The Drosophila actin nucleator DAAM is essential for left-right asymmetry.

Left-Right (LR) asymmetry is essential for organ positioning, shape and function. Myosin 1D (Myo1D) has emerged as an evolutionary conserved chirality determinant in both Drosophila and vertebrates. However, the molecular interplay between Myo1D and the actin cytoskeleton underlying symmetry breaking remains poorly understood. To address this question, we performed a dual genetic screen to identify new cytoskeletal factors involved in LR asymmetry. We identified the conserved actin nucleator DAAM as an essential factor required for both dextral and sinistral development. In the absence of DAAM, organs lose their LR asymmetry, while its overexpression enhances Myo1D-induced de novo LR asymmetry. These results show that DAAM is a limiting, LR-specific actin nucleator connecting up Myo1D with a dedicated F-actin network important for symmetry breaking.

Isolation and cultivation of metabolically competent alveolar epithelial cells from A/J mice.

The A/J mouse strain is used in lung cancer studies. To enable mechanistic investigations the isolation and cultivation of alveolar epithelial cells (AECs) is desirable. Based on four different protocols dispase digestion of lung tissue was best and yielded 9.3 ± 1.5 × 10(6) AECs. Of these 61 ± 13% and 43 ± 5% were positive for AP and NBT staining, respectively. Purification by discontinuous Percoll gradient centrifugation did not change this ratio; however, reduced the total cell yield to 4.4 ± 1.1 × 10(6) AECs. Flow cytometry of lectin bound AECs determined 91 ± 7% and 87 ± 5% as positive for Helix pomatia and Maclura pomifera to evidence type II pneumocytes. On day 3 in culture the ethoxyresorufin-O-demethylase activity was 251 ± 80 pmol/4 h × 1.5 × 10(6) and the production of androstenedione proceed at 243.5 ± 344.4 pmol/24 h × 1.5 × 10(6) AECs. However, 6-α, 6-ß and 16-ß-hydroxytestosterone were produced about 20-fold less as compared to androstenedione and the production of metabolites depended on the culture media supplemented with 2% mouse serum or 10% FCS. Finally, by RT-PCR expression of CYP genes was confirmed in lung tissue and AECs; a link between testosterone metabolism and CYP2A12, 3A16 and 2B9/10 expression was established. Taken collectively, AECs can be successfully isolated and cultured for six days while retaining metabolic competence.

How useful are clinical liver function tests in in vitro human hepatotoxicity assays?

In preclinical hepatotoxicity testing cell based assays are frequently employed. However, prediction of clinical drug induced liver injury (DILI) remains a major challenge. Here we examined the usefulness of frequently employed markers of hepatocellular injury in cultures of primary human hepatocytes (PHH) in response to treatment with either paracetamol, rifampicin, petadolex and/or amiodarone. The changes in the metabolic competency (urea and albumin) and cellular injury (AST, ALT, ALP, LDH, γGT and succinate dehydrogenase) were determined at therapeutic and above drug concentrations as to evaluate the utility of these markers in in vitro systems. Initially, treatment of PHH with any of the drugs caused a statistically significant reduction in enzyme activities to suggest a switch from basic amino acid metabolism towards induced detoxification. However, treatment for prolonged periods of time caused cytolysis, as evidenced by the significant rise in extracellular LDH and the concomitant increase in ALT and AST activity. Notably, amongst the various endpoints studied, urea was best to demonstrate dose dependent metabolic stress, while other markers of hepatocellular injury were highly variable. Taken collectively, urea measurement proofed to be robust in predicting hepatocellular stress; therefore it should be included in preclinical testing strategies for an improved prediction of DILI.

CalpB modulates border cell migration in Drosophila egg chambers.

BACKGROUND: Calpains are calcium regulated intracellular cysteine proteases implicated in a variety of physiological functions and pathological conditions. The Drosophila melanogaster genome contains only two genes, CalpA and CalpB coding for canonical, active calpain enzymes. The movement of the border cells in Drosophila egg chambers is a well characterized model of the eukaryotic cell migration. Using this genetically pliable model we can investigate the physiological role of calpains in cell motility. RESULTS: We demonstrate at the whole organism level that CalpB is implicated in cell migration, while the structurally related CalpA paralog can not fulfill the same function. The downregulation of the CalpB gene by mutations or RNA interference results in a delayed migration of the border cells in Drosophila egg chambers. This phenotype is significantly enhanced when the focal adhesion complex genes encoding for α-PS2 integrin ( if), ß-PS integrin (mys) and talin (rhea) are silenced. The reduction of CalpB activity diminishes the release of integrins from the rear end of the border cells. The delayed migration and the reduced integrin release phenotypes can be suppressed by expressing wild-type talin-head in the border cells but not talin-head(R367A), a mutant form which is not able to bind ß-PS integrin. CalpB can cleave talin in vitro, and the two proteins coimmunoprecipitate from Drosophila extracts. CONCLUSIONS: The physiological function of CalpB in border cell motility has been demonstrated in vivo. The genetic interaction between the CalpB and the if, mys, as well as rhea genes, the involvement of active talin head-domains in the process, and the fact that CalpB and talin interact with each other collectively suggest that the limited proteolytic cleavage of talin is one of the possible mechanisms through which CalpB regulates cell migration.