Three-dimensional analysis of the islet vasculature.

The pancreatic islets of Langerhans are highly vascularized structures scattered throughout the pancreas that contain a capillary network 5-10 times denser than that of the exocrine pancreas. A simple method for three-dimensional (3D) analysis of this intricate intraislet vasculature has been difficult because of the intrinsic opacity of the pancreas. We developed a whole-mount imaging technique that allows relatively easy visualization of the islet vasculature. In combination with confocal microscopy and the use of 3D imaging software, we were able to readily reconstruct the 3D architecture of an islet, allowing delineation of the islet volume, length of the intraislet vessels, and the number of vessel branch-points. This technique allows for straightforward 3D image analysis that may help toward understanding islet function.

Semiquantitative polymerase chain reaction in RNase-producing tissues: Analysis of the developing pancreas.

BACKGROUND/PURPOSE: Many studies in pediatric surgical research use a quantitative analysis of gene expression in microscopic quantities of tissue. The authors describe an analysis of the beta-tubulin mRNA content of the embryonic pancreas, which contains abundant endogenous RNases. A detailed analysis of this RNase-containing system will provide a good template for analysis of other potentially simpler systems. METHODS: Embryonic mouse pancreases were harvested at serial gestational ages. DAPI nuclear staining allowed for counting of cells. cDNA was amplified using a fluoresceinated primer and the normalized fluorescence determined. Known numbers of molecules were amplified in parallel as a standard control. RESULTS: The number of cells increased from 38,000 to 2,700,000 between embryonic day 10.5 (E10.5) and E18.5. mRNA for beta-tubulin did not increase proportionately. Assuming a yield of 100% at E10.5 when no RNases are present, the yield of expected mRNA was 65.3% at E12.5, 13.8% at E15.5, and 0.9% at E18.5, presumably because of the appearance of RNases. CONCLUSIONS: Several parameters must be considered in performing semiquantitative reverse transcription polymerase chain reaction: (1) the yield of RNA based on the projected amount of mRNA, (2) the number of cells in the tissue, and (3) a known number of template molecules amplified in parallel.

Retinoid signaling directs secondary lineage selection in pancreatic organogenesis.

BACKGROUND/PURPOSE: Retinoid signaling plays an important role in many differentiation pathways. Retinoid signaling has been implicated in the induction of differentiation by pancreatic ductal cancer cell lines and in patients with pancreatic cancer. The authors wished to better understand the role of retinoid signaling in pancreatic development. METHODS: Embryonic pancreas was harvested from mice at serial gestational ages and immunohistochemical analysis was performed for retinoic acid receptors (RAR-alpha, RAR-beta, RAR-gamma), and retinoid X receptors (RXR-alpha, RXR-beta, and RXR-gamma). Also, early embryonic pancreases were cultured for 7 days with exogenous 9-cis retinoic acid (9cRA) or all-trans retinoic acid (atRA) and analyzed histologically and immunohistochemically. RESULTS: Retinoid receptors were expressed in a lineage-specific distribution, with stronger expression for many in the exocrine compartment. The receptors were not often expressed until late gestation. Exogenous 9cRA induced predominantly ducts instead of acini, plus more mature endocrine (islet) architecture. Exogenous atRA induced predominantly acini instead of ducts, with no apparent endocrine effect. CONCLUSIONS: Retinoids may have an important role in pancreatic differentiation, with a particular effect on secondary lineage selection between ductal and acinar phenotype. Because the control of ductal versus acinar differentiation has been implicated strongly in the pathogenesis of pancreatic ductal carcinoma, these results may lay the groundwork for studies in the mechanism of induced differentiation of pancreatic ductal cancer by retinoids.

Transfection alters ion transport in MDCK cells.

In the course of an investigation into the effect of Tamm-Horsfall protein (THP) on ion transport, we performed stable transfection of THP into MDCK cells using the SV40 or the cytomegalovirus (CMV) promoter. As controls, we transfected MDCK cells with an "empty" plasmid containing SV40 or CMV promoter but without THP cDNA. In another set of controls, we subjected cells to transfection procedures without DNA (mock transfection). K influx was not altered in cells subjected to mock transfection procedures without DNA, but both ouabain sensitive (OS) and ouabain resistant (OR) components of K influx were diminished in cells transfected with THP cDNA using either SV40 or CMV promoter. However, K influx was also reduced in cells transfected with a control plasmid containing either the SV40 promoter alone, or the CMV promoter alone, without the THP cDNA. Thus, the transport alterations were caused by transfection and not by THP. The reduction in ouabain-sensitive K influx was accompanied by a proportional reduction in the abundance of Na-K pump units as assessed by [3H] ouabain binding. [3H] bumetanide binding, a measure of the number of functioning NaK2Cl cotransporter sites, was reduced pari passu with the reduction in bumetanide-sensitive K influx. These results highlight the possibility that alterations in properties of transfected cells may not be solely due to the presence of transfected protein, but the result of some process associated with transfection itself. Without appropriate controls to evaluate this possibility, results of transfection studies are subject to potentially faulty and misleading interpretation.

Nucleotide sequence and peptide motifs of mouse uromodulin (Tamm-Horsfall protein)--the most abundant protein in mammalian urine.

The mouse uromodulin cDNA sequence was sequenced. The predicted peptide sequence is 642 amino acids long and contains several modular components including four epidermal growth factor like repeats, one betaglycan-like domain (ZP domain), and a consensus sequence for attachment of a glycosyl-phosphatidyl-inositol anchor. An arginine-glycine-aspartate tripeptide reported for rat and human sequence is absent in the mouse. There are several potential sites for post-translational modification.