High level production of secreted proteins: example of the human tissue inhibitor of metalloproteinases 1

The major difficulty for high-throughput screening of therapeutic protein candidates in experimental animal models of pathologies or for structural studies is their fast and efficient production. The tissue inhibitors of metalloproteinases (TIMPs) considered to play a role in many physiological and pathological processes, such as arthritis or cancer, by inhibiting matrix metalloproteinases or acting as signalling molecules, have always been produced with huge difficulties. We hereby propose a new method to overproduce human recombinant TIMP-1 by transient expression in HEK293E cells, followed by a one-step chromatography purification, yielding in only 2 weeks, dozens of milligrams of pure, stable, glycosylated and active protein for in vitro and in vivo studies. This easy to set up, rapid, and efficient method could be applied for any naturally secreted mammalian protein.     

Multiple transgressions of Wallace's Line explain diversity of flightless Trigonopterus weevils on Bali

The fauna of Bali, situated immediately west of Wallace''s Line, is supposedly of recent Javanese origin and characterized by low levels of endemicity. In flightless Trigonopterus weevils, however, we find 100% endemism for the eight species here reported for Bali. Phylogeographic analyses show extensive in situ differentiation, including a local radiation of five species. A comprehensive molecular phylogeny and ancestral area reconstruction of Indo-Malayan–Melanesian species reveals a complex colonization pattern, where the three Balinese lineages all arrived from the East, i.e. all of them transgressed Wallace''s Line. Although East Java possesses a rich fauna of Trigonopterus, no exchange can be observed with Bali. We assert that the biogeographic picture of Bali has been dominated by the influx of mobile organisms from Java, but different relationships may be discovered when flightless invertebrates are studied. Our results highlight the importance of in-depth analyses of spatial patterns of biodiversity.     

Chromosomal mapping of the pel and cel genes in Erwinia chrysanthemi strain B374

Using the RP4::mini-Mu in vivo cloning technique, van Gijsegem et al. (1985) isolated several pel and cel genes of Erwinia chrysanthemi (Ech) B374 strain. We have localized these genes on the Ech chromosome by co-transfer mapping of MudI1734 insertion mutants and refined the map by co-transposition analysis. This analysis has enabled us to identify another cel gene.     

Stress-induced premature senescence in BJ and hTERT-BJ1 human foreskin fibroblasts

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric DNA-binding complex of the subunits alpha and beta with relevance in O(2) and energy homeostasis. The labile component, HIF-1alpha, is not only activated by hypoxia but also by peptides such as insulin and interleukin-1 (IL-1) in normoxia. We investigated whether inhibitors of mitogen-activated protein kinase kinases (MAPKKs: PD 98059, U0126) and phosphatidylinositol 3-kinase (PI3K: LY 294002) do not only lower the hypoxia-induced, but also the insulin- and IL-1-induced HIF-1alpha accumulation and HIF-1 DNA-binding in human hepatoma cell cultures (line HepG2). The results show that LY 294002 suppressed HIF-1 activation in a dose-dependent manner irrespective of the stimulus. With respect to target proteins controlled by HIF-1, the production of erythropoietin was fully blocked and that of vascular endothelial growth factor reduced following inhibition of the PI3K pathway. The role of MAPKKs in this process remained in question, because PD 98059 and U0126 did not significantly reduce HIF-1alpha levels at non-toxic doses. We propose that PI3K signaling is not only important in the hypoxic induction of HIF-1 but it is also crucially involved in the response to insulin and IL-1.     

Isolation and characterization of a recA mutant of Erwinia chrysanthemi B374

Summary Cosmids carrying a rec gene of the Erwinia chrysanthemi strain B374 were identified in a genomic library constructed by Reverchon et al. (1985). One of them was mutagenized with transposon Tn1 and the rec:: Tn1 mutation was introduced into the B374 chromosome, providing the first rec-less E. chrysanthemi strain. The pectinolytic and cellulolytic activities were unaffected by the rec mutation.     

Characterisation of an IS1 induced mutation in the carboxy-terminal end of bacteriophage Mu transposase which affects several functional domains of the protein

We show that a mutation in bacteriophage Mu transposase (pA) which was isolated as a deletion of the C-terminal end of the protein actually consists of the replacement of the last 16 amino acids (which are mostly hydrophilic) by 26 mostly hydrophobic amino acids. This change almost completely inactivates the in vivo enzyme activity as well as its capacity to bind Mu ends in vitro, although the end-binding domain of the protein resides at least 150 amino acids from the C-terminus. This sharply contrasts with the properties of a series of missense mutations and short C-terminal deletions in pA described earlier which only slightly decrease the overall transposase activity.     

Similar slow down in running speed progression in species under human pressure

Running speed in animals depends on both genetic and environmental conditions. Maximal speeds were here analysed in horses, dogs and humans using data sets on the 10 best performers covering more than a century of races. This includes a variety of distances in humans (200–1500 m). Speed has been progressing fast in the three species, and this has been followed by a plateau. Based on a Gompertz model, the current best performances reach 97.4% of maximal velocity in greyhounds to 100.3 in humans. Further analysis based on a subset of individuals and using an ‘animal model’ shows that running speed is heritable in horses (h² = 0.438, P = 0.01) and almost so in dogs (h² = 0.183, P = 0.08), suggesting the involvement of genetic factors. Speed progression in humans is more likely due to an enlarged population of runners, associated with improved training practices. The analysis of a data subset (40 last years in 800 and 1500 m) further showed that East Africans have strikingly improved their speed, now reaching the upper part of the human distribution, whereas that of Nordic runners stagnated in the 800 m and even declined in the 1500 m. Although speed progression in dogs and horses on one side and humans on the other has not been affected by the same genetic/environmental balance of forces, it is likely that further progress will be extremely limited.