Factors Influencing the Measurement of Lysosomal Enzymes Activity in Human Cerebrospinal Fluid

Measurements of the activities of lysosomal enzymes in cerebrospinal fluid have recently been proposed as putative biomarkers for Parkinson''s disease and other synucleinopathies. To define the operating procedures useful for ensuring the reliability of these measurements, we analyzed several pre-analytical factors that may influence the activity of β-glucocerebrosidase, α-mannosidase, β-mannosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, cathepsin D and cathepsin E in cerebrospinal fluid. Lysosomal enzyme activities were measured by well-established fluorimetric assays in a consecutive series of patients (n = 28) with different neurological conditions, including Parkinson''s disease. The precision, pre-storage and storage conditions, and freeze/thaw cycles were evaluated. All of the assays showed within- and between-run variabilities below 10%. At −20°C, only cathepsin D was stable up to 40 weeks. At −80°C, the cathepsin D, cathepsin E, and β-mannosidase activities did not change significantly up to 40 weeks, while β-glucocerebrosidase activity was stable up to 32 weeks. The β-galactosidase and α-fucosidase activities significantly increased (+54.9±38.08% after 4 weeks and +88.94±36.19% after 16 weeks, respectively). Up to four freeze/thaw cycles did not significantly affect the activities of cathepsins D and E. The β-glucocerebrosidase activity showed a slight decrease (−14.6%) after two freeze/thaw cycles. The measurement of lysosomal enzyme activities in cerebrospinal fluid is reliable and reproducible if pre-analytical factors are accurately taken into consideration. Therefore, the analytical recommendations that ensue from this study may contribute to the establishment of actual values for the activities of cerebrospinal fluid lysosomal enzymes as putative biomarkers for Parkinson''s disease and other neurodegenerative disorders.     

The ethics of efficacy in North India's goonda raj (rule of toughs)

This study of goondas (gangsters or toughs) in North Indian politics comes by way of a comment on intellectual method in the anthropology of moralities. More especially, it offers critical remarks on the recent adoption of ‘virtue’ as the cardinal moral co‐ordinate of human life. Drawing on field research conducted across northern India, we show that when people celebrate goondas as leaders, they do so not because they see in them virtuous men, but because they think them capable of ‘getting things done’. This ethics of efficacy is neither merely instrumental nor is it but another variant of virtue ethics. It presents, instead, an altogether different moral teleology orientated towards effective action rather than excellent character. While challenging the self‐centred bent of the late anthropology of ethics, we also make preliminary remarks on the contrast between ‘moral’ and ‘practical’ judgement, and the limits of ‘the moral’ as such.     

To core,or not to core: the impact of coring on tree health and a best‐practice framework for collecting dendrochronological information from living trees

Trees are natural repositories of valuable environmental information that is preserved in the growth and structure of their stems, branches and roots. Dendrochronological analyses, based on the counting, crossdating and characterisation of incrementally formed wood rings, offer powerful insights for diverse fields including ecology, climatology and archaeology. The application of this toolset is likely to increase in popularity over coming decades due to advances in the field and a reduction in the cost of analyses. In research settings where the continued value of living trees subject to dendrochronological investigation is important, the use of an increment bore corer to extract trunk tissue is considered the best option to minimise negative impacts on tree health (e.g. stress and fitness). A small and fragmented body of literature, however, reports significant after‐effects, and in some cases fatal outcomes, from this sampling technique. As it stands, the literature documenting increment bore coring (IBC) impacts lacks experimental consistency and is poorly replicated, making it difficult for prospective users of the method to assess likely tree responses to coring. This paucity of information has the potential to lead to destructive misuse of the method and also limits its safe implementation in circumstances where the risk of impacts may be appropriate. If IBC is to fulfil its potential as a method of choice across research fields, then we must first address our limited understanding of IBC impacts and provide a framework for its appropriate future use. Firstly, we review the historical context of studies examining the impacts of IBC on trees to identify known patterns, focal issues and biases in existing knowledge. IBC wound responses, particularly those that impact on lumber quality, have been the primary focus of prior studies. No universal treatment was identified that conclusively improved wound healing and few studies have linked wound responses to tree health impacts. Secondly, we build on literature insights using a theoretical approach to identify the most important factors to guide future research involving implementation of IBC, including innate tree characteristics and environmental factors. Thirdly, we synthesise and interrogate the quantitative data available through meta‐analysis to identify risk factors for wound reactions. Although poor reporting standards, restricted scopes and a bias towards temperate ecosystems limited quantitative insight, we found that complete cambial wound closure could still harbour high rates of internal trunk decay, and that conditions favouring faster growth generally correlated with reduced indices of internal and external damage in broadleaved taxa. Finally, we propose a framework for guiding best‐practice application of IBC to address knowledge gaps and maximise the utility of this method, including standardised reporting indices for identifying and minimising negative impacts on tree health. While IBC is an underutilised tool of ecological enquiry with broad applicability, the method will always incur some risk of negative impacts on the cored tree. We caution that the decision to core, or not to core, must be given careful consideration on a case‐by‐case basis. In time, we are confident that this choice will be better informed by evidence‐based insight.     

