Improved microscopic observation of mammalian cells on microcarriers by fluorescent staining

Many microcarriers used for the cultivation of animal cells do not allow for convenient microscopic observation of cell morphology and viability due to their optical properties. Using fluorescent viable stain combining fluorescein diacetate and ethidium bromide, we observed the distribution, morphology and viability of cells on various microcarriers.     

Cultivation of anchorage-dependent animal cells in microsphere-induced aggregate culture

Summary Diethylaminoethyl-derivatized dextran microspheres were used to cultivate Chinese hamster ovary, 293, Vero and swine testicular cells. Cells became attached to the microspheres but did not spread out. Instead, they grew in a more spherical shape and eventually formed multiple-cell-layer aggregates. Viability in these aggregates remained high after the cultures reached high cell concentrations. This cultivation method allows a high cell density to be achieved with a low microsphere concentration.Offprint requests to: W.-S. Hu     

The karyotype of Trypanosoma cruzi Dm 28c: comparison with other T. cruzi strains and trypanosomatids

Chromosome-sized DNA molecules from Trypanosoma cruzi clone Dm 28c were analyzed and compared with other T. cruzi strains and monogenetic trypanosomatids by orthogonal field alteration gel electrophoresis. The results showed that T. cruzi Dm 28c displays at least 18 chromosomes ranging from 550 to more than 1500 kb and that in general the trypanosomatids have smaller chromosomes distributed in the size range from 300 to 1500 kb. With the exception of T. cruzi strain G49, there is no evidence of minichromosomes, suggesting they are not widely distributed among different isolates of the parasite. The hybridization of T. cruzi chromosomal Southern blots with probes for T. cruzi-specific genes showed that their location can change from one strain to another, supporting the idea of the plasticity of the parasite genome. Furthermore, the chromosome pattern is strictly conserved during the transformation of T. cruzi Dm 28c epimastigotes to metacyclic trypomastigotes, suggesting that extensive chromosomal rearrangements do not occur during at least part of the life cycle of the parasite.     

Ubiquitin-PCNA fusion as a mimic for mono-ubiquitinated PCNA in Schizosaccharomyces pombe

Translesion synthesis is a major mechanism with which eukaryotic cells deal with DNA damage during replication. Mono-ubiquitinated PCNA is a key regulator of this process. We have investigated whether a ubiquitin-PCNA fusion can mimic ubiquitinated PCNA, by transforming plasmids expressing this fusion protein into different mutants of Schizosaccharomyces pombe. We show that the fusion protein is able to form PCNA trimers and that it can reduce the UV sensitivity and increase translesion synthesis in mutants in which PCNA cannot be ubiquitinated (pcn1-K164R and rhp18), but not of the rad8 mutant in which PCNA can be mono-ubiquitinated but not poly-ubiquitinated. We conclude that the fusion protein is a mimic of mono-ubiquitinated PCNA but it cannot be poly-ubiquitinated. Expression of the fusion protein at levels similar to that of endogenous unmodified protein has little effect on the spontaneous mutation rate of S. pombe. Replacement of the pcn1 locus with PCNA N-terminally tagged with different epitopes resulted in lethality, probably because the tagged proteins were expressed at substantially reduced levels.     

Self-organized asymmetries in ant foraging: a functional response to food type and colony needs

The dominant paradigm to explain asymmetries in the spatialdistribution of foraging animals is that they track the spatialheterogeneity of their environment. However, in social insects,endogenous spatial asymmetries can emerge within a uniformenvironment as an outcome from the self-organizing processof trail recruitment. We studied how self-organized asymmetries contribute to the exploitation of different food sources (carbohydrateor proteins) in colonies of the aphid-tending ant Lasius nigervarying in their nutritional needs (presence or absence ofbrood). Colonies with brood fed on sucrose sources exhibita higher mobilization of foragers than the other experimentalgroups. Foraging patterns differ greatly according to food type: colonies strongly focus their activity on only one dropletof sucrose, whereas they show a rather homogeneous distributionof foragers between proteinaceous sources. In addition, thepresence of brood in the colony enhances the asymmetry of collectiveforaging for both types of food. These spatial differencesin self-organized foraging patterns allow efficient exploitationof natural resources and play a role in the competitive strategy of this widespread palearctic ant.     

Population-scale tissue transcriptomics maps long non-coding RNAs to complex disease

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Under Pressure That Splits a Family in Two. The Case of Lipocalin Family

The lipocalin family is typically composed of small proteins characterized by a range of different molecular recognition properties. Odorant binding proteins (OBPs) are a class of proteins of this family devoted to the transport of small hydrophobic molecules in the nasal mucosa of vertebrates. Among OBPs, bovine OBP (bOBP) is of great interest for its peculiar structural organization, characterized by a domain swapping of its two monomeric subunits. The effect of pressure on unfolding and refolding of native dimeric bOBP and of an engineered monomeric form has been investigated by theoretical and experimental studies under pressure. A coherent model explains the pressure-induced protein structural changes: i) the substrate-bound protein stays in its native configuration up to 330 MPa, where it loses its substrate; ii) the substrate-free protein dissociates into monomers at 200 MPa; and iii) the monomeric substrate-free form unfolds at 120 MPa. Molecular dynamics simulations showed that the pressure-induced tertiary structural changes that accompany the quaternary structural changes are mainly localized at the interface between the monomers. Interestingly, pressure-induced unfolding is reversible, but dimerization and substrate binding can no longer occur. The volume of the unfolding kinetic transition state of the monomer has been found to be similar to that of the folded state. This suggests that its refolding requires relatively large structural and/or hydrational changes, explaining thus the relatively low stability of the monomeric form of this class of proteins.