Carbonic anhydrase and soluble adenylate cyclase regulation of cystic fibrosis cellular phenotypes.

Several aspects of the cell biology of cystic fibrosis (CF) epithelial cells are altered including impaired lipid regulation, disrupted intracellular transport, and impaired microtubule regulation. It is unclear how the loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leads to these differences. It is hypothesized that the loss of CFTR function leads to altered regulation of carbonic anhydrase (CA) activity resulting in cellular phenotypic changes. In this study, it is demonstrated that CA2 protein expression is reduced in CF model cells, primary mouse nasal epithelial (MNE) cells, excised MNE tissue, and primary human nasal epithelial cells (P < 0.05). This corresponds to a decrease in CA2 RNA expression measured by qPCR as well as an overall reduction in CA activity in primary CF MNEs. The addition of CFTR-inhibitor-172 to WT MNE cells for ≥24 h mimics the significantly lower protein expression of CA2 in CF cells. Treatment of CF cells with l-phenylalanine (L-Phe), an activator of CA activity, restores endosomal transport through an effect on microtubule regulation in a manner dependent on soluble adenylate cyclase (sAC). This effect can be blocked with the CA2-selective inhibitor dorzolamide. These data suggest that the loss of CFTR function leads to the decreased expression of CA2 resulting in the downstream cell signaling alterations observed in CF.

Resveratrol restores intracellular transport in cystic fibrosis epithelial cells.

We have demonstrated previously that intracellular transport is impaired in cystic fibrosis (CF) epithelial cells. This impairment is related to both growth and inflammatory regulation in CF cell and animal models. Understanding how transport in CF cells is regulated and identifying means to manipulate that regulation are key to identifying new therapies that can address key CF phenotypes. It was hypothesized that resveratrol could replicate these benefits since it interfaces with multiple pathways identified to affect microtubule regulation in CF. It was found that resveratrol treatment significantly restored intracellular transport as determined by monitoring both cholesterol distribution and the distribution of rab7-positive organelles in CF cells. This restoration of intracellular transport is due to correction of both microtubule formation rates and microtubule acetylation in cultured CF cell models and primary nasal epithelial cells. Mechanistically, the effect of resveratrol on microtubule regulation and intracellular transport was dependent on peroxisome proliferator-activated receptor-γ signaling and its ability to act as a pan-histone deacetylase (HDAC) inhibitor. Resveratrol represents a candidate compound with known anti-inflammatory properties that can restore both microtubule formation and acetylation in CF epithelial cells.

Acetyl-CoA carboxylase inhibition regulates microtubule dynamics and intracellular transport in cystic fibrosis epithelial cells.

The use of high-dose ibuprofen as an anti-inflammatory therapy in cystic fibrosis (CF) has been shown to be an effective intervention although use is limited due to potential adverse events. Identifying the mechanism of ibuprofen efficacy would aid in the development of new therapies that avoid these adverse events. Previous findings demonstrated that ibuprofen treatment restores the regulation of microtubule dynamics in CF epithelial cells through a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent mechanism. The goal of this study is to define the AMPK pathway that leads to microtubule regulation. Here, it is identified that inhibition of acetyl-CoA carboxylase (ACC) is the key step in mediating the AMPK effect. ACC inhibition with 5-(tetradecyloxy)-2-furoic acid (TOFA) increases microtubule reformation rates in cultured and primary CF epithelial cells to wild-type (WT) rates. TOFA treatment also restores microtubule-dependent distribution of cholesterol and Rab7-positive organelles, as well as reduces expression of the proinflammatory signaling molecule RhoA to WT levels. ACC activation with citrate replicates these CF phenotypes in WT cells further supporting the role of AMPK signaling through ACC as a key mediator in CF cell signaling. It is concluded that ACC inhibition is the key step in the efficacy of AMPK activation at the cellular level and could represent a novel site of therapeutic intervention to address inflammation in CF.

