Assembly of amyloid protofibrils via critical oligomers--a novel pathway of amyloid formation

The amyloid formation of phosphoglycerate kinase (PGK) was investigated by static and dynamic light-scattering. The time-course of the scattering intensity and the hydrodynamic radius scale with initial monomer concentration in a linear fashion over a range of about 50 in concentration. This sets limits on theories for aggregation kinetics that can be used, and points towards irreversible, cascade type models. In addition, circular dichroism (CD) was used to monitor the transition between a predominantly alpha-helical spectrum to a beta-sheet enriched one. The time-course of the CD also proves to scale linearly with initial monomer concentration. Electron microscopy shows that small oligomers as well as protofibrils are present during aggregation. The found coupling between growth of intermediates and acquisition of beta-sheet structure is interpreted in terms of a generalized diffusion-collision model, where stabilization of beta-strands takes place by intermolecular interactions.     

Formation of critical oligomers is a key event during conformational transition of recombinant syrian hamster prion protein

We have investigated the conformational transition and aggregation process of recombinant Syrian hamster prion protein (SHaPrP90-232) by Fourier transform infrared spectroscopy, circular dichroism spectroscopy, light scattering, and electron microscopy under equilibrium and kinetic conditions. SHaPrP90-232 showed an infrared absorbance spectrum typical of proteins with a predominant alpha-helical structure both at pH 7.0 and at pH 4.2 in the absence of guanidine hydrochloride. At pH 4.2 and destabilizing conditions (0.3-2 m guanidine hydrochloride), the secondary structure of SHaPrP90-232 was transformed to a strongly hydrogen-bonded, most probably intermolecularly arranged antiparallel beta-sheet structure as indicated by dominant amide I band components at 1620 and 1691 cm-1. Kinetic analysis of the transition process showed that the decrease in alpha-helical structures and the increase in beta-sheet structures occurred concomitantly according to a bimolecular reaction. However, the concentration dependence of the corresponding rate constant pointed to an apparent third order reaction. No beta-sheet structure was formed within the dead time (190 ms) of the infrared experiments. Light scattering measurements revealed that the structural transition of SHaPrP90-232 was accompanied by formation of oligomers, whose size was linearly dependent on protein concentration. Extrapolation to zero protein concentration yielded octamers as the smallest oligomers, which are considered as "critical oligomers." The small oligomers showed spherical and annular shapes in electron micrographs. Critical oligomers seem to play a key role during the transition and aggregation process of SHaPrP90-232. A new model for the structural transition and aggregation process of the prion protein is described.     

Studies on the structure of animal ribosomes. VIII. Application of a digital image processing method to the enhancement of electron micrographs of small ribosomal subunits

Electron micrographs of negatively stained small subunits of rat liver ribosomes have been processed by computer-assisted accumulation. The resulting micrographs are characterized by a significant noise suppression and enhancement of image details.     

Defined sequence segments of the small heat shock proteins HSP25 and alphaB-crystallin inhibit actin polymerization.

The interaction of small heat shock proteins (sHSPs) with the actin cytoskeleton has been described and some members of this family, e.g. chicken and murine HSP25 (HSP27), inhibit the polymerization of actin in vitro. To analyse the molecular basis of this interaction, we synthesized a set of overlapping peptides covering the complete sequence of murine HSP25 and tested the effect of these peptides on actin polymerization in vitro by fluorescence spectroscopy and electron microscopy. Two peptides comprising the sequences W43 to R57 (peptide 6) and I92 to N106 (peptide 11) of HSP25 were found to be potent inhibitors of actin polymerization. Phosphorylation of N-terminally extended peptide 11 at serine residues known to be phosphorylated in vivo resulted in decline of their inhibitory activity. Interestingly, peptides derived from the homologous peptide 11 sequence of murine alphaB-crystallin showed the same behaviour. The results suggest that both HSP25 and alphaB-crystallin have the potential to inhibit actin polymerization and that this activity is regulated by phosphorylation.     

