Hydralazine modifies Aβ fibril formation and prevents modification by lipids in vitro

Lipid oxidative damage and amyloid β (Aβ) misfolding contribute to Alzheimer's disease (AD) pathology. Thus, the prevention of oxidative damage and Aβ misfolding are attractive targets for drug discovery. At present, no AD drugs approved by the Food and Drug Administration (FDA) prevent or halt disease progression. Hydralazine, a smooth muscle relaxant, is a potential drug candidate for AD drug therapy as it reduces Aβ production and prevents oxidative damage via its antioxidant hydrazide group. We evaluated the efficacy of hydralazine, and related hydrazides, in reducing (1) Aβ misfolding and (2) Aβ protein modification by the reactive lipid 4-hydroxy-2-nonenal (HNE) using transmission electron microscopy and Western blotting. While hydralazine did not prevent Aβ aggregation as measured using the protease protection assay, there were more oligomeric species observed by electron microscopy. Hydralazine prevented lipid modification of Aβ, and Aβ was used as a proxy for classes of proteins which either misfold or are modified by HNE. All of the other hydrazides prevented lipid modification of Aβ and also did not prevent Aβ aggregation. Surprisingly, a few of the compounds, carbazochrome and niclosamide, appeared to augment Aβ formation. Thus, hydrazides reduced lipid oxidative damage, and hydralazine additionally reduced Aβ misfolding. While hydralazine would require specific chemical modifications for use as an AD therapeutic itself (to improve blood brain barrier permeability, reduce vasoactive side effects, and optimization for amyloid inhibition), this study suggests its potential merit for further AD drug development.     

Ectoadenylate kinase and plasma membrane ATP synthase activities of human vascular endothelial cells

Formation of ATP from ADP on the external surface of vascular endothelial cells has been attributed to plasma membrane ATP synthase, ectoadenylate kinase (ecto-AK), and/or ectonucleoside diphosphokinase. These enzymes or their catalytic products have been causatively linked to the elaboration of vascular networks and the regulation of capillary function. The amount of ATP generated extracellularly is small, requiring sensitive analytical methods for quantification. Human umbilical vein endothelial cells were used to revisit extracellular ATP synthesis using a reliable tetrazolium reduction assay and multiwell plate cultures. Test conditions compatible with AK stability were established. Extracellular AK activity was found to be <1% of the total (intracellular and extracellular), raising the possibility that the external enzyme could have leaked from living cells and/or a few dying cells. To determine whether AK inadvertently leaked from the cells, the activity of another cytoplasmic enzyme, glucose-6-phosphate dehydrogenase (G6PD), was also measured. G6PD is present in the cytoplasm in similar abundance to AK. The activity ratio of G6PD (extracellular/total) was found to be similar to that of AK. Because G6PD in the medium was probably due to leakage, other cytoplasmic macromolecules, including AK, should be released proportionately from the cells. The role of plasma membrane ATP synthase in extracellular ATP formation was examined using Hanks' balanced salt solution with and without selective inhibitors of AK and ATP synthase activities. With P(1),P(5)-di(adenosine 5')-pentaphosphate (inhibitor of AK activity), no extracellular ATP synthesis was detected, whereas with oligomycin, piceatannol, and aurovertin (inhibitors of F(1)F(0)-ATP synthase and F(1)-ATPase activities), no inhibition of extracellular ATP synthesis was observed. AK activity alone could account for the observed extracellular ATP synthesis. The possible impact of ADP impurity in the assays is discussed.     

Chinch bug (Hemiptera: Blissidae) mouthpart morphology, probing frequencies, and locations on resistant and susceptible germplasm

Chinch bugs are common pests of many agronomic and horticulturally important crops and turfgrasses. Previous research has indicated that some grasses exhibit resistance to multiple chinch bug species, whereas others are resistant to only one species. The objectives of this research were to document differences in the probing frequencies and locations among Blissus species as well as differences in mouthpart morphology as a first step in understanding the differential responses of grasses to chinch bug feeding. Scanning electron microscopy detected differences in the total lengths of proboscises as well as individual mouthpart segments among the four species studied. Blissus occiduus Barber probed significantly more often on buffalograss, Buchlo? dactyloides (Nuttall) Engelmann, than any other plant material. Probing locations of B. occiduus and Blissus leucopterus leucopterus (Say) were similar on both B. occiduus-resistant and susceptible buffalograsses and KS94 sorghum, Sorghum bicolor (L.) Moench (B. occiduus-resistant, B. l. leucopterus-resistant). However, on 'Wheatland' sorghum (B. occiduus-resistant, B. l. leucopterus-susceptible), stylet tracts of B. l. leucopterus most often terminated in the bundle sheath cells, whereas those of B. occiduus generally terminated in the vascular tissues.     

Fitness cost of escape mutations in p24 Gag in association with control of human immunodeficiency virus type 1

Mutational escape by human immunodeficiency virus (HIV) from cytotoxic T-lymphocyte (CTL) recognition is a major challenge for vaccine design. However, recent studies suggest that CTL escape may carry a sufficient cost to viral replicative capacity to facilitate subsequent immune control of a now attenuated virus. In order to examine how limitations can be imposed on viral escape, the epitope TSTLQEQIGW (TW10 [Gag residues 240 to 249]), presented by two HLA alleles associated with effective control of HIV, HLA-B*57 and -B*5801, was investigated. The in vitro experiments described here demonstrate that the dominant TW10 escape mutation, T242N, reduces viral replicative capacity. Structural analysis reveals that T242 plays a critical role in defining the start point and in stabilizing helix 6 within p24 Gag, ensuring that escape occurs at a significant cost. A very similar role is played by Thr-180, which is also an escape residue, but within a second p24 Gag epitope associated with immune control. Analysis of HIV type 1 gag in 206 B*57/5801-positive subjects reveals three principle alternative TW10-associated variants, and each is strongly linked to concomitant additional variants within p24 Gag, suggesting that functional constraints operate against their occurrence alone. The extreme conservation of p24 Gag and the predictable nature of escape variation resulting from these tight functional constraints indicate that p24 Gag may be a critical immunogen in vaccine design and suggest novel vaccination strategies to limit viral escape options from such epitopes.     

