Volatile profiles of human skin cell cultures in different degrees of senescence

It is known that skin releases volatile organic compounds to the environment, and also that its emission pattern changes with aging of the skin. It could be considered, that these compounds are intermediaries in cell metabolism, since many intermediaries of metabolic pathways have a volatile potential. In this work, a simple and non-destructive method consisting of SPME sampling and GC/MS analysis was developed to identify volatile organic emanations from cell cultures. This technique, applied to skin cells culture, indicates that the cells or cell metabolism produce several skin emissions. Chemometric analysis was performed in order to explore the relationship between a volatile profile and the senescence of cell cultures. Volatile profiles were different for cell cultures in different degrees of senescence, indicating that volatile compound patterns could be used to provide information about the age of skin cells.     

Redox-controlled,in vivo and in vitro phosphorylation of the α subunit of the light-harvesting complex I in Rhodobacter capsulatus

Labelling of Rhodobacter capsulatus cells with (³²P)Pi in a phototrophic culture results in phosphorylation of a membrane-bound polypeptide identified as the subunit of the LHI antenna complex of the photosynthetic apparatus. Phosphorylation of the same polypeptide was also observed by incubation of chromatophores with (³²P)ATP or under conditions of photophosphorylation with ADP and (³²P)Pi. The identity of the phosphorylated LHI- subunit was demonstrated by N-terminal protein sequencing of the phosphorylated polypeptide and by failure of labelling in LHI-defective mutants. Pre-aeration of the samples or addition of the oxidant potassium ferrcyanide stimulated the kinase activity whereas the presence of soluble cytoplasmic proteins impaired phosphorylation in an in vitro assay. No effect resulted from addition of reductants to the assay medium. The results indicate the presence of a membrane-bound protein kinase in R. capsulatus that phosphorylates the subunit of the LHI antenna complex under redox control.Abbreviations Pi inorganic phosphate - SDS-PAGE sodium dodecyl-sulfate polyacrylamide gel electrophoresis     

The cysteine proteinase inhibitor, E-64, reduces proteinuria in an experimental model of glomerulonephritis

Proteinuria is a major manifestation of glomerular disease (glomerulonephritis, GN). We examined the effect of trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a specific and irreversible cysteine proteinase inhibitor, on urinary protein excretion in a complement- and neutrophil-independent model of antiglomerular basement membrane (GBM) antibody disease. A single injection of rabbit antirat-GBM IgG produced a marked increase in urinary protein excretion 24hr after injection. In two separate studies using different pools of antiGBM IgG, administration of E-64 (5mg every 6h starting 2hr prior to induction of GN) reduced proteinuria (-45 +/- 7%, and -41 +/- 14%, Mean +/- SEM, n = 6; P less than 0.001) in the 24 hour period following induction of the disease. This reduction in urinary protein excretion was accompanied by a marked decrease in the specific activity of the cysteine proteinases cathepsins B and L in glomeruli (B: -97%; L: -84%) and renal cortex (B: -87%; L: -75%) isolated from the same E-64-treated rats compared to same saline-treated controls. These data, combined with the specificity of E-64 for cysteine proteinases, suggest a potential role for cysteine proteinases in the increased GBM permeability and proteinuria in this experimental model of glomerular disease.     

Ecotropic murine leukemia virus-induced fusion of murine cells.

Extensive fusion occurs upon cocultivation of murine fibroblasts producing ecotropic murine leukemia viruses (MuLVs) with a large variety of murine cell lines in the presence of the polyene antibiotic amphotericin B, the active component of the antifungal agent Fungizone. The resulting polykaryocytes contain nuclei from both infected and uninfected cells, as evidenced by autoradiographic labeling experiments in which one or the other parent cell type was separately labeled with [3H]thymidine and fused with an unlabeled parent. This cell fusion specifically requires the presence of an ecotropic MuLV-producing parent and is not observed for cells producing xenotropic, amphotropic, or dualtropic viruses. Mouse cells infected with nonecotropic viruses retain their sensitivity toward fusion, whereas infection with ecotropic viruses abrogates the fusion of these cells upon cocultivation with other ecotropic MuLV-producing cells. Nonmurine cells lacking the ecotropic gp70 receptor are not fused under similar conditions. Fusion is effectively inhibited by monospecific antisera to gp70, but not by antisera to p15(E), and studies with monoclonal antibodies identify distinct amino- and carboxy-terminal gp70 regions which play a role in the fusion reaction. The enhanced fusion which occurs in the presence of amphotericin B provides a rapid and sensitive assay for the expression of ecotropic MuLVs and should facilitate further mechanistic studies of MuLV-induced fusion of murine cells.     

