Intraspecific tolerance of Metarhizium anisopliae conidia to the upper thermal limits of summer with a description of a quantitative assay system

Conidial tolerance to the upper thermal limits of summer is important for fungal biocontrol agents, whose conidia are formulated into mycoinsecticides for field application. To develop an efficient assay system, aerial conidia of eight Metarhizium anisopliae, four M. anisopliae var. anisopliae, and six M. anisopliae var. acridum isolates with different host and geographic origins were wet-stressed for ≤180 min at 48 °C or incubated for 14 d colony growths at 10-35 °C. The survival ratios (relative to unstressed conidia) of each isolate, examined at 15-min intervals, fit a logistic equation (r² ≥ 0.975), yielding median lethal times (LT₅₀s) of 14.3-150.3 min for the 18 isolates stressed at 48 °C. Seven grasshopper isolates from Africa had a mean LT₅₀ of 110 (73-150) min, but could not grow at 10 or 15 °C. The mean LT₅₀ of five non-grasshopper isolates capable of growing at 10-35 °C was 16 (10-26) min only. Three isolates with typically low (type I), medium (type II), and high (type III) levels of tolerance to 48 °C were further assayed for ≤4-d tolerance of their conidia to the wet stress at 38, 40, 42, or 45 °C. The resultant LT₅₀s decreased to 20, 53 and 167 min at 48 °C from 507, 1612, and 8256 min at 38 °C for types I, II and III, respectively. For the distinguished types, the logarithms of the LT₅₀s were significantly correlated to the temperatures of 38-48 °C with an inverse linearity (r² ≥ 0.88). The method developed to assay quantitatively fungal thermotolerance would be useful for screening of fungal candidates for improved pest control in summer.     

Significant relationships among frost tolerance and net photosynthetic rate,water use efficiency and dehydrin accumulation in cold-treated winter oilseed rapes

Five winter oilseed rape cultivars (Benefit, Californium, Cortes, Ladoga, Navajo) were subjected to 30 days of cold treatment (4 °C) to examine the effect of cold on acquired frost tolerance (FT), dehydrin (DHN) content, and photosynthesis-related parameters. The main aim of this study was to determine whether there are relationships between FT (expressed as LT₅₀ values) and the other parameters measured in the cultivars. While the cultivar Benefit accumulated two types of DHNs (D45 and D35), the other cultivars accumulated three additional DHNs (D97, D47, and D37). The similar-sized DHNs (D45 and D47) were the most abundant; the others exhibited significantly lower accumulations. The highest correlations were detected between LT₅₀ and DHN accumulation (r = −0.815), intrinsic water use efficiency (WUEi; r = −0.643), net photosynthetic rate (r = −0.628), stomatal conductance (r = 0.511), and intracellular/intercellular CO₂ concentration (r = 0.505). Those cultivars that exhibited higher Pn rate in cold (and further a significant increase in WUEi) had higher levels of DHNs and also higher FT. No significant correlation was observed between LT₅₀ and E, PRI, or NDVI. Overall, we have shown the selected physiological parameters to be able to distinguish different FT cultivars of winter oilseed rape.     

Effect of starvation upon baculovirus replication in larval Bombyxmori and Heliothisvirescens

The progression of baculovirus (BmNPV, BmCysPD, AcMNPV or AcAaIT) infection in larval Bombyx mori and Heliothis virescens (1st, 3rd or 5th instar) was investigated following various starvation regimes. When the larvae were starved for 12 or 24 h immediately following inoculation, the median lethal time to death (LT₅₀) was delayed by 9.5-19.2 h in comparison to non-starved controls. This corresponded to a delay of 10-23% depending upon the larval stage and virus that was used for inoculation. When a 24 h-long starvation period was initiated at 1 or 2 days post inoculation (p.i.), a statistically significant difference in LT₅₀ was not found indicating that the early stages of infection are more sensitive to the effects of starvation. Viral titers in the hemolymph of 5th instar B. mori that were starved for 24 h immediately following inoculation were 10-fold lower (p < 0.01) than that found in non-starved control larvae. Histochemical analyses indicated that virus transmission was reduced in 5th instar B. mori that were starved for 24 h immediately following inoculation in comparison to non-starved control larvae. In general, the mass of larvae that were starved immediately after inoculation was 30% lower than that of non-starved control insects. Our findings indicate that starvation of the larval host at the time of baculovirus exposure has a negative effect on the rate baculovirus transmission and pathogenesis.     

