Antioxidant and Antifungal Activities and Characterization of Phenolic Compounds Using High-Performance Liquid Chromatography and Mass Spectrometry (HPLC-MS) in Empetrum rubrum Vahl ex Willd.

In searching for compounds with antioxidant and antifungal activity, our study focused on the subshrub species Empetrum rubrum Vahl ex Willd. (Ericaceae). We measured the antioxidant activity of its methanolic extract (MEE) obtained from the aerial parts (leaves and stems) and of its methanolic extract (MEF) obtained from the lyophilized fruits. The antioxidant activity of the MEE and MEF was evaluated in vitro via a 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical and 2,2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) cationic radical. The results were expressed in gallic acid and Trolox equivalents for the DPPH and ABTS assays, respectively. The antioxidant activities, for the DPPH and ABTS assays, were also evaluated by considering the IC50 values. Concerning the antioxidant activity, the total phenolic content (TPC) in the MEE and MEF was determined using the Folin-Ciocalteu method. Polyphenols contained in the leaves, stems, and fruits of E. rubrum were determined qualitatively by employing high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) analysis. The antifungal activity of the MEE obtained from the aerial parts of E. rubrum was tested against Rhizoctonia solani. The results of IC50 values measured by the DPPH and ABTS methods with MEE were 0.4145 ± 0.0068 mg mL-1 and 0.1088 ± 0.0023 mg mL-1, respectively, and the IC50 values for MEF were 6.4768 ± 0.0218 mg mL-1 and 0.7666 ± 0.0089 mg mL-1 measured by the DPPH and ABTS methods, respectively. The HPLC-MS analysis revealed the presence of anthocyanins, phenolic acids derivatives, and flavonols. In vitro, mycelial growth of this fungus was reduced from 90% to nearly 100% in the presence of MEE. The observed antifungal effect is related to the presence of the abovementioned phenols, detected in the MEE.

Whole Transcriptome Analyses of Apricots and Japanese Plum Fruits after 1-MCP (Ethylene-Inhibitor) and Ethrel (Ethylene-Precursor) Treatments Reveal New Insights into the Physiology of the Ripening Process.

The physiology of Prunus fruit ripening is a complex and not completely understood process. To improve this knowledge, postharvest behavior during the shelf-life period at the transcriptomic level has been studied using high-throughput sequencing analysis (RNA-Seq). Monitoring of fruits has been analyzed after different ethylene regulator treatments, including 1-MCP (ethylene-inhibitor) and Ethrel (ethylene-precursor) in two contrasting selected apricot (Prunus armeniaca L.) and Japanese plum (P. salicina L.) cultivars, 'Goldrich' and 'Santa Rosa'. KEEG and protein-protein interaction network analysis unveiled that the most significant metabolic pathways involved in the ripening process were photosynthesis and plant hormone signal transduction. In addition, previously discovered genes linked to fruit ripening, such as pectinesterase or auxin-responsive protein, have been confirmed as the main genes involved in this process. Genes encoding pectinesterase in the pentose and glucuronate interconversions pathway were the most overexpressed in both species, being upregulated by Ethrel. On the other hand, auxin-responsive protein IAA and aquaporin PIP were both upregulated by 1-MCP in 'Goldrich' and 'Santa Rosa', respectively. Results also showed the upregulation of chitinase and glutaredoxin 3 after Ethrel treatment in 'Goldrich' and 'Santa Rosa', respectively, while photosystem I subunit V psaG (photosynthesis) was upregulated after 1-MCP in both species. Furthermore, the overexpression of genes encoding GDP-L-galactose and ferredoxin in the ascorbate and aldarate metabolism and photosynthesis pathways caused by 1-MCP favored antioxidant activity and therefore slowed down the fruit senescence process.

Plasticity of cardiovascular chemoreflexes after prolonged unilateral carotid body denervation: implications for its therapeutic use.

