Sodium-dependent copper uptake across epithelia: a review of rationale with experimental evidence from gill and intestine

The paper reviews the evidence for apparent sodium-dependent copper (Cu) uptake across epithelia such as frog skin, fish gills and vertebrate intestine. Potential interactions between Na(+) and Cu during transfer through epithelial cells is rationalized into the major steps of solute transfer: (i) adsorption on to the apical/mucosal membrane, (ii) import in to the cell (iii) intracellular trafficking, and (iv) export from the cell to the blood. Interactions between Na(+) and Cu transport are most likely during steps (i) and (ii). These ions have similar mobilities (lambda) in solution (lambda, Na(+), 50.1; Cu(2+), 53.6 cm(2) Int. ohms(-1) equiv(-1)); consequently, Cu(2+) may compete equally with Na(+) for diffusion to membrane surfaces. We present new data on the Na(+) binding characteristics of the gill surface (gill microenvironment) of rainbow trout. The binding characteristics of Na(+) and Cu(2+) to the external surface of trout gills are similar with saturation of ligands at nanomolar concentrations of solutes. At the mucosal/apical membrane of several epithelia (fish gills, frog skin, vertebrate intestine), there is evidence for both a Cu-specific channel (CTR1 homologues) and Cu leak through epithelial Na(+) channels (ENaC). Cu(2+) slows the amiloride-sensitive short circuit current (I(sc)) in frog skin, suggesting Cu(2+) binding to the amiloride-binding site of ENaC. We present examples of data from the isolated perfused catfish intestine showing that Cu uptake across the whole intestine was reduced by 50% in the presence of 2 mM luminal amiloride, with 75% of the overall inhibition attributed to an amiloride-sensitive region in the middle intestine. Removal of luminal Na(+) produced more variable results, but also reduced Cu uptake in catfish intestine. These data together support Cu(2+) modulation of ENaC, but not competitive entry of Cu(2+) through ENaC. However, in situations where external Na(+) is only a few millimoles (fish gills, frogs in freshwater), Cu(2+) leak through ENaC is possible. CTR1 is a likely route of Cu(2+) entry when external Na(+) is higher (e.g. intestinal epithelia). Interactions between Na(+) and Cu ions during intracellular trafficking or export from the cell are unlikely. However, effects of intracellular chloride on the Cu-ATPase or ENaC indicate that Na(+) might indirectly alter Cu flux. Conversely, Cu ions inhibit basolateral Na(+)K(+)-ATPase and may increase [Na(+)](i).     

PACT, a stress-modulated cellular activator of interferon-induced double-stranded RNA-activated protein kinase, PKR

The interferon (IFN)-induced, double-stranded (ds)RNA-activated serine-threonine protein kinase, PKR, is a key mediator of the antiviral activities of IFNs. In addition, PKR activity is also involved in regulation of cell proliferation, apoptosis, and signal transduction. In virally infected cells, dsRNA has been shown to bind and activate PKR kinase function. Implication of PKR activity in normal cellular processes has invoked activators other than dsRNA because RNAs with perfectly duplexed regions of sufficient length that are able to activate PKR are absent in cellular RNAs. We have recently reported cloning of PACT, a novel protein activator of PKR. PACT heterodimerizes with PKR and activates it by direct protein-protein interaction. Overexpression of PACT in mammalian cells leads to phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha), the cellular substrate for PKR, and leads to inhibition of protein synthesis. Here, we present evidence that endogenous PACT acts as a protein activator of PKR in response to diverse stress signals such as serum starvation, and peroxide or arsenite treatment. Following exposure of cells to these stress agents, PACT is phosphorylated and associates with PKR with increased affinity. PACT-mediated activation of PKR leads to enhanced eIF2alpha phosphorylation followed by apoptosis. Based on the results presented here, we propose that PACT is a novel stress-modulated physiological activator of PKR.     