Food allergen detection by mass spectrometry: From common to novel protein ingredients

Food allergens are molecules, mainly proteins, that trigger immune responses in susceptible individuals upon consumption even when they would otherwise be harmless. Symptoms of a food allergy can range from mild to acute; this last effect is a severe and potentially life-threatening reaction. The European Union (EU) has identified 14 common food allergens, but new allergens are likely to emerge with constantly changing food habits. Mass spectrometry (MS) is a promising alternative to traditional antibody-based assays for quantifying multiple allergenic proteins in complex matrices with high sensitivity and selectivity. Here, the main allergenic proteins and the advantages and drawbacks of some MS acquisition protocols, such as multiple reaction monitoring (MRM) and data-dependent analysis (DDA) for identifying and quantifying common allergenic proteins in processed foodstuffs are summarized. Sections dedicated to novel foods like microalgae and insects as new sources of allergenic proteins are included, emphasizing the significance of establishing stable marker peptides and validated methods using database searches. The discussion involves the in-silico digestion of allergenic proteins, providing insights into their potential impact on immunogenicity. Finally, case studies focussing on microalgae highlight the value of MS as an effective analytical tool for ensuring regulatory compliance throughout the food control chain.     

Nitrite exerts potent negative inotropy in the isolated heart via eNOS-independent nitric oxide generation and cGMP-PKG pathway activation

The ubiquitous anion nitrite (NO₂⁻) has recently emerged as an endocrine storage form of nitric oxide (NO) and a signalling molecule that mediates a number of biological responses. Although the role of NO in regulating cardiac function has been investigated in depth, the physiological signalling effects of nitrite on cardiac function have only recently been explored. We now show that remarkably low concentrations of nitrite (1 nM) significantly modulate cardiac contractility in isolated and perfused Langendorff rat heart. In particular, nitrite exhibits potent negative inotropic and lusitropic activities as evidenced by a decrease in left ventricular pressure and relaxation, respectively. Furthermore, we demonstrate that the nitrite-dependent effects are mediated by NO formation but independent of NO synthase (NOS) activity. Specifically, nitrite infusion in the Langendorff system produces NO and cGMP/PKG-dependent negative inotropism, as evidenced by the formation of cellular iron-nitrosyl complexes and inhibition of biological effect by NO scavengers and by PKG inhibitors. These data are consistent with the hypothesis that nitrite represents an eNOS-independent source of NO in the heart which modulates cardiac contractility through the NO-cGMP/PKG pathway. The observed high potency of nitrite supports a physiological function of nitrite as a source of cardiomyocyte NO and a fundamental signalling molecule in the heart.     

Antiproliferative activity of lichen extracts on murine myeloma cells

In the present study we report some preliminary results concerning the evaluation of antiproliferative activity on murine myeloma cells (P3X63-Ag8.653) of crude extracts of two common lichen species, Evernia prunastri and Xanthoria parietina. The results were evaluated by means of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test, which is commonly used to assess the activity of living cells through mitochondrial dehydrogenases. They indicated that extracts of E. prunastri had no effect, while those of X. parietina significantly affected murine myeloma cell proliferation, with a reduction down to 75% for methanolic extracts. This opens perspectives for deeper investigations extended also to other mammalian cell lines.     

