A Contiguous Compartment Functions as Endoplasmic Reticulum and Endosome/Lysosome in Giardia lamblia

The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and functional enzyme assays, we now show that in an early-diverging protozoan, Giardia lamblia, endocytosis and subsequent degradation of exogenous proteins occur in the ER or in an adjacent and communicating compartment. The Giardia endomembrane system is simple compared to those of typical eukaryotes. It lacks peroxisomes, a classical Golgi apparatus, and canonical lysosomes. Giardia orthologues of mammalian lysosomal proteases function within an ER-like tubulovesicular compartment, which itself can dynamically communicate with clathrin-containing vacuoles at the periphery of the cell to receive endocytosed proteins. These primitive characteristics support Giardia''s proposed early branching and could serve as a model to study the compartmentalization of endocytic and lysosomal functions into organelles distinct from the ER. This system also may have functional similarity to the retrograde transport of toxins and major histocompatibility complex class I function in the ER of mammals.A key event in the evolution of eukaryotic cells was the compartmentalization of cellular functions into distinct organelles responsible for protein synthesis, sorting, secretion, endocytosis, and degradation (38). However, it is clear from ultrastructural and biochemical analysis of many eukaryotic cells that these functionally distinct compartments often share common aspects of biogenesis and function and, in some cases, a common tubulovesicular network (TVN) (2). For example, one current debate concerns a putative role for the endoplasmic reticulum (ER) in phagocytosis (11, 42). Gagnon et al. (11) proposed that the ER was involved in direct uptake of material from the extracellular environment via fusion with the plasma membrane. This hypothesis was based on the presence of ER markers at the initial stage of phagosome formation in mammalian macrophages. Touret et al. (42), however, found no evidence for direct ER-plasma membrane communication in either macrophages or dendritic cells. Nevertheless, the concept of pluripotent functions for the ER was left unresolved, and these studies underscore the potential for an ER function in phagocytosis or endocytosis, particularly in regard to antigen processing for major histocompatibility complex (MHC) class I presentation. Furthermore, there are intriguing examples of exogenous toxins and viruses entering mammalian cells via the ER (19, 36). Because cellular compartmentalization is a defining eukaryotic trait, clues from early-diverging eukaryotic cells could provide valuable insights into the way in which compartmentalization and discrete organelle functions evolved.Giardia evolutionary status continues to be a matter of debate. Either Giardia lamblia is one of the earliest branches of the eukaryotic tree, with an estimated point of divergence of 1.7 to 2.1 billion years (3, 16, 17, 35, 38), or it is a very simple cell that has lost endomembrane complexity and classic organelle morphology by evolutionary reduction (6). Giardia has a simple life cycle that includes a replicating trophozoite stage and, under certain environmental conditions, an infectious, environmentally resistant cyst. The cyst form allows the replicative trophozoite to persist under conditions of desiccation outside the host and in harsh chemical environments like the host stomach. Following passage through the acidic stomach into the alkaline duodenum, the trophozoite form excysts and resides in the upper small intestine of its vertebrate host, where it opportunistically scavenges nutrients by uncharacterized endocytic pathways.The endomembrane system of the vegetative trophozoite form of Giardia lacks complexity compared to typical eukaryotic cells. In many extant eukaryotic cells, the majority of secretory and organelle-resident proteins are delivered by cotranslational translocation to the ER lumen, prior to maturation and subsequent targeting to the Golgi apparatus (2). In Giardia, no morphological equivalent of the classic mammalian-cell Golgi apparatus has been identified, and the transient secretory pathway for cyst wall synthesis is induced only under specific conditions (13, 14, 34). Giardia does contain two nuclei, a glycogen-rich cytoplasm, acidified peripheral vacuoles (PVs), and a labyrinthine TVN, segments of which are decorated with ribosomes, consistent with rough ER (5, 21, 22). Giardia also contains a multigene family of cysteine endoproteases that are orthologous to the cathepsin L and cathepsin B found in lysosomes of higher organisms and are therefore useful markers of cell compartments where protein degradation takes place (29, 46; Giardia genome project [http://www.giardiadb.org]). Due to the limited availability of established ER/endocytic subcompartment markers and the lack of classical genetic techniques, the discrete endocytic pathway of Giardia has not been fully characterized. Despite preliminary reports, RNA interference has not been established as a reliable and consistent genetic approach (43). In spite of these limitations, we were able to use reporter gene constructs, organelle-specific markers, and functional protease cytochemistry to show that endocytosis and degradation of exogenous proteins takes place in the ER-like TVN. Such primitive characteristics of Giardia''s endomembrane system support Giardia''s proposed early branching and could be used as an analogous model to study the compartmentalization of endocytic/lysosomal functions into organelles distinct from the ER. Alternatively, Giardia may have had a more complex and “modern” endocytic system that has been lost by evolutionary reduction (6). These observations also have important implications for recent theories of pluripotent mammalian ER functions, including its roles in phagocytosis, entry of toxins and viruses, and MHC class I function (7, 19, 36).     