Identification of proteins of the 40 S ribosomal subunit involved in interaction with initiation factor eIF-2 in the quaternary initiation complex by means of monospecific antibodies

Monospecific polyclonal antibodies against seven proteins of the 40 S subunit of rat liver ribosomes were used to identify ribosomal proteins involved in interaction with initiation factor eIF-2 in the quaternary initiation complex [eIF-2 X GMPPCP X [3H]Met-tRNAf X 40 S ribosomal subunit]. Dimeric immune complexes of 40 S subunits mediated by antibodies against ribosomal proteins S3a, S13/16, S19 and S24 were found to be unable to bind the ternary initiation complex [eIF-2 X GMPPCP X [3H]Met-tRNAf]. In contrast, 40 S dimers mediated by antibodies against proteins S2, S3 and S17 were found to bind the ternary complex. Therefore, from the ribosomal proteins tested, only proteins S3a, S13/16, S19 and S24 are concluded to be involved in eIF-2 binding to the 40 S subunit.     

Influence of sex and genetic background on anxiety-related and stress-induced behaviour of prodynorphin-deficient mice

The role of dynorphin/kappa opioid receptors in epilepsy and addiction are well accepted, but their function in emotional control is not yet fully understood. Data obtained from different strains of prodynorphin (Pdyn)- and kappa opioid receptor (KOP)-deficient mice do not provide a consistent picture of the functions of Dyn/KOP in anxiety, suggesting the influence of testing conditions and/or genetic background. Therefore, we investigated the behaviour and neurochemistry of male and female Pdyn KO mice on the balb/c and C57Bl/6N background. Consistent with our results obtained from male mice on the C57bl/6N background, we observed a less anxious phenotype in the elevated plus maze, open-field and light-dark test in male mice on the balb/c background. Female mice on the balb/c background also displayed less anxiety like behaviour; however these data reflect high trait anxiety and inter-individual differences. In contrast, female mice on the C57Bl/6N background displayed low trait anxiety and a paradigm-dependent reduction of anxiety. No differences were observed in the forced swim test, while balb/c Pdyn KO mice displayed prolonged immobility in the tail suspension test. In line with our previous results, we observed reduced CRH mRNA in the central amygdala in all groups of mice. In contrast, the recently observed CRH mRNA reduction in the hypothalamic paraventricular nucleus appears restricted to male, but not female mice. Our data support previous data suggesting a pronounced impact of endogenous prodynorphin-derived peptides on anxiety. Moreover, our data support the idea that the less anxious phenotype manifests only at elevated stress levels.     

Interaction of huntingtin fragments with brain membranes--clues to early dysfunction in Huntington's disease

Abstract Huntingtin is a large, multi-domain protein of unknown function in the brain. An abnormally elongated polyglutamine stretch in its N-terminus causes Huntington's disease (HD), a progressive neurodegenerative disorder. Huntingtin has been proposed to play a functional role in membrane trafficking via proteins involved in endo- and exocytosis. Here, we supply evidence for a direct association between huntingtin and membranes. In the brains of R6/2 mice with HD pathology, a 64 kDa N-terminal huntingtin fragment accumulated in postsynaptic membranes during the pre-symptomatic period of 4-8 weeks of age. In addition, an oligomeric fragment of approximately 200 kDa was detected at 8 weeks of age. Simultaneous progressive changes in distribution of amphiphysin, synaptojanin, and subunits of NMDA- and AMPA-receptors provide a strong indication of dysfunctional synaptic trafficking. Composition of the major phospholipids in the synaptic membranes was unaffected. In vitro, large unilamellar vesicles of brain lipids readily associated with soluble N-terminal huntingtin exon 1 fragments and stimulated fibrillogenesis of mutant huntingtin aggregates. Moreover, interaction of both mutant and wild-type huntingtin exon 1 fragments with brain lipids caused bilayer perturbation, mediated through a proline-rich region adjacent to the polyglutamines. This suggests that lipid interactions in vivo could influence misfolding of huntingtin and may play an early role in HD pathogenesis.     

