Alzheimer's associated variant ubiquitin causes inhibition of the 26S proteasome and chaperone expression

Intracellular protein inclusions in Alzheimer's disease and progressive supranuclear palsy contain UBB+1, a variant ubiquitin. UBB+1 is able block the 26S proteasome in cell lines. Proteasome inhibition by drug action has previously been shown to induce a heat-shock response and render protection against stress. We investigated UBB+1 by developing a stable, conditional expression model in SH-SY5Y human neuroblastoma cells. Induction of UBB+1 expression caused proteasome inhibition as was confirmed by reduced ability to process misfolded canavanyl proteins, accumulation of GFPu, a proteasome substrate, and reduced cleavage of a fluorogenic substrate. We show that expression of UBB+1 induces expression of heat-shock proteins. This priming of the chaperone system in these cells promotes a subsequent resistance to tert-butyl hydroperoxide-mediated oxidative stress. We conclude that although UBB+1-expressing cells have a compromised ubiquitin-proteasome system, they are protected against oxidative stress conditions.     

Percutaneous cryoablation of colorectal liver metastases: potentiated by two consecutive freeze-thaw cycles

Cryoablation may be beneficial for selected patients with liver tumours. Two freeze-thaw cycles at the same location have been recommended during treatment as this potentiate the effect of ablation in experimental studies. However, single freeze ablations are used by some as double freeze procedures are time-consuming and have been associated with increased risk of complications. Estimation of ice-ball volume is difficult using regularly used monitoring techniques. Magnetic resonance imaging, however, allows excellent and multiplanar visualisation of the frozen region during ablation. We comment on the effect of double freeze cycles in regard to ice-ball volume as estimated from magnetic resonance imaging during percutaneous cryoablation of colorectal liver metastases. The ice-ball volume at the end of the second freeze cycle was median 42% larger than the volume at the end of the first freeze. Double freeze cycles may thus facilitate tumour destruction.     

Interactions of trimeric purine nucleoside phosphorylases with ground state analogues--calorimetric and fluorimetric studies

Binding enthalpies, dissociation constants and stoichiometry of binding for interaction of trimeric calf spleen and Cellulomonas sp. purine nucleoside phosphorylases with their ground state analogues (substrates and inhibitors) were studied by calorimetric and spectrofluorimetric methods. Data for all ligands, with possible exception of hypoxanthine, are consistent with three identical non-interacting binding sites.     

Modulation of cell cycle progression and of antibody production in mouse hybridomas by a nucleotide analogue

The nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) has been identified as a powerful antiproliferative substance when acting on hybridoma cells. In the range of 10 nM to 100 nM concentrations this agent reduces cell growth rate, while its apoptosis-inducing activity is marginal. Marked induction of apoptosis can be observed at micromolar and higher order concentrations. In PMEG-supplemented media the cell cycle progression is perturbed, the flow-cytometric DNA profile shows a higher proportion of cells in the S and G2/M phases of the cell cycle. Concomitantly with the reduction of the growth rate, the specific monoclonal antibody production rate may rise by 20–27%. Addition of PMEG at the end of the exponential phase of a batch culture results in an enhancement of the final monoclonal antibody concentration. This revised version was published online in August 2006 with corrections to the Cover Date.     

The evaluation of photosynthetic parameters in maize inbred lines subjected to water deficiency: Can these parameters be used for the prediction of performance of hybrid progeny?

The response of selected photosynthetic and morphological parameters of plants to drought was examined in 5 inbred lines of maize (Zea mays L.) and their 10 F1 hybrids. The aim of the study was to establish whether the photosynthetic performance of parental genotypes under drought conditions correlates with the performance of their progeny and whether the net photosynthetic rate, the chlorophyll fluorescence parameters or the content of photosynthetic pigments could be used as reliable physiological markers for early breeding generations. The relative importance of the additive and the nonadditive (dominance, maternal) genetic effects in the inheritance of these parameters was also assessed by means of the quantitative genetics analysis. The results showed that the nonadditive genetic effects associated with a particular combination of genotypes or a particular direction of crossing are at least equally and often even more important as the additivity and that these genetic effects almost totally change with the exposure of plants to drought conditions. This was reflected in the inability to predict the response of F1 hybrids to drought on the basis of the photosynthetic performance of their parents, which indicates that the practical usability of such parameters in maize breeding programs is rather limited.     