Cold tolerance of the overwintering larval instars of light brown apple moth Epiphyas postvittana

The light brown apple moth, Epiphyas postvittana, a leafroller native to southeastern Australia was discovered in California in 2006. The highly polyphagous nature of this pest adds to the importance of being able to predict the potential distribution of this invader across the North American continent. The spread of ectothermic species that lack winter diapause, such as E. postvittana, can be limited by their ability to tolerate cold temperature extremes. In this study we examined the cold hardiness of 4th to 6th instar E. postvittana, the only life stages known to overwinter in California, through a combination of supercooling point (SCP) and mortality at low temperatures. Our results showed that the mean SCP for E. postvittana ranged from −14.1 °C for 6th instars to −16.0 °C for 4th instars. Lethal time leading to 50% mortality (LT₅₀) for the three instars combined were 2.5 h at −10.5 °C, 41 h at −6.5 °C and 198 h at −0.9 °C. At 3 °C, the LT₅₀ of 4th instars was significantly lower at 775 h than that for 5th and 6th instars combined at 1029 h. The cold hardiness characteristics of later-instar E. postvittana larvae were comparable to those of pink bollworm, Pectinophora gossypiella, a diapausing invasive with a geographic distribution restricted to southern California. Slightly greater cold hardiness is shown by the indigenous non-diapausing leafroller Argyrotaenia franciscana, which is restricted to the Pacific Coast of North America. We therefore conclude that the moderate cold hardiness of E. postvittana will substantially limit its spread into northern temperate regions of North America.     

Interaction of metal nanoparticles with recombinant arginine kinase from Trypanosoma brucei: Thermodynamic and spectrofluorimetric evaluation

Background

Trypanosoma brucei, responsible for African sleeping sickness, is a lethal parasite against which there is need for new drug protocols. It is therefore relevant to attack possible biomedical targets with specific preparations and since arginine kinase does not occur in humans but is present in the parasite it becomes a suitable target.

Methods

Fluorescence quenching, thermodynamic analysis and FRET have shown that arginine kinase from T. brucei interacted with silver or gold nanoparticles.

Results

The enzyme only had one binding site. At 25 °C the dissociation (K_d) and Stern–Volmer constants (K_SV) were 15.2 nM, 0.058 nM^− 1 [Ag]; and 43.5 nM, 0.052 nM^− 1 [Au] and these decreased to 11.2 nM, 0.041 nM^− 1 [Ag]; and 24.2 nM, 0.039 nM^− 1 [Au] at 30 °C illustrating static quenching and the formation of a non-fluorescent fluorophore–nanoparticle complex. Silver nanoparticles bound to arginine kinase with greater affinity, enhanced fluorescence quenching and easier access to tryptophan molecules than gold. Negative ΔH and ΔG values implied that the interaction of both Ag and Au nanoparticles with arginine kinase was spontaneous with electrostatic forces. FRET confirmed that the nanoparticles were bound 2.11 nm [Ag] and 2.26 nm [Au] from a single surface tryptophan residue.

Conclusions

The nanoparticles bind close to the arginine substrate through a cysteine residue that controls the electrophilic and nucleophilic characters of the substrate arginine–guanidinium group crucial for enzymatic phosphoryl transfer between ADP and ATP.

General significance

The nanoparticles of silver and gold interact with arginine kinase from T. brucei and may prove to have far reaching consequences in clinical trials.     

ATP binding and hydrolysis steps of the uni-site catalysis by the mitochondrial F1-ATPase are affected by inorganic phosphate

The effect of inorganic phosphate (P_i) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F₁-ATPase from beef heart mitochondria (ndMF₁) has been investigated. It is shown for the first time that P_i decreases the apparent rate constant of uni-site ATP binding by ndMF₁ 3-fold with the K_d of 0.38 ± 0.14 mM. During uni-site ATP hydrolysis, P_i also shifts equilibrium between bound ATP and ADP + P_i in the direction of ATP synthesis with the K_d of 0.17 ± 0.03 mM. However, 10 mM P_i does not significantly affect ATP binding during multi-site catalysis.     

