Accessing the biotechnological potential of a novel isolated microalga from a semi-arid region of Brazil.

The use of microalgae as a source of food and pharmaceutical ingredients has garnered growing interest in recent years. Despite the rapid growth of the nutraceutical market, knowledge about the potential of bioactive molecules from microalgae remains insufficient. The present study aimed to investigate the biotechnological potential of the green microalga Desmodesmus armatus isolated from a semi-arid region of Brazil. The algal biomass was characterized in terms of gross biochemical composition, exopolysaccharide content, enzymatic inhibition capacity, and antioxidant, antibacterial, and hemolytic activities from solvents of different polarities (water, ethanol, acetone, and hexane). D armatus biomass had 40% of crude protein content, 25.94% of lipids, and 25.03% of carbohydrates. The prebiotic potential of exopolysaccharides from D armatus was demonstrated, which stimulated the growth of Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum bacteria strains. Moreover, the enzyme inhibition capacity for the proteases chymotrypsin (34.78%-45.8%) and pepsin (16.64%-27.27%), in addition to α-amylase (24.79%) and lipase (31.05%) was confirmed. The antioxidant potential varied between the different extracts, with 2,2-diphenyl-1-picrylhydrazyl sequestration values varying between 17.51% and 63.12%, and those of the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) method between 6.82% and 22.89%. In the antibacterial activity test, only the ethanolic extract showed inhibition against Listeria sp. (at minimum inhibitory concentration [MIC] = 256 µg mL-1). This fraction also presented the highest significant levels of hemolysis (31.88%-52.45%). In summary, the data presented in the study suggest the presence of biocompounds with biotechnological and nutraceutical potential in the D armatus biomass. Future studies may evaluate the inclusion of this biomass in foods in order to increase their biological value.

P2X7 receptor antagonist improves gastrointestinal disorders in spontaneously hypertensive rats.

The purinergic system participates in the control of blood pressure. Hypertension promotes the occurrence of gastrointestinal disorders such as intestinal inflammation and gastric emptying delay. This study aimed i) to investigate the participation of the P2X7 receptor blocker Brilliant Blue G (BBG) on gastric emptying of solids and changes in oxidative stress in the gastric fundus, duodenum, and colon of spontaneously hypertensive rats (SHR) and ii) to study the putative relationship of this effect with the renin-angiotensin system. Rats were divided into five groups: Control, SHR, SHR+BBG, SHR+BBG+ATP, and SHR+BBG+ANG II. In the gastrointestinal tract, we assessed gastric emptying (GE) and oxidative stress markers (NOx, MPO, GSH, SOD). We observed a decrease in the GE rate (P<0.05) in SHR vs control rats (21.8±2.0% vs 42.8±3.5%). The decrease in GE was returned (P<0.05) to control levels by BBG in SHR rats (21.8±2.0% vs 41.6±3.2%). Co-administration of ATP or ANG II together with BBG bypassed the effect of the P2X7 antagonist on GE in SHR (P<0.05) (21.9±5.0% vs 25.6±3.0% vs 41.6±3.2%). The MPO activity increased (P<0.05) in the gastric fundus of SHR compared to control rats (6.12±2.26 vs 0.077±0.02 UMPO/mg tissue); this effect was prevented (P<0.05) by BBG (0.55±0.15 vs 6.12±2.26 UMPO/mg tissue). Data demonstrated that blockage of P2X7 receptors with BBG can improve the GE delay and oxidative stress biomarkers in SHR animals. This preventive effect of BBG on GE delay was abrogated by ANG II and ATP, thus prompting crosstalk between renin-angiotensin and the purinergic signaling systems underlying this phenomenon.

