Effect of water on the molecular mobility of elastin

Purified and hydrated elastin is studied by both thermal and dielectric techniques to have insight into the chain dynamics of this protein. By differential scanning calorimetry, the glassy behavior of elastin is highlighted; the glass transition temperature (T(g)) of elastin is found to be widely dependent on hydration, falling from 200 degrees C in the dehydrated state to 30 degrees C for 30% hydration. A limit of T(g) at around 0 degrees C is found when crystallizable water is present in the system, that is, when the formation of ice prevents motions of some 10 nm along the polypeptidic chains. The technique of thermally stimulated currents, carried out in the -180 to 0 degrees C temperature range, is useful to detect localized motions. In this case, too, the localized motions vary considerably according to hydration: a first relaxation mode is observed at -145 degrees C and it is associated with the reorientation of crystallizable water in ice I; a second relaxation mode, more complex and cooperative, occurs at around -80 degrees C and could be attributed to the complex constituted by the dipolar groups of the polypeptidic chain and noncrystallizable water, behaving as a glassy system.     

Effect of hormones and growth factors on the proliferation of adult cricket neural progenitor cells in vitro

In the adult cricket brain, a cluster of neuroblasts produces new interneurons that integrate into the mushroom body (MB), the main associative structure for multisensory information of the insect brain. In previous study we showed the antagonist role of the two morphogenetic hormones, juvenile hormone (JH) and ecdysone, on the regulation of adult MB neurogenesis in vivo. In order to examine whether these hormones act directly on neural progenitor cells, we developed an organotypic culture of MB cortices. Cell proliferation was assessed by 5-bromo, 2'-deoxyuridine (BrdU) incorporation. We showed that JH increased mushroom body neuroblast (MBNb) proliferation, confirming the mitogenic effect of JH observed in vivo. By contrast, ecdysone did not affect the amount of BrdU-labeled nuclei, suggesting that the inhibitory effect observed in vivo probably proceeded from an indirect pathway. We then examined the role of growth factors known to stimulate neural stem cell/progenitor cell proliferation in vertebrates. As shown by calcium imaging, MBNb only expressed functional receptors for insulin whereas mature interneurons responded to IGF-I and bFGF. Both insulin (10 microg/ml) and IGF-I (10 ng/ml) enhanced MB progenitor cell proliferation in culture, although the insulin effect was more pronounced. This effect was abolished when an inhibitor of polyamine biosynthesis was present in the medium, suggesting a link between polyamines and the insulin signaling pathway. By contrast, bFGF (20-200 ng/ml) failed to stimulate MBNb proliferation. Our results point to conserved and divergent mechanisms between vertebrates and invertebrates in the regulation of adult neural progenitor cell proliferation.     

Mapping of a conformational epitope shared between E1 and E2 on the serum-derived human hepatitis C virus envelope

Monoclonal antibody D32.10 produced by immunizing mice with a hepatitis C virus (HCV)-enriched pellet obtained from plasmapheresis of a chronically HCV1b-infected patient binds HCV particles derived from serum of different HCV1a- and HCV1b-infected patients. Moreover, this monoclonal has been shown to recognize both HCV envelope proteins E1 and E2. In an attempt to provide novel insight into the membrane topology of HCV envelope glycoproteins E1 and E2, we localized the epitope recognized by D32.10 on the E1 and/or E2 sequence using Ph.D.-12 phage display peptide library technology. Mimotopes selected from the phage display dodecapeptide library by D32.10 shared partial similarities with 297RHWTTQGCNC306 of the HCV E1 glycoprotein and with both 613YRLWHYPCT621 and 480PDQRPYCWHYPPKPC494 of the HCV E2 glycoprotein. Immunoreactivity of D32.10 with overlapping peptides corresponding to these three HCV regions confirmed these localizations and suggested that the three regions identified are likely closely juxtaposed on the surface of serum-derived particles as predicted by the secondary model structure of HCV E2 derived from the tick-borne encephalitis virus E protein. This assertion was supported by the detection of specific antibodies directed against these three E1E2 regions in sera from HCV-infected patients.     

