The essential oils of oregano and thyme are active against a number of food-borne pathogens, such as Escherichia coli O157:H7. Carvacrol is one of the major antibacterial components of these oils, and p-cymene is thought to be its precursor in the plant. The effects of carvacrol and p-cymene on protein synthesis in E. coli O157:H7 ATCC 43895 cells were investigated. Bacteria were grown overnight in Mueller-Hinton broth with a sublethal concentration of carvacrol or p-cymene, and their protein compositions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by Western blotting. The presence of 1 mM carvacrol during overnight incubation caused E. coli O157:H7 to produce significant amounts of heat shock protein 60 (HSP60) (GroEL) (P < 0.05) and inhibited the synthesis of flagellin highly significantly (P < 0.001), causing cells to be aflagellate and therefore nonmotile. The amounts of HSP70 (DnaK) were not significantly affected. p-Cymene at 1 mM or 10 mM did not induce HSP60 or HSP70 in significant amounts and did not have a significant effect on flagellar synthesis. Neither carvacrol (0.3, 0.5, 0.8, or 1 mM) nor p-cymene (0.3, 0.5, or 0.8 mM) treatment of cells in the mid-exponential growth phase induced significant amounts of HSP60 or HSP70 within 3 h, although numerical increases of HSP60 were observed. Motility decreased with increasing concentrations of both compounds, but existing flagella were not shed. This study is the first to demonstrate that essential oil components induce HSP60 in bacteria and that overnight incubation with carvacrol prevents the development of flagella in E. coli O157:H7.

IntroductionCells that are going into apoptosis expose phosphatidylserine (PS) on their surface that serves as an "eat me" signal for both professional phagocytes (such as macrophages, B lymphocytes, and immature dendritic cells) and nonprofessional phagocytes (such as fibroblasts and epithelial cells). 1 Phagocytes recognize apoptotic cells directly via the PS receptor or indirectly via ␣ v -integrins that are present on the phagocyte surface. 2 In the latter process, ␣ v ␤ 3 and ␣ v ␤ 5 integrins recognize PS through an opsonin called lactadherin, also known as milk fat globule epidermal growth factor-8 (MFG-E8). 3,4 This opsonin contains a PS-binding C-domain and an RGD-motif present in the epidermal growth factor domain, which binds to ␣ v ␤ 3 and ␣ v ␤ 5 integrins. 2,[5][6][7] Rapid clearance of apoptotic cells prevents the release of potentially harmful or immunogenic intracellular materials from these cells. 8 Lactadherin-deficient mice have shown impaired phagocytosis of apoptotic cells, induction of autoimmune diseases, and autoantibody production. 9,10 ␣ v -Integrins are densely present on macrophages and dendritic cells and play an important role in phagocytosis. 11 Notably, these integrins are also overexpressed on angiogenic endothelium 12 where they play an important role in cell adhesion and migration. 13,14 Recently, Silvestre et al discovered that lactadherin is expressed in and around (angiogenic) blood vessels in an ischemic hind limb model and that the protein seems to play a crucial role in vascular endothelial growth factor (VEGF)-dependent neovascularization. 15 As angiogenesis is also a hallmark of tumor growth and tumor angiogenic endothelium expresses ␣ v -integrins, in this study we have explored the possible role of lactadherin-induced phagocytosis by angiogenic tumor endothelial cells. Methods Cell linesB16.F10 murine melanoma cells (ATCC, Manassas, VA) were cultured on regular FCS-enriched DMEM. Human umbilical vein endothelial cells (HUVECs) were cultured on EGM-2 endothelial cell growth medium-2 (Cambrex, East Rutherford, NJ) consisting of EBM-2 medium supplemented with an EGM-2 bullet kit (containing growth factors, 2% FBS, and antibiotics). HUVECs were used as an ␣ v ␤ 3 -expressing model for proliferating endothelium to a maximal passage number of 8. Lactadherin purificationBovine lactadherin was purified as previously described. 16 Purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequencing and shown to be Submitted June 8, 2007; accepted February 13, 2008. Prepublished online as Blood First Edition paper, February 21, 2008 DOI 10.1182 DOI 10. /blood-2007 The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 12, 2018. by guest www.bloodjournal.org From 97% to...

To study the mode of action of moisturizers on human skin, hydrophilic moisturizers in water and neat lipophilic moisturizers were applied on excised skin for 24 h at 32 degrees C. Samples of the treated skin were subsequently visualized in a cryoscanning electron microscope. The stratum corneum (SC) appeared as a region of swollen corneocytes (the swollen region) sandwiched between two layers of relatively dry corneocytes (the upper and lower non-swelling regions respectively). Lipophilic moisturizers increased the water content of the SC, whereas hydrophilic moisturizers can also reduce the water content of the SC. When focusing on the effect of the moisturizers on the three different regions, it was observed that cells in the swelling region are most sensitive to the application of the moisturizers and that the change in SC thickness is most influenced by the change in the thickness of the swelling region. Summarizing, SC cells are not equally sensitive to moisturizer application: centrally located corneocytes are more sensitive than corneocytes in the upper and the lowest regions of the SC.

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Rab3D is a small GTP-binding protein associated with secretory vesicles in various exocrine and endocrine cells, where it has been implicated in regulated exocytosis. Data obtained previously in pancreas have suggested that rab3D is involved in the coating of secretory granules with filamentous actin. In the present study we employed Western blot analysis, immunofluorescence, and immunoelectron microscopy to examine the distribution of rab3D in rat lung. Rab3D immunoreactivity was detected in bronchiolar Clara cells and alveolar epithelial type II (AET-II) cells. In both cell types, rab3D displayed preferential localization to secretory vesicles that were identified using specific antibodies against Clara Cell Secretory Protein and p180 lamellar body protein, respectively. Interestingly, rab3D was associated with only 24% of the lamellar bodies in AET-II cells. Rab3D-positive lamellar bodies were typically in close proximity of the apical plasma membrane, where exocytosis occurs. Another subpopulation of lamellar bodies, constituting only 2%, was not only rab3D-positive but could also be labeled with the filamentous-actin probe phalloidin. A third subpopulation, constituting 9%, displayed actin coating without rab3D staining. We propose that these three lamellar body subpopulations represent consecutive intermediates along the regulated exocytotic pathway, implying that rab3D release and actin coating are intimately linked processes.