P2X7 receptor antagonist improves gastrointestinal disorders in spontaneously hypertensive rats

The purinergic system participates in the control of blood pressure. Hypertension promotes the occurrence of gastrointestinal disorders such as intestinal inflammation and gastric emptying delay. This study aimed i) to investigate the participation of the P2X7 receptor blocker Brilliant Blue G (BBG) on gastric emptying of solids and changes in oxidative stress in the gastric fundus, duodenum, and colon of spontaneously hypertensive rats (SHR) and ii) to study the putative relationship of this effect with the renin-angiotensin system. Rats were divided into five groups: Control, SHR, SHR+BBG, SHR+BBG+ATP, and SHR+BBG+ANG II. In the gastrointestinal tract, we assessed gastric emptying (GE) and oxidative stress markers (NOx, MPO, GSH, SOD). We observed a decrease in the GE rate (P<0.05) in SHR vs control rats (21.8±2.0% vs 42.8±3.5%). The decrease in GE was returned (P<0.05) to control levels by BBG in SHR rats (21.8±2.0% vs 41.6±3.2%). Co-administration of ATP or ANG II together with BBG bypassed the effect of the P2X7 antagonist on GE in SHR (P<0.05) (21.9±5.0% vs 25.6±3.0% vs 41.6±3.2%). The MPO activity increased (P<0.05) in the gastric fundus of SHR compared to control rats (6.12±2.26 vs 0.077±0.02 UMPO/mg tissue); this effect was prevented (P<0.05) by BBG (0.55±0.15 vs 6.12±2.26 UMPO/mg tissue). Data demonstrated that blockage of P2X7 receptors with BBG can improve the GE delay and oxidative stress biomarkers in SHR animals. This preventive effect of BBG on GE delay was abrogated by ANG II and ATP, thus prompting crosstalk between renin-angiotensin and the purinergic signaling systems underlying this phenomenon.

A thermodynamic-based approach to model the entry into metabolic depression by mammals and birds.

For decades, there was an intense debate in relation to the mechanism behind the entry into metabolic depression (EMD) of mammals and birds. The fulcrum of the argument was whether the depression of metabolic rate ([Formula: see text]) was caused by the drop in body temperature, the so-called "Q10 effect", or whether it was caused by a metabolic downregulation. One present-day model of this process is a qualitative (textual) description: the initial step of EDM would be a downregulation in [Formula: see text] from the value maintaining euthermia at a given ambient temperature to the basal metabolic rate of the animal and, then, Q10 effect would take over and drop [Formula: see text] to its lower levels. Despite widely accepted, this qualitative description still misses a theoretical analysis. Here, we transpose the descriptive model to a formal quantitative one and analyze it under necessary thermodynamic conditions of a system. We, then, compare the results of the formal model to empirical data of EMD by mammals and birds. The comparisons indicate that the metabolic evolution in the course of the entry phase does not follow the descriptive model. Instead, as proposed by alternate models, EMD is a downregulated process as a whole until a new equilibrium Tb is attained.

New 1,3,4-oxadiazole compound with effective antibacterial and antibiofilm activity against Staphylococcus aureus.

Staphylococcus aureus is one of the main aetiological agents causing food-borne diseases. Some strains produce enterotoxins responsible for food poisoning. In addition, they can form biofilms on several surfaces such as plastics, glass and stainless steel making it difficult to eliminate them. The present study evaluated, for the first time, the antibacterial and antibiofilm activities of the synthetic compound LMM6 against S. aureus. The minimum inhibitory concentration was 0·97, 1·95 and 1·95 µg ml-1 against S. aureus ATCC 25923, S. aureus 629/94 and S. aureus FRI S-6, respectively. The time-kill curves showed that 96 h treatment with LMM6 reduced approximately 4 log CFU per ml at all tested concentrations. Furthermore, LMM6 reduced S. aureus preformed biofilm by approximately 1 log CFU per cm2 . During biofilm formation, a reduction of approximately 4 log CFU per cm2 was observed. LMM6 also reduced biofilm biomass during (~60%) and after biofilm formation (~25 to 45%), as shown by the crystal violet assay. Based on these results, we conclude that LMM6 exhibits antibacterial and antibiofilm activity and may be an innovative synthetic molecule for controlling S. aureus.

VIPergic neuroprotection in epileptogenesis: challenges and opportunities.

