Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells

The membrane localization of the plasma membrane Ca²⁺-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.     

Specificity of protein synthesis by bacterial ribosomes and initiation factors: Absence of change after phage T4 infection

Using several natural messenger RNA's—f2 RNA, Qβ RNA, T7 RNA, T4 early mRNA, T4 late mRNA and Escherichia coli RNA—ribosomes isolated from cells either 5 or 12 minutes after T4 infection direct synthesis of only 35 to 70% as much protein as do ribosomes from uninfected cells. However, with poly(U) or formaldehyde-treated f2 RNA message, both types of ribosomes work equally well. Experiments mixing salt-washed ribosomes and initiation factors from these cells show, in agreement with work of others, that the reduction with natural messages is due only to changes in the initiation factors.     

Subcellular localization of enterokinase (Enteropeptidase EC 3.4.21.9) in rat small intestine

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3 in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (M_r, 2·10^?6) and two larger peaks of free enzyme (M_r, 3·10⁵ and 9·10⁵). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.     

Cumulative Risks of Foster Care Placement for Danish Children

Although recent research suggests that the cumulative risk of foster care placement is far higher for American children than originally suspected, little is known about the cumulative risk of foster care placement in other countries, which makes it difficult to gauge the degree to which factor foster care placement is salient in other contexts. In this article, we provide companion estimates to those provided in recent work on the US by using Danish registry data and synthetic cohort life tables to show how high and unequally distributed the cumulative risk of foster care placement is for Danish children. Results suggest that at the beginning of the study period (in 1998) the cumulative risk of foster care placement for Danish children was roughly in line with the risk for American children. Yet, by the end of the study period (2010), the risk had declined to half the risk for American children. Our results also show some variations by parental ethnicity and sex, but these differences are small. Indeed, they appear quite muted relative to racial/ethnic differences in these risks in the United States. Last, though cumulative risks are similar between Danish and American children (especially at the beginning of the study period), the age-specific risk profiles are markedly different, with higher risks for older Danish children than for older American children.