normal glucose homeostasis during pregnancy.

A critical problem within transcription factor families is how diverse regulatory programs are directed by highly related members. Androgen and glucocorticoid receptors (AR, GR) recognize a consensus DNA hormone response element (HRE), but they activate target genes with precise specificity, largely dependent on the promoter and cell context. We have assessed the role of different receptor domains in hormone-specific response by testing chimeras of AR and GR for their ability to activate the androgen-specific enhancer of the mouse sex-limited protein (Slp) gene. Although all of the mutant receptors activated simple HREs, only a few activated the androgen-specific element. One component shared by receptors functional on the AR-specific target was the AR DNA binding domain. Activation was not due to differential DNA affinity but rather to the AR DNA binding domain escaping suppression directed at the GR DNA binding domain in this enhancer context. A further mechanism increasing specific activation was cooperation of receptors at multiple and weak HREs, which was accentuated in the presence of both the AR N terminus and ligand binding domain. These domains together increased recognition of weak HREs, as demonstrated by in vitro DNase I footprinting and transactivation of mutant enhancers. Further, AR N-terminal subdomains reported to interact directly with the ligand binding domain relieved an inhibitory effect imposed by that domain. Therefore, functions intrinsic to AR augment steroid-specific gene activation, by evading negative regulation operating on the domains of other receptors and by enhancing cooperativity through intra-and inter-receptor domain interactions. These subtle distinctions in AR and GR behavior enforce transcriptional specificity established by the context of nonreceptor factors.

Genetically modified mouse strains derived from embryonic stem (ES) cells are powerful tools for gene function analysis. ES cells from the C57BL/6 mouse strain are not widely used to generate mouse models despite the advantage of a defined genetic background. We assessed genetic variation in six such ES cell lines with 275 SSLP markers. Compared to C57BL/6, Bruce4 differed at 34 SSLP markers and had significant heterozygosity on three chromosomes. BL/6#3 and Dale1 ES cell lines differed at only 3 SSLP makers. The C2 and WB6d ES cell lines differed at 6 SSLP markers. It is important to compare the efficiency of producing mouse models with available C57BL/6 ES cells relative to standard 129 mouse strain ES cells. We assessed genetic stability (the tendency of cells to become aneuploid) in 110 gene-targeted ES cell clones from the most widely used C57BL/6 ES cell line, Bruce4, and 710 targeted 129 ES cell clones. Bruce4 clones were more likely to be aneuploid and unsuitable for ES cell-mouse chimera production. Despite their tendency to aneuploidy and consequent inefficiency, use of Bruce4 ES cells can be valuable for models requiring behavioral studies and other mouse models that benefit from a defined C57BL/6 background.