Inducible expression of Bcl-XL restricts apoptosis resistance to the antibody secretion phase in hybridoma cultures

B-cell hybridomas are widely used to produce monoclonal antibodies via large-scale cell culture. Unfortunately, these cells are highly sensitive to apoptotic death under conditions of nutrient deprivation observed at the plateau phase of batch cultures. Previous work has indicated that constitutive high-level expression of antiapoptotic genes in hybridoma cells could delay apoptosis, resulting in higher cell densities and prolonged viability. However, the constitutive high-level expression of antiapoptotic genes has been shown to have detrimental effects on genomic stability of other types of cultured cells. Inducible gene expression may be used to avoid this problem. In the present study, we first constructed an expression vector in which the promoter of a mammalian metallothionein (MT) gene drives the expression of bcl-XL in response to metal exposure. The vector was then used to exogenously control the expression of bcl-XL in D5 hybridoma cells. Our data show that stably transfected D5 cells (4G1.D9) expressed high levels of Bcl-X(L) following overnight exposure to ZnSO(4) concentrations (50 to 100 microM) that did not affect control cells. The level of Bcl-X(L) expressed after ZnSO(4) induction was sufficient to prevent apoptosis experimentally induced by cycloheximide and allowed 4G1.D9 cells to grow at higher densities and remain viable for prolonged periods in suboptimal culture conditions. The use of inducible bcl-XL expression permits extension of the viability of cultured B-cell hybridomas during the antibody secretion phase without the adverse genetic effects associated with constitutive long-term bcl-XL expression.     

The Hsp90 co-chaperones Cdc37 and Sti1 interact physically and genetically

Cdc37 associates with the heat-shock protein 90 (Hsp90) molecular chaperone as one of several auxiliary proteins that are collectively referred to as Hsp90 co-chaperones. Cdc37 has been proposed to be a specificity factor for Hsp90, directing it notably towards kinases. It is not known whether Cdc37 is essential for viability in the budding yeast Saccharomyces cerevisiae because of Hsp90-dependent or -independent functions or both. Sti1 and Cpr7 are non-essential Hsp90 co-chaperones that bind to a common surface on Hsp90 through tetratricopeptide repeats (TPR). We have found that Sti1 is specifically retained from yeast extracts by immobilized Cdc37. Similarly, the endogenous proteins are also found in a complex. Moreover, purified recombinant Sti1 and Cdc37 interact in the complete absence of Hsp90. Complexes between Cdc37 and Sti1 are not unique to this TPR protein since endogenous Cdc37 can be co-purified with exogenously expressed Cpr7 fused to glutathione-S-transferase. The heterogeneity of Cdc37 complexes, both with and without Hsp90, may expand the functional diversity of Cdc37. Here we show that the combination of cdc37 and sti1 mutations is synthetically lethal, suggesting that direct contacts between Cdc37 and Sti1 may at least contribute to vital functions in yeast.     

Human telomeric position effect is determined by chromosomal context and telomeric chromatin integrity

We investigated the influence of telomere proximity and composition on the expression of an EGFP reporter gene in human cells. In transient transfection assays, telomeric DNA does not repress EGFP but rather slightly increases its expression. In contrast, in stable cell lines, the same reporter construct is repressed when inserted at a subtelomeric location. The telomeric repression is transiently alleviated by increasing the dosage of the TTAGGG repeat factor 1 (TRF1). Upon a prolongated treatment with trichostatin A, the derepression of the subtelomeric reporter gene correlates with the delocalization of HP1α and HP1β. In contrast, treating the cells with 5 azacytidin, a demethylating agent, or with sirtinol, an inhibitor of the Sir2 family of deacetylase, has no apparent effect on telomeric repression. Overall, position effects at human chromosome ends are dependent on a specific higher-order organization of the telomeric chromatin. The possible involvement of HP1 isoforms is discussed.     

Innate immunity: the missing link in neuroprotection and neurodegeneration?

Innate immunity was previously thought to be a nonspecific immunological programme that was engaged by peripheral organs to maintain homeostasis after stress and injury. Emerging evidence indicates that this highly organized response also takes place in the central nervous system. Through the recognition of neuronal fingerprints, the long-term induction of the innate immune response and its transition to an adaptive form might be central to the pathophysiology and aetiology of neurodegenerative disorders. Paradoxically, this response also protects neurons by favouring remyelination and trophic support afforded by glial cells.     

