Greening the RDP: people, environment, development: report of proceedings [of the] Environmental Justice Networking Forum Constitutive Conference, Kempton Park Conference Centre, 25-27 November 1994

The Environmental Justice Networking Forum’s first national conference had three major objectives: to constitute the organisation on a national basis; to make policy recommendations regarding the implementation of the RDP both for submission to government and as a guide to action by EJNFparticipants; to build and disseminate the workof thelDRC/ANC/ COSATU/ SACP/ SANCO International Mission on Environmental Policy (referred to here as the Mission). EJNF was in itiated at the Earthlife Africa International Environment Conference in 1992 at Pietermaritzburg. That conference mandated an interim national steering committee to guide a process of establishing an organised voice within civil society for environmental justice. It stipulated that the organisation should be formed on a regional basis leading up to the national constitution of EJNF at a national conference. The EJNF conference is thus the culmination of a two year process. During that time, meetings were held to establish EJNF in six regions: Gauteng, Northern Transvaal, Western Cape, Eastern Cape, KwaZulu- Natal and OFS. Participating organisations include women’s, rural, youth, religious and environmental organisations, unions, civics and service NGOs. Each region sent delegations to the national conference. Two other regions, Eastern Transvaal and Northern Cape, also sent delegations which will form the focus groups for establishing EJNF in those regions. Regional EJNF participant organisations also elected members to the national steering committee. They took office at the constitutive conference. The minutes of the constitutive session of the conference are not included here but are available from the EJNF national office. The EJNF delegates were joined by a number of guest delegates for the conference on Greening the RDP. They included members of national organisations which represent or work with the constitutuencies which EJNF is developing and researchers working in the sectors covered by the conference. Government was represented by Ministers Kader Asmal (Water Affairs) and Derek Hanekom (Land Affairs), by provincial MECs, by members of standing committees in parliament and provincial legislatures andby ministry or department officials

Functional and molecular characterisation of mammary side population cells

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

Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection

Background: Laser-capture microdissection (LCM) that enables the isolation of specific cell populations from complex tissues under morphological control is increasingly used for subsequent gene expression studies in cell biology by methods such as real-time quantitative PCR (qPCR), microarrays and most recently by RNA-sequencing. Challenges are i) to select precisely and efficiently cells of interest and ii) to maintain RNA integrity. The mammary gland which is a complex and heterogeneous tissue, consists of multiple cell types, changing in relative proportion during its development and thus hampering gene expression profiling comparison on whole tissue between physiological stages. During lactation, mammary epithelial cells (MEC) are predominant. However several other cell types, including myoepithelial (MMC) and immune cells are present, making it difficult to precisely determine the specificity of gene expression to the cell type of origin. In this work, an optimized reliable procedure for producing RNA from alveolar epithelial cells isolated from frozen histological sections of lactating goat, sheep and cow mammary glands using an infrared-laser based Arcturus Veritas LCM (Applied Biosystems®) system has been developed. The following steps of the microdissection workflow: cryosectioning, staining, dehydration and harvesting of microdissected cells have been carefully considered and designed to ensure cell capture efficiency without compromising RNA integrity.[br/] Results: The best results were obtained when staining 8 μm-thick sections with Cresyl violet® (Ambion, Applied Biosystems®) and capturing microdissected cells during less than 2 hours before RNA extraction. In addition, particular attention was paid to animal preparation before biopsies or slaughtering (milking) and freezing of tissue blocks which were embedded in a cryoprotective compound before being immersed in isopentane. The amount of RNA thus obtained from ca.150 to 250 acini (300,000 to 600,000 μm2) ranges between 5 to 10 ng. RNA integrity number (RIN) was ca. 8.0 and selectivity of this LCM protocol was demonstrated through qPCR analyses for several alveolar cell specific genes, including LALBA (α-lactalbumin) and CSN1S2 (αs2-casein), as well as Krt14 (cytokeratin 14), CD3e and CD68 which are specific markers of MMC, lymphocytes and macrophages, respectively.[br/] Conclusions: RNAs isolated from MEC in this manner were of very good quality for subsequent linear amplification, thus making it possible to establish a referential gene expression profile of the healthy MEC, a useful platform for tumor biomarker discovery