In mesial temporal lobe epilepsy (MTLE), seizures typically arise in the hippocampus or other mesial temporal lobe structures. The aetiology of MTLE epileptogenesis in still unknown, yet putative precipitating events such as trauma, complex febrile seizures, status epilepticus, inflammatory insults, or ischemia have been implicated. MTLE is commonly associated to a high degree of hippocampal sclerosis (HS) leading to frequent anti-epileptic drug refractoriness. Thus, the aim of recent therapeutic strategies has shifted from control of symptomatic seizures to putative prevention of epileptogenic processes. Vasoactive intestinal peptide (VIP) acts as a neurotransmitter, neurotrophic or neuroprotective factor in the central nervous system (CNS), also displaying anti-inflammatory and neurogenic actions. In the hippocampus, a brain area implicated in learning and memory, VIP released from basket cells and/or interneuron-selective interneurons controls GABAergic transmission and pyramidal cell activity influencing hippocampal-dependent synaptic plasticity (long-term potentiation and long-term depression) and cognition. VPAC1 receptor activation enhances hippocampal synaptic transmission by fostering disinhibition, while stimulation of VPAC2 receptors favours pyramidal cell excitability. Interestingly, VIP released from interneurons has potent anti-inflammatory actions, participates in the maintenance of the blood-brain barrier integrity, and strengthens neurogenesis. VPAC1 and VPAC2 receptors play differential roles in the regulation of the neuro-immune interactions. In this context, we gathered here the available information concerning the impact of VIP on neurotransmission and neuronal excitability in MTLE-HS and discuss the preventive use of selective VIP receptor ligands to abrogate epileptogenesis in MTLE-HS by controlling synaptic plasticity, neurogenesis and neuronal survival, neuroinflammation, and blood-brain barrier damage.

Antibacterial activity of Barbatimão (Stryphnodendron adstringens) against Staphylococcus aureus: in vitro and in silico studies.

We evaluated the activity of the aqueous fraction and the ethyl acetate fraction of Stryphnodendron adstringens against Staphylococcus aureus and proposed their mechanism of action. The antibacterial activity of S. adstringens fractions was evaluated against S. aureus and the cell targets were rated by docking. The fractions showed moderate antibacterial activity against S. aureus without toxicity on two mammalian cell lines. They also showed synergistic antibacterial activity with tannic acid (TA). In silico assays indicated FabG, FabZ and FabI as probable targets. The metabolic pathway for fatty acid biosynthesis in S. aureus was affected by components of S. adstringens. The synergistic effect when combining TA with S. adstringens fractions suggests a natural alternative to S. aureus control. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study describing the possible targets of action of Stryphnodendron adstringens on Staphylococcus aureus. Molecular dynamics simulations showed that the components of S. adstringens affected the metabolic pathway for fatty acid biosynthesis (FAS II) in S. aureus, inhibiting the FabI, FabG and FabZ enzymes. As tannic acid (TA) is a known inhibitor of some targets identified, we showed synergistic antibacterial activity of S. adstringens in combination with TA. This combination did not show toxicity against HaCaT and Vero cells and based on all these results we suggest that S. adstringens can be a natural and sustainable alternative to S. aureus control.

Activation of Prejunctional P2x2/3 Heterotrimers by ATP Enhances the Cholinergic Tone in Obstructed Human Urinary Bladders.

