Inhaled marijuana smoke disrupts mitochondrial energetics in pulmonary epithelial cells in vivo

Habitual marijuana smoking is associated with inflammation and atypia of airway epithelium accompanied by symptoms of chronic bronchitis. We hypothesized that Delta(9)-tetrahydrocannabinol (THC), the primary psychoactive component of marijuana, might contribute to these findings by impairing cellular energetics and mitochondrial function. To test this hypothesis, we examined particulate smoke extracts from marijuana cigarettes, tobacco cigarettes, and placebo marijuana (0% THC) cigarettes for their effects on the mitochondrial function of A549 cells in vitro. Only extracts prepared from marijuana cigarettes altered mitochondrial staining by the potentiometric probe JC-1. With the use of a cross-flow, nose-only inhalation system, rats were then exposed for 20 min to whole marijuana smoke and examined for its effects on airway epithelial cells. Inhalation of marijuana smoke produced lung tissue concentrations of THC that were 8-10 times higher than those measured in blood (75 +/- 38 ng/g wet wt tissue vs. 9.2 +/- 2.0 ng/ml), suggesting high local exposure. Intratracheal infusion of JC-1 immediately following marijuana smoke exposure revealed a diffuse decrease in lung cell JC-1 red fluorescence compared with tissue from unexposed or placebo smoke-exposed rats. Exposure to marijuana smoke in vivo also decreased JC-1 red fluorescence (54% decrease, P < 0.01) and ATP levels (75% decrease, P < 0.01) in single-cell preparations of tracheal epithelial cells. These results suggest that inhalation of marijuana smoke has deleterious effects on airway epithelial cell energetics that may contribute to the adverse pulmonary consequences of marijuana smoking.     

Population Growth of Six Iranian Brachionus Rotifer Strains in Response to Salinity and Food Type

Intrinsic rates of population increase (r) were evaluated as a measure of population dynamics of four strains of Brachionus plicatilis and two strains of B. urceolaris from Iran in response to different salinities and feeding algae. Each rotifer strain was cultured at four salinities: 5, 20, 25 and 30‰ and fed with two microalgal species: Chlorella vulgaris and Nannochloropsis oculata. Salinity of 5‰ was critical for all the examined strains, at which r was at minimum and was different from the other salinities (P < 0.05). For B. plicatilis strains, the maximum r was observed in those fed on Chlorella at salinities of 10 and 30‰ (64 ± 0.01 day⁻¹). While, in B. urceolaris, maximum r was for Nannochloropsis fed rotifers at salinity of 20‰ (0.69 ± 0.01 day⁻¹). Maximum final population density (FD) was obtained for a strain of B. urceolaris fed on Nannochloropsis at 20‰ (329.3 ± 10.9 ind.mL⁻¹). FD was relatively lower in B. plicatilis strains among all examined salinities. ANOVA showed the significant effect of salinity and rotifer strain, and algae × rotifer strain on both r and FD, and salinity × rotifer × algae on FD (P < 0.05). (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Nuclear‐encoded DnaJ homologue of Plasmodium falciparum interacts with replication ori of the apicoplast genome

The apicoplast of Plasmodium falciparum carries a 35 kb circular genome (plDNA) that replicates at the late trophozoite stage of the parasite intraerythocytic cycle. plDNA replication proceeds predominantly via a d ‐loop/bi‐directional ori mechanism with replication ori localized within inverted repeat region. Although replication of the apicoplast genome is a validated drug target, the proteins involved in the replication process are only partially characterized. We analysed DNA–protein interactions at a plDNA replication ori region and report the identification of a nuclear‐encoded DnaJ homologue that binds directly to ori elements of the plDNA molecule. PfDnaJ_A interacted with the minor groove of the DNA double‐helix and recognized a 13 bp sequence within the ori. Inhibition of binding with anti‐PfDnaJ_A antibodies confirmed identity of the protein in DNA‐binding experiments with organellar protein fractions. The DNA‐binding domain of the ～69 kDa PfDnaJ_A lay within the N‐terminal 38 kDa region that carries DnaJ signature motifs. In contrast to PfDnaJ_A in parasite organellar fractions, the recombinant protein interacted with DNA in a sequence non‐specific manner. Our results suggest a role for PfDnaJ_A in replication/repair of the apicoplast genome.     

