CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells

The apoptosis-inducing death receptor CD95 (APO-1/Fas) controls the homeostasis of many tissues. Despite its apoptotic potential, most human tumors are refractory to the cytotoxic effects of CD95 ligand. We now show that CD95 stimulation of multiple apoptosis-resistant tumor cells by CD95 ligand induces increased motility and invasiveness, a response much less efficiently triggered by TNFalpha or TRAIL. Three signaling pathways resulting in activation of NF-kappaB, Erk1/2 and caspase-8 were found to be important to this novel activity of CD95. Gene chip analyses of a CD95-stimulated tumor cell line identified a number of potential survival genes and genes that are known to regulate increased motility and invasiveness of tumor cells to be induced. Among these genes, urokinase plasminogen activator was found to be required for the CD95 ligand-induced motility and invasiveness. Our data suggest that CD95L, which is found elevated in many human cancer patients, has tumorigenic activities on human cancer cells. This could become highly relevant during chemotherapy, which can cause upregulation of CD95 ligand by both tumor and nontumor cells.     

How physiological and cultural conditions influence heterologous protein production in Kluyveromyces lactis

The optimization and scale-up of a specific protein production process have to take into account cultural conditions as well as cell physiology of growth and influence of foreign protein expression on host cell metabolism. Growth on cheap substrates, efficient secretion ability and a weaker tendency to hypermannosilate proteins than S. cerevisiae, make K. lactis an excellent and well-accepted host for heterologous protein production, even for human use. A fairly good heterologous glucoamylase yield and the setting of the optimal conditions to produce it were obtained expressing the Arxula adeninivorans glucoamylase in a strain of K. lactis and its isogenic mutant, which seems to have higher secretion ability. We performed batch cultures of both strains to analyze the influence of different physiological and environmental parameters on glucoamylase production/secretion. Interestingly, the maintenance of pH in the range of neutrality causes the consumption of a larger amount of carbon source, a longer time of production and a better stability of the active form of the enzyme, thus increasing biomass and glucoamylase production. Furthermore, the enrichment of the culture medium adds up to the action of pH control, forcing the mutant production/secretion to higher levels.     

Kluyveromyces lactis cells entrapped in Ca-alginate beads for the continuous production of a heterologous glucoamylase

Viable cells of Kluyveromyces lactis, transformed with the glucoamylase gene from Arxula adeninivorans, were entrapped in beads of Ca-alginate and employed on a lab scale in a continuous stirred and a fluidised bed reactor (FBR), both fed with a rich medium (YEP) containing lactose as carbon source. Experiments with freely suspended cells in batch and chemostat had demonstrated that glucoamylase production was favoured in the presence of lactose and YEP medium. Employing controlled-sized beads having a 2.13 mm diameter, specific glucoamylase productivity was higher in the stirred reactor (CSTR) than in the FBR; in the latter a higher volumetric productivity was achieved, due to the lower void degree. The performance of the immobilised cell systems, in terms of specific glucoamylase productivity, was strongly affected by mass transfer limitations occurring throughout the gel due to the high molecular weight of the product. In the perspective to improve and scale-up the immobilised cell system proposed, a mathematical model, which takes into account substrate transfer limitations throughout the gel, has been developed. The effective lactose diffusivity was related to the bead reactive efficiency by means of the Thiele modulus. The regression of the model parameters on the experimental data of substrate consumption obtained both in the CSTR and in the FBR allowed to estimate lactose diffusivity and the kinetic parameters of the immobilised yeast.     

