Genome-Wide Microarray Expression and Genomic Alterations by Array-CGH Analysis in Neuroblastoma Stem-Like Cells

Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells.     

Characterization of the ribosomal rrnD operon of the cephamycin-producer 'Nocardia lactamdurans' shows that this actinomycete belongs to the genus Amycolatopsis

The cephamycin producer strain 'Nocardia lactamdurans' contains four ribosomal RNA (rrn) operons. One of them (rrnD) was cloned from a DNA library in the bifunctional cosmid pJAR4. A 2229 bp region of rrnD has been sequenced. The 'N. lactamdurans' rrnD operon maintains the canonical order 5'-16S-23S-5S-3'. Four of the consensus Gürtler-Stanisch sequences were found in the 16S rRNA gene and a fifth one in the sequenced 5' region of the 23S rRNA gene. The anti Shine-Dalgarno sequence of 'N. lactamdurans' (located in the 3'-end of the 16S rRNA gene) was found to be 5'-CCUCCUUUCU-3' and is identical to that of Corynebacterium lactofermentum and Mycobacterium tuberculosis. A phylogenetic analysis of 'N. lactamdurans' by the neighbor-joining method using the entire 16S rRNA nucleotide sequence revealed that this actinomycete is closely related to Amlycolatopsis orientalis subsp orientalis, Amycolatopsis coloradensis, Amycolatopsis alba, Amycolatopsis sulphurea and other Amycolatopsis sp. but only distantly related to species of the genus Nocardia. The cephamycin producer 'N. lactamdurans' NRRL 3802 should be, therefore, classified as Amycolatopsis lactamdurans. The deduced secondary structure of the 16S rRNA is very similar to that of A. colorandensis and A. alba but different from those of species of the Nocardia genus supporting the incorporation of 'N. lactamdurans' into the genus Amycolatopsis.     

Chemotaxonomic study on Thymus villosus from Portugal

The composition of the essential oils of four populations of Thymus villosus subsp. lusitanicus (Boiss.) Coutinho from Portugal was investigated by GC and GC-MS. To study the chemical polymorphism the results obtained from GC analyses of the volatile oils from individual plants from four populations were submited to Principal Component and Cluster analyses. A comparision with the essential oil of T. villosus subsp. villosus, previously studied by us was done. Important differences with regard to the major constituents in these two taxa were found. Linalool, geranyl acetate, geraniol and terpinen-4-ol were the main components of the essential oils of T. villosus subsp. lusitanicus, whereas in the oil of T. villosus subsp. villosus p-cymene, myrcene and alpha-terpineol were the major ones. Although, both taxa showed chemical polymorphism, different types of essential oils were characterized in each one: linalool; linalool/ terpinen-4-ol/trans-sabinene hydrate; linalool/1,8-cineole; geranyl acetate/geraniol; geranyl acetate/geraniol/1,8-cineole in T. villosus subsp. lusitanicus and p-cymene/camphor/linalool; p-cymene/borneol; linalool/geraniol/geranyl acetate; alpha-terpineol/camphor/myrcene in T. villosus subsp. villosus. Thus, the two subspecies of T. villosus can be easely differenciated by the composition of their essential oils.     

The Heterogeneity of Structural and Functional Photosynthetic Characteristics of Mesophyll Chloroplasts in Various Parts of Mature or Senescing Leaf Blade of Two Maize (Zea Mays L.) Genotypes