Unilateral carotid body denervation has been proposed as treatment for sympathetic-related human diseases such as systolic heart failure, hypertension, obstructive sleep apnea, and cardiometabolic diseases. The long-term therapeutic effects of carotid body removal will be maintained if the remnant "buffer nerves," that is, the contralateral carotid nerve and the aortic nerves that innervate second-order neurons at the solitary tract nuclei (NTS), do not modify their contributions to the cardiovascular chemoreflexes. Here, we studied the cardiovascular chemoreflexes 1 mo after unilateral carotid body denervation either by excision of the petrosal ganglion (petrosal ganglionectomy, which eliminates central carotid afferents) or exeresis of a segment of one carotid nerve (carotid neurectomy, which preserves central afferents). Cardiovascular chemoreflexes were induced by intravenous (iv) injections of sodium cyanide in pentobarbitone-anesthetized adult cats. After 1 mo of unilateral petrosal ganglionectomy, without significant changes in basal arterial pressure, the contribution of the contralateral carotid nerve to the chemoreflex increases in arterial pressure was enhanced without changes in the contribution provided by the aortic nerves. By contrast, after 1 mo of unilateral carotid neurectomy, the contribution of remnant buffer nerves to cardiovascular chemoreflexes remained unmodified. These results indicate that a carotid nerve interruption involving denervation of second-order chemosensory neurons at the NTS will trigger cardiovascular chemoreflex plasticity on the contralateral carotid pathway. Then, unilateral carotid body denervation as therapeutic tool should consider the maintenance of the integrity of carotid central chemoafferents to prevent plasticity on remnant buffer nerves.NEW & NOTEWORTHY Unilateral carotid body denervation has been proposed as treatment for sympathetic hyperactivity-related human disorders. Its therapeutic effectiveness for maintaining a persistent decrease in the sympathetic outflow activity will depend on the absence of compensatory chemoreflex plasticity in the remnant carotid and aortic afferents. Here, we suggest that the integrity of central afferents after carotid body denervation is essential to prevent the emergence of plastic functional changes on the contralateral "intact" carotid nerve.

Genotyping by Sequencing for SNP-Based Linkage Analysis and Identification of QTLs Linked to Fruit Quality Traits in Japanese Plum (Prunus salicina Lindl.).

Marker-assisted selection (MAS) in stone fruit (Prunus species) breeding is currently difficult to achieve due to the polygenic nature of the most relevant agronomic traits linked to fruit quality. Genotyping by sequencing (GBS), however, provides a large quantity of useful data suitable for fine mapping using Single Nucleotide Polymorphisms (SNPs) from a reference genome. In this study, GBS was used to genotype 272 seedlings of three F1 Japanese plum (Prunus salicina Lindl) progenies derived from crossing "98-99" (as a common female parent) with "Angeleno," "September King," and "September Queen" as male parents. Raw sequences were aligned to the Peach genome v1, and 42,909 filtered SNPs were obtained after sequence alignment. In addition, 153 seedlings from the "98-99" × "Angeleno" cross were used to develop a genetic map for each parent. A total of 981 SNPs were mapped (479 for "98-99" and 502 for "Angeleno"), covering a genetic distance of 688.8 and 647.03 cM, respectively. Fifty five seedlings from this progeny were phenotyped for different fruit quality traits including ripening time, fruit weight, fruit shape, chlorophyll index, skin color, flesh color, over color, firmness, and soluble solids content in the years 2015 and 2016. Linkage-based QTL analysis allowed the identification of genomic regions significantly associated with ripening time (LG4 of both parents and both phenotyping years), fruit skin color (LG3 and LG4 of both parents and both years), chlorophyll degradation index (LG3 of both parents in 2015) and fruit weight (LG7 of both parents in 2016). These results represent a promising situation for GBS in the identification of SNP variants associated to fruit quality traits, potentially applicable in breeding programs through MAS, in a highly heterozygous crop species such as Japanese plum.

Shifting from hypoxia to hyperoxia to assess the peripheral chemosensory drive of ventilation.

The study of the initial effects of a sudden and brief replacement of air by pure oxygen has been proposed as a tool (Dejours' test) to determine the tonic influence that arterial (peripheral) chemoreceptors were exerting upon ventilation under previous normoxic conditions. Therefore, the acute ventilatory response to transient hyperoxia should be used to assess the level of hypoxic chemosensory drive. In spontaneously ventilated pentobarbitone-anesthetized cats, we observed that the degree of ventilatory depression provoked by hyperoxia was correlated to the degree of previous hypoxia. Minimal tidal volumes (V(T)) or transient apnea were reached between second to fourth cycles after switching from 5% to 100% O(2) breathing. Continuous recordings of chemosensory discharges from one carotid (sinus) nerve allowed correlation of the falls in frequency of chemosensory discharges to the degree of hyperoxia-induced ventilatory depression and provided an accurate measure of the prevailing chemosensory drive of ventilation exerted during hypoxic steady-state conditions.

