The peculiar structural features of kiwi fruit pectin methylesterase: amino acid sequence, oligosaccharides structure, and modeling of the interaction with its natural proteinaceous inhibitor

Pectin methylesterase (PME) from kiwi fruit (Actinidia deliciosa) is a glycoprotein, showing an apparent molecular mass of 50 kDa upon size exclusion chromatography and SDS-PAGE. The primary structure, elucidated by direct sequencing of the protein, comprises 321 amino acid residues providing a molecular mass of 35 kDa. The protein has an acetylated Thr residue at the amino terminus and five N-glycosylation consensus sequences, four of which are actually glycosylated. A careful investigation of the oligosaccharide structures demonstrated that PME glycans belong to complex type oligosaccharides essentially consisting of xylosylated polyfucosylated biantennary structures. Alignment with known mature plant PME sequences indicates that the postulated active site residues are conserved. Kiwi PME activity is inhibited following the interaction with the proteinaceous inhibitor PMEI, isolated from the same source. Gel-filtration experiments show that kiwi PME/PMEI complex is stable in a large pH range and dissociates only at pH 10.0. Modeling of the interaction with the inhibitor was performed by using the crystal structure of the complex between kiwi PMEI and tomato PME as a template. The model shows that the binding site is the same reported for tomato PME. However, additional salt link interactions are found to connect the external loops of kiwi PME to PMEI. This finding may explain the higher pH stability of the complex formed by the two kiwi proteins respect to that formed by PMEI and tomato PME

Allergen Micro-Bead Array for IgE Detection: A Feasibility Study Using Allergenic Molecules Tested on a Flexible Multiplex Flow Cytometric Immunoassay

Background: Allergies represent the most prevalent non infective diseases worldwide. Approaching IgE-mediated sensitizations improved much by adopting allergenic molecules instead of extracts, and by using the micro-technology for multiplex testing. Objective and Methods: To provide a proof-of-concept that a flow cytometric bead array is a feasible mean for the detection of specific IgE reactivity to allergenic molecules in a multiplex-like way. A flow cytometry Allergenic Moleculebased micro-bead Array system (ABA) was set by coupling allergenic molecules with commercially available micro-beads. Allergen specific polyclonal and monoclonal antibodies, as well as samples from 167 allergic patients, characterized by means of the ISAC microarray system, were used as means to show the feasibility of the ABA. Three hundred and thirty-six sera were tested for 1 or more of the 16 selected allergens, for a total number of 1,519 tests on each of the two systems. Results: Successful coupling was initially verified by detecting the binding of rabbit polyclonal IgG, mouse monoclonal, and pooled human IgE toward three allergens, namely nDer s 1, nPen m 1, and nPru p 3. The ABA assay showed to detect IgE t

Allergens in allergy diagnosis: a glimpse at emerging new concepts and methodologies

Allergic diseases are important concern of public health. A reliable diagnosis is of utmost importance for the management of allergic patients both when immunotherapy is planned and when the treatment is essentially based on the avoidance of the allergy source. However, the available diagnostic systems sometimes fail to detect specific IgE antibodies thus impairing the correct diagnosis. The traditional test systems are generally based on the use of protein extracts derived from the allergenic sources whose composition is very variable and cannot be standardized. The development of a new methodology combining the so-called allergenic molecule-based diagnosis with the multiplex microarray technology and allowing the analysis of multiple purified allergens in a single test represents an important improvement in allergy diagnosis. In addition, the biochemical and immunological characterisation of individual allergens has provided new insights into the understanding of allergen-IgE recognition that could be exploited for further improvements of allergy diagnostic tests

