Hsc70 Rapidly Engages Tau after Microtubule Destabilization

The microtubule-associated protein Tau plays a crucial role in regulating the dynamic stability of microtubules during neuronal development and synaptic transmission. In a group of neurodegenerative diseases, such as Alzheimer disease and other tauopathies, conformational changes in Tau are associated with the initial stages of disease pathology. Folding of Tau into the MC1 conformation, where the amino acids at residues 7–9 interact with residues 312–342, is one of the earliest pathological alterations of Tau in Alzheimer disease. The mechanism of this conformational change in Tau and the subsequent effect on function and association to microtubules is largely unknown. Recent work by our group and others suggests that members of the Hsp70 family play a significant role in Tau regulation. Our new findings suggest that heat shock cognate (Hsc) 70 facilitates Tau-mediated microtubule polymerization. The association of Hsc70 with Tau was rapidly enhanced following treatment with microtubule-destabilizing agents. The fate of Tau released from the microtubule was found to be dependent on ATPase activity of Hsc70. Microtubule destabilization also rapidly increased the MC1 folded conformation of Tau. An in vitro assay suggests that Hsc70 facilitates formation of MC1 Tau. However, in a hyperphosphorylating environment, the formation of MC1 was abrogated, but Hsc70 binding to Tau was enhanced. Thus, under normal circumstances, MC1 formation may be a protective conformation facilitated by Hsc70. However, in a diseased environment, Hsc70 may preserve Tau in a more unstructured state, perhaps facilitating its pathogenicity.     

Weapons of mass destruction: virulence factors of the global killer enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of food and water-borne E. coli-mediated human diarrhoea worldwide. The incidence in developing countries is estimated at 650 million cases per year, resulting in 800 000 deaths, primarily in children under the age of five. ETEC is also the most common cause of diarrhoea among travellers, including the military, from industrialized nations to less developed countries. In addition, ETEC is a major pathogen of animals, being responsible for scours in cattle and neonatal and postweaning diarrhoea in pigs and resulting in significant financial losses. Studies on the pathogenesis of ETEC infections have concentrated on the plasmid-encoded heat-stable and heat-labile enterotoxins and on the plasmid-encoded antigenically variable colonization factors. Relatively little work has been carried out on chromosomally encoded virulence factors. Here, we review the known virulence factors of ETEC and highlight the future for combating this major disease.     

Multiple approaches to a complete inventory of Pseudomonas syringae pv. tomato DC3000 type III secretion system effector proteins

Pseudomonas syringae pv. tomato DC3000 is a pathogen of tomato and Arabidopsis that translocates virulence effector proteins into host cells via a type III secretion system (T3SS). Many effector-encoding hypersensitive response and pathogenicity (Hrp) outer protein (hop) genes have been identified previously in DC3000 using bioinformatic methods based on Hrp promoter sequences and characteristic N-terminal amino acid patterns that are associated with T3SS substrates. To approach completion of the Hop/effector inventory in DC3000, 44 additional candidates were tested by the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter assay; 10 of the high-probability candidates were confirmed as T3SS substrates. Several previously predicted hop genes were tested for their ability to be expressed in an HrpL-dependent manner in culture or to be expressed in planta. The data indicate that DC3000 harbors 53 hop/avr genes and pseudogenes (encoding both injected effectors and T3SS substrates that probably are released to the apoplast); 33 of these genes are likely functional in DC3000, 12 are nonfunctional members of valid Hop families, and 8 are less certain regarding their production at functional levels. Growth of DC3000 in tomato and Arabidopsis Col-0 was not impaired by constitutive expression of repaired versions of two hops that were disrupted naturally by transposable elements or of hop genes that are naturally cryptic. In summary, DC3000 carries a complex mixture of active and inactive hop genes, and the hop genes in P. syringae can be identified efficiently by bioinformatic methods; however, a precise inventory of the subset of Hops that are important in pathogenesis awaits more knowledge based on mutant phenotypes and functions within plants.     