Near anoxia and sulfide as possible factors influencing the spatial distribution of Acartia tonsa and Acartia clausi: Comparative evaluation of egg tolerance

In many coastal and estuarine areas the planktonic copepods Acartia tonsa and Acartia clausi show a spatial separation with A. tonsa restricted to brackish waters and confined environments and A. clausi inhabiting areas more influenced by sea water. The hatching and viability of A. tonsa and A. clausi eggs exposed to anoxia and anoxia/sulfide (conditions that are frequent in bottom waters of the most confined areas) was evaluated to determine if these stress factors play a role in the distribution of these species. Subitaneous eggs, spawned by laboratory reared organisms, were incubated in near anoxia (< 7.59 × 10^− 3 mmol O₂ L^− 1) or anoxia/sulfide (∼ 1 mmol L^− 1) for different periods (1, 4, 8 and 15 days), then transferred to normoxic conditions. The exposure of the eggs to near anoxia and sulfide appears to induce the same response (quiescence) in both species. Exposure times ≤ 8 days to near anoxia or anoxia/sulfide did not affect egg viability, while 15 day exposure caused significant declines in hatching success of both species. A significant difference between the effects of near anoxia and anoxia/sulfide was observed when incubation lasted 15 days; hatching of eggs exposed to sulfide being higher than that of eggs exposed to near anoxia for both species. No significant differences were observed between the two species in hatching success of eggs exposed to both near anoxia and anoxia/sulfide (with the exception of eggs incubated in near anoxia for 4 days). The results indicate that the impact of anoxia and sulfide on the eggs of the two Acartia species cannot be a factor explaining the spatial distribution in coastal and brackish environments of these copepods.Feeding experiments on A. clausi were also performed. Suitability of different algal species to rear this copepod was evaluated and the results were compared with data previously obtained for A. tonsa. Differences in feeding needs between A. clausi and A. tonsa are discussed.     

Phagocytic activity,respiratory burst,cytoplasmic free-Ca concentration and apoptotic cell ratio of haemocytes from the black tiger shrimp, Penaeus monodon under acute copper stress

The aim of this study was to investigate the cellular toxicity of copper-induced injury to the black tiger shrimp Penaeus monodon. The 24 h, 48 h, 72 h and 96 h LC₅₀ (median lethal concentration) of Cu²⁺ on P. monodon (11.63 ± 1.14 g) were found to be 3.49, 1.54, 0.73 and 0.40 mg L^− 1, respectively. Total haemocyte count (THC), phagocytic activity, respiratory burst (RB), cytoplasmic free-Ca²⁺ (cf-Ca²⁺) concentration and apoptotic cell ratio of shrimp were determined after exposure to different concentrations of Cu²⁺ (0, 0.05, 0.5, 1.5 and 3.5 mg L^− 1) for 0, 6, 12, 24 and 48 h. There was no significant effect on the analytic indicator of shrimp exposed to 0.05 mg L^− 1 Cu²⁺. THC decreased after Cu-exposure to 0.5 mg L^− 1 for 48 h, 1.5 mg L^− 1 for 24 h and 3.5 mg L^− 1 for 12 h. Phagocytic activity decreased in P. monodon following 48 h exposure to 3.5 mg L^− 1 Cu²⁺. RB was induced after 6 h exposure to 0.5, 1.5 and 3.5 mg L^− 1 Cu²⁺. cf-Ca²⁺ concentration increased after 48 h exposure to 0.5 mg L^− 1 Cu²⁺, and 12 h exposure to 1.5 and 3.5 mg L^− 1 Cu²⁺. The percentage of apoptotic cells increased to 9.5%, 16.3% and 18.6% respectively following 48 h exposure to 0.5, 1.5 and 3.5 mg L^− 1 Cu²⁺. These results indicate that Cu can induce oxidative stress, elevation of cf-Ca²⁺ and cell apoptosis, and inhibit phagocytic activity in the shrimp P. monodon, and the lethal injury of Cu²⁺ to P. monodon may be mainly due to the sharp reduction of THC caused by ROS-induced apoptosis.     