Quantification of changing Pseudomonas aeruginosa sodA, htpX and mt gene abundance in response to trace metal toxicity: a potential in situ biomarker of environmental health

Sediment-dwelling prokaryotes play a vital role in determining the fate and speciation of metals, yet are also susceptible to the biological effects of trace metals. In this article, optimized DNA extraction and purification techniques and species-specific primers are used to assess the genetic incidence and abundance of metal detoxification and general stress genes of Pseudomonas aeruginosa to complement chemical analysis in inferring the severity of metal-contaminated sites along the Clark Fork River, Montana. Results show the highest incidence of candidate genes related to bacterial stress at the most polluted site, while multiple regression analysis demonstrated significant correlations (P<0.05, r(2)=0.9) between in situ metal concentrations (As, Cu and Zn), total gene incidence, and the incidence of metal detoxification genes. Furthermore, principal components plotting the incidence of genes related to metal resistance show clear separation of sites giving clear clusters on the basis of contamination. Quantification of three genes (sodA, htpX and mt) from surveyed sites found significantly higher (anova, P<0.05) copy numbers at the more contaminated sites compared with reference sites. The development of rapid microbial biomarker tools represents a significant advance in the field of environmental biomonitoring and the prediction of metal bioavailability.     

Antifungal activity of substituted aurones

Novel antifungals are in high demand as there is a growing resistance to antifungals currently in use. In particular, opportunistic fungal infections caused by Candida spp. are on the rise with infections by this genus accounting for the most severe fungal infections following chemotherapy, implantation procedures, and in patients with HIV/AIDS. A series of simple aurone analogs were synthesized and screened for antifungal activity versus Candida spp. Several compounds displayed activity at 100 μM, with two having IC₅₀ values below 20 μM for three species of Candida. One of the compounds tested here also exhibits anti-biofilm activity for mid-maturation growth.     

Preclinical Comparison of Mechanistically Different Antiseizure,Antinociceptive, and/or Antidepressant Drugs in a Battery of Rodent Models of Nociceptive and Neuropathic Pain

The series of experiments herein evaluated prototype drugs representing different mechanisms of antiseizure, antinociceptive or antidepressant action in a battery of preclinical pain models in adult male CF#1 mice (formalin, writhing, and tail flick) and Sprague Dawley rats partial sciatic nerve ligation (PSNL). In the formalin assay, phenytoin (PHT, 6 mg/kg), sodium valproate (VPA, 300 mg/kg), amitriptyline (AMI, 7.5 and 15 mg/kg), gabapentin (GBP, 30 and 70 mg/kg), tiagabine (TGB, 5 and 15 mg/kg), and acetominophen (APAP, 250 and 500 mg/kg) reduced both phases of the formalin response to ≤?25% of vehicle-treated mice. In the acetic acid induced writhing assay, VPA (300 mg/kg), ethosuximide (ETX, 300 mg/kg), morphine (MOR, 5 & 10 mg/kg), GBP (10, 30, and 60 mg/kg), TGB (15 mg/kg), levetiracetam (LEV, 300 mg/kg), felbamate (FBM, 80 mg/kg) and APAP (250 mg/kg) reduced writhing to ≤?25% of vehicle-treated mice. In the tail flick test, MOR (1.25-5 mg/kg), AMI (15 mg/kg) and TGB (5 mg/kg) demonstrated significant antinociceptive effects. Finally, carbamazepine (CBZ, 20 and 50 mg/kg), VPA, MOR (2 and 4 mg/kg), AMI (12 mg/kg), TPM (100 mg/kg), lamotrigine (LTG, 40 mg/kg), GBP (60 mg/kg), TGB (15 mg/kg), FBM (35 mg/kg), and APAP (250 mg/kg) were effective in the PSNL model. Thus, TGB was the only prototype compound with significant analgesic effects in each of the four models, while AMI, GBP, APAP, and MOR each improved three of the four pain phenotypes. This study highlights the importance evaluating novel targets in a variety of pain phenotypes.     

