Synergism among lysophosphatidic acid, beta1A integrins, and epidermal growth factor or platelet-derived growth factor in mediation of cell migration.

GD25 cells lacking the beta1 integrin subunit or expressing beta1A with certain cytoplasmic mutations have poor directed cell migration to platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), ligands of receptor tyrosine kinases, or to lysophosphatidic acid (LPA), a ligand of G-protein-coupled receptors (Sakai, T., Zhang, Q., F?ssler, R., and Mosher, D. F. (1998) J. Cell Biol. 141, 527-538 and Sakai, T., Peyruchaud, O., F?ssler, R., and Mosher, D. F. (1998) J. Biol. Chem. 273, 19378-19382). We demonstrate here that LPA synergizes with signals induced by beta1A integrins and ligated EGF or PDGF receptors to modulate migration. When LPA was mixed with EGF or PDGF, migration was greater than with EGF or PDGF alone. The enhancement was greater for beta1A-expressing cells than for beta1-null cells. Cells expressing beta1A with mutations of prolines or tyrosines in conserved cytoplasmic NPXY motifs had blunted migratory responses to mixtures of LPA and EGF or PDGF. The major effects on beta1A-expressing cells of LPA when combined with EGF or PDGF were to sensitize cells so that maximal responses were obtained with >10-fold lower concentrations of growth factor and increase the chemokinetic component of migration. Sensitization by LPA was lost when cells were preincubated with pertussis toxin or C3 exotransferase. There was no evidence for transactivation or sensitization of receptors for EGF or PDGF by LPA. EGF or PDGF and LPA caused activation of mitogen-activated protein kinase by pertussis toxin-insensitive and -sensitive pathways respectively, but activation was not additive. These findings indicate that signaling pathways initiated by the cytoplasmic domains of ligated beta1A integrins and tyrosine kinase receptors interact with signaling pathways initiated by LPA to facilitate directed cell migration.     

Expression of Mitochondrial Regulators PGC1α and TFAM as Putative Markers of Subtype and Chemoresistance in Epithelial Ovarian Carcinoma

Epithelial ovarian carcinoma (EOC), the major cause of gynaecological cancer death, is a heterogeneous disease classified into five subtypes. Each subtype has distinct clinical characteristics and is associated with different genetic risk factors and molecular events, but all are treated with surgery and platinum/taxane regimes. Tumour progression and chemoresistance is generally associated with major metabolic alterations, notably altered mitochondrial function(s). Here, we report for the first time that the expression of the mitochondrial regulators PGC1α and TFAM varies between EOC subtypes; furthermore, we have identified a profile in clear-cell carcinoma consisting of undetectability of PGC1α/TFAM, and low ERα/Ki-67. By contrast, high-grade serous carcinomas were characterised by a converse state of PGC1α/TFAM, ERα positivity and a high Ki-67 index. Interestingly, loss of PGC1α/TFAM and ERα was found also in a non-clear cell EOC cell line made highly resistant to platinum in vitro. Similar to clear-cell carcinomas, these resistant cells also showed accumulation of glycogen. Altogether, our data provide mechanistic insights into the chemoresistant nature of ovarian clear-cell carcinomas. Furthermore, these findings corroborate the need to take into account the diversity of EOC and to develop subtype specific treatment strategies.     

Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies.

Bispecific single-chain diabodies (scDb) consist of the variable heavy and light chain domains of two antibodies connected by three linkers. The structure of an scDb in the V(H)-V(L) orientation is V(H)A-linkerA-V(L)B-linkerM-V(H)B-linkerB-V(L)A, with linkers A and B routinely chosen to be 5-6 residues and linker M 15-20 residues. Here, we applied display of scDb on filamentous phage to analyse the composition of optimal linker sequences. The three linkers were randomized in length and sequence using degenerated triplets coding for only six hydrophilic or aliphatic amino acids (Thr, Ser, Asp, Asn, Gly, Ala). Antigen-binding clones were then isolated by one to two rounds of selection on the two different antigens recognized by the bispecific scDb. Using an scDb directed against carcinoembryonic antigen (CEA) and beta-galactosidase (Gal), we found that monomeric scDb had a preferred length of 15 or more amino acid residues for the middle linker M and of 3-6 residues for the linkers A and B. No obvious bias towards a preferred linker sequence was observed. Reduction of the middle linker below 13 residues led to the formation of dimeric scDb, which most likely results from interchain pairing between all the V(H) and V(L) domains. Dimeric scDb were also formed by fragments possessing a long linker M and linkers A and B of 0 or 1 residue. We assume that these dimeric scDb are formed by intrachain pairing of the central variable domains and interchain pairing of the flanking variable domains. Thus, the latter molecules represent a novel format of bispecific and tetravalent molecules. The described strategy allows for the isolation of both optimized and minimal linker sequences for the assembly of monomeric or dimeric single-chain diabodies.     