Influence of ionic interactions on essential oil and phenolic diterpene composition of Dalmatian sage (Salvia officinalis L.)

The potential of four essential cations (K⁺, Ca²⁺, Mg²⁺ and Fe²⁺) to alleviate salt toxicity was studied in sage (Salvia officinalis L.) plants grown in pots. Two concentrations of the following chloride salts: KCl, CaCl₂, MgCl₂ and FeCl_3, were used together with 100 mM NaCl to study the effects of these nutrients on plant growth, leaf essential oils (EOs) and phenolic diterpenes composition. The sage plants accumulated Na⁺ in their leaves (includers); this has affected secondary metabolites’ biosynthesis. Treatment with 100 mM NaCl slightly decreased borneol and viridiflorol, while increased manool concentrations. Addition of KCl, CaCl₂ and MgCl₂ increased considerably in a dose-dependent manner the oxygen-containing monoterpenes (1.8-cineole, camphor, β-thujone and borneol) in 100 mM NaCl-treated sage. Whereas, the contents of viridiflorol decreased further with the addition of KCl in 100 mM NaCl-treated sage. Our results suggest that the changes in EOs composition were more related to K⁺ and Ca²⁺ availability than to Na⁺ toxicity. Furthermore, treatment with NaCl decreased by 50% carnosic acid (CA), a potent antioxidant, content in the leaves. K⁺ and Ca²⁺ promoted the accumulation of CA and its methoxylated form (MCA) in the leaves. The concentration of CA was positively correlated with leaf K⁺ (r = 0.56, P = 0.01) and Ca²⁺ (r = 0.44, P = 0.05) contents. It appears that different salt applications in combination with NaCl treatments had a profound effect on EOs and phenolic diterpene composition in sage. Therefore, ionic interactions may be carefully considered in the cultivation of this species to get the desired concentrations of these secondary metabolites in leaf extracts.     

Solid-phase synthesis of 5'-deoxy-5'-amino-clitocine analogues

Several 6-substituted-amino-5'-deoxy-5'-amino-clitocine analogues were synthesized in a parallel fashion in solid phase. The desired scaffold was generated by coupling 2,3-O-bis-(t-butyldimethylsilyl)-5-N-(monomethoxytrityl-polystyrene-resin)-1,5-diamino-5-deoxy-beta-D-ribofuranose and 4, 6-dichloro-5-nitropyrimidine. The scaffold was then reacted with a variety of amines to generate a small library of 14 analogues of 5'-deoxy-5'-amino-clitocine following a protocol developed earlier.     

Sodium azide dilates coronary arterioles via activation of inward rectifier K+ channels and Na+-K+-ATPase

Sodium azide (NaN(3)), a potent vasodilator, causes severe hypotension on accidental exposure. Although NaN(3) has been shown to increase coronary blood flow, the direct effect of NaN(3) on coronary resistance vessels and the mechanism of the NaN(3)-induced response remain to be established. To address these issues without confounding influences from systemic parameters, subepicardial coronary arterioles were isolated from porcine hearts for in vitro study. Arterioles developed basal tone at 60 cmH(2)O intraluminal pressure and dilated acutely, in a concentration-dependent manner, to NaN(3) (0.1 microM to 50 microM). The NaN(3) response was not altered by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester or endothelial removal. Neither inhibition of phosphoinositol 3-kinase and tyrosine kinases nor blockade of ATP-sensitive, Ca(2+)-activated, and voltage-dependent K(+) channels affected NaN(3)-induced dilation. However, the vasomotor action of NaN(3) was significantly attenuated in a similar manner by the inward rectifier K(+) (K(IR)) channel inhibitor Ba(2+), the Na(+)-K(+) ATPase inhibitor ouabain, or the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Ba(2+), in combination with either ouabain or ODQ, nearly abolished the vasodilatory response. However, there was no additive inhibition by combining ouabain and ODQ. The NaN(3)-mediated vasodilation was also attenuated by morin, an inhibitor of phosphatidylinositolphosphate (PIP) kinase, which can regulate K(IR) channel activity. With the use of whole cell patch-clamp methods, NaN(3) acutely enhanced Ba(2+)-sensitive K(IR) current in isolated coronary arteriolar smooth muscle cells. Collectively, this study demonstrates that NaN(3), at clinically toxic concentrations, dilates coronary resistance vessels via activation of both K(IR) channels and guanylyl cyclase/Na(+)-K(+)-ATPase in the vascular smooth muscle. The K(IR) channels appear to be modulated by PIP kinase.     

