Photodynamic activity of anthraquinones isolated from Heterophyllaea pustulata Hook f. (Rubiaceae) on MCF-7c3 breast cancer cells

Searching for agents that could be effective in the treatment of cancer, special highlight has focused on the study of numerous plant-derived compounds. We previously demonstrated that anthraquinones (AQs) isolated from a vegetal species: Heterophyllaea pustulata Hook f. (Rubiaceae), such as rubiadin, rubiadin-1-methyl ether, soranjidiol, soranjidiol-1-methyl ether exhibit photosensitizing properties without antecedents as photodynamic agents in malignant cells. In the present study, we investigated the potential role of these AQs as a phototoxic agent against human breast carcinoma using MCF-7c3 cells. All AQs exhibited significant photocytotoxicity on cancer cells at the concentration of 100 μM with 1 J/cm² light dose, resulting soranjidiol-1-methyl ether in complete cell destruction. The observed cellular killing by photoactivated AQs exhibited close relation with singlet oxygen production, except for soranjidiol-1-methyl ether, where cell viability decrease is in relation to uptake by tumor cells.     

Differential N-glycosylation of a monoclonal antibody expressed in tobacco leaves with and without endoplasmic reticulum retention signal apparently induces similar in vivo stability in mice

Plant cells are able to perform most of the post-translational modifications that are required by recombinant proteins to achieve adequate bioactivity and pharmacokinetics. However, regarding N-glycosylation the processing of plant N-glycans in the Golgi apparatus displays major differences when compared with that of mammalian cells. These differences in N-glycosylation are expected to influence serum clearance rate of plant-derived monoclonal antibodies. The monoclonal antibody against the hepatitis B virus surface antigen expressed in Nicotiana tabacum leaves without KDEL endoplasmic reticulum (ER) retention signal (CB.Hep1(-)KDEL) and with a KDEL (Lys-Asp-Glu-Leu) fused to both IgG light and heavy chains (CB.Hep1(+)KDEL) were tested for in vivo stability in mice. Full characterization of N-glycosylation and aggregate formation in each monoclonal antibody batch was determined. The mouse counterpart (CB.Hep1) was used as control. Both (CB.Hep1(-)KDEL) and (CB.Hep1(+)KDEL) showed a faster initial clearance rate (first 24 h) compared with the analogous murine antibody while the terminal phase was similar in the three antibodies. Despite the differences between CB.Hep1(+)KDEL and CB.Hep1(-)KDEL N-glycans, the in vivo elimination in mice was indistinguishable from each other and higher than the murine monoclonal antibody. Molecular modelling confirmed that N-glycans linked to plantibodies were oriented away from the interdomain region, increasing the accessibility of the potential glycan epitopes by glycoprotein receptors that might be responsible for the difference in stability of these molecules.     

Identification of amino acids that account for long-range interactions in two triosephosphate isomerases from pathogenic trypanosomes

For a better comprehension of the structure-function relationship in proteins it is necessary to identify the amino acids that are relevant for measurable protein functions. Because of the numerous contacts that amino acids establish within proteins and the cooperative nature of their interactions, it is difficult to achieve this goal. Thus, the study of protein-ligand interactions is usually focused on local environmental structural differences. Here, using a pair of triosephosphate isomerase enzymes with extremely high homology from two different organisms, we demonstrate that the control of a seventy-fold difference in reactivity of the interface cysteine is located in several amino acids from two structurally unrelated regions that do not contact the cysteine sensitive to the sulfhydryl reagent methylmethane sulfonate, nor the residues in its immediate vicinity. The change in reactivity is due to an increase in the apparent pKa of the interface cysteine produced by the mutated residues. Our work, which involved grafting systematically portions of one protein into the other protein, revealed unsuspected and multisite long-range interactions that modulate the properties of the interface cysteines and has general implications for future studies on protein structure-function relationships.     