Immunosensory signalling by carotid body chemoreceptors.

Injections of lipopolysaccharide (LPS) have been used to produce the signs of sepsis and study their underlying mechanisms. Intravenous (IV) injections of LPS in anesthetized cats induce tachypnea, tachycardia and hypotension, but ventilatory changes are suppressed after sectioning carotid and aortic nerves. Otherwise, LPS increases the basal frequency of carotid chemosensory discharges, but reduces ventilatory and chemosensory responses to hypoxia and nicotine injections. Increases in cytokines (IL-1ß, IL-6 and TNF-α) are observed in plasma and tissues after injecting LPS. In carotid bodies perfused in vitro, TNF-α reduces chemosensory discharges induced by hypoxia. The rat carotid body and its sensory ganglion constitutively express LPS canonical receptor, TLR4, as well as TNF-α and its receptors (TNF-R1 and TNF-R2). Increases of TNF-α and TNF-R2 expression occur after LPS administration. The activation of peripheral and central autonomic pathways induced by LPS or IL's is partly dependent on intact vagus nerves. Thus, the carotid and vagus nerves provide routes between the immune system and CNS structures involved in systemic inflammatory responses.

Cardiovascular responses to hyperoxic withdrawal of arterial chemosensory drive.

Searching for an arterial chemosensory drive exerted upon the cardiovascular system under eucapnic normoxia, we performed experiments on spontaneously ventilated, pentobarbitone-anesthetized cats, in which ventilatory flow through a pneumo-tachograph, instantaneous respiratory frequency, end-tidal pressure of CO(2), arterial pressure, and instantaneous heart frequency were simultaneously recorded. Repeated exposures to 100% O(2) breathing for 5 to 60 s caused the well-known transient decreases in tidal ventilatory volume and instantaneous respiratory frequency, after which minor decreases in systolic, diastolic and mean arterial pressures, as well as in instantaneous heart frequency were observed. After selective bilateral denervation of carotid sinuses (barodenervation), hyperoxia-induced falls in arterial pressure and heart rate became more evident. Subsequent bilateral section of the carotid nerves (with or without section of the aortic nerves) suppressed these effects. Present results indicate the presence of a chemosensory drive of the cardiovascular system under eucapnic normoxia, although considerably smaller than that exerted upon ventilation. The small magnitude of the decreases in arterial pressure and heart rate observed under control conditions suggests that cardiovascular effects elicited by hyperoxic challenges are normally buffered by carotid baroreflexes.

Lipopolysaccharide-induced carotid body inflammation in cats: functional manifestations, histopathology and involvement of tumour necrosis factor-alpha.

In the absence of information on functional manifestations of carotid body (CB) inflammation, we studied an experimental model in which lipopolysaccharide (LPS) administration to pentobarbitone-anaesthetized cats was performed by topical application upon the CB surface or by intravenous infusion (endotoxaemia). The latter caused: (i) disorganization of CB glomoids, increased connective tissue, and rapid recruitment of polymorphonuclear cells into the vascular bed and parenchyma within 4 h; (ii) increased respiratory frequency and diminished ventilatory chemoreflex responses to brief hypoxia (breathing 100% N(2) for 10 s) and diminished ventilatory chemosensory drive (assessed by 100% O(2) tests) during normoxia and hypoxia; (iii) tachycardia, increased haematocrit and systemic hypotension in response to LPS i.v.; and (iv) increased basal frequency of carotid chemosensory discharges during normoxia, but no change in maximal chemoreceptor responses to brief hypoxic exposures. Lipopolysaccharide-induced tachypnoea was prevented by prior bilateral carotid neurotomy. Apoptosis was not observed in CBs from cats subjected to endotoxaemia. Searching for pro-inflammatory mediators, tumour necrosis factor-alpha (TNF-alpha) was localized by immunohistochemistry in glomus and endothelial cells; reverse transcriptase-polymerase chain reaction revealed that the CB expresses the mRNAs for both type-1 (TNF-R1) and type-2 TNF-alpha receptors (TNF-R2); Western blot confirmed a band of the size expected for TNF-R1; and histochemistry showed the presence of TNF-R1 in glomus cells and of TNF-R2 in endothelial cells. Experiments in vitro showed that the frequency of carotid nerve discharges recorded from CBs perfused and superfused under normoxic conditions was not significantly modified by TNF-alpha, but that the enhanced frequency of chemosensory discharges recorded along responses to hypoxic stimulation was transiently diminished in a dose-dependent manner by TNF-alpha injections. The results suggest that the CB may operate as a sensor for immune signals, that the CB exhibits histological features of acute inflammation induced by LPS, that TNF-alpha may participate in LPS-induced changes in chemosensory activity and that some pathophysiological reactions to high levels of LPS in the bloodstream may originate from changes in CB function.