The objective of this study was to investigate the role of ATP in cholinergic neurotransmission in the urinary bladder of control men and of patients obstructed as a result of benign prostatic hyperplasia (BPH). Human detrusor samples were collected from 41 patients who submitted to transvesical prostatectomy resulting from BPH and 26 male organ donors. The release of [3H]acetylcholine ([3H]ACh) was evoked by electrical field stimulation (10 Hz, 200 pulses) in urothelium-denuded detrusor strips. Myographic recordings were performed to test detrusor strip sensitivity to ACh and ATP. Nerve-evoked [3H]ACh release was 1.5-fold higher in detrusor strips from BPH patients compared with controls. This difference was abolished after desensitization of ionotropic P2X1-3 receptors with an ATP analog, α,ß-methylene ATP (30 µM, applied for 15 minutes). TNP-ATP (10 nM, a preferential P2X2/3 antagonist) and A317491 (100 nM, a selective P2X3 antagonist) were about equipotent in decreasing nerve-evoked [3H]ACh release in control detrusor strips, but the selective P2X1 receptor antagonist NF023 (3 µM) was devoid of effect. The inhibitory effect of TNP-ATP (10 nM) increased from 27% ± 9% to 43% ± 6% in detrusor strips of BPH patients, but the effect of A317491 (100 nM) [3H]ACh release unaltered (20% ± 2% vs. 24% ± 4%). The amplitude of ACh (0.1-100 µM)-induced myographic recordings decreased, whereas sensitivity to ATP (0.01-3 mM) increased in detrusor strips from BPH patients. Besides the well characterized P2X1 receptor-mediated contractile activity of ATP in pathologic human bladders, we show here for the first time that cholinergic hyperactivity in the detrusor of BPH patients is facilitated by activation of ATP-sensitive P2X2/3 heterotrimers. SIGNIFICANCE STATEMENT: Bladder outlet obstruction often leads to detrusor overactivity and reduced bladder compliance in parallel to atropine-resistant increased purinergic tone. Our data show that P2X1 purinoceptors are overexpressed in the detrusor of patients with benign prostatic hyperplasia. Besides the P2X1 receptor-mediated detrusor contractions, ATP favors nerve-evoked acetylcholine release via the activation of prejunctional P2X2/3 excitatory receptors in these patients Thus, our hypothesis is that manipulation of the purinergic tone may be therapeutically useful to counteract cholinergic overstimulation in obstructed patients.

Photodynamic inactivation of foodborne bacteria by eosin Y.

AIMS: The aim of this study was evaluate the effect of photodynamic inactivation mediated by eosin Y in Salmonella enterica serotype Typhimurium ATCC 14028, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923 and Bacillus cereus ATCC 11778. METHODS AND RESULTS: Bacteria (107 CFU per ml) were incubated with eosin Y at concentrations ranging from 0·1 to 10 µmol l-1 , irradiated by green LED (λmax 490-570 nm) for 5, 10 and 15 min and the cellular viability was determined. Pseudomonas aeruginosa was completely inactivated when treated with 10 µmol l-1 eosin Y for 10 min. Treatments reduced B. cereus and Salm. Typhimurium counts to 2·7 log CFU per ml and 1·7 log CFU per ml, respectively. Escherichia coli counts were slightly reduced. Staphylococcus aureus presented the highest sensitivity, being completely inactivated by eosin Y at 5 µmol l-1 and 5 min of illumination. The reduction of cellular viability of photoinactivated Staph. aureus was also demonstrated by flow cytometry and morphological changes were observed by scanning electron microscopy. CONCLUSIONS: Eosin Y in combination with LED produced bacterial inactivation, being a potential candidate for photodynamic inactivation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study evidenced the efficacy of photodynamic inactivation as a novel and promising alternative to bacterial control.

Sialolitíase em equino: relato de caso / Sialolithiasis in equine: case report

Sialolitíase é uma afecção que afeta as glândulas salivares ou seus ductos, caracterizada pela presença de estruturas calcificadas, denominadas de sialolitos, com crescimento lento e gradual, geralmente assintomático, dificultando ou impedindo o fluxo normal de saliva. Devido à ausência de relatos na literatura nacional, descreve-se o caso de uma égua de 15 anos, que apresentava um sialolito de 13cm no ducto parotídico havia mais de dois anos, próximo à crista facial. O diagnóstico foi realizado por meio do exame clínico: visualização do aumento de volume, palpação do sialolito, avaliação odontológica; e de exames complementares: radiografia e ultrassonografia. Optou-se pelo tratamento cirúrgico, através do acesso percutâneo, pois é o mais indicado para cálculos grandes, realizando-se sutura do ducto de Stenon, sem presença de fístulas no pós-operatório. Foi de extrema importância a avaliação e os cuidados odontológicos durante a realização do procedimento, pois as pontas dentárias facilitam a formação dos cálculos.(AU)