Synthesis of a series of ruthenium and rhodium complexes with tridentate pyridine-based ligands containing hydrogen bonding groups

A series of ruthenium and rhodium complexes with a urea-disubstituted pyridine ligand are reported. The X-ray crystal structures of three of these species, RuCl₂(L¹)(PPh₃) (1), [Ru(MeCN)₂(L¹)(PPh₃)][BF₄]₂ (3) and Rh(CH₂Cl)Cl₂(L¹) (9) (where L¹ = N,N′-(2,2′-(1E,1′E)-(1,1′-(pyridine-2,6-diyl)bis(ethan-1-yl-1-ylidene))bis(azan-1-yl-1-ylidene)bis(ethane-2,1-diyl))diacetamide) have shown that the disubstituted pyridine acts as a tridentate ligand and its urea substituents engage in hydrogen bonding interactions with species coordinated to the metal centres. The reactivity of the ruthenium complexes towards coordination of other anions such as NCS⁻ has been investigated, as well as the oxidative-addition of alkyl chlorides to rhodium(I) centres (to yield species such as 9).     

A Target Repurposing Approach Identifies N-myristoyltransferase as a New Candidate Drug Target in Filarial Nematodes

Myristoylation is a lipid modification involving the addition of a 14-carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of proteins, a modification that promotes their binding to cell membranes for varied biological functions. The process is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT), an enzyme which has been validated as a drug target in human cancers, and for infectious diseases caused by fungi, viruses and protozoan parasites. We purified Caenorhabditis elegans and Brugia malayi NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and peptide substrates. Biochemical and structural analyses both revealed that the nematode enzymes are canonical NMTs, sharing a high degree of conservation with protozoan NMT enzymes. Inhibitory compounds that target NMT in protozoan species inhibited the nematode NMTs with IC₅₀ values of 2.5–10 nM, and were active against B. malayi microfilariae and adult worms at 12.5 µM and 50 µM respectively, and C. elegans (25 µM) in culture. RNA interference and gene deletion in C. elegans further showed that NMT is essential for nematode viability. The effects observed are likely due to disruption of the function of several downstream target proteins. Potential substrates of NMT in B. malayi are predicted using bioinformatic analysis. Our genetic and chemical studies highlight the importance of myristoylation in the synthesis of functional proteins in nematodes and have shown for the first time that NMT is required for viability in parasitic nematodes. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against nematode diseases including filariasis.     

Characterization of lipopolysaccharides from four Pasteurella haemolytica serotype strains: evidence for presence of sialic acid in serotypes 1 and 5

Highly purified lipopolysaccharides (LPS) obtained from four strains of Pasteurella haemolytica representative of four different serotypes were studied to ascertain their overall structural elements and sugar and fatty acid compositions. SDS-PAGE analysis revealed that each LPS was of the smooth-type although they differed in migration patterns. Somewhat unusual features of these LPS included the presence of: (a) rhamnose in the core oligosaccharides of serotypes 2 and 3; and (b) sialic acid in the LPS of serotypes 1 and 5. The fatty acids, myristic, hydroxymyristic and palmitic occur in essentially equivalent amounts in each of these LPS. In addition, stearic acid was present in small amounts of serotypes 1 and 5.     

Persistent social interactions beget more pronounced personalities in a desert-dwelling social spider

The social niche specialization hypothesis predicts that repeated social interactions will generate social niches within groups, thereby promoting consistent individual differences in behaviour. Current support for this hypothesis is mixed, probably because the importance of social niches is dependent upon the ecology of the species. We test whether repeated interactions among group mates generate consistent individual differences in boldness in the social spider, Stegodyphus dumicola. In support of the social niche specialization hypothesis, we found that consistent individual differences in boldness increased with longer group tenure. Interestingly, these differences took longer to appear than in previous work suggesting this species needs more persistent social interactions to shape its behaviour. Recently disturbed colonies were shyer than older colonies, possibly reflecting differences in predation risk. Our study emphasizes the importance of the social environment in generating animal personalities, but also suggests that the pattern of personality development can depend on subtle differences in species'' ecologies.     

Molecular evidence for the identity of the Magenta petrel

A lone petrel was shot from the decks of an Italian warship (the ‘Magenta’) while it was sailing the South Pacific Ocean in 1867, far from land. The species, unknown to science, was named the ‘Magenta petrel’ (Procellariiformes, Procellariidae, Pterodroma magentae). No other specimens of this bird were collected and the species it represented remained a complete enigma for over 100 years. We compared DNA sequence of the mitochondrial cytochrome b gene from the Magenta petrel to that of other petrels using phylogenetic methods and ancient DNA techniques. Our results strongly suggest that the Magenta petrel specimen is a Chatham Island taiko. Furthermore, given the collection location of the Magenta petrel, our finding indicates that the Chatham Island taiko forages far into the Pacific Ocean (near South America). This has implications for the conservation of the taiko, one of the world's rarest seabirds.