Are there interactions between acetylcholine- and ATP-induced responses at the level of a visceral sensory ganglion?

We investigate possible interactions between acetylcholine (ACh)- and adenosine 5'-triphosphate (ATP)-induced responses of petrosal ganglion, where the perikarya of most sensory neurons of the glossopharyngeal nerve are located. Experiments were performed on petrosal ganglia excised from pentobarbitone-anesthetized cats, desheathed and perfused in vitro. Separate applications of ACh and ATP to the exposed surface of the ganglion induced bursts of antidromic potentials recorded from the carotid (sinus) nerve branch of the glossopharyngeal nerve, which frequencies were dependent on the dose of the applied agonists. The simultaneous application of previously determined ED50s of ACh and ATP provoked responses corresponding closely to the simple addition of the responses elicited by the separate application of each agent. Responses usually subsided within 1 min of stimuli application but were followed by periods of refractoriness to subsequent application of the same agent. After determining the timing for recovering from desensitization to the ED50s of ACh and ATP applied separately, ACh was applied while the preparation had been desensitized to ATP and then ATP was applied during desensitization to ACh, but responses obtained were similar to control responses induced by each agent separately. In summary, ACh- and ATP-induced responses of petrosal ganglion neurons are simply additive, followed by a few minute lasting desensitization, but cross-desensitization was not observed. Thus, ACh and ATP seem to operate through independent receptors, activating separate ionic channels, whose coincident currents do not interfere each other.

How the carotid body works: different strategies and preparations to solve different problems.

This is a review of the different experimental approaches developed to solve the problems in our progress towards a comprehensive understanding of how arterial chemoreceptors operate. An analysis is performed oi the bases, advantages and limits of the following preparations: studies of ventilatory reflexes originated from carotid bodies (CBs) in the entire animal; recordings of CB chemosensory discharges in situ; CB preparations perfused in situ; CB explants in oculo; CB explants in ovo; CB preparations incubated in vitro; CB preparations superfused in vitro; CB preparations perfused and superfused in vitro: CB tissue slices in vitro; cells acutely dissociated from CBs; CB cells in tissue culture; petrosal ganglia superfused in vitro; petrosal ganglion cells in tissue culture; and co-cultures of CB and sensory ganglion cells. A brief historical account is given of the passage from one preparation to the next one. Emphasis is placed on personal experience with the different preparations whenever possible. Examples are given of the importance of selecting the appropriate experimental preparation for solving each particular theoretical problem. In fact, brilliant ideas on how the CB works have been unproductive until finding the adequate experimental approach to explore the validity of such ideas.

How the carotid body works: Different strategies and preparations to solve different problems

This is a review of the different experimental approaches developed to solve the problems in our progress towards a comprehensive understanding of how arterial chemoreceptors operate. An analysis is performed of the bases, advantages and limits of the following preparations: studies of ventilatory reflexes originated from carotid bodies (CBs) in the entire animal; recordings of CB chemosensory discharges in situ; CB preparations perfused in situ; CB explants in oculo; CB explants in ovo; CB preparations incubated in vitro; CB preparations superfused in vitro; CB preparations perfused and superfused in vitro; CB tissue slices in vitro; cells acutely dissociated from CBs; CB cells in tissue culture; petrosal ganglia superfused in vitro; petrosal ganglion cells in tissue culture; and co-cultures of CB and sensory ganglion cells. A brief historical account is given of the passage from one preparation to the next one. Emphasis is placed on personal experience with the different preparations whenever possible. Examples are given of the importance of selecting the appropriate experimental preparation for solving each particular theoretical problem. In fact, brilliant ideas on how the CB works have been unproductive until finding the adequate experimental approach to explore the validity of such ideas.

