Extracellular Vesicles Secreted from Cancer Cell Lines Stimulate Secretion of MMP-9, IL-6, TGF-β1 and EMMPRIN

Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-β1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles.     

Streptococcus pneumoniae G5 domains bind different ligands

Many bacterial pathogens express small G5 domains that exist in the context of various membrane‐anchored proteins and these G5 domains have been associated with colonization, cellular adhesion, and biofilm formation. However, despite over a decade since the computational prediction of these G5 domains, many remain uncharacterized, particularly those from Streptococcus pneumoniae. Of five previously predicted G5 domains we found that four of these, all derived from S. pneumoniae, are independently folded modules. As one of these exhibits extreme line broadening due to self‐association, we were able to use NMR solution studies to probe the potential ligand interactions of the remaining three G5 domains. None of these G5 domains engage N‐acetylglucosamine (NAG) as previously predicted but do interact with other small molecules that may modulate adherence to both bacteria and host cells. Specifically, while all G5 domains tested engage Zn, only one of these G5 domains engage heparin. NMR solution structural studies of the IgA1 Protease G5 (IgA1P‐G5) and endo‐beta‐N‐acetylglucosaminidase‐D G5 (ENDD‐G5) also facilitated identification of the ligand binding sites and confirm the typical G5 fold that comprises two connected β‐sheets with no canonical core. NMR relaxation experiments indicate flexibility on both ends and within the connecting regions between the β‐sheets. Our studies thus establish a basis for future biological experiments to test whether the ligands presented here are involved in bacterial adherence, either to bacteria or to host cells.     

Contribution of inorganic cations and organic compounds to osmotic adjustment in root cultures of two <Emphasis Type="Italic">Centaurium</Emphasis> species differing in tolerance to salt stress

The effect of reduced availability of sugars on growth and essential metabolic processes in roots, resulting from decreased photosynthesis under salinity, was excluded by establishing a non-photosynthetic model-system in this study: root cultures of Centaurium maritimum (L.) Fritch and Centaurium spicatum (L.) Fritch. The contribution of inorganic cations and organic compounds (e.g. carbohydrates and amino acids) to the osmotic adjustment (OA) in roots during short-term exposure to various salt concentrations (0, 50, 100 or 200 mM NaCl) was emphasized. Observed morphological and histological changes in roots were species specific, and were dependent on salinity level. Although C. spicatum appears to be more tolerant to salt stress, both species employed similar strategies in response to elevated salinity to different extents, and displayed effective OA mechanisms. Under low and moderate salinity, inorganic cations were the major contributors to OA in roots of both species, followed by soluble sugars, while the relative contribution of proline (Pro) and free amino acids was insignificant. Osmotic adjustment under severe stress appears to be mediated by increased accumulation of organic compounds. The analysis of the intraspecies variability in salt response of C. spicatum and C. maritimum roots enabled the identification of some organic compounds which could be used as potential biochemical markers in screening for salt tolerance, including Pro in C. spicatum, and trehalose and polyols in C. maritimum.     

Sugars and acid invertase mediate the physiological response of Schenkia spicata root cultures to salt stress

A heterotrophic model system was established in our studies in order to differentiate the effect of high salt concentrations in external medium on growth and sugar metabolism in roots from the effect of reduced sugar availability resulting from decreased photosynthesis under salinity. Soluble sugar content and the activity of acid invertase in root cultures of salt-tolerant (ST) and salt-sensitive (SS) Schenkia spicata (L.) Mansion genotypes were investigated during exposure to different NaCl concentrations (0-200mM). Their response to severe salinity was characterized by a metabolic adjustment that led to the accumulation of sucrose (Suc) in root tissues. There was clear evidence that cell wall invertase (CW-Inv) is the major contributor to the Suc/hexose ratio in roots during exposure to elevated salinity. The results of CW-Inv activity and immunodetection assays in our study suggest that the regulation of CW-Inv expression is most likely achieved in a salt stress dependent manner. Also, NaCl modulated soluble acid invertase (SA-Inv) expression differentially in SS and ST genotypes of S. spicata. Regardless of the salt treatment, genotype, or the amount of enzyme, SA-Inv activity was generally low, indicating regulation at the posttranslational level. The results suggest no direct role of SA-Inv in the regulation of the root tissue carbohydrate pool and therefore in the control of the availability of glucose and fructose for the primary metabolism and/or osmotic adjustment in the present heterotrophic model system.     

Amplification of DNA Encoding Entire Type I Polyketide Synthase Domains and Linkers from Streptomyces Species

Polyketides are a group of bioactive compounds from bacteria, plants, and fungi. To increase the availability of analogs for testing, the active sites of polyketide synthases are often substituted with homologous domains having altered substrate specificities. This study reports the design of polymerase chain reaction primers that enables isolation of entire active site domains from type I polyketide synthases with native interdomain linkers. This bypasses the need for further genetic screening to obtain functional units for use in genetic engineering. This is especially important in bioprospecting projects exploring new environments for bioresources.     

