Stress-induced expression of the gamma subunit (FXYD2) modulates Na,K-ATPase activity and cell growth

In kidney, the Na,K-ATPase is associated with a single span protein, the gamma subunit (FXYD2). Two splice variants are differentially expressed along the nephron and have a differential influence on Na,K-ATPase when stably expressed in mammalian cells in culture. Here we used a combination of gene induction and gene silencing techniques to test the functional impact of gamma by means other than transfection. NRK-52E cells (of proximal tubule origin) do not express gamma as a protein under regular tissue culture conditions. However, when they were exposed to hyperosmotic medium, induction of only the gammaa splice variant was observed, which was accompanied by a reduction in the rate of cell division. Kinetic analysis of stable enzyme properties from control (alpha1beta1) and hypertonicity-treated cultures (alpha1beta1gammaa) revealed a significant reduction (up to 60%) of Na,K-ATPase activity measured under V(max) conditions with little or no change in the amounts of alpha1beta1. This effect as well as the reduction in cell growth rate was practically abolished when gamma expression was knocked down using specific small interfering RNA duplexes. Surprisingly, a similar induction of endogenous gammaa because of hypertonicity was seen in rat cell lines of other than renal origin: C6 (glioma), PC12 (pheochromocytoma), and L6 (myoblasts). Furthermore, exposure of NRK-52E cells to other stress inducers such as heat shock, exogenous oxidation, and chemical stress also resulted in a selective induction of gammaa. Taken together, the data imply that induction of gammaa may have adaptive value by being a part of a general cellular response to genotoxic stress.     

The early light-inducible protein (ELIP) gene is expressed during the chloroplast-to-chromoplast transition in ripening tomato fruit

Chloroplast-to-chromoplast transitions during fruit ripening require massive transformation of the plastid internal membrane structure as the photosynthetic apparatus is disassembled. Early Light-Inducible Proteins (ELIPs) are known to accumulate in chloroplasts during thylakoid biogenesis and under stressful conditions. To determine if ELIP may also play a role in thylakoid disassembly during the chloroplast-to-chromoplast transition, ELIP mRNA expression was measured in tomato, Lycopersicon esculentum Mill. cv. Rutgers. An EST clone was identified in the Tomato Genome Project/Solanaceae Genomics Network database that has high sequence similarity with the amino acid sequence of Arabidopsis ELIP1 and ELIP2. It has complete identity in the two conserved regions of the protein. Genomic Southern blots indicate that the gene is a single copy in tomato. The genomic sequence shows the three-exon structure typical of ELIP sequences from other species. mRNA for this gene is barely detectable on northern blots from etiolated seedlings, but transiently accumulates to high levels 2 h after transfer to the light. Greenhouse-grown tomatoes were used to measure ELIP mRNA accumulation during fruit development and ripening. Tomato ELIP mRNA is detectable in all stages of fruit ripening, but is most abundant in the breaker/turning stage of development. A survey of tomato EST databases revealed that ELIP cDNA is also relatively abundant in developing flowers, which contain yellow chromoplasts. Combined, these results suggest that ELIP may play a newly-recognized role in the chloroplast-to-chromoplast transition process.     

Aβ(1-40) Forms Five Distinct Amyloid Structures whose β-Sheet Contents and Fibril Stabilities Are Correlated

The ability of a single polypeptide sequence to grow into multiple stable amyloid fibrils sets these aggregates apart from most native globular proteins. The existence of multiple amyloid forms is the basis for strain effects in yeast prion biology, and might contribute to variations in Alzheimer's disease pathology. However, the structural basis for amyloid polymorphism is poorly understood. We report here five structurally distinct fibrillar aggregates of the Alzheimer's plaque peptide Aβ(1-40), as well as a non-fibrillar aggregate induced by Zn²⁺. Each of these conformational forms exhibits a unique profile of physical properties, and all the fibrillar forms breed true in elongation reactions under a common set of growth conditions. Consistent with their defining cross-β structure, we find that in this series the amyloid fibrils containing more extensive β-sheet exhibit greater stability. At the same time, side chain packing outside of the β-sheet regions contributes to stability, and to differences of stability between polymorphic forms. Stability comparison is facilitated by the unique feature that the free energy of the monomer (equivalent to the unfolded state in a protein folding reaction) does not vary, and hence can be ignored, in the comparison of ΔG° of elongation values for each polymorphic fibril obtained under a single set of conditions.     