Cytotherapy with naive rat umbilical cord matrix stem cells significantly attenuates growth of murine pancreatic cancer cells and increases survival in syngeneic mice

Background aimsPancreatic cancer, sometimes called a ‘silent killer’, is one of the most aggressive human malignancies, with a very poor prognosis. It is the fourth leading cause of cancer-related morbidity and mortality in the USA.MethodsA mouse peritoneal model was used to test the ability of unengineered rat umbilical cord matrix-derived stem cells (UCMSC) to control growth of pancreatic cancer. In vivo results were supported by various in vitro assays, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), direct cell count, [³H]thymidine uptake and soft agar colony assays.ResultsCo-culture of rat UCMSC with PAN02 murine pancreatic carcinoma cells (UCMSC:PAN02, 1:6 and 1:3) caused G0/G1 arrest and significantly attenuated the proliferation of PAN02 tumor cells, as monitored by MTT assay, direct cell counts and [³H]thymidine uptake assay. Rat UCMSC also significantly reduced PAN02 colony size and number, as measured by soft agar colony assay. The in vivo mouse studies showed that rat UCMSC treatment significantly decreased the peritoneal PAN02 tumor burden 3 weeks after tumor transplantation and increased mouse survival time. Histologic study revealed that intraperitoneally administered rat UCMSC survived for at least 3 weeks, and the majority were found near or inside the tumor.ConclusionsThese results indicate that naive rat UCMSC alone remarkably attenuate the growth of pancreatic carcinoma cells in vitro and in a mouse peritoneal model. This implies that UCMSC could be a potential tool for targeted cytotherapy for pancreatic cancer.     

Genetic alterations in caspase-10 may be causative or protective in autoimmune lymphoproliferative syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by lymphadenopathy, elevated numbers of T cells with αβ-T cell receptors but neither CD4 nor CD8 co-receptors, and impaired lymphocyte apoptosis in vitro. Defects in the Fas receptor are the most common cause of ALPS (ALPS Ia), but in rare cases other apoptosis proteins have been implicated, including caspase-10 (ALPS II). We investigated the role of variants of caspase-10 in ALPS. Of 32 unrelated probands with ALPS who did not have Fas defects, two were heterozygous for the caspase-10 missense mutation I406L. Like the previously reported ALPS II-associated mutation L285F, I406L impaired apoptosis when transfected alone and dominantly inhibited apoptosis mediated by wild type caspase-10 in a co-transfection assay. Other variants in caspase-10, V410I and Y446C, were found in 3.4 and 1.6% of chromosomes in Caucasians, and in 0.5 and <0.5% of African Americans, respectively. In contrast to L285F and I406L, these variants had no dominant negative effect in co-transfection assays into the H9 lymphocytic cell line. We found healthy individuals homozygous for V410I, challenging the earlier suggestion that homozygosity for V410I alone causes ALPS. Moreover, an association analysis suggested protection from severe disease by caspase-10 V410I in 63 families with ALPS Ia due to dominant Fas mutations (P<0.05). Thus, different genetic variations in caspase-10 can produce contrasting phenotypic effects.     