Sexual size dimorphism in parasitic oxyurid nematodes

As in many invertebrates, female oxyurids are larger than male. Sexual size dimorphism (SSD) of oxyurid nematodes (the hosts of which are both invertebrate and vertebrate), is investigated regarding body size of both host and parasite. SSD of parasites appeared to be weakly, but not significandy, correlated with invertebrate and vertebrate host body size. However, this study reveals a different pattern for SSD with respect to either type of host. SSD does not increase in tandem with body size in vertebrate parasites either at the level of species or genus. SSD is much more pronounced in Syphaciidae than in Heteroxynematidae, two families of vertebrate parasites exhibiting different modes of transmission (members of the Syphaciidae are transmitted through perianal contamination). SSD is investigated in one monophyletic group of parasites of primates, for which a phylogeny is known. Independent comparisons method is used and we find that the body size of female parasite is strongly correlated with that of the male. The hypoallometry (slope<1) of the relationship suggests that the SSD is not linked to an increase of parasite body size. Moreover, there is no influence of host body size on parasite SSD. The pattern in parasites of invertebrates is different. First, SSD has been found to increase with parasite body size in two groups of invertebrate parasites: the oxyurids of Dictyoptera and Coleoptera. Second, female body size of invertebrate parasites is not correlated with male body size either at genus or species level. Finally, the evolution of SSD is discussed in relation to the demographic patterns of invertebrate parasites and the haplodiploid mode of reproduction of these parasitic nematodes.     

Zooplankton assemblages and influence of environmental parameters on them in a Mediterranean coastal area

Temporal dynamics of zooplankton communities and assemblages,as well as the influence of environmental factors on them, werestudied in Saronikos Gulf (Aegean Sea, Greece). Different multivariatetechniques (hierarchical clustering, multidimensional scalingand correspondence analysis) were applied on a 2 year data setof zooplankton species composition, based on samples collectedat five stations of the study area. A clear discrimination oftwo communities was revealed, the first one in the semi-enclosedpolluted northern part (Elefsis Bay) andthe second in SaronikosGulf proper. Within the latter community, five assemblages weredistinguished: (i) the coastal winter assemblage characterizedby Ctenocalanus vanus, Oithona similis, Clausocalanus pergensand Fritillaria pellucida; (ii) the psychrophilic assemblageinfluenced by the open sea and characterized by Oithona plumiferaand Clausocalanus jobei; (iii) the spring assemblage characterizedby Evadne nordmanni and Centropages typicus; (iv) the coastalthermophilic assemblage characterized by Penilia avirostis,Evadne ergesrina and Temora stylifera; (v) the thermophilicassemblage influenced by the open sea and characterized by Clausocalanusfurcatus. Both zooplankton and environmental data were treatedby multiple correspondence analysis which revealed the importanceof some environmental factors on zooplankton community composition(eutrophication-pollution, temperature, water mass circulation,hydrology and topography).     

Length polymorphism and allele structure of trinucleotide microsatellites in natural accessions of Arabidopsis thaliana

 The objective of this work was to assess the degree of trinucleotide microsatellite length polymorphism in the selfing species Arabidopsis thaliana. PCR amplifications of 12 microsatellite loci among 49 natural populations revealed between one to eight length variants (alleles) for each locus. The average number of alleles per locus was four and the average genetic diversity index was 0.43. Divergence between length variants was investigated at the nucleotide level. Several observations emerge from the sequence data: (1) for most loci, length polymorphism results only from variations in the number of trinucleotide repeats; (2) for a few others, some variability was noted in the flanking sequences; (3) for compound and interrupted loci containing two arrays of trinucleotide repeats, length variations preferentially affect the longest one. Five of the Arabidopsis thaliana accessions were clearly composed of two sublines. In 2 other accessions, some heterozygous individual plants, probably resulting from recent outcrosses, were found. A phylogenetic tree constructed on the basis of trinucleotide microsatellite allelic diversity shows that genetic relationships among the accessions are not correlated with their geographic origin. Received: 4 November 1997 / Accepted: 3 March 1998     

Choroidal readaptation to gravity in rats after spaceflight and head-down tilt

Davet, Julien, Benoit Clavel, Lucien Datas, LaurenceMani-Ponset, Daniel Maurel, Serge Herbuté, Michel Viso, WilliamHinds, Joellen Jarvi, and Jacqueline Gabrion.Choroidal readaptation to gravity in rats after spaceflight andhead-down tilt. J. Appl. Physiol.84(1): 19-29, 1998.To determine when choroidal structures wererestored after readaptation to Earth gravity or orthostatic position,fine structure and protein distribution were studied in rat choroidplexus dissected either 6 h [Space Life Sciences-2 (SLS-2)experiments] or 2 days [National Institutes ofHealth-Rodent 1 (NIH-R1) experiments] after a spaceflight, or 6 hafter head-down tilt (HDT) experiments. Apical alterations were notedin choroidal cells from SLS-2 and HDT animals, confirming thatweightlessness impaired choroidal structures and functions. However,the presence of small apical microvilli and kinocilia and the absenceof vesicle accumulations showed that the apical organization began tobe restored rapidly after landing. Very enlarged apical microvilli appeared after 2 days on Earth, suggesting increased choroidal activity. However, as distributions of ezrin and carbonic anhydrase IIremained altered in both flight and suspended animals after readaptation to Earth gravity, it was concluded that choroidal structures and functions were not completely restored, even after 2 days in Earth's gravity.