A DNA Damage Checkpoint Pathway Coordinates the Division of Dikaryotic Cells in the Ink Cap Mushroom Coprinopsis cinerea

The fungal fruiting body or mushroom is a multicellular structure essential for sexual reproduction. It is composed of dikaryotic cells that contain one haploid nucleus from each mating partner sharing the same cytoplasm without undergoing nuclear fusion. In the mushroom, the pileus bears the hymenium, a layer of cells that includes the specialized basidia in which nuclear fusion, meiosis, and sporulation occur. Coprinopsis cinerea is a well-known model fungus used to study developmental processes associated with the formation of the fruiting body. Here we describe that knocking down the expression of Atr1 and Chk1, two kinases shown to be involved in the response to DNA damage in a number of eukaryotic organisms, dramatically impairs the ability to develop fruiting bodies in C. cinerea, as well as other developmental decisions such as sclerotia formation. These developmental defects correlated with the impairment in silenced strains to sustain an appropriated dikaryotic cell cycle. Dikaryotic cells in which chk1 or atr1 genes were silenced displayed a higher level of asynchronous mitosis and as a consequence aberrant cells carrying an unbalanced dose of nuclei. Since fruiting body initiation is dependent on the balanced mating-type regulator doses present in the dikaryon, we believe that the observed developmental defects were a consequence of the impaired cell cycle in the dikaryon. Our results suggest a connection between the DNA damage response cascade, cell cycle regulation, and developmental processes in this fungus.     

Control of the growth of Alicyclobacillus acidoterrestris in industrialized orange juice using rosemary essential oil and nisin

The use of rosemary essential oil (RO) and its combination with nisin (RO+N) in preventing the multiplication of Alicyclobacillus acidoterrestris in orange juice was evaluated. The minimum inhibitory and bactericidal concentrations (MIC and MBC) for RO were both 125 μg ml⁻¹ while RO+N displayed a synergistic effect. The use of RO and RO+N at concentrations of 1, 4 and 8× MIC in orange juice for 96 h was evaluated in terms of their sporicidal effectiveness. With regard to the action against A. acidoterrestris spores, RO at 8× MIC was sporostatic, whereas RO+N at 1× MIC was sporicidal. Morphological changes in the structure of the micro-organism after treatment were also observed by microscopy. Furthermore, flow cytometric analysis showed that most cells were damaged or killed after treatment. In general, the antioxidant activity after addition of RO+N decreased with time. The results demonstrate that using the combination of RO and nisin can prevent the A. acidoterrestris growth in orange juice.     

Fate of biomedical research protocols and publication bias in France: retrospective cohort study

Objectives To describe the fate of protocols approved by the French research ethics committees, a national system created by the French 1988 Huriet-Sérusclat Act; to assess publication bias at a national level.Design Retrospective cohort study.Setting Representative sample of 25/48 French research ethics committees in 1994.Protocols 649 research protocols approved by committees, with follow-up information.Main outcome measures Protocols'' initial characteristics (design, study size, investigator) abstracted from committees'' archives; follow-up information (rates of initiation, completion, and publication) obtained from mailed questionnaire to principal investigators.Results Completed questionnaires were available for 649/976 (69%) protocols. Of these, 581 (90%) studies were initiated, 501/581 (86%) were completed, and 190/501 (38%) were published. Studies with confirmatory results were more likely to be published as scientific papers than were studies with inconclusive results (adjusted odds ratio 4.59, 95% confidence interval 2.21 to 9.54). Moreover, studies with confirmatory results were published more quickly than studies with inconclusive results (hazard ratio 2.48, 1.36 to 4.55).Conclusion At a national level, too many research studies are not completed, and among those completed too many are not published. We suggest capitalising on research ethics committees to register and follow all authorised research on human participants on a systematic and prospective basis.     

Effects of cytochalasin B on Na+ content and cell volume of Entamoeba histolytica

Cells of Entamoeba histolytica accumulated K+ and extruded Na+ compared to the concentrations of those ions present in the growth medium. Pinocytic activity, measured by the uptake of horseradish peroxidase of 125I-polyvinylpyrrolidone, was high (up to 0.3 ml/ml cells per h). Upon addition of cytochalasin B, at a concentration (20 microM) that completely blocked pinocytosis, cells lost up to 40% of their Na+ content within 90 min; K+ content was not affected or increased slightly compared to control cells without the inhibitor. Cation loss was associated with cell shrinkage. The dose-response curves for the effects of cytochalasin B on pinocytosis and Na+ content were identical. These data provide direct evidence that pinocytosis is an important component of the homeostatic system for Na+.     

Morphological taxonomy of little-differentiated Hyphomycetes

Morphological taxonomy of simple Hyphomycetes is complicated by the frequent occurrence of pleoanamorphism. In some groups of yeast-like fungi, uncommon synanamorphs are diagnostic. Differences in conidiogenesis do not always delimit natural groups. Some nomenclatural problems are mentioned, with an emphasis on the need of neotypification. Prospects are sketched for future taxonomic research.