Expression of membrane-bound carbonic anhydrases IV, IX, and XIV in the mouse heart.

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-alpha-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.     

Alanine scanning mutagenesis of Abeta(1-40) amyloid fibril stability

We describe here an alanine scanning mutational analysis of the Abeta(1-40) amyloid fibril monitored by fibril elongation thermodynamics derived from critical concentration values for fibril growth. Alanine replacement of most residues in the amyloid core region, residues 15-36, leads to destabilization of the elongation step, compared to wild-type, by about 1kcal/mol, consistent with a major role for hydrophobic packing in Abeta(1-40) fibril assembly. Where comparisons are possible, the destabilizing effects of Ala replacements are generally in very good agreement with the effects of Ala replacements of the same amino acid residues in an element of parallel beta-sheet in the small, globular protein Gbeta1. We utilize these Ala-WT DeltaDeltaG values to filter previously described Pro-WT DeltaDeltaG values, creating Pro-Ala DeltaDeltaG values that specifically assess the sensitivity of a sequence position, in the structural context of the Abeta fibril, to replacement by proline. The results provide a conservative view of the energetics of Abeta(1-40) fibril structure, indicating that positions 18-21, 25-26, and 32-33 within amyloid structure are particularly sensitive to the main-chain disrupting effects of Pro replacements. In contrast, residues 14-17, 22, 24, 27-31, and 34-39 are relatively insensitive to Pro replacements; most N-terminal residues were not tested. The results are discussed in terms of amyloid fibril structure and folding energetics, in particular focusing on how the data compare to those from other structural studies of Abeta(1-40) amyloid fibrils grown in phosphate-buffered saline at 37 degrees C under unstirred ("quiescent") conditions.     

Clonal dissemination of Escherichia coli O157:H7 subtypes among dairy farms in northeast Ohio

To ascertain the extent to which indistinguishable strains of Escherichia coli O157:H7 are shared between farms, molecular characterization was performed on E. coli O157:H7 isolates recovered during a longitudinal study of 20 dairy farms in northeast Ohio. Of the 20 dairy farms sampled, 16 were located in a primary area and 4 were located in two other distant geographical areas. A total of 92 E. coli O157:H7 isolates obtained from bovine fecal samples, water trough sediment samples, free-stall bedding, and wild-bird excreta samples were characterized. Fifty genetic subtypes were observed among the isolates using XbaI and BlnI restriction endonucleases. Most restriction endonuclease digestion profiles (REDPs) were spatially and temporally clustered. However, four REDPs from multiple sources were found to be indistinguishable by pulsed-field gel electrophoresis between four pairs of farms. The geographical distance between farms which shared an indistinguishable E. coli O157:H7 REDP ranged from 9 to 50 km, and the on-farm sources sharing indistinguishable REDPs included cattle and wild bird feces and free-stall bedding. Within the study population, E. coli O157:H7 REDP subtypes were disseminated with considerable frequency among farms in close geographic proximity, and nonbovine sources may contribute to the transmission of this organism between farms.     

Molecular determinants of proteolytic disassembly of the reovirus outer capsid

Following attachment and internalization, mammalian reoviruses undergo intracellular proteolytic disassembly followed by viral penetration into the cytoplasm. The initiating event in reovirus disassembly is the cathepsin-mediated proteolytic degradation of viral outer capsid protein σ3. A single tyrosine-to-histidine mutation at amino acid 354 (Y354H) of strain type 3 Dearing (T3D) σ3 enhances reovirus disassembly and confers resistance to protease inhibitors such as E64. The σ3 amino acid sequence of strain type 3 Abney (T3A) differs from that of T3D at eight positions including Y354H. However, T3A displays disassembly kinetics and protease sensitivity comparable with T3D. We hypothesize that one or more additional σ3 polymorphisms suppress the Y354H phenotype and restore T3D disassembly characteristics. To test this hypothesis, we engineered a panel of reovirus variants with T3A σ3 polymorphisms introduced individually into T3D-σ3Y354H. We evaluated E64 resistance and in vitro cathepsin L susceptibility of these viruses and found that one containing a glycine-to-glutamate substitution at position 198 (G198E) displayed disassembly kinetics and E64 sensitivity similar to those properties of T3A and T3D. Additionally, viruses containing changes at positions 233 and 347 (S233L and I347T) developed de novo compensatory mutations at position 198, strengthening the conclusion that residue 198 is a key determinant of σ3 proteolytic susceptibility. Variants with Y354H in σ3 lost infectivity more rapidly than T3A or T3D following heat treatment, an effect abrogated by G198E. These results identify a regulatory network of residues that control σ3 cleavage and capsid stability, thus providing insight into the regulation of nonenveloped virus disassembly.     

