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The normal products of the murine Steel (Sl) and Dominant white spotting (W) genes are essential for the development of melanocyte precursors, germ cells, and hematopoietic cells. The Sl locus encodes stem cell factor (SCF), which is the ligand of c-kit, a receptor tyrosine kinase encoded by the W locus. One allele of the Sl mutation, Sl17H, exhibits minor hematopoietic defects, sterility only in males, and a complete absence of coat pigmentation. The Sl17H gene encodes SCF protein which exhibits an altered cytoplasmic domain due to a splicing defect. In this paper we analyzed the mechanism by which the pigmentation phenotype in Sl17H mutant mice occurs. We show that in embryos homozygous for Sl17H the number of melanocyte precursors is severely reduced on the lateral neural crest migration pathway by e11.5 and can no longer be detected by e13.5 when they would enter the epidermis in wildtype embryos. The reduced number of dispersing melanocyte precursors correlates with a reduction of SCF immunoreactivity in mutant embryos in all tissues examined. Regardless of the reduced amount, functional SCF is present at the cell surface of fibroblasts transfected with Sl17H mutant SCF cDNA. Since SCF immunoreactivity normally accumulates in basolateral compartments of SCF-expressing embryonic epithelial tissues, we analyzed the localization of wildtype and Sl17H mutant SCF protein in transfected epithelial (MDCK) cells in vitro. As expected, wildtype forms of SCF localize to and are secreted from the basolateral compartment. In contrast, mutant forms of SCF, which either lack a membrane anchor or exhibit the Sl17H altered cytoplasmic tail, localize to and are secreted from the apical compartment of the cultured epithelium. We suggest, therefore, that the loss of melanocyte precursors prior to epidermal invasion, and the loss of germ cells from mature testis, can be explained by the inability of Sl17H mutant SCF to be targeted to the basolateral compartment of polarized epithelial keratinocytes and Sertoli cells, respectively.

Phospholipase A 2 mediates immediate early genes in cultured renal epithelial cells: Possible role of lysophospholipid. Background Exposure to high levels of oxalate induces oxidant stress in renal epithelial cells and produces diverse changes in cell function, ranging from cell death to cellular adaptation, as evidenced by increased DNA synthesis, cellular proliferation, and induction of genes associated with remodeling and repair. These studies focused on cellular adaptation to this oxidant stress, examining the manner by which oxalate exposure leads to increased expression of immediate early genes (IEGs). Specifically, our studies assessed the possibility that oxalate-induced changes in IEG expression are mediated by phospholipase A 2 (PLA 2 ), a common pathway in cellular stress responses. Methods Madin-Darby canine kidney (MDCK) cells were exposed to oxalate in the presence or absence of PLA 2 inhibitors: mepacrine and arachidonyl trifluoromethyl ketone (AACOCF 3 ). Expression of IEG (c- jun , egr -1, and c- myc ) mRNA was assessed by Northern blot analysis. PLA 2 activity was determined by measuring the release of [ 3 H]arachidonic acid (AA) from prelabeled cells. Results Oxalate exposure (1 to 1.5 mmol/L) induced time- and concentration-dependent increases in IEG mRNA. Treatment with mepacrine resulted in a 75 to 113% reduction of oxalate-induced c- jun , egr -1, and c- myc mRNA, while AACOCF 3 caused a 41 to 46% reduction of oxalate-induced c- jun and egr -1 mRNA. Of the two major byproducts of PLA 2 , only lysophosphatidylcholine (20 μmol/L) increased c- jun and egr -1 mRNA. In contrast, AA (25 μmol/L) attenuated the oxalate-induced increase in c- jun and egr -1 mRNA, presumably by inhibiting PLA 2 activity. Conclusions These findings suggest that PLA 2 plays a major role in oxalate-induced IEG expression in renal epithelial cells and that lysophospholipids might be a possible lipid mediator in this pathway.

