Edelfosine-induced metabolic changes in cancer cells that precede the overproduction of reactive oxygen species and apoptosis

Background  

Metabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells.     

Injection of highly supersaturated oxygen solutions without nucleation

It is possible to inject highly supersaturated aqueous solutions of gas through a small capillary into an aqueous environment without the formation of significant gas bubbles. Such a technique has considerable potential therapeutic value in the treatment, for example, of heart attacks and strokes. The present paper is the second in a series (see Brereton et al. [1]) investigating the basic phenomenon behind this surprising effect. Recent experiments clearly demonstrate that the nucleation, when it does occur, results from heterogeneous nucleation on the interior surface of the distal end of the capillary. This paper describes the effects of the treatment of this interior surface on the nucleation processes and the results of high speed video observations of the phenomena. A heterogeneous nucleation model is presented which is in accord with the experimental observations.     

Lumped parameter model for computing the minimum pressure during mechanical heart valve closure

The cavitation inception threshold of mechanical heart valves has been shown to be highly variable. This is in part due to the random distribution of the initial and final conditions that characterize leaflet closure. While numerous hypotheses exist explaining the mechanisms of inception, no consistent scaling laws have been developed to describe this phenomenon due to the complex nature of these dynamic conditions. Thus in order to isolate and assess the impact of these varied conditions and mechanisms on inception, a system of ordinary differential equations is developed to describe each system component and solved numerically to predict the minimum pressure generated during valve closure. In addition, an experiment was conducted in a mock circulatory loop using an optically transparent size 29 bileaflet valve over a range of conditions to calibrate and validate this model under physiological conditions. High-speed video and high-response pressure measurements were obtained simultaneously to characterize the relationship between the valve motion, fluid motion, and negative pressure transients during closure. The simulation model was calibrated using data from a single closure cycle and then compared to other experimental flow conditions and to results found in the literature. The simulation showed good agreement with the closing dynamics and with the minimum pressure trends in the current experiment. Additionally, the simulation suggests that the variability observed experimentally (when using dP/dt alone as the primary measure of cavitation inception) is predictable. Overall, results from the current form of this lumped parameter model indicate that it is a good engineering assessment tool.     

Fibroblast activation protein peptide substrates identified from human collagen I derived gelatin cleavage sites

A highly consistent trait of tumor stromal fibroblasts is the induction of the membrane-bound serine protease fibroblast activation protein-alpha (FAP), which is overexpressed on the surface of reactive stromal fibroblasts present within the stroma of the majority of human epithelial tumors. In contrast, FAP is not expressed by tumor epithelial cells or by fibroblasts or other cell types in normal tissues. The proteolytic activity of FAP, therefore, represents a potential pan-tumor target that can be exploited for the release of potent cytotoxins from inactive prodrugs consisting of an FAP peptide substrate coupled to a cytotoxin. To identify FAP peptide substrates, we used liquid chromatography tandem mass spectroscopy based sequencing to generate a complete map of the FAP cleavage sites within human collagen I derived gelatin. Positional analysis of the frequency of each amino acid at each position within the cleavage sites revealed FAP consensus sequences PPGP and (D/E)-(R/K)-G-(E/D)-(T/S)-G-P. These studies further demonstrated that ranking cleavage sites based on the magnitude of the LC/MS/MS extracted ion current predicted FAP substrates that were cleaved with highest efficiency. Fluorescence-quenched peptides were synthesized on the basis of the cleavage sites with the highest ion current rankings, and kinetic parameters for FAP hydrolysis were determined. The substrate DRGETGP, which corresponded to the consensus sequence, had the lowest Km of 21 microM. Overall the Km values were relatively similar for both high and low ranked substrates, whereas the kcat values differed by up to 100-fold. On the basis of these results, the FAP consensus sequences are currently being evaluated as FAP-selective peptide carriers for incorporation into FAP-activated prodrugs.     

Fairy, tadpole, and clam shrimps (Branchiopoda) in seasonally inundated clay pans in the western Mojave Desert and effect on primary producers

Background

Fairy shrimps (Anostraca), tadpole shrimps (Notostraca), clam shrimps (Spinicaudata), algae (primarily filamentous blue-green algae [cyanobacteria]), and suspended organic particulates are dominant food web components of the seasonally inundated pans and playas of the western Mojave Desert in California. We examined the extent to which these branchiopods controlled algal abundance and species composition in clay pans between Rosamond and Rogers Dry Lakes. We surveyed branchiopods during the wet season to estimate abundances and then conducted a laboratory microcosm experiment, in which dried sediment containing cysts and the overlying algal crust were inundated and cultured. Microcosm trials were run with and without shrimps; each type of trial was run for two lengths of time: 30 and 60 days. We estimated the effect of shrimps on algae by measuring chlorophyll content and the relative abundance of algal species.

Results

We found two species of fairy shrimps (Branchinecta mackini and B. gigas), one tadpole shrimp (Lepidurus lemmoni), and a clam shrimp (Cyzicus setosa) in our wet-season field survey. We collected Branchinecta lindahli in a pilot study, but not subsequently. The dominant taxa were C. setosa and B. mackini, but abundances and species composition varied greatly among playas. The same species found in field surveys also occurred in the microcosm experiment. There were no significant differences as a function of experimental treatments for either chlorophyll content or algal species composition (Microcoleus vaginatus dominated all treatments).

Conclusions

The results suggest that there was no direct effect of shrimps on algae. Although the pans harbored an apparently high abundance of branchiopods, these animals had little role in regulating primary producers in this environment.     

Treatment with the {alpha}-glucosidase 1 inhibitor 6-O-butanoyl castanospermine reduces the detection of LFA-1 (CD18/CD11a) by monoclonal antibodies

The antiviral clinical candidate 6-O-butanoyl castano-spermine(MDL 28,574), an -glucosidase 1 inhibitor, was examined forits effect on elementary parameters of immune function. It didnot affect the mitogenic response of uninfected human mononuclearleukocytes or the detection of a range of cell surface markers,with the exception of the integrin LFA-1 (CD18/CD11a), whichwas reduced, after cell growth in vitro. The detection of LFA-1was also reduced on both human and murine cells after oral administrationof the compound to xenochimaeric or normal mice, respectively.Altered LFA-1 expression or function may contribute to reducedcell adhesion and the observed reduction in the in vitro allogeneicresponse by uninfected cells, as well as the previously describedprevention of cell conjugate and HTV-induced syncytium formation. adhesion 6-O-butanoyl castanospermine CD18 CD11a LFA-1     

Seneca Valley Virus 3Cpro Substrate Optimization Yields Efficient Substrates for Use in Peptide-Prodrug Therapy

The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3C^pro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3C^pro, we measured cleavage kinetics of predicted SVV-001 3C^pro substrates. An efficient substrate, L/VP4 (k_cat/K_M = 1932 ± 183 M^-1s^-1), was further optimized by a P2’ N→P substitution yielding L/VP4.1 (k_cat/K_M = 17446 ± 2203 M^-1s^-1). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3C^pro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3C^pro, with direct implications for anti-cancer therapeutic development.