Effects of drying pretreatment and particle size adjustment on the composting process of discarded flue-cured tobacco leaves.

The main characteristic of discarded flue-cured tobacco leaves is their high nicotine content. Aerobic composting is an effective method to decrease the nicotine level in tobacco leaves and stabilize tobacco wastes. However, high levels of nicotine in discarded flue-cured tobacco leaves complicate tobacco waste composting. This work proposes a drying pretreatment process to reduce the nicotine content in discarded flue-cured tobacco leaves and thus enhance its carbon-to-nitrogen ratio to a suitable level for composting. The effect of another pretreatment method, particle size adjustment, on composting efficiency was also tested in this work. The results indicated that the air-dried (nicotine content: 1.35%) and relatively long discarded flue-cured tobacco leaves (25 mm) had a higher composting efficiency than damp (nicotine content: 1.57%) and short discarded flue-cured tobacco leaves (15 mm). When dry/25 mm discarded flue-cured tobacco leaves mixed with tobacco stems in an 8:2 ratio was composted at a temperature above 55 °C for 9 days, the nicotine content dropped from 1.29% to 0.28%. Since the discarded flue-cured tobacco leaves was successfully composted to a fertile and harmless material, the germination index values increased to 85.2%. The drying pretreatment and particle size adjustment offered ideal physical and chemical conditions to support microbial growth and bioactivity during the composting process, resulting in efficient conversion of discarded flue-cured tobacco leaves into a high quality and mature compost.

Trace metals in resuspended fraction of settled bus dust and assessment of non-occupational exposure.

Trace metals (TMs) within urban public transportation systems have rarely been studied and information on related health risks is scant. This study measured TM (arsenic, chromium, cadmium, nickel, zinc, copper and lead) concentrations in resuspended fractions of settled bus dust in Harbin, China, and estimated the exposure and health risks. The incremental lifetime cancer risk (ILCR) for commuters was estimated for TM exposures. The average concentration of total TMs was 559µg/g (ranges from 312 to 787) among 45 bus routes in Harbin. The hazard quotient of three selected commuter groups increased in the following order: teenagersdermal contact>inhalation.

Adipose knockout of H-ferritin improves energy metabolism in mice.

OBJECTIVE: Ferritin, the principal iron storage protein, is essential to iron homeostasis. How iron homeostasis affects the adipose tissue is not well understood. We investigated the role of ferritin heavy chain in adipocytes in energy metabolism. METHODS: We generated adipocyte-specific ferritin heavy chain (Fth, also known as Fth1) knockout mice, herein referred to as FthAKO. These mice were analyzed for iron homeostasis, oxidative stress, mitochondrial biogenesis and activity, adaptive thermogenesis, insulin sensitivity, and metabolic measurements. Mouse embryonic fibroblasts and primary mouse adipocytes were used for in vitro experiments. RESULTS: In FthAKO mice, the adipose iron homeostasis was disrupted, accompanied by elevated expression of adipokines, dramatically induced heme oxygenase 1(Hmox1) expression, and a notable decrease in the mitochondrial ROS level. Cytosolic ROS elevation in the adipose tissue of FthAKO mice was very mild, and we only observed this in the brown adipose tissue (BAT) but not in the white adipose tissue (WAT). FthAKO mice presented an altered metabolic profile and showed increased insulin sensitivity, glucose tolerance, and improved adaptive thermogenesis. Interestingly, loss of ferritin resulted in enhanced mitochondrial respiration capacity and a preference for lipid metabolism. CONCLUSIONS: These findings indicate that ferritin in adipocytes is indispensable to intracellular iron homeostasis and regulates systemic lipid and glucose metabolism.

Electrochemical measurement of membrane cholesterol correlates with CFTR function and is HDAC6-dependent.