An attempt was made to develop a method for the morphological and morphometric evaluation of fat inclusions in monolayer cell cultures. For this purpose we used electron microscopy (SEM and TEM) and Sudan black staining methods followed by morphometric evaluation techniques. MDCK (Madin-Darby-caninekidney) cells were treated with valproic acid (VPA; 10, 100, 1000 pglml medium for 22 hrs) and gentamicin (1, 10,50 pglml for 60 min). Sudan black staining revealed a dose-dependent increase in fat deposits after application of the two substances. SEM inspection showed differences in the size and distribution of the fat inclusions. TEM findings only partly allowed us to differentiate clearly between VPA- and gentamicin-induced fat inclusions.

We have used scanning electron microscopy (SEM) to examine the surface morphology of the renal epithelial cell lines MDCK and LLC-PK1 to determine the influence of alternative culture substrate conditions on cell polarity. We observed that regardless of physical culture conditions, cells establishes and maintained polarity, expressed by the characteristics of apical and basal surfaces. culture conditions did, however, influence the orientation of cell polarity in vitro. MDCK cells were grown within collagen gel, in which individual cells exhibited clonal growth to form fluid-filled epithelial cysts. The cells of MDCK-cysts were polarized with apical surface facing the lumen and basal surface against the surrounding collagen gel. This configuration made it possible to gain direct visual access, by SEM, to the basal surface by removing the supportive collagen lattice. The apical surface of MDCK-cysts was lined by short microvilli. Each cell possessed a solitary cilium. In comparison, the basal surface had few appendages, although cell boundaries were marked by interdigitating short processes. LLC-PK1 cells in monolayer culture bore solitary cilia and long microvilli at their apical surface. The basal surface of cells involved in dome formation was observed to possess only a sparse population of short, blunt processes. When LLC-PK1 cells were raised in stationary suspension culture or in monolayer atop non-culture grade plastic, they formed cysts with the cell apex facing the surrounding medium. These cells showed variable apical morphology. The cells of large, highly expanded cysts were often attenuated and had a relatively smooth apical surface. The basal surface of cells of fractured LLC-PK1 cysts commonly was also smooth, without prominent appendages.

The fate of pluripotent cells in early mouse embryos is controlled by graded Nodal signals that are activated by the endoproteases Furin and Pace4. Soluble forms of Furin and Pace4 cleave proNodal in vitro and after secretion in transfected cells, but direct evidence for paracrine activity in vivo is elusive. Here, we show that Furin and Pace4 are released by the extraembryonic microenvironment, and that they cleave a membrane-bound reporter substrate in adjacent epiblast cells and activate Nodal to maintain pluripotency. Secreted Pace4 and Furin also stimulated mesoderm formation, whereas endoderm was only induced by Pace4, correlating with a difference in the spatiotemporal distribution of these proteolytic activities. Our analysis of paracrine Furin and Pace4 activities and their in vivo functions significantly advances our understanding of how the epiblast is patterned by its microenvironment. Adding cell–cell communication to the pleiotropic portfolio of these proteases provides a new framework to study proprotein processing also in other relevant contexts.

Objective: Monitoring of influenza virus shedding and optimization of multiplicities of infection (MOI) is important in the investigation of a virus one step growth cycle and for obtaining a high yield of virus in vaccine development and conventional basic diagnostic methods. However, eluted infectious viruses may still be present immediately after virus inoculation and when cells are washed following virus cultivation which may lead to a false positive virus infectivity assay. Materials and Methods: In this experimental study, we investigated influenza virus progeny production in Madin-Darby canine kidney (MDCK) cells with five different MOI at determined time points. The results were analyzed by end point titration tests and immunofluorescence assay. Results: Higher titers of eluted virus were observed following a high MOI inoculation of virus in cell culture. Most probably, this was the result of sialic acid residues from viral hemagglutin in proteins that were cleaved by neuraminidase glycoproteins on the surface of the influenza virus, which promoted viral spread from the host cell to the culture supernatant or during endocytosis, where viruses recycle to the cell surface by recycling endosomes which culminated in virus shedding without replication. Conclusion: We demonstrated that the pattern of influenza virus progeny production was dose-dependent and not uniform. This production was influenced by several factors, particularly MOI. Understanding the exact features of viral particle propagation has a major impact in producing high virus yields in the development of vaccines. Use of lower MOI (0.01) could result in accurate, precise quantitative assays in virus diagnosis and titration methods.