Thoracic radiography of pet macaques in Sulawesi, Indonesia

This report describes the results from a study of thoracic radiographs taken on a sample (n = 20) of pet macaque monkeys (Macaca) on the Indonesian island of Sulawesi. We present findings relating to general thoracic health, as well as describe our field methods and outline some of the challenges of conducting thoracic radiography in remote areas of the developing world. Examination of the radiographic images identified six possible cases of cardiac enlargement, and one case of minor lung consolidation possibly consistent with tuberculosis. The study did not identify major radiographic evidence of significant respiratory illness in the monkeys as might be expected based on the intense exposure to human respiratory diseases. These largely negative findings may be in part a consequence of image quality which seems to be influenced by power output and incomplete inspiration during film exposure.     

Mutations of beta-arrestin 2 that limit self-association also interfere with interactions with the beta2-adrenoceptor and the ERK1/2 MAPKs: implications for beta2-adrenoceptor signalling via the ERK1/2 MAPKs

FRET (fluorescence resonance energy transfer) and co-immunoprecipitation studies confirmed the capacity of beta-arrestin 2 to self-associate. Amino acids potentially involved in direct protein-protein interaction were identified via combinations of spot-immobilized peptide arrays and mapping of surface exposure. Among potential key amino acids, Lys(285), Arg(286) and Lys(295) are part of a continuous surface epitope located in the polar core between the N- and C-terminal domains. Introduction of K285A/R286A mutations into beta-arrestin 2-eCFP (where eCFP is enhanced cyan fluorescent protein) and beta-arrestin 2-eYFP (where eYFP is enhanced yellow fluorescent protein) constructs substantially reduced FRET, whereas introduction of a K295A mutation had a more limited effect. Neither of these mutants was able to promote beta2-adrenoceptor-mediated phosphorylation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases). Both beta-arrestin 2 mutants displayed limited capacity to co-immunoprecipitate ERK1/2 and further spot-immobilized peptide arrays indicated each of Lys(285), Arg(286) and particularly Lys(295) to be important for this interaction. Direct interactions between beta-arrestin 2 and the beta2-adrenoceptor were also compromised by both K285A/R286A and K295A mutations of beta-arrestin 2. These were not non-specific effects linked to improper folding of beta-arrestin 2 as limited proteolysis was unable to distinguish the K285A/R286A or K295A mutants from wild-type beta-arrestin 2, and the interaction of beta-arrestin 2 with JNK3 (c-Jun N-terminal kinase 3) was unaffected by the K285A/R286A or L295A mutations. These results suggest that amino acids important for self-association of beta-arrestin 2 also play an important role in the interaction with both the beta2-adrenoceptor and the ERK1/2 MAPKs. Regulation of beta-arrestin 2 self-association may therefore control beta-arrestin 2-mediated beta2-adrenoceptor-ERK1/2 MAPK signalling.     

Globular and pre-fibrillar prion aggregates are toxic to neuronal cells and perturb their electrophysiology

Prion diseases are characterised at autopsy by neuronal loss and accumulation of amorphous protein aggregates and/or amyloid fibrils in the brains of humans and animals. These protein deposits result from the conversion of the cellular, mainly alpha-helical prion protein (PrP(C)) to the beta-sheet-rich isoform (PrP(Sc)). Although the pathogenic mechanism of prion diseases is not fully understood, it appears that protein aggregation is itself neurotoxic and not the product of cell death. The precise nature of the neurotoxic species and mechanism of cell death are yet to be determined, although recent studies with other amyloidogenic proteins suggest that ordered pre-fibrillar or oligomeric forms may be responsible for cellular dysfunction. In this study we have refolded recombinant prion protein (rPrP) to two distinct forms rich in beta-sheet structure with an intact disulphide bond. Here we report on the structural properties of globular aggregates and pre-fibrils of rPrP and show that both states are toxic to neuronal cells in culture. We show that exogenous rPrP aggregates are internalised by neuronal cells and found in the cytoplasm. We also measured the changes in electrophysiological properties of cultured neuronal cells on exposure to exogenous prion aggregates and discuss the implications of these findings.     