Analysis of NK cells and chemokine receptors in tumor infiltrating CD4 T lymphocytes in human renal carcinomas

Recent data suggest that chemokines and chemokine receptors mediate leukocyte recruitment of all components of the antitumor response. This study aimed to phenotypically characterize the immune lymphocyte infiltrate in human renal cell carcinomas (RCCs) and at the invasive margin (tumor–host interface) and to define the association of these findings with established prognostic indicators. Tumor infiltrating lymphocytes (TILs) were obtained from 24 patients with RCC undergoing radical nephrectomy. Peripheral blood cells from 37 patients were also obtained before surgery. Our findings are consistent with the preferential recruitment of CD4+ Th1-polarized effector memory cells that express CXCR3/CCR5. These cells were the main component of TILs and expressed as CXCR3, CCR5, CD45RO, and CD95. Natural killer (NK) cells were found in significantly higher proportions in TILs of RCCs than in peripheral blood lymphocytes (PBLs) or in other tumors studied (colorectal and breast cancers), where these cells were found in small proportions. No differences in nuclear grade or other studied parameters were observed between the TILs and the lymphocytes present at the invasive margin, which showed a similar composition. However, differences were found according to the tumor stage. First, significantly fewer NK cells were observed in PBLs from metastatic patients. Second, a significantly lower proportion of CCR5/CXCR3/CD4+ cells and a higher proportion of CCR4/CD4+ cells were observed in metastatic patients, suggesting that preferential Th1-polarization may gradually change during the progression of renal cancer cells. Finally, the frequency of CD25/CD4+ cells was higher in metastatic patients. Although the sample of patients with metastasis was small, the overall results suggest a change in composition of the TILs that may potentially confer a selective advantage for tumor growth and may account for the suppression of an effective cytotoxic response.     

Microbiological control in stem cell banks: approaches to standardisation

The transplant of cells of human origin is an increasingly complex sector of medicine which entails great opportunities for the treatment of a range of diseases. Stem cell banks should assure the quality, traceability and safety of cultures for transplantation and must implement an effective programme to prevent contamination of the final product. In donors, the presence of infectious micro-organisms, like human immunodeficiency virus, hepatitis B virus, hepatitis C virus and human T cell lymphotrophic virus, should be evaluated in addition to the possibility of other new infectious agents (e.g. transmissible spongiform encephalopathies and severe acute respiratory syndrome). The introduction of the nucleic acid amplification can avoid the window period of these viral infections. Contamination from the laboratory environment can be achieved by routine screening for bacteria, fungi, yeast and mycoplasma by European pharmacopoeia tests. Fastidious micro-organisms, and an adventitious or endogenous virus, is a well-known fact that will also have to be considered for processes involving in vitro culture of stem cells. It is also a standard part of current good practice in stem cell banks to carry out routine environmental microbiological monitoring of the cleanrooms where the cell cultures and their products are prepared. The risk of viral contamination from products of animal origin, like bovine serum and mouse fibroblasts as a “feeder layer” for the development of embryonic cell lines, should also be considered. Stem cell lines should be tested for prion particles and a virus of animal origin that assure an acceptable quality.     

Production of yam microtubers using a temporary immersion system

Yam clones ‚Pacala Duclos’ and ‚Belep’ in temporary immersion system culture showed favourable results on shoot growth stage and in the development of microtubers in comparison with solid culture media. Cultures in temporary immersion systems in both clones obtained a higher microtuber number per plant, with greater fresh weight and diameter in comparison with solid culture media. Besides, 45 and 47% of microtubers greater than 3.0 gFW for ‚Belep’ and ‚Pacala Duclos’ clones respectively, were obtained. Those tubers may be planted without acclimatization and may be stored for a prolonged period of time.     