Sialolithiasis is a condition that affects the salivary glands or their ducts, characterized by the presence of calcified structures, called sialolites, with slow and gradual growth, usually asymptomatic, hindering or impeding the normal flow of saliva. Due to the absence of reports in the national literature, the case of a 15-year-old mare who had a 13cm sialolite in the parotid duct near the face ridge for more than 2 years is described. The diagnosis was made through clinical examination: with visualization and palpation of the sialolite, dental evaluation; and complementary exams: radiography and ultrasonography. We chose surgical treatment through percutaneous access, which is the most appropriate for large stones, and Stenon's duct suture was performed, without postoperative fistulas. The assessment and dental care during the procedure was extremely important, since the dental tips facilitate the formation of the stones.(AU)

A moderate metal-binding hydrazone meets the criteria for a bioinorganic approach towards Parkinson's disease: Therapeutic potential, blood-brain barrier crossing evaluation and preliminary toxicological studies.

Alzheimer's and Parkinson's diseases share similar amyloidogenic mechanisms, in which metal ions might play an important role. In this last neuropathy, misfolding and aggregation of α-synuclein (α-Syn) are crucial pathological events. A moderate metal-binding compound, namely, 8-hydroxyquinoline-2-carboxaldehyde isonicotinoyl hydrazone (INHHQ), which was previously reported as a potential 'Metal-Protein Attenuating Compound' for Alzheimer's treatment, is well-tolerated by healthy Wistar rats and does not alter their major organ weights, as well as the tissues' reduced glutathione and biometal levels, at a concentration of 200mgkg-1. INHHQ definitively crosses the blood-brain barrier and can be detected in the brain of rats so late as 24h after intraperitoneal administration. After 48h, brain clearance is complete. INHHQ is able to disrupt, in vitro, anomalous copper-α-Syn interactions, through a mechanism probably involving metal ions sequestering. This compound is non-toxic to H4 (human neuroglioma) cells and partially inhibits intracellular α-Syn oligomerization. INHHQ, thus, shows definite potential as a therapeutic agent against Parkinson's as well.

Pyrazinamide susceptibility testing in Mycobacterium tuberculosis using the fast resazurin microtiter assay plate.

SETTING: Department of Clinical Analysis and Biomedicine, State University of Maringa, Maringa, PR, Brazil. OBJECTIVE: To evaluate the performance of the resazurin microtiter assay (REMA) plate at pH 5.5 in detecting Mycobacterium tuberculosis susceptibility to pyrazinamide (PZA). DESIGN: The minimal inhibitory concentration (MIC) of PZA in M. tuberculosis H37Rv and M. bovis AN5 reference strains and in 34 clinical M. tuberculosis isolates (26 PZA-susceptible and eight PZA-resistant) was determined using REMA at pH 5.5 and compared to REMA at pH 6.0. RESULTS: REMA at pH 5.5 was helpful in discriminating PZA-susceptible from resistant M. tuberculosis isolates when â©¿50 µg/ml PZA was considered as the cut-off for PZA susceptibility. Furthermore, it provided results in 8 days. However, two PZA-resistant isolates failed to grow at pH 5.5. CONCLUSION: As the REMA method is rapid, inexpensive, easy to perform and read, it would be of great usefulness in low-income countries for detecting PZA-resistant M. tuberculosis. REMA at pH 5.6-5.9 should be evaluated on an extended panel of clinical M. tuberculosis isolates with a greater range of MIC values in different laboratories for a better understanding of its utility in differentiating PZA-resistant from PZA-susceptible isolates.