Characterizing and controlling the inherent dynamics of cyclophilin-A

With the recent advances in NMR relaxation techniques, protein motions on functionally important timescales can be studied at atomic resolution. Here, we have used NMR-based relaxation experiments at several temperatures and both 600 and 900 MHz to characterize the inherent dynamics of the enzyme cyclophilin-A (CypA). We have discovered multiple chemical exchange processes within the enzyme that form a “dynamic continuum” that spans 20–30 Å comprising active site residues and residues proximal to the active site. By combining mutagenesis with these NMR relaxation techniques, a simple method of counting the dynamically sampled conformations has been developed. Surprisingly, a combination of point mutations has allowed for the specific regulation of many of the exchange processes that occur within CypA, suggesting that the dynamics of an enzyme may be engineered.     

Proteasome regulation,plant growth and stress tolerance

Plant cells contain a mixture of 26S and 20S proteasomes that mediate ubiquitin-dependent and ubiquitin-independent proteolysis, respectively. The 26S proteasome contains the 20S proteasome and one or two regulatory particles that are required for ubiquitin-dependent degradation. Comparative analyses of Arabidopsis proteasome mutants revealed that a decrease in 26S proteasome biogenesis causes heat shock hypersensitivity and reduced cell division rates that are compensated by increased cell expansion. Loss of 26S proteasome function also leads to an increased 20S proteasome biogenesis, which in turn enhances the cellular capacity to degrade oxidized proteins and thus increases oxidative stress tolerance. These findings suggest the intriguing possibility that 26S and 20S proteasome activities are regulated to control plant development and stress responses. This mini-review highlights some of the recent studies on proteasome regulation in plants.Key words: proteasome, cell division, ubiquitin-dependent proteolysis, ubiquitin-independent proteolysis, stress responses     

Biochemical characterization of soluble nucleotide pyrophosphatase/phosphodiesterase activity in rat serum

Biochemical properties of nucleotide pyrophosphatase/phosphodiesterase (NPP) in rat serum have been described by assessing its nucleotide phosphodiesterase activity, using p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate. It was demonstrated that NPP activity shares some typical characteristics described for other soluble NPP, such as divalent cation dependence, strong alkaline pH optimum (pH 10.5), inhibition by glycosaminoglycans, and K _m for p-Nph-5′-TMP hydrolysis of 61.8 ± 5.2 μM. In order to characterize the relation between phosphodiesterase and pyrophosphatase activities of NPP, we have analyzed the effects of different natural nucleotides and nucleotide analogs. ATP, ADP, and AMP competitively inhibited p-Nph-5′-TMP hydrolysis with K _i values ranging 13–43 μM. Nucleotide analogs, α,β-metATP, BzATP, 2-MeSATP, and dialATP behaved as competitive inhibitors, whereas α,β-metADP induced mixed inhibition, with K _i ranging from 2 to 20 μM. Chromatographic analysis revealed that α,β-metATP, BzATP, and 2-MeSATP were catalytically degraded in the serum, whereas dialATP and α,β-metADP resisted hydrolysis, implying that the former act as substrates and the latter as true competitive inhibitors of serum NPP activity. Since NPP activity is involved in generation, breakdown, and recycling of extracellular adenine nucleotides in the vascular compartment, the results suggest that both hydrolyzable and non-hydrolyzable nucleotide analogs could alter the amplitude and direction of ATP actions and could have potential therapeutic application.     

Puroindoline-a and alpha1-purothionin form ion channels in giant liposomes but exert different toxic actions on murine cells

Puroindoline-a (PIN-a) and alpha1-purothionin (alpha1-PTH), isolated from wheat endosperm of Triticum aestivum sp., have been suggested to play a role in plant defence mechanisms against phytopathogenic organisms. We investigated their ability to form pores when incorporated into giant liposomes using the patch-clamp technique. PIN-a formed cationic channels (approximately 15 pS) with the following selectivity K(+) > Na(+) > Cl(-). Also, alpha1-PTH formed channels of approximately 46 pS and 125 pS at +100 mV, the selectivity of which was Ca(2+) > Na(+) approximately K(+) > Cl(-) and Cl(-) > Na(+), respectively. In isolated mouse neuromuscular preparations, alpha1-PTH induced muscle membrane depolarization, leading to blockade of synaptic transmission and directly elicited muscle twitches. Also, alpha1-PTH caused swelling of differentiated neuroblastoma NG108-15 cells, membrane bleb formation, and disorganization of F-actin. In contrast, similar concentrations of PIN-a had no detectable effects. The cytotoxic actions of alpha1-PTH on mammalian cells may be explained by its ability to induce cationic-selective channels.     

Development of an HPLC method for the determination of nifedipine in human plasma by solid-phase extraction

Nifedipine, a dihydropyridine calcium channel antagonist, is widely used in the treatment of hypertension and other cardiovascular disorders. A selective, sensitive and accurate high-performance liquid chromatographic method has been developed, validated and applied for determination of nifedipine in human plasma samples. A series of studies were conducted in order to investigate the effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers, and to develop a convenient and easy-to-use method for quantitative analysis of nifedipine. The method involves solid-phase extraction on C18 cartridges. The chromatographic separation was accomplished on a Lichrocart Lichrospher 60 RP selectB column with a mobile phase composed of 0.020 mol/L KH2PO4 (pH 4.8) and acetonitrile (42:58, v/v). UV detection was set at 240 nm. The calibration curve was linear in the concentration range of 5.0-200.0 ng/mL for nifedipine in plasma and the limit of quantification was 5.0 ng/mL.