Slow amyloid nucleation via α-helix-rich oligomeric intermediates in short polyglutamine-containing huntingtin fragments

The 17-amino-acid N-terminal segment (htt(NT)) that leads into the polyglutamine (polyQ) segment in the Huntington's disease protein huntingtin (htt) dramatically increases aggregation rates and changes the aggregation mechanism, compared to a simple polyQ peptide of similar length. With polyQ segments near or above the pathological repeat length threshold of about 37, aggregation of htt N-terminal fragments is so rapid that it is difficult to tease out mechanistic details. We describe here the use of very short polyQ repeat lengths in htt N-terminal fragments to slow this disease-associated aggregation. Although all of these peptides, in addition to htt(NT) itself, form α-helix-rich oligomeric intermediates, only peptides with Q(N) of eight or longer mature into amyloid-like aggregates, doing so by a slow increase in β-structure. Concentration-dependent circular dichroism and analytical ultracentrifugation suggest that the htt(NT) sequence, with or without added glutamine residues, exists in solution as an equilibrium between disordered monomer and α-helical tetramer. Higher order, α-helix rich oligomers appear to be built up via these tetramers. However, only htt(NT)Q(N) peptides with N=8 or more undergo conversion into polyQ β-sheet aggregates. These final amyloid-like aggregates not only feature the expected high β-sheet content but also retain an element of solvent-exposed α-helix. The α-helix-rich oligomeric intermediates appear to be both on- and off-pathway, with some oligomers serving as the pool from within which nuclei emerge, while those that fail to undergo amyloid nucleation serve as a reservoir for release of monomers to support fibril elongation. Based on a regular pattern of multimers observed in analytical ultracentrifugation, and a concentration dependence of α-helix formation in CD spectroscopy, it is likely that these oligomers assemble via a four-helix assembly unit. PolyQ expansion in these peptides appears to enhance the rates of both oligomer formation and nucleation from within the oligomer population, by structural mechanisms that remain unclear.     

Toxoplasma gondii actively inhibits neuronal function in chronically infected mice

Upon infection with the obligate intracellular parasite Toxoplasma gondii, fast replicating tachyzoites infect a broad spectrum of host cells including neurons. Under the pressure of the immune response, tachyzoites convert into slow-replicating bradyzoites, which persist as cysts in neurons. Currently, it is unclear whether T. gondii alters the functional activity of neurons, which may contribute to altered behaviour of T. gondii-infected mice and men. In the present study we demonstrate that upon oral infection with T. gondii cysts, chronically infected BALB/c mice lost over time their natural fear against cat urine which was paralleled by the persistence of the parasite in brain regions affecting behaviour and odor perception. Detailed immunohistochemistry showed that in infected neurons not only parasitic cysts but also the host cell cytoplasm and some axons stained positive for Toxoplasma antigen suggesting that parasitic proteins might directly interfere with neuronal function. In fact, in vitro live cell calcium (Ca(2+)) imaging studies revealed that tachyzoites actively manipulated Ca(2+) signalling upon glutamate stimulation leading either to hyper- or hypo-responsive neurons. Experiments with the endoplasmatic reticulum Ca(2+) uptake inhibitor thapsigargin indicate that tachyzoites deplete Ca(2+) stores in the endoplasmatic reticulum. Furthermore in vivo studies revealed that the activity-dependent uptake of the potassium analogue thallium was reduced in cyst harbouring neurons indicating their functional impairment. The percentage of non-functional neurons increased over time In conclusion, both bradyzoites and tachyzoites functionally silence infected neurons, which may significantly contribute to the altered behaviour of the host.