Glycerophosphoinositol 4-phosphate, a putative endogenous inhibitor of adenylylcyclase.

In a continuous line of rat thyroid cells transformed by the k-ras oncogene (KiKi), the expression of ras-p21 correlates with an increased activity of a phosphoinositide-specific phospholipase A2, which leads to elevated levels of glycerophosphoinositols. In this study we have characterized the biological activities of these compounds. Growth and differentiation in thyroid cells are mainly regulated by the activation of adenylylcyclase. Therefore, we have studied the effects of glycerophosphoinositols on the activity of this enzyme using a normal thyroid cell line (FRTL5). Micromolar concentrations of glycerophosphoinositol 4-phosphate (GroPIns-4-P) caused a approximately 50% inhibition of the adenylylcyclase activity in FRTL5 membranes stimulated by the GTP-binding protein activator fluoroaluminate. Similar concentrations of GroPIns-4-P were detected in KiKi cells but not in the normal FRTL5 line. Micromolar GroPIns-4-P was found to be taken up by intact FRTL5 cells and to induce nearly 50% inhibition of the thyrotropin- and cholera toxin-induced increase in cAMP levels. Similar results were also observed in other cell lines (smooth muscle, pituitary cells, and pneumocytes). GroPIns-4-P inhibited cAMP-dependent cellular functions such as iodide uptake and thymidine incorporation in FRTL5 cells when stimulated by thyrotropin and cholera toxin but not when induced by forskolin. These results are consistent with GroPIns-4-P exerting an inhibitory effect on the GTP-binding protein that stimulates adenylycyclase. We propose that GroPIns-4-P might mediate a mechanism of cross-talk between adenylylcyclase and phospholipase A2 in thyroid as well as in other cell systems

Raw milk from vending machines: Effects of boiling, microwave treatment, and refrigeration on microbiological quality

Abstract In Italy, the sale of raw milk from vending machines has been allowed since 2004. Boiling treatment before its use is mandatory for the consumer, because the raw milk could be an important source of foodborne pathogens. This study fits into this context with the aim to evaluate the microbiological quality of 30 raw milk samples periodically collected (March 2013 to July 2013) from 3 vending machines located in Molise, a region of southern Italy. Milk samples were stored for 72h at 4°C and then subjected to different treatments, such as boiling and microwaving, to simulate domestic handling. The results show that all the raw milk samples examined immediately after their collection were affected by high microbial loads, with values very close to or even greater than those acceptable by Italian law. The microbial populations increased during refrigeration, reaching after 72h values of about 8.0 log cfu/mL for Pseudomonas spp., 6.5 log cfu/mL for yeasts, and up to 4.0 log cfu/mL for Enterobacteriaceae . Boiling treatment, applied after 72h to refrigerated milk samples, caused complete decontamination, but negatively affected the nutritional quality of the milk, as demonstrated by a drastic reduction of whey proteins. The microwave treatment at 900 W for 75s produced microbiological decontamination similar to that of boiling, preserving the content in whey proteins of milk. The microbiological characteristics of raw milk observed in this study fully justify the obligation to boil the raw milk from vending machines before consumption. However, this study also showed that domestic boiling causes a drastic reduction in the nutritional value of milk. Microwave treatment could represent a good alternative to boiling, on the condition that the process variables are standardized for safe domestic application

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

NUTRITIONAL EVALUATION OF FRESH AND DRIED GOJI BERRIES CULTIVATED IN ITALY

The nutritional profile of fresh and dried goji berries cultivated in Italy was investigated. The obtained data confirm goji berries as a source of nutritional and healthy components, such as vitamin E, minerals and fibre. Taking into account the Recommended Daily Allowance (RDA) for minerals and vitamins established by the Commission of the European Communities, Goji berries provide significant amounts of dietary fibre and zeaxanthin and can be declared on the label as a potential source of vitamins E and C. Moreover, dried goji berries can be declared as a source of K, P, Cu, Fe Mn, Zn

Complex I function in mitochondrial supercomplexes

This review discusses the functional properties of mitochondrial Complex I originating from its presence in an assembled form as a supercomplex comprising Complex III and Complex IV in stoichiometric ratios. In particular several lines of evidence are presented favouring the concept that electron transfer from Complex I to Complex III is operated by channelling of electrons through Coenzyme Q molecules bound to the supercomplex, in contrast with the hypothesis that the transfer of reducing equivalents from Complex I to Complex III occurs via random diffusion of the Coenzyme Q molecules in the lipid bilayer. Furthermore, another property provided by the supercomplex assembly is the control of generation of reactive oxygen species by Complex I. This article is part of a Special Issue entitled Respiratory Complex I, edited by Volker Zickermann and Ulrich Brandt

Two separate though interconneted route underlie NADH and succinate oxidation:kinetic evidence for different functional compartments of Coenzyme Q and/or Complex III.

The discovery of respiratory supercomplexes (SCs) led to the proposal that electron transfer between complexes I and III (CI, CIII) is mediated by channelling of Coenzyme Q (Q), with a kinetic advantage on the transfer based on random collisions, whereas electron transfer from CII to CIII obeys to the random collision model. The evidence for Q channelling, however, is highly controversial [1, 2]. We have approached the problem in bovine heart submitochondrial particles and in reconstituted proteoliposomes in which CI and CIII are preserved as SC I1III2. We restricted electron transfer to the Q area by studying NADH and succinate oxidation by exogenous cytochrome c (cyt. c) as acceptor, thus avoiding the bottleneck of endogenous cyt. c. Using this system we found the rates of NADH and succinate oxidation by cyt. c to be almost completely additive. The rate obtained by simultaneous addition of NADH and succinate was much higher than that predicted for a homogeneous Q pool [3], thus suggesting that NADH and succinate oxidation by cyt. c follow two different routes. The NADH route presumably operates through Q channelling in the SC I1III2. However Qpool molecules may exchange with Qbound in SC, approaching the rates predicted for a single pool, when the reducing pressure increases by strong CIII inhibition or when detergents destabilize the SCs. The accessibility of Qpool to SC I1III2 may be a physiological device to control electron fluxes from different substrates and implies a dissociation equilibrium of Qbound with the Q pool, by which the size of the pool determines saturation of the binding site(s) in the SC. Thus bulk Qpool has a role also in oxidation of NAD-linked substrates, providing a rationale for the beneficial effect of exogenous Q supplementation on mitochondrial bioenergetics. References 1. JN Blaza et al. Proc Natl Acad Sci USA 111 (2014) 15735-40. 2. G Lenaz et al. BBA Bioenerg. (2016) Epub ahead of print. 3. A Kröger, M Klingenberg. Eur J Biochem. 34 (1973) 358-68

Two separate though interconnected routes underlie NADH and succinate oxidation: kinetic evidence for different functional compartments of Coenzyme Q and/or Complex III

The discovery of respiratory supercomplexes (SCs) led to the proposal that electron transfer between complexes I and III (CI, CIII) is mediated by channelling of Coenzyme Q (Q), with a kinetic advantage on the transfer based on random collisions, whereas electron transfer from CII to CIII obeys to the random collision model. The evidence for Q channelling, however, is highly controversial [1, 2]. We have approached the problem in bovine heart submitochondrial particles and in reconstituted proteoliposomes in which CI and CIII are preserved as SC I1III2. We restricted electron transfer to the Q area by studying NADH and succinate oxidation by exogenous cytochrome c (cyt. c) as acceptor, thus avoiding the bottleneck of endogenous cyt. c. Using this system we found the rates of NADH and succinate oxidation by cyt. c to be almost completely additive. The rate obtained by simultaneous addition of NADH and succinate was much higher than that predicted for a homogeneous Q pool [3], thus suggesting that NADH and succinate oxidation by cyt. c follow two different routes. The NADH route presumably operates through Q channelling in the SC I1III2. However Qpool molecules may exchange with Qbound in SC, approaching the rates predicted for a single pool, when the reducing pressure increases by strong CIII inhibition or when detergents destabilize the SCs. The accessibility of Qpool to SC I1III2 may be a physiological device to control electron fluxes from different substrates and implies a dissociation equilibrium of Qbound with the Q pool, by which the size of the pool determines saturation of the binding site(s) in the SC. Thus bulk Qpool has a role also in oxidation of NAD-linked substrates, providing a rationale for the beneficial effect of exogenous Q supplementation on mitochondrial bioenergetics. References 1. JN Blaza et al. Proc Natl Acad Sci USA 111 (2014) 15735-40. 2. G Lenaz et al. BBA Bioenerg. (2016) Epub ahead of print. 3. A Kr\uf6ger, M Klingenberg. Eur J Biochem. 34 (1973) 358-68

Respiratory Supercomplexes in Mitochondria

Table of contents 12.1. INTRODUCTION 12.1.1. The respiratory chain of mitochondria 12.1.2. Organization of the respiratory chain: historical outline 12.2. DISTRIBUTION AND COMPOSITION OF RESPIRATORY SUPERCOMPLEXES 12.2.1. Distribution in different organisms 12.2.2. Composition of respiratory supercomplexes 12.3. SUPERCOMPLEX ASSOCIATION PROVIDES A KINETIC ADVANTAGE 12.3.1. Structural evidence 12.3.1.1. Molecular structure of supercomplexes 12.3.1.2. Dynamic nature of supercomplexes: the plasticity model 12.3.1.3. The role of lipids: cardiolipin in supercomplexes 12.3.1.4. Standing uncertainties 12.3.2. Evidence for channelling in the Coenzyme Q region 12.3.2.1. Rate advantage in the Coenzyme Q region 12.3.2.2. Evidence for channelling by metabolic flux control analysis 12.3.2.3. Separate compartments of Coenzyme Q? 12.3.2.4. The function of the Coenzyme Q pool 12.3.2.4.1. Dissociation equilibrium of bound Coenzyme Q 12.3.2.4.2. Electron transfer between individual complexes not involved in supercomplex organization 12.3.2.5. Concluding evidence about channelling in the Coenzyme Q region 12.3.3. Electron transfer through cytochrome c 12.4. SUPERCOMPLEXES AND REACTIVE OXYGEN SPECIES 12.5. PHYSIOLOGICAL AND PATHOLOGICAL IMPLICATIONS 12.5.1. Supercomplexes and regulation of metabolic fluxes 12.5.2. Supercomplexes and ROS signalling 12.5.3. Supercomplexes in pathology and agin

Glycerophosphoinositol 4-phosphate, a putative endogenous inhibitor of adenylyl cyclase.

In a continuous line of rat thyroid cells transformed by the k-ras oncogene (KiKi), the expression of rasp21 correlates with an increased activity of a phosphoinositide- specific phospholipase AB, which leads to elevated levels of glycerophosphoinositols. In this study we have characterized bthioel ogical activities of these compounds. Growth and differentiation in thyroid cells are mainly regulated by the activation of adenylylcyclase. Therefore,w e haves tudied the effects of glycerophosphoinositols on the activity of this enzyme using a normal thyroid cell line (FRTL5M).i cromolar concentrations of glycerophosphoinositol 4- phosphate (GroPIns-4-P) caused a -50% inhibition of the adenylylcyclase activity in FRTL5 membranes stimulated by the GTP-binding protein activaftlouro - roaluminate. Similar concentrations of GroPIns-4-P were detected in KiKi cells but not in the normal FRTLS line. Micromolar GroPIns-4-P was found to be taken up by intact FRTL5 cells and to induce nearly 50% inhibition of the thyrotropin- and cholera toxin-induced increase in cAMP levels. Similar results were also observed in other cell lines (smooth muscle, pituitary cells, and pneumocytes). GroPIns-4-P inhibitedC AMPdependent cellular functions sucahs iodide uptake and thymidine incorporation in FRTL5 cells when stimulated by thyrotropin and cholera toxin but not when induced by forskolin. Theser esults arec onsistent with GroPIns-4-P exerting an inhibitoerfyf ect on the GTPbinding protein that stimulateasd enylylcyclase. We propose that GroPIns-4-Pm ight mediatea mechanism of cross-talk between adenylylcyclase anphdo spholipase A2 in thyroida s well as in otherc ell systems