Cytogenetic evaluation of extractable agents from airborne particulate matter generated in the city of Catania (Italy)

In order to document cytogenetic damage associated with air pollution and, possibly, with health effects in the city of Catania, Sicily (Italy), we analyzed the induction of chromosomal aberrations by extractable agents from airborne particulate matter in a Chinese hamster epithelial liver (CHEL) cells. These cells retain their metabolic competence to activate different classes of promutagens/procarcinogens into biologically active metabolites. Airborne particulate matter was obtained from two stationary samplers (stations I and II) in two areas endowed by an elevated car transit in the centre of Catania. The results obtained clearly indicated that airborne particulate matter from both stations I and II proved to be clastogens in CHEL cells but not in Chinese hamster ovary (CHO) cells without metabolic activation, indicating that airborne particulate mixtures need to be metabolically converted before exerting their genotoxic potential. On the basis of these results we can assert that the test system employed to identify the cytogenetic potential of airborne particulate matter is useful and profitable for environmental control, and helpful to plan specific actions aimed at reducing the hazards derived from exposure to polluted air.     

Modeling the acid–base properties of glutathione in different ionic media, with particular reference to natural waters and biological fluids

The acid-base properties of γ-L-glutamyl-L-cysteinyl-glycine (glutathione, GSH) were determined by potentiometry (ISE-H(+), glass electrode) in pure NaI((aq)) and in NaCl((aq))/MgCl(2(aq)), and NaCl((aq))/CaCl(2(aq)) mixtures, at T = 298.15 K and different ionic strengths (up to I(c) ~ 5.0 mol L(-1)). In addition, the activity coefficients of glutathione were also determined by the distribution method at the same temperature in various ionic media (LiCl((aq)), NaCl((aq)), KCl((aq)), CsCl((aq)), MgCl(2(aq)), CaCl(2(aq)), NaI((aq))). The results obtained were also used to calculate the Specific ion Interaction Theory (SIT) and Pitzer coefficients for the dependence on medium and ionic strength of glutathione species, as well as the formation constants of weak Mg(j)H( i )(GSH)((i+2j-3)) and Ca(j)H(i)(GSH)((i+2j-3)) complexes. Direct calorimetric titrations were also carried out in pure NaCl((aq)) and in NaCl((aq))/CaCl(2(aq)) mixtures at different ionic strengths (0.25 ≤ I (c )/mol L(-1) ≤ 5.0) in order to determine the enthalpy changes for the protonation and complex formation equilibria in these media at T = 298.15 K. Results obtained are useful for the definition of glutathione speciation in any aqueous media containing the main cations of natural waters and biological fluids, such as Na(+), K(+), Mg(2+), and Ca(2+). Finally, this kind of systematic studies, where a series of ionic media (e.g., all alkali metal chlorides) is taken into account in the determination of various thermodynamic parameters, is useful for the definition of some trends in the thermodynamic behavior of glutathione in aqueous solution.     

Vertical Distribution of Archaea and Bacteria in a Meromictic Lake as Determined by Fluorescent In Situ Hybridization

The prokaryotic cells distribution in the water column of the coastal saline meromictic Lake Faro (Messina, Italy) was investigated by microscopic counting techniques. Water samples were collected at a central station from the surface to the bottom, when waters were characterized by a marked stratification. A “red-water” layer, caused by a dense growth of photosynthetic sulfur bacteria, was present at a depth of 15 m, defining a transition area between oxic (mixolimnion) and anoxic (monimolimnion) layers. Fluorescently labeled 16S rRNA oligonucleotide, group-specific probes were used to determine the abundance of Bacteria and Archaea, and their subgroups, Green Sulfur Bacteria (GSB), Sulfate Reducing Bacteria (SRB), Cyanobacteria and Chromatium okenii, and Crenarchaeota and Euryarchaeota, as key elements of the microbial community. Bacteria decreased from surface to bottom, while Archaea increased with depth and reached the maximum value at 30 m, where they outnumbered the Bacteria. Bacteria and picophytoplankton prevailed in the mixolimnion. At the chemocline high numbers of prokaryotic cells were present, mainly represented by Cyanobacteria, Chromatium okenii and Euryarchaeota. GSB, SRB, and Crenarchaeota prevailed below the chemocline. Although Archaea constitute a minor fraction of microbial community, they could represent active contributors to the meromictic Lake Faro ecosystem.