Phosphatidylserine is localized exclusively to the inner leaflet of the membrane lipid bilayer of most cells, including erythrocytes. This asymmetric distribution is critical for the survival of erythrocytes in circulation since externalized phosphatidylserine is a phagocytic signal for splenic macrophages. Flippases are P-IV ATPase family proteins that actively transport phosphatidylserine from the outer to inner leaflet. It has not yet been determined which of the 14 members of this family of proteins is the flippase in human erythrocytes. Herein, we report that ATP11C encodes a major flippase in human erythrocytes, and a genetic mutation identified in a male patient caused congenital hemolytic anemia inherited as an X-linked recessive trait. Phosphatidylserine internalization in erythrocytes with the mutant ATP11C was decreased 10-fold compared to that of the control, functionally establishing that ATP11C is a major flippase in human erythrocytes. Contrary to our expectations phosphatidylserine was retained in the inner leaflet of the majority of mature erythrocytes from both controls and the patient, suggesting that phosphatidylserine cannot be externalized as long as scramblase is inactive. Phosphatidylserine-exposing cells were found only in the densest senescent cells (0.1% of total) in which scramblase was activated by increased Ca 2+ concentration: the percentage of these phosphatidylserine-exposing cells was increased in the patient's senescent cells accounting for his mild anemia. Furthermore, the finding of similar extents of phosphatidylserine exposure by exogenous Ca 2+ -activated scrambling in both control erythrocytes and the patient's erythrocytes implies that suppressed scramblase activity rather than flippase activity contributes to the maintenance of phosphatidylserine in the inner leaflet of human erythrocytes.

The expression of granulocyte colony-stimulating factor (G-CSF) mRNA was studied in human non-hematopoietic tumors, including 18 cases of lung cancers 10 cases of stomach cancers, three cases of glioblastomas, and one case each of breast phyllode sarcoma, thyroid cancer, and hepatocellular carcinoma. Northern blot analysis detected G-CSF mRNA in two of the lung cancer cases, in one of the glioblastoma cases, and in both the breast phyllode sarcoma and hepatocellular carcinoma cases. Since G-CSF receptors were not detected on the tumor cells by 125I-G-CSF binding assay, G-CSF autocrine loop are probably not involved in the growth of these G-CSF-producing tumors. Interestingly, granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA was concomitantly expressed in most of these G-CSF-producing tumors. No major gene deletions or rearrangements of G-CSF and GM-CSF genes were demonstrated by Southern blot analysis in the tumors expressing G-CSF and GM-CSF mRNAs except for one of the glioblastomas (G3) in which one chromosome 17 allele was deleted. Although the mechanism of the concomitant expression of G-CSF and GM-CSF mRNA is unknown, relatively high frequency of this phenomenon suggests the presence of common transcriptional factors acting on regulatory regions of G-CSF and GM-CSF genomes.

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein hormone that specifically stimulates both production and functional activation of neutrophils, while interferon-a (IFN-a) is known to suppress myelopoiesis, including neutrophil production in vivo and in vitro. On a possibility that IFN-a may operate as one of the inhibitoryfeedback factors in neutropoiesis, we examined whether neutrophils produce IFN-a in response to G-CSF. Northern blot analysis showed that messenger RNA (mRNA) for human IFN-a 1 became detectable time-dependently in highly purified human neutrophils incubated with purified recombinant human G-CSF (rhG-CSF). But such transcription was not by the conventional method^.'^ Pooled culture supernatant was assayed for IFN content using a human IFN-a radioimmunoassay kit (DAINADOT Co Ltd, Tokyo, Japan) according to its instructions. Cell culture. Northern blot analysis.IFN-a assay.

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Background: Allergen sensitization through a disrupted skin barrier appears to play a prominent role in the development of atopic diseases, including allergic asthma. The role of the genetic background in immunological and physiological phenotypes induced by epicutaneous sensitization is undetermined. Methods: BALB/c and C57BL/6 mice were sensitized either epicutaneously by patch application of ovalbumin (OVA) or systemically by intraperitoneal injection of OVA with alum before exposure to aerosolized OVA. The concentrations of OVA-specific immunoglobulin in serum and cytokines in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay. The severity of airway inflammation was evaluated by cell counts in BALF, and bronchial responsiveness to methacholine was measured by the flexiVent system. Results: The production of OVA-specific IgG1 and IgE was greater in the epicutaneously sensitized BALB/c than C57BL/6 mice. In contrast, both eosinophilic airway inflammation and bronchial responsiveness to methacholine were more prominent in the C57BL/6 than in the BALB/c mice. The concentrations of interleukin-4 increased significantly in the BALF from C57BL/6 mice only. No between-strain differences were observed after intraperitoneal sensitization. Conclusions: The C57BL/6 mouse is a more appropriate model than the BALB/c mouse to study the relationship between skin barrier dysfunction and the pathogenesis of allergic asthma.