Manual lymphatic drainage for lymphedema following breast cancer treatment

Background More than one in five of breast cancer patients will develop breast cancer-related lymphedema (BCRL). BCRL is a swelling that can occur in the arm, breast, or chest wall as a result of breast cancer surgery and/or radiation therapy. BCRL can negatively impact comfort, function, and quality of life Manual lymphatic drainage (MLD) is a hands-on therapy that is commonly used for BCRL and often as part of complex decongestive therapy (CDT). CDT consists of MLD, compression bandaging, lymph-reducing exercises (LREs), and skin care. The Review Questions Is MLD safe and effective in treating BCRL? Study Characteristics We found six trials published through May, 2013, totaling 208 participants. Key Results When women were treated with a course of intensive compression bandaging, their swelling went down about 30% to 37%. When MLD was added to the intensive course of compression bandaging, their swelling went down another 7.11%. Thus, MLD may offer benefit when added to compression bandaging. Examining this finding more closely showed that this significant reduction benefit was observed in people with mild-to-moderate lymphedema when compared to participants with moderate-to-severe lymphedema. Thus, our findings suggest that individuals with mild-to-moderate BCRL are the ones who may benefit from adding MLD to an intensive course of treatment with compression bandaging. This finding, however, needs to be confirmed by further research. When women were given a standard elastic compression sleeve plus MLD and compared to women who received a standard compression sleeve plus a nonMLD treatment, results were mixed (sometimes favoring MLD and sometimes favoring neither treatment.) One-year follow-up suggests that once swelling had been reduced, participants were likely to keep their swelling down if they continued to use a custom-made sleeve. MLD is safe and well tolerated. Findings were contradictory for function (range of motion), with one trial showing benefit and the other not. Two trials measured quality of life, but neither trial presented results comparing the treatment group to the control, so findings are inconclusive. No trial measured cost of care. Quality of the Evidence Trials were small ranging from 24 to 45 participants. Most trials appeared to randomize participants adequately. However, in four trials the person measuring the swelling knew what treatment the participants were receiving, and this could have biased results. Authors' conclusions: MLD is safe and may offer additional benefit to compression bandaging for swelling reduction. Compared to individuals with moderate-to-severe BCRL, those with mild-to-moderate BCRL may be the ones who benefit from adding MLD to an intensive course of treatment with compression bandaging. This finding, however, needs to be confirmed by randomized data. In trials where MLD and sleeve were compared with a nonMLD treatment and sleeve, volumetric outcomes were inconsistent within the same trial. Research is needed to identify the most clinically meaningful volumetric measurement, to incorporate newer technologies in LE assessment, and to assess other clinically relevant outcomes such as fibrotic tissue formation. Findings were contradictory for function (range of motion), and inconclusive for quality of life. For symptoms such as pain and heaviness, 60% to 80% of participants reported feeling better regardless of which treatment they received. One-year follow-up suggests that once swelling had been reduced, participants were likely to keep their swelling down if they continued to use a custom-made sleeve.sch_nurpub4539pu

Astrocytes are important mediators of Aβ-induced neurotoxicity and tau phosphorylation in primary culture

Alzheimer's disease (AD) is pathologically characterised by the age-dependent deposition of β-amyloid (Aβ) in senile plaques, intraneuronal accumulation of tau as neurofibrillary tangles, synaptic dysfunction and neuronal death. Neuroinflammation, typified by the accumulation of activated microglia and reactive astrocytes, is believed to modulate the development and/or progression of AD. We have used primary rat neuronal, astrocytic and mixed cortical cultures to investigate the contribution of astrocyte-mediated inflammatory responses during Aβ-induced neuronal loss. We report that the presence of small numbers of astrocytes exacerbate Aβ-induced neuronal death, caspase-3 activation and the production of caspase-3-cleaved tau. Furthermore, we show that astrocytes are essential for the Aβ-induced tau phosphorylation observed in primary neurons. The release of soluble inflammatory factor(s) from astrocytes accompanies these events, and inhibition of astrocyte activation with the anti-inflammatory agent, minocycline, reduces astrocytic inflammatory responses and the associated neuronal loss. Aβ-induced increases in caspase-3 activation and the production of caspase-3-truncated tau species in neurons were reduced when the astrocytic response was attenuated with minocycline. Taken together, these results show that astrocytes are important mediators of the neurotoxic events downstream of elevated Aβ in models of AD, and suggest that mechanisms underlying pro-inflammatory cytokine release might be an important target for therapy