Transforming the sapstaining fungus Ophiostoma piceae with Agrobacterium tumefaciens

A transformation protocol mediated by Agrobacterium tumefaciens is described for the sapstaining fungus Ophiostoma piceae. We compared transformants obtained from Agrobacterium with those obtained from yeast-like cells made into spheroplasts and treated with CaCl2. For all putative transformants analyzed, Southern hybridization confirmed that the hygromycin resistance gene had been integrated into the genomic DNA. While all transformants obtained from the treated spheroplasts had multiple copy vector insertion, 85% of the Agrobacterium-mediated transformants had single copy vector insertion.     

Increasing amounts of dietary myristic acid modify the plasma cholesterol level and hepatic mass of scavenger receptor BI without affecting bile acid biosynthesis in hamsters

The purpose of this study was to analyze the effects of increasing amounts of dietary myristic acid (0.03 to 4.2% of the total dietary energy) on the plasma and hepatic cholesterol metabolism. Six groups of hamsters received semi-purified diets containing 0.05% cholesterol and 12.5% lipids and differing only by the nature of the triglycerides (Safflower oil, lard, lard/coconut oil (1:1), milk fat, milk fat/coconut oil (1:1), coconut oil) for 3 weeks. A positive regression between the plasma cholesterol level and the dietary myristic acid level was observed (r = 0.60, P < 0.0001). However, it is noteworthy that the increase in plasma total cholesterol only reflects an increase in the level of HDL-cholesterol. In parallel, the mass SR-BI decreased linearly with the increased level of myristic acid in the diet, whereas the LDL-R did not change. This study shows that increasing amounts of myristic acid (0.03 to 4.2%) do not alter the cholesterol or bile acid metabolism and increase only the HDL-C.     

Molecular mobility of elastin: effect of molecular architecture

The thermal and dielectric properties of elastin and two soluble derivatives (kappa-elastin and derived elastin peptides from enzymatic elastolysis) were investigated in the freeze-dried state in a wide temperature range (from -180 to +220 degrees C). The glass transition of these amorphous proteins was studied by differential scanning calorimetry (DSC). The dielectric relaxations of both proteins were followed by thermally stimulated currents (TSC), an isochronal dielectric spectrometry running at variable temperature, analogous to a low-frequency spectroscopy (10(-3)-10(-2) Hz) and by dynamic dielectric spectroscopy (DDS), performed isothermally with the frequency varying from 10(-2) to 3 x 10(6) Hz. The combination of TSC and DDS experiments and the determination of the activation parameters of the relaxation times inform about the molecular mobility of the proteins, both in the glassy state and in the liquid state. Major differences between the relaxation behavior of elastin and its soluble derivatives have been discussed and correlated with the molecular architecture of the proteins.     

Exploring the active site of Trypanosoma brucei phosphofructokinase by inhibition studies: specific irreversible inhibition

This work deals with the phosphofructokinase enzyme (PFK) of the parasite Trypanosoma brucei. Inhibitors which are analogues of fructose-6-phosphate (F6P) derived from 2,5-anhydromannitol and therefore blocked in a closed conformation, both nonphosphorylated and phosphorylated, were designed. They provided information on this class of ATP-dependent PFK (structurally more similar to PPi-dependent PFKs revealing (i) an ordered mechanism, ATP binding first, inducing an essential conformational change to increase the affinity for F6P, and (ii) a rather hydrophobic environment at the ATP binding site. Nonphosphorylated mannitol derivatives bind at both the ATP and F6P binding sites, whereas the phosphorylated derivatives only bind at the ATP binding site. The inhibitors bearing an aromatic ring substituted at the meta position indicate a polar interaction with lysine 227, which is specific to T. brucei PFK and is replaced by a glycine in human PFK. This lysine can be irreversibly bound, leading to inhibition when an electrophilic carbon atom is beta to the meta position on the ring. This lysine was identified by site-directed mutagenesis. This first example of a specific irreversible inactivation of T. brucei PFK offers an opportunity to develop biologically active compounds against the sleeping sickness, the causative agent of which is the trypanosome.