Inactivation of Rhodospirillum rubrum coupling factor by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Modification of a tyrosine protected by phosphate

Chemical modification of Rhodospirillum rubrum chromatophores by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) results in inactivation of photophosphorylation, Mg²⁺-ATPase, oxidative phosphorylation and ATP-driven transhydrogenase, with apparent first-order kinetics. Other energy-linked reactions such as light-driven transhydrogenase and light-dependent proton uptake were insensitive to NBD-Cl. The Ca²⁺-ATPase activity of the soluble coupling factor from chromatophores (R. rubrum F₁) was inactivated by NBD-Cl with kinetics resembling those described for Mg²⁺-ATPase and photophosphorylation activities of chromatophores. Both NBD-chromatophores and NBD-R. rubrum F₁ fully recovered their activities when subjected to thiolysis by dithioerythritol. Phosphoryl transfer reactions of chromatophores and Ca²⁺-ATPase activity of R. rubrum F₁ were fully protected by 5 mM P_i against modification by NBD-Cl. ADP or ATP afforded partial protection. Analysis of the protection of Ca²⁺-ATPase activity by P_i indicated that NBD-Cl and P_i are mutually exclusive ligands. Spectroscopic studies revealed that tyrosine and sulfhydryl residues in R. rubrum F₁ underwent modification by NBD-Cl. However, the inactivation was only related to the modification of tyrosine groups.     

Structural and signaling requirements for BCR-ABL-mediated transformation and inhibition of apoptosis.

BCR-ABL is a deregulated tyrosine kinase expressed in Philadelphia chromosome-positive human leukemias. Prolongation of hematopoietic cell survival by inhibition of apoptosis has been proposed to be an integral component of BCR-ABL-induced chronic myelogenous leukemia. BCR-ABL elicits transformation of both fibroblast and hematopoietic cells and blocks apoptosis following cytokine deprivation in various factor-dependent cells. To elucidate the mechanisms whereby BCR-ABL induces transformation and blocks apoptosis in hematopoietic cells, we examined the biological effects of expression of a series of BCR-ABL mutants. Single amino acid substitutions in the GRB2 binding site (Y177F), Src homology 2 domain (R552L), or an autophosphorylation site in the tyrosine kinase domain (Y793F) do not diminish the antiapoptotic and transforming properties of BCR-ABL in hematopoietic cells, although these mutations were previously shown to drastically reduce the transforming activity of BCR-ABL in fibroblasts. A BCR-ABL molecule containing all three mutations (Y177F/R552L/Y793F) exhibits a severe decrease in transforming and antiapoptotic activities compared with the wild-type BCR-ABL protein in 32D myeloid progenitor cells. Ras is activated, the SHC adapter protein is tyrosine phosphorylated and binds GRB2, and myc mRNA levels are increased following expression of all kinase active BCR-ABL proteins with the exception of the Y177F/R552L/Y793F BCR-ABL mutant in 32D cells. We propose that BCR-ABL uses multiple pathways to activate Ras in hematopoietic cells and that this activation is necessary for the transforming and antiapoptotic activities of BCR-ABL. However, Ras activation is not sufficient for BCR-ABL-mediated transformation. A BCR-ABL deletion mutant (delta 176-427) that activates Ras and blocks apoptosis but has severely impaired transforming ability in 32D cells has been identified. These data suggest that BCR-ABL requires additional signaling components to elicit tumorigenic growth which are distinct from those required to block apoptosis.     

A Design Pattern for Decentralised Decision Making

The engineering of large-scale decentralised systems requires sound methodologies to guarantee the attainment of the desired macroscopic system-level behaviour given the microscopic individual-level implementation. While a general-purpose methodology is currently out of reach, specific solutions can be given to broad classes of problems by means of well-conceived design patterns. We propose a design pattern for collective decision making grounded on experimental/theoretical studies of the nest-site selection behaviour observed in honeybee swarms (Apis mellifera). The way in which honeybee swarms arrive at consensus is fairly well-understood at the macroscopic level. We provide formal guidelines for the microscopic implementation of collective decisions to quantitatively match the macroscopic predictions. We discuss implementation strategies based on both homogeneous and heterogeneous multiagent systems, and we provide means to deal with spatial and topological factors that have a bearing on the micro-macro link. Finally, we exploit the design pattern in two case studies that showcase the viability of the approach. Besides engineering, such a design pattern can prove useful for a deeper understanding of decision making in natural systems thanks to the inclusion of individual heterogeneities and spatial factors, which are often disregarded in theoretical modelling.     

Survival of Lactobacillus paracasei subsp. paracasei LBC 81 in Fermented Milk Enriched with Green Banana Pulp Under Acid Stress and in the Presence of Bile Salts

This study evaluated the in vitro effect of 3.0, 6.0, and 9.0% of green banana pulp (GBP) incorporation in fermented milk on the survival of Lactobacillus paracasei subsp. paracasei LBC 81 subjected to acid stress conditions and in the presence of bile salts. Tolerance to acid stress in pH 2.0 and in the presence of 0.30% of bile salts was evaluated right after the incorporation of the fermented milk in each of these conditions, and also during 3 and 4 h of exposure, respectively. The addition of GBP (3.0%) gives a protective effect on L. paracasei LBC 81 when exposed to stress conditions evaluated, while of 9.0% there is a marked decrease of L. paracasei LBC 81. In the absence of GBP, a decrease of L. paracasei LBC 81 is observed, but lower in the presence of GBP (9.0%).