Mesenchymal Stem Cells Ageing: Targeting the "Purinome" to Promote Osteogenic Differentiation and Bone Repair.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming cells. Such ability is compromised in elderly individuals resulting in bone disorders such as osteoporosis, also limiting their clinical usage for cell transplantation and bone tissue engineering strategies. In bone marrow niches, adenine and uracil nucleotides are important local regulators of osteogenic differentiation of MSCs. Nucleotides can be released to the extracellular milieu under both physiological and pathological conditions via (1) membrane cell damage, (2) vesicle exocytosis, (3) ATP-binding cassette transporters, and/or (4) facilitated diffusion through maxi-anion channels, hemichannels or ligand-gated receptor pores. Nucleotides and their derivatives act via adenosine P1 (A1 , A2A , A2B , and A3 ) and nucleotide-sensitive P2 purinoceptors comprising ionotropic P2X and G-protein-coupled P2Y receptors. Purinoceptors activation is terminated by membrane-bound ecto-nucleotidases and other ecto-phosphatases, which rapidly hydrolyse extracellular nucleotides to their respective nucleoside 5'-di- and mono-phosphates, nucleosides and free phosphates, or pyrophosphates. Current knowledge suggests that different players of the "purinome" cascade, namely nucleotide release sites, ecto-nucleotidases and purinoceptors, orchestrate to fine-tuning regulate the activity of MSCs in the bone microenvironment. Increasing studies, using osteoprogenitor cell lines, animal models and, more recently, non-modified MSCs from postmenopausal women, raised the possibility to target chief components of the purinergic signaling pathway to regenerate the ability of aged MSCs to differentiate into functional osteoblasts. This review summarizes the main findings of those studies, prompting for novel therapeutic strategies to control ageing disorders where bone destruction exceeds bone formation, like osteoporosis, rheumatoid arthritis, and fracture mal-union. J. Cell. Physiol. 231: 1852-1861, 2016. © 2016 Wiley Periodicals, Inc.

Inverse gradient of nitrergic and purinergic inhibitory cotransmission in the mouse colon.

AIM: Gastrointestinal smooth muscle relaxation is accomplished by the neural corelease of ATP or a related purine and nitric oxide. Contractions are triggered by acetylcholine and tachykinins. The aim of this work was to study whether regional differences in neurotransmission could partially explain the varied physiological roles of each colonic area. METHODS: We used electrophysiological and myography techniques to evaluate purinergic (L-NNA 1 mm incubated tissue), nitrergic (MRS2500 0.3 µm incubated tissue) and cholinergic neurotransmission (L-NNA 1 mm and MRS2500 0.3 µm incubated tissue) in the proximal, mid and distal colon of CD1 mice (n = 42). RESULTS: Purinergic electrophysiological responses elicited by single pulses (28 V) were greater in the distal (IJPfMAX = -35.3 ± 2.2 mV), followed by the mid (IJPfMAX = -30.6 ± 1.0 mV) and proximal (IJPfMAX = -11.7 ± 1.1 mV) colon. In contrast, nitrergic responses decreased from the proximal colon (IJPsMAX = -11.4 ± 1.1 mV) to the mid (IJPsMAX = -9.1 ± 0.4 mV), followed by the distal colon (IJPsMAX = -1.8 ± 0.3 mV). A similar rank of order was observed in neural mediated inhibitory mechanical responses including electrical field stimulation-mediated responses and neural tone. ADPßs concentration-response curve was shifted to the left in the distal colon. In contrast, NaNP responses did not differ between regions. Cholinergic neurotransmission elicited contractions of a similar amplitude throughout the colon. CONCLUSION: An inverse gradient of purinergic and nitrergic neurotransmission exists through the mouse colon. The proximal and mid colon have a predominant nitrergic neurotransmission probably due to the fact that their storage function requires sustained relaxations. The distal colon, in contrast, has mainly purinergic neurotransmission responsible for the phasic relaxations needed to propel dehydrated faeces.

Impairment of ATP hydrolysis decreases adenosine A1 receptor tonus favoring cholinergic nerve hyperactivity in the obstructed human urinary bladder.