Morphology engineering of Aspergillus niger for improved enzyme production

Supplementation with silicate microparticles was used as novel approach to control the morphological development of Aspergillus niger, important as the major world source of citric acid and higher‐value enzymes, in submerged culture. With careful variation of size and concentration of the micromaterial added, a number of distinct morphological forms including pellets of different size, free dispersed mycelium, and short hyphae fragments could be reproducibly created. Aluminum oxide particles similarly affected morphology, showing that this effect is largely independent of the chemical particle composition. Image analysis of morphological development of A. niger during the cultivation process showed that the microparticles influence the morphology by collision‐induced disruption of conidia aggregates and probably also the hindrance of new spore–spore interactions in the very early stage of the process. Exemplified for different recombinant A. niger strains enzyme production could be strongly enhanced by the addition of microparticles. Linked to the formation of freely dispersed mycelium, titers for glucoamylase (GA) expressed as intracellular enzyme (88 U/mL) and fructofuranosidase secreted into the supernatant (77 U/mL), were up to fourfold higher in shake flasks. Moreover, accumulation of the undesired by‐product oxalate was suppressed by up to 90%. The microparticle strategy could be successfully transferred to fructofuranosidase production in bioreactor, where a final titer of 160 U/mL could be reached. Using co‐expression of GA with green fluorescent protein, enzyme production was localized in the cellular aggregates of A. niger. For pelleted growth, protein production was maximal only within a thin layer at the pellet surface and markedly decreased in the pellet interior, whereas the interaction with the microparticles created a highly active biocatalyst with the dominant fraction of cells contributing to production. Biotechnol. Bioeng. 2010;105: 1058–1068. © 2009 Wiley Periodicals, Inc.     

Inhibitors of catalase-amyloid interactions protect cells from beta-amyloid-induced oxidative stress and toxicity

Compelling evidence shows a strong correlation between accumulation of neurotoxic β-amyloid (Aβ) peptides and oxidative stress in the brains of patients afflicted with Alzheimer disease (AD). One hypothesis for this correlation involves the direct and harmful interaction of aggregated Aβ peptides with enzymes responsible for maintaining normal, cellular levels of reactive oxygen species (ROS). Identification of specific, destructive interactions of Aβ peptides with cellular anti-oxidant enzymes would represent an important step toward understanding the pathogenicity of Aβ peptides in AD. This report demonstrates that exposure of human neuroblastoma cells to cytotoxic preparations of aggregated Aβ peptides results in significant intracellular co-localization of Aβ with catalase, an anti-oxidant enzyme responsible for catalyzing the degradation of the ROS intermediate hydrogen peroxide (H(2)O(2)). These catalase-Aβ interactions deactivate catalase, resulting in increased cellular levels of H(2)O(2). Furthermore, small molecule inhibitors of catalase-amyloid interactions protect the hydrogen peroxide-degrading activity of catalase in Aβ-rich environments, leading to reduction of the co-localization of catalase and Aβ in cells, inhibition of Aβ-induced increases in cellular levels of H(2)O(2), and reduction of the toxicity of Aβ peptides. These studies, thus, provide evidence for the important role of intracellular catalase-amyloid interactions in Aβ-induced oxidative stress and propose a novel molecular strategy to inhibit such harmful interactions in AD.     

Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals

Epigenetic information can be inherited through the mammalian germline and represents a plausible transgenerational carrier of environmental information. To test whether transgenerational inheritance of environmental information occurs in mammals, we carried out an expression profiling screen for genes in?mice that responded to paternal diet. Offspring of?males fed a low-protein diet exhibited elevated hepatic expression of many genes involved in lipid and cholesterol biosynthesis and decreased levels of cholesterol esters, relative to the offspring of males fed a control diet. Epigenomic profiling of offspring livers revealed numerous modest (～20%) changes in cytosine methylation depending on paternal diet, including reproducible changes in methylation over a likely enhancer for the key lipid regulator Ppara. These results, in conjunction with recent human epidemiological data, indicate that parental diet can affect cholesterol and lipid metabolism in offspring and define a model system to study environmental reprogramming of the heritable epigenome.