Sugar signalling and gene expression in relation to carbohydrate metabolism under abiotic stresses in plants

Sucrose is required for plant growth and development. The sugar status of plant cells is sensed by sensor proteins. The signal generated by signal transduction cascades, which could involve mitogen-activated protein kinases, protein phosphatases, Ca²⁺ and calmodulins, results in appropriate gene expression. A variety of genes are either induced or repressed depending upon the status of soluble sugars. Abiotic stresses to plants result in major alterations in sugar status and hence affect the expression of various genes by down- and up-regulating their expression. Hexokinase-dependent and hexokinase-independent pathways are involved in sugar sensing. Sucrose also acts as a signal molecule as it affects the activity of a proton-sucrose symporter. The sucrose transporter acts as a sucrose sensor and is involved in phloem loading. Fructokinase may represent an additional sensor that bypasses hexokinase phosphorylation especially when sucrose synthase is dominant. Mutants isolated on the basis of response of germination and seedling growth to sugars and reporter-based screening protocols are being used to study the response of altered sugar status on gene expression. Commoncis-acting elements in sugar signalling pathways have been identified. Transgenic plants with elevated levels of sugars/sugar alcohols like fructans, raffinose series oligosaccharides, trehalose and mannitol are tolerant to different stresses but have usually impaired growth. Efforts need to be made to have transgenic plants in which abiotic stress responsive genes are expressed only at the time of adverse environmental conditions instead of being constitutively synthesized.     

Structural changes of the prion protein in lipid membranes leading to aggregation and fibrillization

Prion diseases are associated with a major refolding event of the normal cellular prion protein, PrP(C), where the predominantly alpha-helical and random coil structure of PrP(C) is converted into a beta-sheet-rich aggregated form, PrP(Sc). Under normal physiological conditions PrP(C) is attached to the outer leaflet of the plasma membrane via a GPI anchor, and it is plausible that an interaction between PrP and lipid membranes could be involved in the conversion of PrP(C) into PrP(Sc). Recombinant PrP can be refolded into an alpha-helical structure, designated alpha-PrP isoform, or into beta-sheet-rich states, designated beta-PrP isoform. The current study investigates the binding of beta-PrP to model lipid membranes and compares the structural changes in alpha- and beta-PrP induced upon membrane binding. beta-PrP binds to negatively charged POPG membranes and to raft membranes composed of DPPC, cholesterol, and sphingomyelin. Binding of beta-PrP to raft membranes results in substantial unfolding of beta-PrP. This membrane-associated largely unfolded state of PrP is slowly converted into fibrils. In contrast, beta-PrP and alpha-PrP gain structure with POPG membranes, which instead leads to amorphous aggregates. Furthermore, binding of beta-PrP to POPG has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents, whereas raft vesicles are not destabilized upon binding of beta-PrP.     

Binding of prion protein to lipid membranes and implications for prion conversion

The binding of the Syrian hamster prion protein, SHaPrP(90-231), to model lipid membranes was investigated by tryptophan fluorescence. Membranes composed of negatively charged or zwitterionic lipids, and raft-like membranes containing dipalmitoylphosphatidylcholine(1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol and sphingomyelin, were investigated. It was found that SHaPrP(90-231) binds to negatively charged lipid membranes and raft-like membranes. Binding of PrP to negatively charged lipid membranes involves both electrostatic and hydrophobic lipid-protein interactions and results in partial insertion of PrP into the lipid bilayer. This membrane-inserted conformation of PrP is richer in beta-sheet structure and has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents. In contrast, the binding of PrP to raft-like membranes is driven by hydrophobic lipid-protein interactions and induces the formation of alpha-helical structure. This conformation of PrP with a high content of alpha-helix is formed only at pH 7 and does not destabilize the lipid bilayer. Our findings support the view that an interaction of PrP with lipid membranes could play a role in PrP conversion.     

MHC-based vaccination approaches: progress and perspectives

The major histocompatibility complex (MHC) harbours genes whose primary function in regulating immune responsiveness to infection is to present foreign antigens to cytotoxic T lymphocytes (CTLs) and T helper cells. In the case of infection by human immunodeficiency virus (HIV), defining the optimal HIV epitopes that are recognised by CTLs is important for vaccine design, and this in turn will depend on the characteristics of the predominant infecting virus. Moreover, the particular MHC human leukocyte antigens (HLAs) expressed by a geographical population is important since these are likely to determine which HIV epitopes are immunodominant in the anti-HIV immune response. Consideration of these aspects has lead to the dawn of a new era of MHC-based vaccine design, in which the CTL epitopes are selected on the basis of the frequency of restricting MHC alleles. This article reviews data on the distribution patterns of molecular subtypes of HLA class I and class II extended haplotypes, discussing distribution among Asian Indians but with reference to global distributions. These data provide a genetic basis for the possible predisposition and fast progression of HIV infections in the Indian population. Since there is selective predominance of different HLA alleles and haplotypes in different populations, a dedicated screening effort is required at the global level to develop MHC-based vaccines against infectious diseases. It is hoped that this might lead to the development of multivalent, poly-epitope, subtype-specific HIV vaccines that are specific for the target geographical location.     