This study was designed to investigate whether reduced adenosine formation linked to deficits in extracellular ATP hydrolysis by NTPDases contributes to detrusor neuromodulatory changes associated with bladder outlet obstruction in men with benign prostatic hyperplasia (BPH). The kinetics of ATP catabolism and adenosine formation as well as the role of P1 receptor agonists on muscle tension and nerve-evoked [(3)H]ACh release were evaluated in mucosal-denuded detrusor strips from BPH patients (n = 31) and control organ donors (n = 23). The neurogenic release of ATP and [(3)H]ACh was higher (P < 0.05) in detrusor strips from BPH patients. The extracellular hydrolysis of ATP and, subsequent, adenosine formation was slower (t (1/2) 73 vs. 36 min, P < 0.05) in BPH detrusor strips. The A(1) receptor-mediated inhibition of evoked [(3)H]ACh release by adenosine (100 µM), NECA (1 µM), and R-PIA (0.3 µM) was enhanced in BPH bladders. Relaxation of detrusor contractions induced by acetylcholine required 30-fold higher concentrations of adenosine. Despite VAChT-positive cholinergic nerves exhibiting higher A(1) immunoreactivity in BPH bladders, the endogenous adenosine tonus revealed by adenosine deaminase is missing. Restoration of A1 inhibition was achieved by favoring (1) ATP hydrolysis with apyrase (2 U mL(-1)) or (2) extracellular adenosine accumulation with dipyridamole or EHNA, as these drugs inhibit adenosine uptake and deamination, respectively. In conclusion, reduced ATP hydrolysis leads to deficient adenosine formation and A(1) receptor-mediated inhibition of cholinergic nerve activity in the obstructed human bladder. Thus, we propose that pharmacological manipulation of endogenous adenosine levels and/or A(1) receptor activation might be useful to control bladder overactivity in BPH patients.

P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 µM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 µM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 µM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 µM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability.

Amplification of neuromuscular transmission by methylprednisolone involves activation of presynaptic facilitatory adenosine A2A receptors and redistribution of synaptic vesicles.

The mechanisms underlying improvement of neuromuscular transmission deficits by glucocorticoids are still a matter of debate despite these compounds have been used for decades in the treatment of autoimmune myasthenic syndromes. Besides their immunosuppressive action, corticosteroids may directly facilitate transmitter release during high-frequency motor nerve activity. This effect coincides with the predominant adenosine A2A receptor tonus, which coordinates the interplay with other receptors (e.g. muscarinic) on motor nerve endings to sustain acetylcholine (ACh) release that is required to overcome tetanic neuromuscular depression in myasthenics. Using myographic recordings, measurements of evoked [(3)H]ACh release and real-time video microscopy with the FM4-64 fluorescent dye, results show that tonic activation of facilitatory A2A receptors by endogenous adenosine accumulated during 50 Hz bursts delivered to the rat phrenic nerve is essential for methylprednisolone (0.3 mM)-induced transmitter release facilitation, because its effect was prevented by the A2A receptor antagonist, ZM 241385 (10 nM). Concurrent activation of the positive feedback loop operated by pirenzepine-sensitive muscarinic M1 autoreceptors may also play a role, whereas the corticosteroid action is restrained by the activation of co-expressed inhibitory M2 and A1 receptors blocked by methoctramine (0.1 µM) and DPCPX (2.5 nM), respectively. Inhibition of FM4-64 loading (endocytosis) by methylprednisolone following a brief tetanic stimulus (50 Hz for 5 s) suggests that it may negatively modulate synaptic vesicle turnover, thus increasing the release probability of newly recycled vesicles. Interestingly, bulk endocytosis was rehabilitated when methylprednisolone was co-applied with ZM241385. Data suggest that amplification of neuromuscular transmission by methylprednisolone may involve activation of presynaptic facilitatory adenosine A2A receptors by endogenous adenosine leading to synaptic vesicle redistribution.

Infusion of Life: patient perceptions of expressive therapy during chemotherapy sessions.

Through a quanti-qualitative study, we observed the effects of group expressive therapy (ET) sessions on patients' feelings and sense of well-being, as part of the Infusion of Life project. This project is part of a broader programme to improve integral care, developed by an interdisciplinary team headed by a medical doctor who is also an artist and expert in ET. We offered 48 group ET sessions to a total of 253 outpatients with cancer or autoimmune disorders receiving venous infusions in the chemotherapy room of University Hospital Antonio Pedro, Rio de Janeiro, Brazil. The qualitative analysis showed that the programme was a pleasant way to spend time, revived their sense of humour, relieved symptoms, provided meaningful experiences, improved their relationships with staff, enabled expression of their feelings, stimulated them to be creative, improved coping resources and reorganisation of the psyche, and renewed their perspective on life. Family and spirituality were major sources of support. Expressive therapy was shown to be flexible and applicable in small spaces, using recycled materials, even with patients with restrained movements; it can also offer great benefits with relatively small investments if a qualified team is in charge of planning, executing, and auditing the work.