Challenges in the clinical utility of the serum test for HER2 ECD

Approximately 15-30% of breast cancers over-express the HER2/neu receptor. Historically, over-expression of HER2/neu has been identified using IHC or FISH, both of which are invasive approaches requiring tissue samples. Recent evidence has shown that some tumors identified as "negative" using these methods can respond to HER2/neu targeted therapy. Shedding of the extracellular domain (ECD) of the receptor into the circulation has led to the development of a serum test of HER2 ECD as an additional approach to probe HER2/neu overexpression. The serum test will be able to monitor the dynamic changes of HER2 status over the course of disease progression. Some studies further suggest that the serum HER2 ECD level and its change may serve as a biomarker to reflect patients' response to therapy. Yet more than 10years after the first serum HER2 ECD test was approved by the FDA, serum HER2 testing has yet to be widely used in clinical practice. In this article we will review the progress of the serum HER2 ECD test and discuss some obstacles impeding its incorporation into broad clinical practice. We will also discuss recent improvements in the sensitivity and specificity of the assay that offer some hope for the future of serum HER2 test.     

Progress of molecular targeted therapies for prostate cancers

Prostate cancer remains the most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in men in the United States. The current standard of care consists of prostatectomy and radiation therapy, which may often be supplemented with hormonal therapies. Recurrence is common, and many develop metastatic prostate cancer for which chemotherapy is only moderately effective. It is clear that novel therapies are needed for the treatment of the malignant forms of prostate cancer that recur after initial therapies, such as hormone refractory (HRPC) or castration resistant prostate cancer (CRPC). With advances in understanding of the molecular mechanisms of cancer, we have witnessed unprecedented progress in developing new forms of targeted therapy. Several targeted therapeutic agents have been developed and clinically used for the treatment of solid tumors such as breast cancer, non-small cell lung cancer, and renal cancer. Some of these reagents modulate growth factors and/or their receptors, which are abundant in cancer cells. Other reagents target the downstream signal transduction, survival pathways, and angiogenesis pathways that are abnormally activated in transformed cells or metastatic tumors. We will review current developments in this field, focusing specifically on treatments that can be applied to prostate cancers. Finally we will describe aspects of the future direction of the field with respect to discovering biomarkers to aid in identifying responsive prostate cancer patients.     

Tracking dispersal across a patchy landscape reveals a dynamic interaction between genotype and habitat structure

Theoretical and empirical studies often show that within populations, individuals vary in their propensity to disperse. We aspired to understand how this behavioural variation is impacted by the distribution and pattern of food patches across a landscape. In a series of experiments we examined how inter-patch distance and the distribution of food patches influenced dispersal in wild-type strains of Drosophila melanogaster with natural allelic variants of the foraging (for) gene known to influence dispersal in this species. The ‘rover' strain was homozygous for the for^R allele (more dispersive) whereas the ‘sitter' strain was homozygous for for^s (less dispersive). We also assessed an outbred population of flies with an unknown dispersal propensity. Dispersal was assayed in a multi-patch lab arena (25 cells, 5 × 5 array). In the inter-patch distance trials, landscapes of two different sizes (small versus large) were used, both with food in all 25 cells. Dispersal was reduced in the large landscape relative to the small landscape for all three fly strains. Sitter dispersal was lowest relative to both rovers and the outbred flies, whose dispersal tendencies were similar. In the patch distribution trials, flies were assayed in landscapes with varying distribution and number of cells containing food. Dispersal generally increased as the number of patches with food increased, however, rovers and sitters adopted similar dispersal strategies when food was fixed and limited. Conversely, their strategies differed when the total amount of food increased with the number of patches. We find that both the inter-patch distance and distribution can influence dispersal. However, the effect of inter-patch distance and distribution on dispersals depends on genotype × environment interaction. Our findings highlight the importance of considering G × E when assessing how dispersal strategies and landscape dynamics influence the distribution of animal communities.