The effects of brassinosteroids on photosynthetic parameters in leaves of two field-grown maize inbred lines and their F1 hybrid

The effect of foliar spray with 10⁻¹² M aqueous solutions of 24-epibrassinolide or a synthetic androstane analogue of castasterone on the activity of photosystem (PS) 1, the Hill reaction activity, the content of photosynthetic pigments and the specific leaf mass was examined for three different leaves developed after brassinosteroid (BR) treatment in two inbred lines of field-grown maize and their F1 hybrid. The brassinosteroids significantly affected neither the efficiency of photosynthetic electron transport, nor the content of chlorophylls or carotenoids.     

Modeling the interaction between aldolase and the thrombospondin-related anonymous protein, a key connection of the malaria parasite invasion machinery

A complex molecular motor empowers substrate-dependent motility and host cell invasion in malaria parasites. The interaction between aldolase and the transmembrane adhesin thrombospondin-related anonymous protein (TRAP) transduces the motor force across the parasite surface. Here, we analyzed this interaction by using state-of-the-art flexible docking. Besides algorithms to account for induced fit in the side-chains of the Plasmodium falciparum aldolase (PfAldo) structure, we used additional in silico receptors modeled upon crystallographic structures of evolutionarily related aldolases to incorporate enzyme backbone flexibility, and to overcome structure inaccuracies due to the relatively low resolution (3.0 A) of the genuine PfAldo structure. Our results indicate that, in spite of multiple intermolecular contacts, only the six C-terminal residues of the TRAP cytoplasmic tail bind in an ordered manner to PfAldo. This portion of TRAP targets the PfAldo active site, with its n-1 Trp residue, which is essential for this interaction, buried within the PfAldo catalytic pocket. Docking of a TRAP peptide bearing a Trp to Ala mutation rendered the lower energy configurations either bound weakly outside the active site or not bound to PfAldo at all. The position of the bound TRAP peptide, and particularly the close proximity between the carbonyl of its n-2 Asp residue and the experimentally determined position of the phosphate-6 group of fructose 1,6-phosphate bound to mammalian aldolases, predicts an inhibitory effect of TRAP on catalysis. Enzymatic and TRAP-binding assays using mutant PfAldo molecules strongly support the overall structural model. These results might provide the initial framework for the identification of novel antiparasitic compounds.     

Secretins: dynamic channels for protein transport across membranes

Secretins form megadalton bacterial-membrane channels in at least four sophisticated multiprotein systems that are crucial for translocation of proteins and assembled fibers across the outer membrane of many species of bacteria. Secretin subunits contain multiple domains, which interact with numerous other proteins, including pilotins, secretion-system partner proteins, and exoproteins. Our understanding of the structure of secretins is rapidly progressing, and it is now recognized that features common to all secretins include a cylindrical arrangement of 12-15 subunits, a large periplasmic vestibule with a wide opening at one end and a periplasmic gate at the other. Secretins might also play a key role in the biogenesis of their cognate secretion systems.     

Structural and functional studies on the interaction of GspC and GspD in the type II secretion system

Type II secretion systems (T2SSs) are critical for secretion of many proteins from Gram-negative bacteria. In the T2SS, the outer membrane secretin GspD forms a multimeric pore for translocation of secreted proteins. GspD and the inner membrane protein GspC interact with each other via periplasmic domains. Three different crystal structures of the homology region domain of GspC (GspC(HR)) in complex with either two or three domains of the N-terminal region of GspD from enterotoxigenic Escherichia coli show that GspC(HR) adopts an all-β topology. N-terminal β-strands of GspC and the N0 domain of GspD are major components of the interface between these inner and outer membrane proteins from the T2SS. The biological relevance of the observed GspC-GspD interface is shown by analysis of variant proteins in two-hybrid studies and by the effect of mutations in homologous genes on extracellular secretion and subcellular distribution of GspC in Vibrio cholerae. Substitutions of interface residues of GspD have a dramatic effect on the focal distribution of GspC in V. cholerae. These studies indicate that the GspC(HR)-GspD(N0) interactions observed in the crystal structure are essential for T2SS function. Possible implications of our structures for the stoichiometry of the T2SS and exoprotein secretion are discussed.