Ratiometric fluorescent scaffold giving discrete response towards iodide ion: a combined experimental and DFT study

A novel ratiometric fluorescent receptor (Z)‐2‐(4‐[diethylamino]‐2‐hydroxybenzylideneamino) pyridine‐3‐carbaldehyde (3) bearing one phenolic OH and one aldehyde group as recognition sites was synthesized and characterized. The anion recognition behaviour of receptor 3 was evaluated by various spectroscopic (UV‐visible, fluorescence and ¹H nuclear magnetic resonance) methods and was validated by computational studies. The receptor showed fast response time, excellent selectivity and reproducibility towards iodide ion detection among the other surveyed anions, with a binding constant of 6.12 × 10⁴ M^?1 and a detection limit of 0.24 μM, thus confirming its potential applicability as a fluorescent sensor for iodide Copyright © 2014 John Wiley & Sons, Ltd.     

p53 Dependent Apoptotic Cell Death Induces Embryonic Malformation in Carassius auratus under Chronic Hypoxia

Hypoxia is a global phenomenon affecting recruitment as well as the embryonic development of aquatic fauna. The present study depicts hypoxia induced disruption of the intrinsic pathway of programmed cell death (PCD), leading to embryonic malformation in the goldfish, Carrasius auratus. Constant hypoxia induced the early expression of pro-apoptotic/tumor suppressor p53 and concomitant expression of the cell death molecule, caspase-3, leading to high level of DNA damage and cell death in hypoxic embryos, as compared to normoxic ones. As a result, the former showed delayed 4 and 64 celled stages and a delay in appearance of epiboly stage. Expression of p53 efficiently switched off expression of the anti-apoptotic Bcl-2 during the initial 12 hours post fertilization (hpf) and caused embryonic cell death. However, after 12 hours, simultaneous downregulation of p53 and Caspase-3 and exponential increase of Bcl-2, caused uncontrolled cell proliferation and prevented essential programmed cell death (PCD), ultimately resulting in significant (p<0.05) embryonic malformation up to 144 hpf. Evidences suggest that uncontrolled cell proliferation after 12 hpf may have been due to downregulation of p53 abundance, which in turn has an influence on upregulation of anti-apoptotic Bcl-2. Therefore, we have been able to show for the first time and propose that hypoxia induced downregulation of p53 beyond 12 hpf, disrupts PCD and leads to failure in normal differentiation, causing malformation in gold fish embryos.     

Differential response of the antioxidant system in wild and cultivated genotypes of chickpea

Two chickpea cultivars PBG-1 and PDG-3 along with a wild species Cicer judaicum were investigated to compare the activities of their antioxidant enzymes in mature seeds and roots, as well as shoots and cotyledons of seedlings germinated under dark and continuous illumination of 40 μmol m⁻² s⁻¹ photosynthetically active radiation (PAR). Seedling biomass of C. judaicum was lower as compared to cultivars of PBG-1 and PDG-3 both under dark and light conditions. Light reduced the biomass of seedlings. Activities of glutathione reductase (GR) and ascorbate peroxidase (APX) were higher in shoots and roots of C. judaicum compared to the cultivars PBG-1 and PDG-3. In mature seeds, the activities of GR and APX were higher in the cultivated genotypes whereas catalase (CAT) and peroxidase were higher in C. judaicum. Under illumination, a general upregulation of CAT in both shoots and cotyledons and of GR in shoots was observed in all the three genotypes. However, superoxide dismutase (SOD) increased in C. judaicum and APX in PBG-1 and PDG-3. The differences in antioxidant enzyme system between wild and cultivated genotypes possibly contribute to better tolerance of wild Cicer species against abiotic and biotic stresses.