Kinetics of an extracellular exo-inulinase production from a 5-flourocytosine resistant mutant of Geotrichum candidum using two-factorial design

In the present study, eight different strains of Geotrichum candidum were isolated and screened for an extracellular exo-inulinase production using chemically enriched sucrose–mineral media. The isolate (Zool-3i) with a better enzyme activity (1.38 IU/ml) was subjected to induced mutagenesis using methyl methane sulphonate (MMS) and a mutant with an enzyme activity of 32.06 IU/ml was obtained. Further exposure to ethyl methane sulphonate (EMS) and ultraviolet (UV) radiations yielded a mutant exhibiting an improved activity of 39.34 IU/ml. The potential mutant was cultured overnight and plated on 5fc–YPR agar medium and thus made resistant against 5-flourocytocine. Over 50-fold enhancement in enzyme production (71.85 IU/ml) was achieved when the process parameters including incubation period (48 h), sucrose concentration (5.0 g/L), pH (6.0), inoculum size (2.0%, 16 h old) and urea (0.2%) were identified using Plackett–Burman design. On the basis of kinetic variables, notably Q_p (0.723 U/g/h), Y_p/s (2.036 U/g) and q_p (0.091 U/g cells/h), the mutant MEU-5fc-6 was found to be a hyper producer of exo-inulinase (HS, LSD 0.045, p ? 0.05).     

Impaired cardiac mitochondrial function and contractile reserve following an acute exposure to environmental particulate matter

Background

It has been suggested that mitochondrial function plays a central role in cardiovascular diseases associated with particulate matter inhalation. The aim of this study was to evaluate this hypothesis, with focus on cardiac O₂ and energetic metabolism, and its impact over cardiac contractility.

Methods

Swiss mice were intranasally instilled with either residual oil fly ash (ROFA) (1.0 mg/kg body weight) or saline solution. After 1, 3 or 5 h of exposure, O₂ consumption was evaluated in heart tissue samples. Mitochondrial respiration, respiratory chain complexes activity, membrane potential and ATP content and production rate were assessed in isolated mitochondria. Cardiac contractile reserve was evaluated according to the Langendorff technique.

Results

Three hours after ROFA exposure, tissue O₂ consumption was significantly decreased by 35% (from 1180 ± 70 to 760 ± 60 ng-at O/min g tissue), as well as mitochondrial rest (state 4) and active (state 3) respiration, by 30 and 24%, respectively (control state 4: 88 ± 5 ng-at O/min mg protein; state 3: 240 ± 20 ng-at O/min mg protein). These findings were associated with decreased complex II activity, mitochondrial depolarization and deficient ATP production. Even though basal contractility was not modified (control: 75 ± 5 mm Hg), isolated perfused hearts failed to properly respond to isoproterenol in ROFA-exposed mice. Tissue O₂ consumption rates positively correlated with cardiac contractile state in controls (r² = 0.8271), but not in treated mice (r² = 0.1396).

General Significance

The present results show an impaired mitochondrial function associated with deficient cardiac contractility, which could represent an early cardiovascular alteration after the exposure to environmental particulate matter.     

Multigenerational analysis of temperature and salinity variability affects on metabolic rate,generation time,and acute thermal and salinity tolerance in Daphnia pulex

Climate change, sea level rise, and human freshwater demands are predicted to result in elevated temperature and salinity variability in upper estuarine ecosystems. Increasing levels of environmental stresses are known to induce the cellular stress response (CSR). Energy for the CSR may be provided by an elevated overall metabolic rate. However, if metabolic rate is constant or lower under elevated stress, energy for the CSR is taken from other physiological processes, such as growth or reproduction. This study investigated the examined energetic responses to the combination of temperature and salinity variability during a multigenerational exposure of partheogenetically reproducing Daphnia pulex. We raised D. pulex in an orthogonal combination of daily fluctuations in temperature (15, 15–25, 15–30 °C) and salinity (0, 0–2, 0–5). Initially metabolic rates were lower under all variable temperature and variable salinity treatments. By the 6th generation there was little metabolic variation among low and intermediate temperature and salinity treatments, but metabolic suppression persisted at the most extreme salinity. When grown in the control condition for the 6th generation, metabolic suppression was only observed in D. pulex from the most extreme condition (15–30 °C, 0–5 salinity). Generation time was influenced by acclimation temperature but not salinity and was quickest in specimens reared at 15–25 °C, likely due to Q₁₀ effects at temperatures closer to the optima for D. pulex, and slowest in specimens reared at 15–30 °C, which may have reflected elevated CSR. Acute tolerance to temperature (LT₅₀) and salinity (LC₅₀) were both highest in D. pulex acclimated to 15–30 °C and salinity 0. LT₅₀ and LC₅₀ increased with increasing salinity in specimens raised at 15 °C and 15–25 °C, but decreased with increasing salinity in specimens raised at 15–30 °C. Thus, increasing temperature confers cross-tolerance to salinity stress, but the directionality of synergistic effects of temperature and salinity depend on the degree of environmental variability. Overall, the results of our study suggest that temperature is a stronger determinant of metabolism, growth, and tolerance thresholds, and assessment of the ecological impacts of environmental change requires explicit information regarding the degree of environmental variability.     