Effects of acrylamide, latrunculin, and nocodazole on intracellular transport and cytoskeletal organization in melanophores

The effects of acrylamide (ACR), nocodazole, and latrunculin were studied on intracellular transport and cytoskeletal morphology in cultured Xenopus laevis melanophores, cells that are specialized for regulated and bidirectional melanosome transport. We used three different methods; light microscopy, fluorescence microscopy, and spectrophotometry. ACR affected the morphology of both microtubules and actin filaments in addition to inhibiting retrograde transport of melanosomes but leaving dispersion unaffected. Using the microtubule-inhibitor nocodazole and the actin filament-inhibitor latrunculin we found that microtubules and actin filaments are highly dependent on each other, and removing either component dramatically changed the organization of the other. Both ACR and latrunculin induced bundling of microtubules, while nocodazole promoted formation of filaments resembling stress fibers organized from the cell center to the periphery. Removal of actin filaments inhibited dispersion of melanosomes, further concentrated the central pigment mass in aggregated cells, and induced aggregation even in the absence of melatonin. Nocodazole, on the other hand, prevented aggregation and caused melanosomes to cluster and slowly disperse. Dispersion of nocodazole-treated cells was induced upon addition of alpha-melanocyte-stimulating hormone (MSH), showing that dispersion can proceed in the absence of microtubules, but the distribution pattern was altered. It is well established that ACR has neurotoxic effects, and based on the results in the present study we suggest that ACR has several cellular targets of which the minus-end microtubule motor dynein and the melatonin receptor might be involved. When combining morphological observations with qualitative and quantitative measurements of intracellular transport, melanophores provide a valuable model system for toxicological studies.     

Discriminating formation of HNO from other reactive nitrogen oxide species

Nitroxyl (HNO) exhibits unique pharmacological properties that often oppose those of nitric oxide (NO), in part due to differences in reactivity toward thiols. Prior investigations suggested that the end products arising from the association of HNO with thiols were condition-dependent, but were inconclusive as to product identity. We therefore used HPLC techniques to examine the chemistry of HNO with glutathione (GSH) in detail. Under biological conditions, exposure to HNO donors converted GSH to both the sulfinamide [GSONH2] and the oxidized thiol (GSSG). Higher thiol concentrations generally favored a higher GSSG ratio, suggesting that the products resulted from competitive consumption of a single intermediate (GSNHOH). Formation of GSONH2 was not observed with other nitrogen oxides (NO, N2O3, NO2, or ONOO(-)),indicating that it is a unique product of the reaction of HNO with thiols. The HPLC assay was able to detect submicromolar concentrations of GSONH2. Detection of GSONH2 was then used as a marker for HNO production from several proposed biological pathways, including thiol-mediated decomposition of S-nitrosothiols and peroxidase-driven oxidation of hydroxylamine (an end product of the reaction between GSH and HNO) and NG-hydroxy-l-arginine (an NO synthase intermediate). These data indicate that free HNO can be biosynthesized and thus may function as an endogenous signaling agent that is regulated by GSH content.     

Computational and Empirical Trans-hydrogen Bond Deuterium Isotope Shifts Suggest that N1–N3 A:U Hydrogen Bonds of RNA are Shorter than those of A:T Hydrogen Bonds of DNA

Density functional theory calculations of isolated Watson–Crick A:U and A:T base pairs predict that adenine ¹³C2 trans-hydrogen bond deuterium isotope shifts due to isotopic substitution at the pyrimidine H3, ^2hΔ¹³C2, are sensitive to the hydrogen-bond distance between the N1 of adenine and the N3 of uracil or thymine, which supports the notion that ^2hΔ¹³C2 is sensitive to hydrogen-bond strength. Calculated ^2hΔ¹³C2 values at a given N1–N3 distance are the same for isolated A:U and A:T base pairs. Replacing uridine residues in RNA with 5-methyl uridine and substituting deoxythymidines in DNA with deoxyuridines do not statistically shift empirical ^2hΔ¹³C2 values. Thus, we show experimentally and computationally that the C7 methyl group of thymine has no measurable affect on ^2hΔ¹³C2 values. Furthermore, ^2hΔ¹³C2 values of modified and unmodified RNA are more negative than those of modified and unmodified DNA, which supports our hypothesis that RNA hydrogen bonds are stronger than those of DNA. It is also shown here that ^2hΔ¹³C2 is context dependent and that this dependence is similar for RNA and DNA. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.