Pseudomonas aeruginosa survives in epithelia by ExoS‐mediated inhibition of autophagy and mTOR

One major factor that contributes to the virulence of Pseudomonas aeruginosa is its ability to reside and replicate unchallenged inside airway epithelial cells. The mechanism by which P. aeruginosa escapes destruction by intracellular host defense mechanisms, such as autophagy, is not known. Here, we show that the type III secretion system effector protein ExoS facilitates P. aeruginosa survival in airway epithelial cells by inhibiting autophagy in host cells. Autophagy inhibition is independent of mTOR activity, as the latter is also inhibited by ExoS, albeit by a different mechanism. Deficiency of the critical autophagy gene Atg7 in airway epithelial cells, both in vitro and in mouse models, greatly enhances the survival of ExoS‐deficient P. aeruginosa but does not affect the survival of ExoS‐containing bacteria. The inhibitory effect of ExoS on autophagy and mTOR depends on the activity of its ADP‐ribosyltransferase domain. Inhibition of mTOR is caused by ExoS‐mediated ADP ribosylation of RAS, whereas autophagy inhibition is due to the suppression of autophagic Vps34 kinase activity.     

A survey of Dinophysis spp. and their potential to cause diarrhetic shellfish poisoning in coastal waters of the United States

Multiple species of the genus Dinophysis produce diarrhetic shellfish toxins (okadaic acid and Dinophysis toxins, OA/DTXs analogs) and/or pectenotoxins (PTXs). Only since 2008 have DSP events (illnesses and/or shellfish harvesting closures) become recognized as a threat to human health in the United States. This study characterized 20 strains representing five species of Dinophysis spp. isolated from three US coastal regions that have experienced DSP events: the Northeast/Mid-Atlantic, the Gulf of Mexico, and the Pacific Northwest. Using a combination of morphometric and DNA-based evidence, seven Northeast/Mid-Atlantic isolates and four Pacific Northwest isolates were classified as D. acuminata, a total of four isolates from two coasts were classified as D. norvegica, two isolates from the Pacific Northwest coast were identified as D. fortii, and three isolates from the Gulf of Mexico were identified as D. ovum and D. caudata. Toxin profiles of D. acuminata and D. norvegica varied by their geographical origin within the United States. Cross-regional comparison of toxin profiles was not possible with the other three species; however, within each region, distinct species-conserved profiles for isolates of D. fortii, D. ovum, and D. caudata were observed. Historical and recent data from various State and Tribal monitoring programs were compiled and compared, including maximum recorded cell abundances of Dinophysis spp., maximum concentrations of OA/DTXs recorded in commercial shellfish species, and durations of harvesting closures, to provide perspective regarding potential for DSP impacts to regional public health and shellfish industry.     

Effects of dietary caloric restriction and aging on thyroid hormones of rhesus monkeys.

Plasma levels of thyroid hormones - triiodothyronine (T 3 ), thyroxin (T 4 ), and thyroid-stimulating hormone (TSH) were measured in male and female rhesus monkeys (Macaca mulatta) fed either ad libitum or a 30 % calorie-restricted (CR) diet (males for 11 years; females for 6 years). The same hormones were measured in another group of young male rhesus monkeys during adaptation to the 30 % CR regimen. Both long- and shorter-term CR diet lowered total T 3 in plasma of the monkeys. The effect appeared to be greater in younger monkeys than in older counterparts. No effects of CR diet were detected for either free or total T 4, although unlike T 3, levels of this hormone decreased with age. TSH levels also decreased with age, and were increased by long-term CR diet in older monkeys only. No consistent effects of shorter-term CR diet were observed for TSH. In the light of the effects of the thyroid axis on overall metabolism, these results suggest a possible mechanism by which CR diets may elicit their well-known beneficial 'anti-aging' effects in mammals.