Drug Development for Alzheimer's Disease: Recent Progress

Alzheimer's disease, the most common cause of dementia, is characterized by two major pathological hallmarks: amyloid plaques and neurofibrillary tangles. Based on these two indicators, an amyloid cascade hypothesis was proposed, and accordingly, most current therapeutic approaches are now focused on the removal of β-amyloid peptides (Aβ from the brain. Additionally, strategies for blocking tau hyperphosphorylation and aggregation have been suggested, including the development of drugs that can block the formation of tangles. However, there are no true disease-modifying drugs in the current market, though many drugs based on theories other than Aβ and tau pathology are under development. The purpose of this review was to provide information on the current development of AD drugs and to discuss the issues related to drug development

The Toll→NFκB Signaling Pathway Mediates the Neuropathological Effects of the Human Alzheimer's Aβ42 Polypeptide in Drosophila

Alzheimer's (AD) is a progressive neurodegenerative disease that afflicts a significant fraction of older individuals. Although a proteolytic product of the Amyloid precursor protein, the Αβ42 polypeptide, has been directly implicated in the disease, the genes and biological pathways that are deployed during the process of Αβ42 induced neurodegeneration are not well understood and remain controversial. To identify genes and pathways that mediated Αβ42 induced neurodegeneration we took advantage of a Drosophila model for AD disease in which ectopically expressed human Αβ42 polypeptide induces cell death and tissue degeneration in the compound eye. One of the genes identified in our genetic screen is Toll (Tl). It encodes the receptor for the highly conserved Tl→NFkB innate immunity/inflammatory pathway and is a fly homolog of the mammalian Interleukin-1 (Ilk-1) receptor. We found that Tl loss-of-function mutations dominantly suppress the neuropathological effects of the Αβ42 polypeptide while gain-of-function mutations that increase receptor activity dominantly enhance them. Furthermore, we present evidence demonstrating that Tl and key downstream components of the innate immunity/inflammatory pathway play a central role in mediating the neuropathological activities of Αβ42. We show that the deleterious effects of Αβ42 can be suppressed by genetic manipulations of the Tl→NFkB pathway that downregulate signal transduction. Conversely, manipulations that upregulate signal transduction exacerbate the deleterious effects of Aβ42. Since postmortem studies have shown that the Ilk-1→NFkB innate immunity pathway is substantially upregulated in the brains of AD patients, the demonstration that the Tl→NFkB signaling actively promotes the process of Αβ42 induced cell death and tissue degeneration in flies points to possible therapeutic targets and strategies

Body Fluid Cytokine Levels in Mild Cognitive Impairment and Alzheimer’s Disease: a Comparative Overview

This article gives a comprehensive overview of cytokine and other inflammation associated protein levels in plasma, serum and cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD) and mild cognitive impairment (MCI). We reviewed 118 research articles published between 1989 and 2013 to compare the reported levels of 66 cytokines and other proteins related to regulation and signaling in inflammation in the blood or CSF obtained from MCI and AD patients. Several cytokines are evidently regulated in (neuro-) inflammatory processes associated with neurodegenerative disorders. Others do not display changes in the blood or CSF during disease progression. However, many reports on cytokine levels in MCI or AD are controversial or inconclusive, particularly those which provide data on frequently investigated cytokines like tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6). The levels of several cytokines are possible indicators of neuroinflammation in AD. Some of them might increase steadily during disease progression or temporarily at the time of MCI to AD conversion. Furthermore, elevated body fluid cytokine levels may correlate with an increased risk of conversion from MCI to AD. Yet, research results are conflicting. To overcome interindividual variances and to obtain a more definite description of cytokine regulation and function in neurodegeneration, a high degree of methodical standardization and patients collective characterization, together with longitudinal sampling over years is essential