Direct Detection of Disease-Associated Prions in Brain and Lymphoid Tissue Using Antibodies Recognizing the Extreme N-terminus of PrPC

A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrP^C). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrP^Sc followed by detection with a europium-labelled anti-PrP^C antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrP^Sc in cases of vCJD.     

Protein Misfolding and Aggregation in Ageing and Disease

Although intensively researched, the fundamental mechanism of protein misfolding that leads to protein aggregation and associated diseases remains somewhat enigmatic. The failure of a protein to correctly fold de novo or to remain correctly folded can have profound consequences on a living system especially when the cellular quality control processes fail to eliminate the rogue proteins. Over 20 different human diseases have now been designated as ‘conformational diseases’ and include neurodegenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD) and Creutzfeldt Jakob disease (CJD) that are becoming increasingly prevalent in an ageing human population. Such diseases are usually characterised by the deposition of specific misfolded proteins as amyloid fibrils and hence are often referred to as the amyloidoses.     

α-Synuclein facilitates endocytosis by elevating the steady-state levels of phosphatidylinositol 4,5-bisphosphate

α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson''s disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for α-Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of α-Syn in these mechanisms is currently unclear. Here we investigate the associations of α-Syn with the acidic phosphoinositides (PIPs), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P₂). Our results show that α-Syn colocalizes with PIP₂ and the phosphorylated active form of the clathrin adaptor protein 2 (AP2) at clathrin-coated pits. Using endocytosis of transferrin as an indicator for clathrin-mediated endocytosis (CME), we find that α-Syn involvement in endocytosis is specifically mediated through PI(4,5)P₂ levels on the plasma membrane. In accord with their effects on PI(4,5)P₂ levels, the PD associated A30P, E46K, and A53T mutations in α-Syn further enhance CME in neuronal and nonneuronal cells. However, lysine to glutamic acid substitutions at the KTKEGV repeat domain of α-Syn, which interfere with phospholipid binding, are ineffective in enhancing CME. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by the α-Syn mutations and associates with their effects on PI(4,5)P₂ levels, however, with the exception of the A30P mutation. This study provides evidence for a critical involvement of PIPs in α-Syn–mediated membrane trafficking.     

Dual character of Toll-like receptor signaling: Pro-tumorigenic effects and anti-tumor functions

As a major class of pattern-recognition receptors, Toll-like receptors (TLRs) play a critical role in defense against invading pathogens. Increasing evidence demonstrates that, in addition to infection, TLRs are involved in other important pathological processes, such as tumorigenesis. Activation of TLRs results in opposing outcomes, pro-tumorigenic effects and anti-tumor functions. TLR signaling can inhibit apoptosis and promote chronic inflammation-induced tumorigenesis. TLR activation in tumor cells and immune cells can induce production of cytokines, increase tumor cell proliferation and apoptosis resistance, promote invasion and metastasis, and inhibit immune cell activity resulting in tumor immune escape. In contrast, the engagement of other TLRs directly induces growth inhibition and apoptosis of tumor cells and triggers activation of immune cells enhancing anti-tumor immune responses. Thus, the interpretation of the precise function of each TLR in tumors is very important for targeting TLRs and using TLR agonists in tumor therapy. We review the role of TLR signaling in tumors and discuss the factors that affect outcomes of TLR activation.     

Effects of corticotropin-releasing hormone on proopiomelanocortin derivatives and monocytic HLA-DR expression in patients with septic shock

Little is known about interactions between immune and neuro-endocrine systems in patients with septic shock. We therefore evaluated whether the corticotropin-releasing hormone (CRH) and/or proopiomelanocortin (POMC) derivatives [ACTH, β-endorphin (β-END), β-lipotropin (β-LPH), α-melanocyte stimulating hormone (α-MSH) or N-acetyl-β-END (Nac-β-END)] have any influences on monocyte deactivation as a major factor of immunosuppression under septic shock conditions. Sixteen patients with septic shock were enrolled in a double-blind, cross-over and placebo controlled clinical study; 0.5 μg/(kg_bodyweight h) CRH (or placebo) were intravenously administered for 24 h. Using flow cytometry we investigated the immunosuppression in patients as far as related to the loss of leukocyte surface antigen-DR expression on circulating monocytes (mHLA-DR). ACTH, β-END immunoreacive material (IRM), β-LPH IRM, α-MSH and Nac-β-END IRM as well as TNF-α and mHLA-DR expression were determined before, during and after treatment with CRH (or placebo). A significant correlation between plasma concentration of α-MSH and mHLA-DR expression and an inverse correlation between mHLA-DR expression and TNF-α plasma level were found. Additionally, a significant increase of mHLA-DR expression was observed 16 h after starting the CRH infusion; 8 h later, the mHLA-DR expression had decreased again. Our results indicate that the up-regulation of mHLA-DR expression after CRH infusion is not dependent on the release of POMC derivatives. From the correlation between plasma concentration of α-MSH and mHLA-DR expression, we conclude that in patients with septic shock the down-regulation of mHAL-DR expression is accompanied by the loss of monocytic release of α-MSH into the cardiovascular compartment.     