Catecholamine release from isolated sensory neurons of cat petrosal ganglia in tissue culture.

The petrosal ganglion (PG) is entirely constituted by the perikarya of primary sensory neurons, part of which innervates the carotid body via the carotid sinus nerve (CSN). Application of acetylcholine (ACh) or nicotine (Nic) as well as adenosine 5'-triphosphate (ATP) to the PG in vitro increases the frequency of CSN discharges, an effect that is modified by the concomitant application of dopamine (DA). Since a population of PG neurons expresses tyrosine hydroxylase, and DA is released from the cat carotid body in response to electrical stimulation of C-fibers in the CSN, it is possible that DA may be released from the perikarya of PG neurons. Therefore, we studied whether ACh or Nic, ATP and high KCl could induce DA release from PG neurons in culture. Petrosal ganglia were excised from pentobarbitone-anesthetized adult cats, dissociated and their neurons maintained in culture for 7-21 days. Catecholamine release was measured by amperometry via carbon-fiber microelectrodes. In response to KCl, Nic, ACh or ATP application, about 25% of neurons exhibited electrochemical signals compatible with DA release. This percentage increased to 41% after loading the neurons with exogenous DA. The present results suggest that DA release may be induced from the perikarya of a population of PG neurons.

Dopamine inhibits ATP-induced responses in the cat petrosal ganglion in vitro.

The petrosal ganglion (PG) provides sensory innervation to the carotid sinus and carotid body through the carotid (sinus) nerve (CN). Application of either acetylcholine (ACh) or adenosine 5'-triphosphate (ATP) to the PG superfused in vitro activates CN fibers. Dopamine (DA) modulates the effects of ACh. We have previously shown that DA when applied to the PG modulates the effects of ACh on carotid sinus nerve fibers. We currently report the effects of DA on the ATP-induced responses in the isolated PG in vitro. While DA had no effect on the basal activity recorded from the CN, it reduced ATP-induced responses in a dose-dependent manner, when preceding ATP applications by 30 s. Our results suggest that DA-a transmitter present in a group of PG neurons and in carotid body cells-may act as an inhibitory modulator of ATP-evoked responses in PG neurons.

Acute ventilatory and circulatory reactions evoked by nicotine: are they excitatory or depressant?

Either excitatory or inhibitory cardio-respiratory responses induced by nicotine have been reported. We evaluated the joint and separate contributions of peripheral arterial chemoreceptors and pulmonary vagal afferences to nicotine-induced cardio-respiratory responses in 11 pentobarbitone-anaesthetized cats. Nicotine, given i.v. in doses of from 1 to 200 microg/kg, evoked dose-dependent transient increases in tidal volume (VT) and arterial blood pressure (BP), but the highest doses evoked brief apnoea, immediately followed by intense hyperventilation, as well as discrete early hypotension followed by late hypertension. Bilateral section of the aortic and carotid nerves abolished all hyperventilatory responses to nicotine, giving way to apnoea followed by few cycles of reduced VT and profound hypotension followed by slight hypertension in response to intermediate doses (50-100 microg/kg). Subsequent bilateral vagotomy (BV) suppressed apnoeic and hypotensive responses. In other cats initially subjected to BV, only increases in VT and BP were observed in response to nicotine, effects which were no longer observed after additional carotid and aortic deafferentation. These data suggest that excitatory effects of nicotine on respiration and BP are reflexes evoked by stimulation of peripheral arterial chemoreceptors, while inhibitory effects are also reflex responses but evoked from stimulation of pulmonary vagal afferences.

A model of internal control may improve the response time of an automatic arterial pressure controller

A simplified model for the arterial pressure control system was implemented on a personal computer using Matlab Simulink. Model responsees to variations of systemic vascular resistance were comparable to those predicted by physiology. Computer simulation suggested that including this model of the internal pressure control system within the design of an external controller would achieve better arterial pressure control and faster response than previous systems.

A report on the evolution of this journal and an analysis of its present state and perspectives

This journal appeared as Archivos de Biología y Medicina Experimentales between 1964 and 1992, its name being changed into Biological Research in 1992. This is a report on the past and present of the journal, analyzing the problems of a scientific journal published in English but produced in Latin America, those that its contributors must deal with in the preparation of the manuscripts, those involved in the evaluation procedures and editing of material for the press, and those about coverage by indexing services and the international recognition of the journal quality