Differences in ultrastructural parameters of mesophyll cell (MC) chloroplasts, contents of photosynthetic pigments, and photochemical activities of isolated MC chloroplasts were studied in the basal, middle, and apical part of mature or senescing leaf blade of two maize genotypes. A distinct heterogeneity of leaf blade was observed both for structural and functional characteristics of chloroplasts. In both mature and senescing leaves the shape of MC chloroplasts changed from flat one in basal part of leaf to nearly spherical one in leaf apex. The volume density of granal thylakoids decreased from leaf base to apex in both types of leaves examined, while the amount of intergranal thylakoids increased in mature leaves but decreased in senescing leaves. The most striking heterogeneity was found for the quantity of plastoglobuli, which strongly increased with the increasing distance from leaf base. The differences in chloroplast ultrastructure were accompanied by differences in other photosynthetic characteristics. The Hill reaction activity and activity of photosystem 1 of isolated MC chloroplasts decreased from leaf base to apex in mature leaves. Apical part of senescing leaf blade was characterised by low contents of chlorophyll (Chl) a and Chl b, whereas in mature leaves, the content of Chls as well as the content of total carotenoids (Car) slightly increased from basal to apical leaf part. This was reflected also in the ratio Chl (a+b)/total Car; the ratio of Chl a/b did not significantly differ between individual parts of leaf blade. Both genotypes examined differed in the character of developmental gradient observed along whole length of leaf blade.     

EGb761 Blocks MPP+-Induced Lipid Peroxidation in Mouse Corpus Striatum

EGb761 has been suggested to be an antioxidant and free radical scavenger. Excess generation of free radicals, leading to lipid peroxidation (LP), has been proposed to play a role in the damage to striatal neurons induced by 1-methyl-4-phenylpyridinium (MPP⁺). We investigated the effects of EGb761 pretreatment on MPP⁺ neurotoxicity. C-57 black mice were pretreated with EGb761 for 17 days at different doses (0.63, 1.25, 2.5, 5 or 10 mg/kg) followed by administration of MPP⁺, (0.18, 0.36 or 0.72 mg/kg). LP was analyzed in corpus striatum at 30 min, 1 h, 2 h and 24 h after MPP⁺ administration. Striatal dopamine content was analyzed by HPLC at the highest EGb761 dose at 2 h and 24 h after MPP⁺ administration. MPP⁺-induced LP was blocked (100%) by EGb761 (10 mg/kg). Pretreatment with EGb761 partially prevented (32%) the dopamine-depleting effect of MPP⁺ at 24 h. These results suggest that supplements of EGb761 may be effective at preventing MPP⁺-induced oxidative stress.     

Development of a homologous transformation system for the opportunistic human pathogen Aspergillus fumigatus based on the sC gene encoding ATP sulfurylase

The development of a homologous transformation system for the opportunistic human pathogenic fungus Aspergillus fumigatus is described. The system is based on the sC gene encoding ATP sulfurylase. Several A. fumigatus sC mutant strains were readily isolated by strong selection for selenate resistance. The coding region plus upstream and downstream regulatory sequences of the A. fumigatus sC gene were cloned by inverse PCR and then sequenced. Sequencing of the sC cDNA revealed the presence of five introns located within the first half of the gene. The A. fumigatus sC gene encodes a protein of 574 amino acids which is highly similar to ATP sulfurylases from the filamentous fungal species Aspergillus nidulans, Aspergillus terreus and Penicillium chrysogenum. By contrast, ATP sulfurylases from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the C-terminal adenosine-5'-phosphosulfate kinase-like domain present in the filamentous fungal orthologues. A 3.8-kb DNA fragment amplified by PCR and containing the sC gene plus 5' and 3' flanking regions was cloned into pUC19 to give the vector pSCFUM. Transformation of two different sC mutant isolates with the plasmid pSCFUM established the functionality of this new homologous transformation system. Molecular analysis of sC+ transformants showed that up to 44% of transformed clones contained one or more copies of the entire plasmid integrated at the sC locus. This result also demonstrates the utility of the sC marker for targeting specific genetic constructs to the A. fumigatus sC locus, facilitating studies of gene regulation and function.     