Role of ecto-NTPDases on UDP-sensitive P2Y(6) receptor activation during osteogenic differentiation of primary bone marrow stromal cells from postmenopausal women.

This study aimed at investigating the expression and function of uracil nucleotide-sensitive receptors (P2Y(2), P2Y(4), and P2Y(6)) on osteogenic differentiation of human bone marrow stromal cells (BMSCs) in culture. Bone marrow specimens were obtained from postmenopausal female patients (68 ± 5 years old, n = 18) undergoing total hip arthroplasty. UTP and UDP (100 µM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration-dependently increased [Ca(2+)](i) in BMSCs; their effects became less evident with time (7 > 21 days) of the cells in culture. Selective activation of P2Y(6) receptors with the stable UDP analog, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPγS was devoid of effect. Selective blockade of P2Y(6) receptors with MRS 2578 prevented [Ca(2+)](i) rises and osteogenic differentiation caused by UDP at all culture time points. BMSCs are immunoreactive against P2Y(2), P2Y(4), and P2Y(6) receptors. While the expression of P2Y(6) receptors remained fairly constant (7∼21 days), P2Y(2) and P2Y(4) became evident only in less proliferative and more differentiated cultures (7 < 21 days). The rate of extracellular UTP and UDP inactivation was higher in less proliferative and more differentiated cell populations. Immunoreactivity against NTPDase1, -2, and -3 rises as cells differentiate (7 < 21 days). Data show that uracil nucleotides are important regulators of osteogenic cells differentiation predominantly through the activation of UDP-sensitive P2Y(6) receptors coupled to increases in [Ca(2+)](i) . Endogenous actions of uracil nucleotides may be balanced through specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation.

Presynaptic muscarinic and adenosine receptors are involved in 2 Hz-induced train-of-four fade caused by antinicotinic neuromuscular relaxants in the rat.

1. Train-of-four fade (TOF(fade) ) is a clinically useful parameter to monitor the degree of block of neuromuscular transmission in curarized patients. Experimentally, TOF(fade) has been attributed to the blockade of facilitatory nicotinic receptors on motor nerve terminals. There is less information regarding the involvement of coexistent presynaptic receptors (e.g. muscarinic M(1) and M(2) , adenosine A(1) and A(2A) ) in the TOF(fade) produced by antinicotinic agents. 2. In the present study, we evaluated the TOF(fade) caused by antinicotinic neuromuscular relaxants (hexamethonium, d-tubocurarine, vecuronium and rocuronium) as the ratio of the muscle tension produced in the rat diaphragm by the fourth to the first stimulus (T(4) /T(1) ) of a train-of-four stimuli delivered to the phrenic nerve trunk at a frequency of 2 Hz. 3. All antinicotinic agents, except hexamethonium, decreased the amplitude of muscle tension during the first stimulus. Hexamethonium, (5.47 mmol/L), d-tubocurarine- (1.1 µmol/L), vecuronium (4.7 µmol/L)- and rocuronium (9.8 µmol/L)-induced TOF(fade) was attenuated by 10 nmol/L pirenzepine (an M(1) receptor antagonist), 1 µmol/L methoctramine (an M(2) receptor antagonist) and 2.5 nmol/L 1,3-dipropyl-8-cyclopentylxanthine (an A(1) receptor antagonist). Blockade of the A(2A) receptor with 10 nmol/L ZM241385 partially reversed the TOF(fade) induced by d-tubocurarine, vecuronium and rocuronium, but not that caused by the 'pure' neuronal nicotinic receptor antagonist hexamethonium, unless one increased the concentration of ZM241385 to 50 nmol/L. 4. The data indicate that presynaptic M(1) , M(2) , A(1) and A(2A) receptors play a role in neuromuscular TOF(fade) caused by antinicotinic neuromuscular relaxants. Such interplay depends on adenosine tonus and on the affinity of neuromuscular blocking agents for neuronal versus muscular nicotinic receptors.