Influence of abiotic factors on bacterial proliferation and anoxic survival of the sea mussel Mytilus edulis L.

The effect of several abiotic factors (salinity, temperature and pH) on bacterial proliferation and survival time of the sea mussel Mytilus edulis L. were studied under anoxic incubations. In addition, the presence in the incubation media of ammonium and the volatile fatty acids propionate and acetate, both excreted fermentation products of the bivalve, was tested.Anoxic incubations with seawater diluted with demineralised water showed at the lowest salinity (50% seawater, SW) a significant increase in the capacity of M. edulis to survive anoxia as compared to both 75% SW and control [100% SW, corresponding to 32 practical salinity units (psu)]. Formation of biotic sulphide and ammonium occurred in all incubations. However, bacterial proliferation was postponed by 2-3 days at lowest salinity and accordingly, concentrations of both compounds were lower. Anoxic survival profiles of mussels collected from different habitats in the Dutch Scheldt area, characterised by differences in salinity (range from 17 to 31 psu), corresponded with the above salinity effect. Walsoorden mussels (17 psu) showed the longest (P<0.001) survival time under anoxia (LT₅₀=17.2 days) as compared with Paulina (27 psu) and Wemeldinge (31 psu) mussels (LT₅₀=12.8 and 9.8 days, respectively). Condition index (ratio of soft body weight to shell volume) was not correlated with anoxic survival time in untreated mussels, although this was clearly the case when the antibiotic chloramphenicol was added to the anoxic seawater.Acidification of the anoxic incubation medium had a positive effect on survival time. LT₅₀ values significantly (P<0.001) increased from 10.2 days at pH 8.1 to 11.6 and 11.5 days at pH 7.3 and 6.5, respectively. Biotic sulphide and ammonium accumulation as well as bacterial numbers were significantly lower at pH 7.3 and 6.5 as compared with pH 8.1. Anoxic incubations at 10 °C (LT₅₀=12.0 days) strongly increased survival time as compared to 18 °C (LT₅₀=5.9 days). The benefit of antibiotic addition was also stronger at lower temperature (10 °C).Addition of both propionate and acetate (0.5 mM) displayed no effect on mortality of mussels under anoxia, but ammonium (0.5 mM) caused a negative effect (P<0.001). Biotic sulphide and ammonium concentrations measured in both volatile fatty acid incubations were lower than the control situation, as well as total bacterial numbers.This study shows that environmental factors play a significant role in determining the course of bacterial infection and death of bivalves exposed to anoxia.     

Tolerance of copepods to short-term thermal stress caused by coastal power stations

The tolerance of marine copepods to short-term thermal stress was measured by the median lethal temperature (LT₅₀) tests in laboratory. Experiments on LT₅₀ of copepods from different acclimation and acclimatization conditions collected from the Yueqing Bay were carried out under heat exposure for 15, 30 and 45 min. The LT₅₀ of copepods decreased with increasing exposure time but increased with rising acclimation and acclimatization temperatures. However, the differences in copepod LT₅₀ decreased with rising acclimatization temperatures, which suggested that entrained copepod mortality increased with raised water temperature due to the acute thermal stress of coastal power stations. Results also revealed that the thermal tolerance of Labidocera euchaeta was much higher than that of Calanus sinicus in spring. The thermal tolerances of different copepod species in summer were in the order, Pseudodiaptomus marinus, Acartia spinicauda, Acartia pacifica and L. euchaeta.     

