No abstract

Cancer stem cells (CSCs) are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133(+)] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB) (Celdyne, Houston, TX). For comparison, another bioreactor, the rotary cell culture system (RCCS) manufactured by Synthecon (Houston, TX) was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133(+) cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a (+)15-fold proliferation of the CD133(+) cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (−)4.8-fold decrease in the CD133(+)cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133(+) cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates.

A number of genes have been shown to be transcribed specifically during sporulation in Saccharomyces cerevisiae, yet their developmentalifnction is unknown. The SPRP gene is transcribed during only the late stages of sporulation. We have sequenced the SPRi gene and found that it has extensive DNA and protein sequence homology to the S. cerevisiae EXG1 gene which encodes an exo-1,3-0-glucanase expressed during vegetative growth (C. R. Vasquez de Aldana, J. Correa, P. San Segundo, A. Bueno, A. R. Nebrada, E.Mendez, and F. del Ray, Gene 97:173-182, 1991). We show that spri mutant cells do not hydrolyze p-nitrophenyl-1-D-glucoside or laminarin in a whole-cell assay for exo-1,3-13-glucanases. In addition to the absence of this enzymatic activity, sprl mutant spores exhibit reduced thermoresistance relative to isogenic wild-type spores. These observations are consistent with the notion that SPRI encodes a sporulation-specific exo-1,3-0-glucanase.In Saccharomyces cerevisiae, sporulation is initiated when a/a diploids are deprived of glucose and nitrogen. This complex pathway, which includes premeiotic DNA synthesis, high levels of genetic recombination, and two meiotic divisions, culminates in the formation of an ascus containing four haploid ascospores (reviewed in reference 11). Molecular and genetic analyses of this pathway are important because sporulation can serve as a model system for eukaryotic differentiation, i.e., for the coordination of a complex series of events that result in the formation of a new cell type. Furthermore, the process of recombination and meiosis is fundamental to eukaryotic biology.Genetic analysis has identified more than 30 complementation groups that are required for the completion of specific sporulation events (11,45); several of these genes have been cloned either by direct complementation of spo mutants, e.g., spoll (1)

Structural genes have been identified for all of the enzymes involved in the biosynthesis of pantothenic acid in Salmonella typhimurium and Escherichia coli K-12, with the exception of ketopantoic acid reductase, which catalyzes the conversion of a-ketopantoate to pantoate. The acetohydroxy acid isomeroreductase from S. typhimurium efficiently bound a-ketopantoate (Km = 0.25 mM) and catalyzed its reduction at 1/20 the rate at which ot-acetolactate was reduced. Since two enzymes could apparently participate in the synthesis of pantoate, a S. typhimurium ilvC8 strain was mutagenized to derive strains completely blocked in the conversion of a-ketopantoate to pantoate. Several isolates were obtained that grew in isoleucine-valine medium supplemented with either pantoate or pantothenate, but not in the same medium supplemented with a-ketopantoate or P-alanine.

Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensis invades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU, or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks.