Cardiac influence of the β3-adrenoceptor in the goldfish (Carassius auratus): a protective role under hypoxia?

The goldfish (Carassius auratus) exhibits a remarkable capacity to survive and remain active under prolonged and severe hypoxia, representing a well-suited model to study cardiac function when oxygen availability represents a limiting factor. Under hypoxia, the goldfish heart increases its performance, this representing a putative component of hypoxia tolerance; however, underlying mechanisms have not been yet elucidated. We aimed to investigate the role of β3-ARs in the mechanisms which modulate the goldfish heart performance, also in relation to oxygen levels. By western blotting analysis, we found that the goldfish heart expresses β3-ARs, and this expression increases under hypoxia. Effects of β3-ARs stimulation were analysed by using an ex vivo working heart preparation. Under normoxia, the β3-ARs selective agonist BRL37344 (10−12-10−7M) elicited a concentration-dependent increase of contractility abolished by the specific β3-AR antagonist (SR59230A; 10−8M), but not by α/β1/β2-ARs inhibitors (phentolamine, nadolol, and ICI118,551; 10−7M). Under acute hypoxia, BRL37344 did not affect the goldfish heart performance. However, SR59230A, but not phentolamine, nadolol, and ICI118,551, abolished the time-dependent enhancement of contractility which characterizes the hypoxic goldfish heart. Under both normoxia and hypoxia, adenylate cyclase and cAMP were found to be involved in the β3-ARs-dependent downstream transduction pathway. Our findings suggest the presence of functional β3-ARs in the goldfish heart, whose activation modulates the basal performance and contributes to the hypoxia-dependent increase of contractility

Survival of Listeria monocytogenes in uncooked Italian dry sausage (salami).

This study was undertaken to supplement existing information on the survival of Listeria monocytogenes in Italian salami. The fact that Italian salami is frequently consumed by a large number of people poses some serious health implications. Some raw materials have been found to be microbiologically contaminated, for their production occurs without any thermic treatment, and these are in circulation throughout Italy all year round. We selected the product for its microbiological, technological, and commercial characteristics. We analyzed 1,020 samples taken during the autumn and winter 2002 and spring and summer 2003 periods and immediately before selling. The samples were collected from 17 plants with an annual production of between 1 and 2,000 metric tons and with a distribution of products in over 80% of Italy in geographic terms. To detect and enumerate L. monocytogenes, we followed International Organization for Standardization (ISO) 11290 part 1 and 2: 1996 (modified using chromogenic medium Agar Listeria according to Ottarviani and Agosti [ALOA]). L. monocytogenes was found in 22.7% of samples, but the contamination level was less than 10 CFU/g. Contamination prevalence ranged from 1.6 to 58.3% and was lower than 10% in 5 of the 17 plants checked. The most frequently isolated serotypes were 1/2c, 1/2a, 1/2b, and 4b. Additional studies are necessary to establish if the exposure to a small number of L. monocytogenes cells through the consumption of salami represents a significant health risk and, in light of the future introduction of the SANCO/4198/2001 revision 21 "Commission Regulation on Microbiological Criteria for Foodstuffs," is a necessary investigation

Selenoprotein T as a new positive inotrope in the goldfish, Carassius auratus.

Selenoprotein T (SELENOT) is a thioredoxin-like protein, which mediates oxidoreductase functions via its redox active motif Cys-X-X-Sec. In mammals, SELENOT is expressed during ontogenesis and progressively decreases in adult tissues. In the heart, it is re-expressed after ischemia and induces cardioprotection against ischemia/reperfusion (I/R) injury. SELENOT is present in teleost fish, including the goldfish Carassius auratus. This study aimed to evaluate the cardiac expression of SELENOT, and the effects of exogenous PSELT (a 43-52 SELENOT derived-peptide) on the heart function of C. auratus, a hypoxia tolerance fish model. We found that SELENOT was expressed in cardiac extracts of juvenile and adult fish, located in the sarcoplasmic reticulum (SR) together with calsequestrin-2. Expression increased under acute hypoxia. On ex vivo isolated and perfused goldfish heart preparations, under normoxia, PSELT dose-dependently increased Stroke Volume (SV), Cardiac Output (Q̇), and Stroke Work (SW), by involving cAMP, PKA, L-type calcium channels, SERCA2a pumps, and pAkt. Under hypoxia, PSELT did not affect myocardial contractility. Only at higher concentrations (10−8 -10−7 M) an increase of SV and Q̇ was observed. It also reduced the cardiac expression of 3-NT, a tissue marker of nitrosative stress which increases under low oxygen availability. These data are the first to propose SELENOT 43-52, PSELT, as a cardiac modulator in fish, with a potential protective role under hypoxia

Effect of different formulations of magnesium chloride, used as anaesthetic agents, on the performance of the isolated heart of Octopus vulgaris

Scuola di Dottorato Life Sciences, Ciclo XVIII, a.a. 2015-2016Il cloruro di magnesio (MgCl2) rappresenta l'anestetico più comunemente utilizzato per i cefalopodi; tuttavia, i suoi effetti fisiologici compresi quelli a livello cardiaco non sono ben caratterizzati. Nel presente studio, utilizzando come modello sperimentale il polpo comune Octopus vulgaris, è stato valutato mediante la tecnica del cuore isolato e perfuso: a) se l'esposizione dell’animale in vivo (immersione) a diverse formulazioni di MgCl2 induce effetti sulla funzione cardiaca in vitro che potrebbero danneggiare il recupero dall'anestesia; b) l’effetto diretto della perfusione con diverse formulazioni di MgCl2 sulla funzione cardiaca. Per gli esperimenti in vivo, sono state utilizzate le seguenti formulazioni con relativi tempi di esposizione: i) MgCl2 3,5% in acqua di mare (MgCl2 sw, 20min); ii) MgCl2 3,5% (1:1, acqua di mare:acqua distillata, 20min); miscela di MgCl2 (1,12%) + etanolo (1%) (Mix, 20min e 45min); ipotermia (4°C, 5-10min). Alle condizioni basali, i cuori prelevati dopo esposizione ai vari trattamenti anestetici, inclusa l’ipotermia, sono stati in grado di lavorare con carichi emodinamici simili a quelli in vivo e di produrre valori fisiologici di gittata sistolica. In condizioni di carico emodinamico (incrementi di precarico), i cuori rimossi da animali esposti in vivo ad MgCl2 sw (20min) o al Mix (20min) hanno mostrato una performance cardiaca (in termini di risposta di Frank-Starling) comparabile a quella di animali anestetizzati mediante ipotermia. Al contrario, sia l'esposizione a MgCl2 (1:1, 20min), che al Mix (45min) ha mostrato un significativo deterioramento della risposta di Frank-Starling in termini di ridotta capacità di rispondere agli incrementi di precarico. Dopo esposizione ad ipotermia, la perfusione dei cuori isolati con concentrazioni crescenti delle diverse formulazioni di MgCl2, ha prodotto un effetto bradicardico dose-dipendente (e in alcuni casi arresto cardiaco) ed una riduzione della gittata cardiaca e del volume sistolico, indicativi di un effetto diretto dei trattamenti anestetici sul cuore. Al contrario, la perfusione con dosi crescenti di solo etanolo non ha causato alcuna variazione della frequenza cardiaca, indicando che l’effetto bradicardico evidenziato con la soluzione Mix è attribuibile al cloruro di magnesio. L'espressione di HSP70 e caspasi-3, e la fosforilazione di JNK e p38MAPK sono stati determinati in omogenati cardiaci, ottenuti da esemplari di O. vulgaris esposti alle diverse formulazioni di cloruro di magnesio o ad ipotermia (4°C). I risultati hanno evidenziato un incremento dell’espressione di HSP70 (ma non della caspasi-3); in particolare, l’espressione di HSP70 è risultata essere inferiore nei cuori prelevati da animali esposti ad ipotermia, rispetto a quelli esposti alle diverse formulazioni di MgCl2. Tra questi ultimi, nessuna differenza quantitativa è stata evidenziata tra le diverse formulazioni, ad eccezione del Mix 45min, effetto dovuto probabilmente al maggior tempo di esposizione. L'aumento della fosforilazione di JNK e p38MAPK, parallelo all'aumentata espressione di HSP70, lascia supporre il coinvolgimento di queste MAPKs nell'espressione di HSP70. Nell'insieme i dati dimostrano che, a condizione che l'esposizione in vivo al 3,5% di MgCl2 in acqua di mare o ad una miscela di MgCl2+etanolo sia limitato a ~ 20min, gli effetti residui sulla funzione cardiaca non influenzano il recupero post-anestesia. Inoltre, l’espressione di HSP70 che può giocare un ruolo citoprotettivo nella risposta allo stress durante l'anestesia, parallela alla mancata espressione della caspasi-3 in tutti i tessuti analizzati, permette di escludere eventi pro-apoptotici da parte di tutti i trattamenti anestetici. Nell’insieme questi risultati possono contribuire alla discussione circa le pratiche anestesiologiche da utilizzare anche in relazione all'applicazione della nuova Direttiva 2010/63 / UE per i cefalopodi.Università della Calabri

Azione cardiaca della catestatina in Rana esculenta

Dottorato di Ricerca in Biologia Animale, XXI Ciclo,a.a. 2007-2008Università della Calabri

Hypoxic and Thermal Stress: Many Ways Leading to the NOS/NO System in the Fish Heart

Teleost fish are often regarded with interest for the remarkable ability of several species to tolerate even dramatic stresses, either internal or external, as in the case of fluctuations in O2 availability and temperature regimes. These events are naturally experienced by many fish species under different time scales, but they are now exacerbated by growing environmental changes. This further challenges the intrinsic ability of animals to cope with stress. The heart is crucial for the stress response, since a proper modulation of the cardiac function allows blood perfusion to the whole organism, particularly to respiratory organs and the brain. In cardiac cells, key signalling pathways are activated for maintaining molecular equilibrium, thus improving stress tolerance. In fish, the nitric oxide synthase (NOS)/nitric oxide (NO) system is fundamental for modulating the basal cardiac performance and is involved in the control of many adaptive responses to stress, including those related to variations in O2 and thermal regimes. In this review, we aim to illustrate, by integrating the classic and novel literature, the current knowledge on the NOS/NO system as a crucial component of the cardiac molecular mechanisms that sustain stress tolerance and adaptation, thus providing some species, such as tolerant cyprinids, with a high resistance to stress

Hypoxia Tolerance in Teleosts: Implications of Cardiac Nitrosative Signals

Changes in environmental oxygen (O2) are naturally occurring phenomena which ectotherms have to face on. Many species exhibit a striking capacity to survive and remain active for long periods under hypoxia, even tolerating anoxia. Some fundamental adaptations contribute to this capacity: metabolic suppression, tolerance of pH and ionic unbalance, avoidance and/or repair of free-radical-induced cell injury during reoxygenation. A remarkable feature of these species is their ability to preserve a normal cardiovascular performance during hypoxia/anoxia to match peripheral (tissue pO2) requirements. In this review, we will refer to paradigms of hypoxia- and anoxia-tolerant teleost fish to illustrate cardiac physiological strategies that, by involving nitric oxide and its metabolites, play a critical role in the adaptive responses to O2 limitation. The information here reported may contribute to clarify the molecular and cellular mechanisms underlying heart vulnerability vs. resistance in relation to O2 availability

Application of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of <i>Listeria monocytogenes</i> in Cooked Ham

Changing eating habits and rising demand of food have increased the incidence of foodborne diseases, particularly in industrialized countries. In this context, contaminated ready-to-eat food (RTE) may be a vehicle for the transmission of Listeria monocytogenes (L. monocytogenes), a foodborne pathogen responsible of listeriosis, a severe infectious disease involving humans and animals. It would be useful to have rapid detection methods to screen the presence of L. monocytogenes in food. In this study, a colorimetric Loop-mediated isothermal amplification (LAMP) assay was applied to the detection of L. monocytogenes in 37 experimentally contaminated RTE meat samples. The LAMP primers consisted of a set of six primers targeting eight regions on the hlyA gene; the assay was carried out in 30 min at 65 °C in a water bath. Amplification products were visualized by color change assessment. The results of colorimetric LAMP assays based on the hly gene obtained in this study were compared to microbiological cultural methods, real-time PCR and real-time LAMP PCR, which show 100% specificity and sensitivity. These data suggest that colorimetric LAMP assays can be used as a screen to detect L. monocytogenes in ready-to-eat meat food

The Hypoxia Tolerance of the Goldfish (Carassius auratus) Heart: The NOS/NO System and Beyond

The extraordinary capacity of the goldfish (Carassius auratus) to increase its cardiac performance under acute hypoxia is crucial in ensuring adequate oxygen supply to tissues and organs. However, the underlying physiological mechanisms are not yet completely elucidated. By employing an ex vivo working heart preparation, we observed that the time-dependent enhancement of contractility, distinctive of the hypoxic goldfish heart, is abolished by the Nitric Oxide Synthase (NOS) antagonist L-NMMA, the Nitric Oxide (NO) scavenger PTIO, as well as by the PI3-kinase (PI3-K) and sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) pumps&rsquo; inhibition by Wortmannin and Thapsigargin, respectively. In goldfish hearts exposed to hypoxia, an ELISA test revealed no changes in cGMP levels, while Western Blotting analysis showed an enhanced expression of the phosphorylated protein kinase B (pAkt) and of the NADPH oxidase catalytic subunit Nox2 (gp91phox). A significant decrease of protein S-nitrosylation was observed by Biotin Switch assay in hypoxic hearts. Results suggest a role for a PI3-K/Akt-mediated activation of the NOS-dependent NO production, and SERCA2a pumps in the mechanisms conferring benefits to the goldfish heart under hypoxia. They also propose protein denitrosylation, and the possibility of nitration, as parallel intracellular events

Cardiac Hypoxia Tolerance in Fish: From Functional Responses to Cell Signals

Aquatic animals are increasingly challenged by O2 fluctuations as a result of global warming, as well as eutrophication processes. Teleost fish show important species-specific adaptability to O2 deprivation, moving from intolerance to a full tolerance of hypoxia and even anoxia. An example is provided by members of Cyprinidae which includes species that are amongst the most tolerant hypoxia/anoxia teleosts. Living at low water O2 requires the mandatory preservation of the cardiac function to support the metabolic and hemodynamic requirements of organ and tissues which sustain whole organism performance. A number of orchestrated events, from metabolism to behavior, converge to shape the heart response to the restricted availability of the gas, also limiting the potential damages for cells and tissues. In cyprinids, the heart is extraordinarily able to activate peculiar strategies of functional preservation. Accordingly, by using these teleosts as models of tolerance to low O2, we will synthesize and discuss literature data to describe the functional changes, and the major molecular events that allow the heart of these fish to sustain adaptability to O2 deprivation. By crossing the boundaries of basic research and environmental physiology, this information may be of interest also in a translational perspective, and in the context of conservative physiology, in which the output of the research is applicable to environmental management and decision making