Effect of environmental conditions on aggregation and fibril formation of barstar

The dependence on environmental conditions of the assembly of barstar into amyloid fibrils was investigated starting from the nonnative, partially folded state at low pH (A-state). The kinetics of this process was monitored by CD spectroscopy and static and dynamic light scattering. The morphology of the fibrils was visualized by electron microscopy, while the existence of the typical cross- structure substantiated by solution X-ray scattering. At room temperature, barstar in the A-state is unable to form amyloid fibrils, instead amorphous aggregation is observed at high ionic strength. Further destabilization of the structure is required to transform the polypeptide chain into an ensemble of conformations capable of forming amyloid fibrils. At moderate ionic strength (75 mM NaCl), the onset and the rate of fibril formation can be sensitively tuned by increasing the temperature. Two types of fibrils can be detected differing in their morphology, length distribution and characteristic far UV CD spectrum. The formation of the different types depends on the particular environmental conditions. The sequence of conversion: A-statefibril type Ifibril type II appears to be irreversible. The transition into fibrils is most effective when the protein chain fulfills particular requirements concerning secondary structure, structural flexibility and tendency to cluster.Abbreviations CD circular dichroism - DLS dynamic light scattering - EM electron microscopy - SLS static light scattering - SAXS small-angle X-ray scattering - SOXS solution X-ray scattering     

Immunogenicity and Safety in Adults of a New Chromatographically Purified Vero-cell Rabies Vaccine (CPRV): a Randomized, Double-blind Trial with Purified Vero-cell Rabies Vaccine (PVRV)

Recent improvements in chromatographic purification procedures have made it possible to develop a new chromatographically purified rabies vaccine (CPRV) by further purifying the current rabies vaccine prepared from Vero-cell culture (Verorab; Pasteur Mérieux Connaught). The immunogenicity and safety of primary immunization, followed by a booster at one year, with CPRV was compared to that of the purified Vero cell vaccine (PVRV) in a randomized, double-blind study carried out at four veterinary schools in France. A total of 330 healthy, male and female, first-year veterinary students, aged at least 18 years and who required pre-exposure rabies prophylaxis, were enrolled in this study. Included subjects were randomly assigned either CPRV (n = 163) or PVRV (n = 167) to be given as a primary immunization series of three intramuscular injections (D0, D7, D28), followed by a booster after 1 year (D365). Blood samples for serological analysis were taken at D0 (before first injection), D28, D42, D180, D365 (before booster) and D379. All subjects developed a strong immune response to the primary series, and at D42, all subjects had seroconverted for rabies neutralizing antibody (serum titre > or = 0.5 IU/ml). The rabies virus-neutralizing antibody GMT value at D42 in the CPRV group (23.0 IU/ml) was non-inferior to that in the PVRV group (29.6 IU/ml), according to a one-sided non-inferiority test. While antibody titres tended to decrease over the period of follow-up, at D365 (before booster), 97.5% subjects in the CPRV group and 98.8% of subjects in the PVRV group remained seroconverted. After booster, although the rabies antibody GMT value in the CPRV group was lower than that in the PVRV group, all subjects in both groups were seroconverted, and the difference is probably not clinically important. The incidence of local and systemic reactions tended to decrease with each dose during the primary immunization series, followed by a slight increase after booster (significant time-effect in an exploratory logistic regression analysis). Although mild or moderate local reactions tended to be more frequent after injection with CPRV compared to PVRV, systemic reactions were reported less often (significant group-effects in exploratory logistic regression analyses). One serious adverse event possibly related to vaccine occurred during this study (severe asthenia after the third dose of PVRV). This comparative study in healthy young adults demonstrates that the new chromatographically purified rabies vaccine is as immunogenic as PVRV, and seems to be associated with fewer systemic reactions.