Optimization of the production of poly-γ-glutamic acid by Bacillus amyloliquefaciens C1 in solid-state fermentation using dairy manure compost and monosodium glutamate production residues as basic substrates

Poly-γ-glutamic acid (γ-PGA) is a polymer with uses in foods, cosmetics, medicine and agriculture. The medium for the production of γ-PGA by Bacillusamyloliquefaciens C1 was optimized by response surface methodology using agro-industrial wastes in solid-state fermentation (SSF). The optimal SSF medium (20 g substrates with 50% initial moisture) for producing γ-PGA was determined to contain 5.51 g dairy manure compost, 1.91 g soybean cake, 0.57 g corn flour, 2.15 g monosodium glutamate production residues, 1.5 g wheat bran, 0.5 g rapeseed cake, 0.1 g citric acid, 0.05 g MgSO₄·7H₂O and 0.03 g MnSO₄·H₂O. In this medium the strain produced up to 0.0437 g γ-PGA per gram of substrates when cultured for 48 h at 37 °C. SDS-PAGE showed that the molecular weight of the γ-PGA was more than 130 kDa. Due to the high-yields observed and the low-cost nature of the optimal medium, this study indicates a possibility to establish economical large-scale production of γ-PGA.     

Direct conversion of inulin and extract of tubers of Jerusalem artichoke into single cell oil by co-cultures of Rhodotorula mucilaginosa TJY15a and immobilized inulinase-producing yeast cells

In this study, it was found that the immobilized inulinase-producing cells of Pichia guilliermondii M-30 could produce 169.3 U/ml of inulinase activity while the free cells of the same yeast strain only produced 124.3 U/ml of inulinase activity within 48 h. When the immobilized inulinase-producing yeast cells were co-cultivated with the free cells of Rhodotorula mucilaginosa TJY15a, R. mucilaginosa TJY15a could accumulate 53.2% oil from inulin in its cells and cell dry weight reached 12.2 g/l. Under the similar conditions, R. mucilaginosa TJY15a could accumulate 55.4% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 12.8 g/l within 48 h. When the co-cultures were grown in 2 l fermentor, R. mucilaginosa TJY15a could accumulate 56.6% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 19.6 g/l within 48 h. Over 90.0% of the fatty acids from the yeast strain TJY15a grown in the extract of Jerusalem artichoke tubers was C_16:0, C_18:1 and C_18:2, especially C_18:1 (50.6%).     

Stimulation of oxidative phosphorylation by calcium in cardiac mitochondria is not influenced by cAMP and PKA activity

Cardiac oxidative ATP generation is finely tuned to match several-fold increases in energy demand. Calcium has been proposed to play a role in the activation of ATP production via PKA phosphorylation in response to intramitochondrial cAMP generation. We evaluated the effect of cAMP, its membrane permeable analogs (dibutyryl-cAMP, 8-bromo-cAMP), and the PKA inhibitor H89 on respiration of isolated pig heart mitochondria. cAMP analogs did not stimulate State 3 respiration of Ca^2 +-depleted mitochondria (82.2 ± 3.6% of control), in contrast to the 2-fold activation induced by 0.95 μM free Ca^2 +, which was unaffected by H89. Using fluorescence and integrating sphere spectroscopy, we determined that Ca^2 + increased the reduction of NADH (8%), and of cytochromes b_H (3%), c₁ (3%), c (4%), and a (2%), together with a doubling of conductances for Complex I + III and Complex IV. None of these changes were induced by cAMP analogs nor abolished by H89. In Ca^2 +-undepleted mitochondria, we observed only slight changes in State 3 respiration rates upon addition of 50 μM cAMP (85 ± 9.9%), dibutyryl-cAMP (80.1 ± 5.2%), 8-bromo-cAMP (88.6 ± 3.3%), or 1 μM H89 (89.7 ± 19.9%) with respect to controls. Similar results were obtained when measuring respiration in heart homogenates. Addition of exogenous PKA with dibutyryl-cAMP or the constitutively active catalytic subunit of PKA to isolated mitochondria decreased State 3 respiration by only 5–15%. These functional studies suggest that alterations in mitochondrial cAMP and PKA activity do not contribute significantly to the acute Ca^2 + stimulation of oxidative phosphorylation.     

Functional effects of alcohol withdrawal syndrome on peripheral sympathetic neurotransmission in vas deferens of adult rats

Aims

Alcohol withdrawal syndrome (AWS) is characterized by a set of physiological modifications triggered by abrupt withdrawal and/or decreasing consumption of ethanol (EtOH), which may manifest 16–48 h after ceasing consumption. The relationship between the effects of AWS and central and peripheral sympathetic neurotransmission is unknown. This study investigates the possible mechanisms on the sympathetic system during periods of AWS.