Residues in the alternative reading frame tumor suppressor that influence its stability and p53-independent activities

The Alternative Reading Frame (ARF) protein suppresses tumorigenesis through p53-dependent and p53-independent pathways. Most of ARF's anti-proliferative activity is conferred by sequences in its first exon. Previous work showed specific amino acid changes occurred in that region during primate evolution, so we programmed those changes into human p14ARF to assay their functional impact. Two human p14ARF residues (Ala¹⁴ and Thr³¹) were found to destabilize the protein while two others (Val²⁴ and Ala⁴¹) promoted more efficient p53 stabilization and activation. Despite those effects, all modified p14ARF forms displayed robust p53-dependent anti-proliferative activity demonstrating there are no significant biological differences in p53-mediated growth suppression associated with simian versus human p14ARF residues. In contrast, p53-independent p14ARF function was considerably altered by several residue changes. Val²⁴ was required for p53-independent growth suppression whereas multiple residues (Val²⁴, Thr³¹, Ala⁴¹ and His⁶⁰) enabled p14ARF to block or reverse the inherent chromosomal instability of p53-null MEFs. Together, these data pinpoint specific residues outside of established p14ARF functional domains that influence its expression and signaling activities. Most intriguingly, this work reveals a novel and direct role for p14ARF in the p53-independent maintenance of genomic stability.     

Spinophilin: from partners to functions

Spinophilin/neurabin 2 has been isolated independently by two laboratories as a protein interacting with protein phosphatase 1 (PP1) and F-actin. Gene analysis and biochemical approaches have contributed to define a number of distinct modular domains in spinophilin that govern protein-protein interactions such as two F-actin-, three potential Src homology 3 (SH3)-, a receptor- and a PP1-binding domains, a PSD95/DLG/zo-1 (PDZ) and three coiled-coil domains, and a potential leucine/isoleucine zipper (LIZ) motif. More than 30 partner proteins of spinophilin have been discovered, including cytoskeletal and cell adhesion molecules, enzymes, guanine nucleotide exchange factors (GEF) and regulator of G-protein signalling protein, membrane receptors, ion channels and others proteins like the tumour suppressor ARF. The physiological relevance of some of these interactions remains to be demonstrated. However, spinophilin structure suggests that the protein is a multifunctional protein scaffold that regulates both membrane and cytoskeletal functions. Spinophilin plays important functions in the nervous system where it is implicated in spine morphology and density regulation, synaptic plasticity and neuronal migration. Spinophilin regulates also seven-transmembrane receptor signalling and may provide a link between some of these receptors and intracellular mitogenic signalling events dependent on p70(S6) kinase and Rac G protein-GEF. Strikingly a role for spinophilin in cell growth was demonstrated and this effect was enhanced by its interaction with ARF. Here we review the current knowledge of the protein partners of spinophilin and present the available data that are contributing to the appreciation of spinophilin functions.     

Evolution: the paradox of sperm leviathans

Sexual selection theory predicts that sperm competition will push males to produce more, smaller sperm. Paradoxically, in the fruitfly Drosophila bifurca sperm competition is rife but males produce few, giant sperm--the largest known. A recent study reconciles the evolution of giant sperm with theory.     

Spare interactions of highly potent [Arg14,Lys15]nociceptin for cooperative induction of ORL1 receptor activation

[Arg¹⁴,Lys¹⁵]Nociceptin is a very potent for ORL1 receptor, showing a few times stronger binding activity and much more enhanced biological activity than endogenous nociceptin. This synergistic outcome has been suggested to be due to the interaction with the receptor aromatic and/or acidic amino acid residues crucial to receptor activation. In order to identify such receptor residues in the second ORL1 extracellular loop, we prepared a series of recombinant mutant receptors. The mutant receptor Gln205Ala was found to be as active as wild-type ORL1 for both nociceptin and [Arg¹⁴,Lys¹⁵]nociceptin. In contrast, Asp206Ala and Tyr207Ala exhibited considerably reduced activity for [Arg¹⁴,Lys¹⁵]nociceptin, exhibiting no synergistic activity enhancement. These results suggest that Asp206 and Tyr207 are directly involved in the interaction with nociceptin-[Arg¹⁴,Lys¹⁵]. Trp208Ala was found to bind strongly both nociceptin and [Arg¹⁴,Lys¹⁵]nociceptin, although it elicited no biological activity. All these results indicate that the consecutive amino acid residues Asp206, Tyr207, and Trp208 are critical to the activation of the ORL1 receptor, but not to nociceptin-binding.