[Abstract unavailable

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

Mitogen-activated protein kinases (MAPK) are serine-threonine protein kinases that are activated by diverse stimuli ranging from cytokines, growth factors, neurotransmitters, hormones, cellular stress, and cell adherence. Mitogen-activated protein kinases are expressed in all eukaryotic cells. The basic assembly of MAPK pathways is a three-component module conserved from yeast to humans. The MAPK module includes three kinases that establish a sequential activation pathway comprising a MAPK kinase kinase (MKKK), MAPK kinase (MKK), and MAPK. Currently, there have been 14 MKKK, 7 MKK, and 12 MAPK identified in mammalian cells. The mammalian MAPK can be subdivided into five families: MAPKerk1/2, MAPKp38, MAPKjnk, MAPKerk3/4, and MAPKerk5. Each MAPK family has distinct biological functions. In Saccharomyces cerevisiae, there are five MAPK pathways involved in mating, cell wall remodelling, nutrient deprivation, and responses to stress stimuli such as osmolarity changes. Component members of the yeast pathways have conserved counterparts in mammalian cells. The number of different MKKK in MAPK modules allows for the diversity of inputs capable of activating MAPK pathways. In this review, we define all known MAPK module kinases from yeast to humans, what is known about their regulation, defined MAPK substrates, and the function of MAPK in cell physiology.

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Ferroptosis is an iron-dependent, oxidative cell death, and is distinct from apoptosis, necrosis and autophagy. In this study, we demonstrated that lysosome disrupting agent, siramesine and a tyrosine kinase inhibitor, lapatinib synergistically induced cell death and reactive oxygen species (ROS) in MDA MB 231, MCF-7, ZR-75 and SKBr3 breast cancer cells over a 24 h time course. Furthermore, the iron chelator deferoxamine (DFO) significantly reduced cytosolic ROS and cell death following treatment with siramesine and lapatinib. Furthermore, we determined that FeCl3 levels were elevated in cells treated with siramesine and lapatinib indicating an iron-dependent cell death, ferroptosis. To confirm this, we treated cells with a potent inhibitor of ferroptosis, ferrastatin-1 that effectively inhibited cell death following siramesine and lapatinib treatment. The increase levels of iron could be due to changes in iron transport. We found that the expression of transferrin, which is responsible for the transport of iron into cells, is increased following treatment with lapatinib alone or in combination with siramesine. Knocking down of transferrin resulted in decreased cell death and ROS after treatment. In addition, ferroportin-1 (FPN) is an iron transport protein, responsible for removal of iron from cells. We found its expression is decreased after treatment with siramesine alone or in combination with lapatinib. Overexpression FPN resulted in decreased ROS and cell death whereas knockdown of FPN increased cell death after siramesine and lapatinib treatment. This indicates a novel induction of ferroptosis through altered iron regulation by treating breast cancer cells with a lysosome disruptor and a tyrosine kinase inhibitor.

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T lymphocytes require two signals to be activated. The antigen-specific T-ceUl receptor can deliver the first signal, while ligation of the T-oeU surface molecule CD28 by antibodies or its cognate ligands B7-1 (CD80) or B7-2 has been demonstrated to be sufficient for the delivery of the second signal. Signaling via CD28 and the T-cell receptor results (a) in their costimulation ofT cells to produce numerous lymphokines induding interleukin 2 and (u) Antigen-specific activation of T lymphocytes is under stringent control, which is achieved by the requirement for an antigen-specific signal and the delivery of a costimulatory signal (the two-signal hypothesis) (1). The antigen specificity is conferred by the antigen-specific T-cell receptor (TcR), which recognizes antigenic peptides in the context of the major histocompatibility complex class I and class II gene products (2). The recognition of antigen by the TcR results in the activation of a number of tyrosine kinases including members of the Src and Syk family, leading to downstream effects, including lymphokine production (3,4).Since the concentration of antigen is usually limiting, and as an additional mechanism to prevent aberrant activation, T cells require costimulation. This costimulation can be delivered by the binding of T-cell surface protein CD28 to its cognate ligands B7-1 (CD80) (5) and B7-2 (6-8) on antigenpresenting cells. This second signal serves to enhance production of key lymphokines required for the growth of activated T cells (5,9) and also can prevent anergy induction (10) and human immunodeficiency virus-primed apoptosis (11). The primary structure of CD28 indicates that this molecule has a short cytoplasmic tail (41 residues) with no obvious enzymatic activity (12). While there is an outline of the pathway by which the TcR signals, very little is known about signal transduction resulting from ligation of the costimulatory molecule CD28. However, it is clear that the signal delivered by CD28 differs from that given by the TcR in a number of ways, including sensitivity to cyclosporin A and rapamycin (5, 9). Since both pathways result in proteintyrosine phosphorylation (3,4,13,14), there may be subtle differences that are manifest in the differences in drug sensitivity.The Tec family of protein tyrosine kinases is a newly emerging family that includes the prototypical members mouse Tec I and Tec II and the products of the X-linked agammaglobulinemia gene BTK (Btk in the mouse), Drosophila Dsrc28 gene, and mouse Itk (formerly called Tsk and Emt), the human homologue of which has been assigned the name ITK (15)(16)(17)(18)(19)(20)(21)(22)(23). All the members of this family contain Src homology (SH) domains SH2 and SH3 but lack the negative regulatory tyrosine found in the Src family members. They also contain extensive N-terminal regions, which differ from that seen in Src family members (15)(16)(17)(18)(19)(20)(21)(22)(23). While the product of the BTK gene has been shown genetically to be essential for proper B-cell development (1...