Background: Breast cancer is thought to arise in mammary epithelial stem cells. However, the identity of these stem cells is unknown. Methods:Studies in the haematopoetic and muscle systems show that stem cells have the ability to efflux the dye Hoechst 33342. Cells with this phenotype are referred to as the side population (SP). We have adapted the techniques from the haematopoetic and muscle systems to look for a mammary epithelial SP. Results:Of mammary epithelial cells isolated from both the human and mouse mammary epithelia, 0.2-0.45% formed a distinct SP. The SP was relatively undifferentiated but grew as typical differentiated epithelial clones when cultured. Transplantation of murine SP cells at limiting dilution into cleared mammary fat pads generated epithelial ductal and lobuloalveolar structures.Conclusion: These data demonstrate the existence of an undifferentiated SP in human and murine mammary epithelium. Purified SP cells are a live single-cell population that retain the ability to differentiate in vitro and in vivo. Studies of haematopoetic cells have suggested that the SP phenotype constitutes a universal stem cell marker. This work therefore has implications for mammary stem cell biology.

We have investigated the cellular basis for the effects of oxidative stress on stomatal behavior using stomatal bioassay and ratio photometric techniques. Two oxidative treatments were employed in this study: (a) methyl viologen, which generates superoxide radicais, and (b) H202. Both methyl viologen and H 2 0 2 inhibited stomatal opening and promoted stomatal closure. At concentrations 510-5 M, the effects of methyl viologen and H 2 0 2 on stomatal behavior were reversible and were abolished by 2 mM ECTA or 10 PM verapamil. I n addition, at 10-5 M, i.e. the maximum concentration at which the effects of the treatments were prevented by ECTA or verapamil, methyl viologen and H 2 0 2 caused an increase in guard cell cytosolic free Ca2+ ([Ca'+l,), which was abolished in the presence of ECTA. Therefore, at low concentrations of methyl viologen and H202, removal of extracellular Ca2+ prevented both the oxidative stress-induced changes in stomatai aperture and the associated increases i n [Ca2+],. This suggests that in this concentration range the effects of the treatments are Ca'+-dependent and are mediated by changes in [Ca2+],. I n contrast, at concentrations of methyl viologen and H,O, >10-5 M, ECTA and verapamil had no effect. However, in this concentration range the effects of the treatments were irreversible and correlated with a marked reduction in membrane integrity and guard cell viability. This suggests that at high concentrations the effects of methyl viologen and H202 may be due to changes in membrane integrity. The implications of oxidative stress-induced increases in [Ca2+Ii and the possible disruption of guard-cell ca'+ homeostasis are discussed in relation t o the processes of Ca'+-based signal transduction in stomatal guard cells and the control of stomatal aperture.

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