Genetic mapping of QTLs controlling horticultural traits in diploid roses

A segregating progeny set of 96 F₁ diploid hybrids (2n=2x=14) between Blush Noisette (D10), one of the first seedlings from the original Champneys Pink Cluster, and Rosa wichurana (E15), was used to construct a genetic linkage map of the rose genome following a pseudo-testcross mapping strategy. A total of 133 markers (130 RAPD, one morphological and two microsatellites) were located on the 14 linkage groups (LGs) of the D10 and E15 maps, covering total map lengths of 388 and 260 cM, respectively. Due to the presence of common biparental markers the homology of four LGs between parental maps (D10-1/E15-1 to D10-4/E15-4) could be inferred. Four horticulturally interesting quantitative traits, flower size (FS), days to flowering (DF), leaf size (LS), and resistance to powdery mildew (PM) were analysed in the progeny in order to map quantitative trait loci (QTLs) controlling these traits. A total of 13 putative QTLs (LOD>3.0) were identified, four for FS, two for flowering time, five for LS, and two for resistance to PM. Possible homologies between QTLs detected in the D10 and E15 maps could be established between Fs1 and Fs3, Fs2 and Fs4, and Ls1 and Ls3. Screening for pairwise epistatic interactions between loci revealed additional, epistatic QTLs (EQTLs) for DF and LS that were not detected in the original QTL analysis. The genetic maps developed in this study will be useful to add new markers and locate genes for important traits in the genus providing a practical resource for marker-assisted selection programs in roses.     

Mapping sugar beet pectin acetylation pattern

Homogalacturonan-derived partly methylated and/or acetylated oligogalacturonates were recovered after enzymatic hydrolysis (endo-polygalacturonase+pectin methyl esterase+side-chain degrading enzymes) of sugar beet pectin followed by anion-exchange and size exclusion chromatography. Around 90% of the GalA and 75% of the acetyl groups present in the initial sugar beet pectin were recovered as homogalacturonan-derived oligogalacturonates, the remaining GalA and acetyl belonging to rhamnogalacturonic regions. Around 50% of the acetyl groups present in sugar beet homogalacturonans were recovered as partly methylated and/or acetylated oligogalacturonates of degree of polymerisation 5 whose structures were determined by electrospray ionization ion trap mass spectrometry (ESI-IT-MSn). 2-O-acetyl- and 3-O-acetyl-GalA were detected in roughly similar amounts but 2,3-di-O-acetylation was absent. Methyl-esterified GalA residues occurred mainly upstream 2-O-acetyl GalA. Oligogalacturonates containing GalA residues that are at once methyl- and acetyl-esterified were recovered in very limited amounts. A tentative mapping of the distribution of acetyl and methyl esters within sugar beet homogalacturonans is proposed. Unsubstituted GalA residues are likely to be present in limited amounts (approximately 10% of total GalA residues), due to the fact that methyl and acetyl groups are assumed to be most often not carried by the same residues.     

Structural elements in IGP synthase exclude water to optimize ammonia transfer

In the complex pathway of histidine biosynthesis, a key branch point linking amino acid and purine biosynthesis is catalyzed by the bifunctional enzyme imidazole glycerol phosphate (IGP) synthase. The first domain of IGP synthase, a triad glutamine amidotransferase, hydrolyzes glutamine to form glutamate and ammonia. Its activity is tightly regulated by the binding of the substrate PRFAR to its partner synthase domain. Recent crystal structures and molecular dynamics simulations strongly suggest that the synthase domain, a (beta/alpha)(8) barrel protein, mediates the insertion of ammonia and ring formation in IGP by channeling ammonia from one remote active site to the other. Here, we combine both mutagenesis experiments and computational investigations to gain insight into the transfer of ammonia and the mechanism of conduction. We discover an alternate route for the entrance of ammonia into the (beta/alpha)(8) barrel and argue that water acts as both agonist and antagonist to the enzymatic function. Our results indicate that the architecture of the two subdomains, most notably the strict conservation of key residues at the interface and within the (beta/alpha)(8) barrel, has been optimized to allow the efficient passage of ammonia, and not water, between the two remote active sites.