Relevance of signaling molecules for apoptosis induction on influenza A virus replication

Apoptosis is an important mechanism to maintain homeostasis in mammals, and disruption of the apoptosis regulation mechanism triggers a range of diseases, such as cancer, autoimmune diseases, and developmental disorders. The severity of influenza A virus (IAV) infection is also closely related to dysfunction of apoptosis regulation. In the virus infected cells, the functions of various host cellular molecules involved in regulation of induction of apoptosis are modulated by IAV proteins to enable effective virus replication. The modulation of the intracellular signaling pathway inducing apoptosis by the IAV infection also affects extracellular mechanisms controlling apoptosis, and triggers abnormal host responses related to the disease severity of IAV infections. This review focuses on apoptosis related molecules involved in IAV replication and pathogenicity, the strategy of the virus propagation through the regulation of apoptosis is also discussed.     

Effect of age at the beginning of the free-range fattening period on growth and carcass and fat quality in Iberian pigs

Abstract

This experiment was carried out to study the influence of age at the beginning of the free-range fattening period (traditional pigs, TP, age 12 months vs. young pigs, YP, age 8 months) on the performance of Iberian pigs. During 152 days prior to the fattening period, TP and YP pigs received 1.7 and 2.6 kg feed per day, respectively. During fattening, TP pigs had a higher average daily gain (p < 0.05) than YP pigs. The proportions of PUFA and n-3 fatty acids of the outer and inner layers of subcutaneous backfat were higher in TP than in YP pigs (p < 0.05), while the proportions of C16:0 and SFA in the inner layer of subcutaneous backfat were greater in YP than in TP pigs (p < 0.05). The ratio of n-6/n-3 in subcutaneous backfat was lower in TP than in YP pigs (p < 0.05). The percentage of intramuscular fat in longissimus dorsi muscle was higher in TP than in YP pigs (p < 0.05). The relationship between the percentage of intramuscular fat in longissimus dorsi muscle and average daily gain during the free-range fattening period adjusted to a quadratic function (p < 0.05). The concentration of α- and γ-tocopherol in subcutaneous backfat at slaughter was significantly higher in TP than in YP pigs (p < 0.05). It is concluded that Iberian pigs that have 8 months of age at the beginning of free-range feeding have adequate commercial quality.     

Upregulation of the p53-p21 pathway by G2019S LRRK2 contributes to the cellular senescence and accumulation of α-synuclein

Parkinson’s disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies (LB) in neurons. α-Synuclein (αSyn) is a major component of LB and promote the PD pathogenesis via its accumulation by the impaired proteasomal or autophagic clearance. Numerous studies have revealed that the reduction of proteasome activity and autophagy is accelerated by cellular senescence. Leucine-rich repeat kinase 2 (LRRK2) contributes to PD progression and its most prevalent mutation, G2019S LRRK2, increases its activity. Our previous report has shown that the G2019S LRRK2 mutant promoted p53-induced p21 expression and neuronal cytotoxicity. The p53-p21 pathway plays a role in cellular senescence. We hypothesized that the loss of dopaminergic neurons by the stimulated p53-p21 pathway via the G2019S LRRK2 mutation might be associated with cellular senescence, thereby promoting the accumulation of αSyn. We confirmed that the ectopic expression of the phosphomimetic p53 mutant, p21, or G2019 in differentiated SH-SY5Y cells increased the following: 1) the expression of β-galactosidase, a marker of cellular senescence, and the activity of senescence-associated β-galactosidase, 2) endogenous αSyn protein level, but not its mRNA level, and 3) αSyn fibril accumulation in dSH-SY5Y via low proteasome and cathepsin D activities. Elevated oligomeric αSyn and the increase in β-galactosidase with induced p21 were observed in brain lysates of G2019S transgenic mice. Our results suggest that cellular senescence is promoted via the p53-p21 pathway due to the G2019S LRRK2 mutation. Eventually, decreased protein degradation by G2019S-mediated senescence could accelerate αSyn aggregate formation.