Main methods

Male Wistar rats were treated with EtOH (6–10 g/kg/day/v.o. 5 days). Subsequently, 1 h, 24 h, 48 h and 120 h after administration of the last dose of EtOH, the animals were sacrificed, and their vas deferens (VD) were removed to perform the following evaluations: (a) concentration–effect curves of sympathetic agonist; (b) activity of α₂-adrenoreceptor; (c) function of voltage-dependent calcium channels (Cav); and (d) release of endogenous catecholamines measured in real time coupled to HPLC.

Key findings

The results showed that the maximum effects of contraction were increased by agonists tested in at 24 h and 48 h EtOH withdrawal. The inhibitory affinity (pIC₅₀) of guanfacine was decreased 24 h EtOH withdrawal. The function of Cav was also decreased as pIC₅₀ values dropped 24 h and 48 h EtOH withdrawal. The release of catecholamines increased 48 h after EtOH withdrawal. It is suggested that AWS triggers hyperactivity in peripheral sympathetic neurotransmission.

Significance

The mechanisms underlying hyperactivity are possibly explained by a failure of autoregulation from catecholamines released by α₂-adrenoreceptors and/or an increase of Cav function, which may be potential targets to attenuate the symptoms of AWS at the peripheral level.     

Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molybdate

Over the last few decades there has been increasing interest in oxometalate and polyoxometalate applications to medicine and pharmacology. This interest arose, at least in part, due to the properties of these classes of compounds as anti-cancer, anti-diabetic agents, and also for treatment of neurodegenerative diseases, among others. However, our understanding of the mechanism of action would be improved if biological models could be used to clarify potential toxicological effects in main cellular processes. Sarcoplasmic reticulum (SR) vesicles, containing a large amount of Ca^2 +-ATPase, an enzyme that accumulates calcium by active transport using ATP, have been suggested as a useful model to study the effects of oxometalates on calcium homeostasis. In the present article, it is shown that decavanadate, decaniobate, vanadate, tungstate and molybdate, all inhibited SR Ca²⁺-ATPase, with the following IC₅₀ values: 15, 35, 50, 400 μM and 45 mM, respectively. Decaniobate (Nb₁₀), is the strongest P-type enzyme inhibitor, after decavanadate (V₁₀). Atomic-absorption spectroscopy (AAS) analysis, indicates that decavanadate binds to the protein with a 1:1 decavanadate:Ca^2 +-ATPase stoichiometry. Furthermore, V₁₀ binds with similar extension to all the protein conformations, which occur during calcium translocation by active transport, namely E1, E1P, E2 and E2P, as analysed by AAS. In contrast, it was confirmed that the binding of monomeric vanadate (H₂VO₄^2 −; V₁₎ to the calcium pump is favoured only for the E2 and E2P conformations of the ATPase, whereas no significant amount of vanadate is bound to the E1 and E1P conformations. Scatchard plot analysis, confirmed a 1:1 ratio for decavanadate-Ca^2 +-ATPase, with a dissociation constant, k_d of 1 μM^− 1. The interaction of decavanadate V₁₀O₂₈^6 − (V₁₀) with Ca^2 +-ATPase is prevented by the isostructural and isoelectronic decaniobate Nb₁₀O₂₈^6 − (Nb₁₀), whereas no significant effects were detected with ATP or with heparin, a known competitive ATP binding molecule, suggesting that V₁₀ binds non-competitively, with respect to ATP, to the protein. Finally, it was shown that decaniobate inhibits SR Ca^2 +-ATPase activity in a non competitive type of inhibition, with respect to ATP. Taken together, these data demonstrate that decameric niobate and vanadate species are stronger inhibitors of the SR calcium ATPase than simple monomeric vanadate, tungstate and molybdate oxometalates, thus affecting calcium homeostasis, cell signalling and cell bioenergetics, as well many other cellular processes. The ability of these oxometalates to act either as phosphate analogues, as a transition-state analogue in enzyme-catalysed phosphoryl group transfer processes and as potentially nucleotide-dependent enzymes modulators or inhibitors, suggests that different oxometalates may reveal different mechanistic preferences in these classes of enzymes.