Photosynthetic acclimation to photon irradiance and its relation to chlorophyll fluorescence and carbon assimilation in the halotolerant green alga Dunaliella viridis

This work describes the long-term acclimation of the halotolerant microalga Dunaliella viridis to different photon irradiance, ranging from darkness to 1500 μmol m⁻² s⁻¹. In order to assess the effects of long-term photoinhibition, changes in oxygen production rate, pigment composition, xanthophyll cycle and in vivo chlorophyll fluorescence using the saturating pulse method were measured. Growth rate was maximal at intermediate irradiance (250 and 700 μmol m⁻² s⁻¹). The increase in growth irradiance from 700 to 1500 μmol m⁻² s⁻¹ did not lead to further significant changes in pigment composition or EPS, indicating saturation in the pigment response to high light. Changes in Photosystem II optimum quantum yield (F_v/F_m) evidenced photoinhibition at 700 and especially at 1500 μmol m⁻² s⁻¹. The relation between photosynthetic electron flow rate and photosyntetic O₂ evolution was linear for cultures in darkness shifting to curvilinear as growth irradiance increased, suggesting the interference of the energy dissipation processes in oxygen evolution. Carbon assimilation efficiencies were studied in relation to changes in growth rate, internal carbon and nitrogen composition, and organic carbon released to the external medium. All illuminated cultures showed a high capability to maintain a C:N ratio between 6 and 7. The percentage of organic carbon released to the external medium increased to its maximum under high irradiance (1500 μmol m⁻² s⁻¹). These results suggest that the release of organic carbon could act as a secondary dissipation process when the xanthophyll cycle is saturated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Continuous enantioselective esterification of trans-2-phenyl-1-cyclohexanol using a new Candida rugosa lipase in a packed bed bioreactor

Enantioselective resolution of trans-2-phenyl-1-cyclohexanol (TPCH) by a Candida rugosa lipase, obtained by fermentation in the laboratory, and immobilised on EP100 polypropylene powder has been carried out using isooctane as solvent and propionic acid as esterifying agent. The study have included the utilisation of this biocatalyst in a batch process and the optimisation of the esterification conditions by means of a Box-Hunter-based experimental design. The main variables controlling the process, concentration of acid and alcohol, have been numerically optimised using initial esterification rate as objective function. The optimal concentrations for the batch process were 50 mM for the alcohol and 71 mM for the acid. This esterification reaction kinetics corresponded to a reversible Michaelis-Menten kinetic law for the optimal conditions, which has permitted to select a plug-flow packed bed bioreactor as the most appropriate configuration to minimise the residence time and to avoid shear stress effect on the biocatalyst. The behaviour of the continuous packed bed bioreactor at two different residence times (302 and 582 min) was in accordance with predictions from batch experiments, with slightly deviations (less than 10%). Continuous experiments maintained high values of enantioselectivity (enantiomeric factor was practically 1) and conversion near equilibrium value (35%) when long-time operation was carried out. Besides, long-time stability of biocatalyst has permitted to scale-up the production of enantioenriched (1R,2S)-TPCH propionate to yield gram quantities.     

Induction of cancer cell apoptosis by alpha-tocopheryl succinate: molecular pathways and structural requirements.

The vitamin E analog alpha-tocopheryl succinate (alpha-TOS) can induce apoptosis. We show that the proapoptotic activity of alpha-TOS in hematopoietic and cancer cell lines involves inhibition of protein kinase C (PKC), since phorbol myristyl acetate prevented alpha-TOS-triggered apoptosis. More selective effectors indicated that alpha-TOS reduced PKCalpha isotype activity by increasing protein phosphatase 2A (PP2A) activity. The role of PKCalpha inhibition in alpha-TOS-induced apoptosis was confirmed using antisense oligonucleotides or PKCalpha overexpression. Gain- or loss-of-function bcl-2 mutants implied modulation of bcl-2 activity by PKC/PP2A as a mitochondrial target of alpha-TOS-induced proapoptotic signals. Structural analogs revealed that alpha-tocopheryl and succinyl moieties are both required for maximizing these effects. In mice with colon cancer xenografts, alpha-TOS suppressed tumor growth by 80%. This epitomizes cancer cell killing by a pharmacologically relevant compound without known side effects.