BACKGROUND: Previous studies have demonstrated that CF epithelial cells exhibit increased cholesterol content at the plasma membrane compared to wild type controls as measured by electrochemical methods. Microtubule dysregulation that impacts intracellular transport has also been identified in CF cells and is reversible with histone deacetylase 6 (HDAC6) inhibition, a regulator of tubulin acetylation. The hypothesis of this study is that increased membrane cholesterol content in CF cells is dependent on HDAC6 regulation. METHODS: Electrochemical measurement of membrane cholesterol in mouse trachea and in primary human CF bronchial epithelial cells is used to monitor CFTR correction and manipulation of cholesterol processing by HDAC6 inhibition. RESULTS: Data demonstrate that induction of Cftr expression in an inducible CF mouse model restores tubulin acetylation levels and normalizes membrane cholesterol content. To test the relationship between tubulin acetylation, membrane cholesterol levels were measured in a CF mouse model depleted of Hdac6 expression (CF/HDA). CF/HDA mouse trachea have WT membrane cholesterol levels while CF mice have approximately two-fold increase in membrane cholesterol compared to WT consistent with previous studies. Pharmacological inhibition of HDAC6 in primary human CF bronchial epithelial cells also reduces membrane cholesterol levels. CONCLUSIONS: This study demonstrates that elevated membrane cholesterol in CF epithelium is regulated by HDAC6 function and that the electrochemical measure of membrane cholesterol correlates with both genetic and pharmacological CFTR correction.

Taraxerol Induces Cell Apoptosis through A Mitochondria-Mediated Pathway in HeLa Cells.

OBJECTIVES: Taraxerol acetate has potent anti-cancer effects via the induction of apoptosis, autophagy, cell cycle arrest, and inhibition of cell migration. However, whether taraxerol induced apoptosis and its underlying mechanisms of action is not clear. In the present study, we assess the effects of taraxerol on the mitochondrial apoptotic pathway and determine the release of cytochrome c to the cytosol and activation of caspases. MATERIALS AND METHODS: In this experimental study, we mainly investigated the effect of taraxerol on HeLa cells. We tested cell viability by the MTT assay and morphologic changes, analyzed apoptosis by DAPI staining and flow cytometry. We also determined reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) using a Microplate Reader. In addition, the apoptotic proteins were tested by Western blot. RESULTS: Taraxerol enhanced ROS levels and attenuated the MMP (Δψm) in HeLa cells. Taraxerol induced apoptosis mainly via the mitochondrial pathway including the release of cytochrome c to the cytosol and activation of caspases 9 and 3, and anti-poly (ADPribose) polymerase (PARP). Taraxerol could induce the down-regulation of the anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax. It suppressed the PI3K/ Akt signaling pathway. CONCLUSIONS: These results demonstrated that taraxerol induced cell apoptosis through a mitochondria-mediated pathway in HeLa cells. Thus, taraxerol might be a potential anticervical cancer candidate.

Cell Plasma Membrane Cholesterol as a Diagnostic.

Cholesterol is a tightly regulated major structural component of the cell plasma membrane (PM) where it forms stoichiometric complexes with phospholipids and sphingolipids. The amount of cholesterol in the PM exhibits a regulatory role in basal activity of several biomolecular processes by direct binding to proteins and by indirect local environmental effects within the PM that are also coupled to overall cellular cholesterol homeostasis. The term "active cholesterol" refers to PM cholesterol not complexed to lipids, a cholesterol state that arises above a threshold mole fraction of cholesterol in the PM. Active cholesterol level in the PM provides a control mechanism for cellular cholesterol homeostasis through its recognition by membrane bound proteins that activate genes of cholesterol synthesis enzymes. Uptake of LDL, production and release of HDL as well as reversible storage of cholesterol in the cytosol by covalent modification are also regulated and dependent on PM cholesterol (thermodynamic) activity: active cholesterol. A number of human disease states have been found to have associated alterations in PM cholesterol and thus a method for its determination is described.

Solanesol induces the expression of heme oxygenase-1 via p38 and Akt and suppresses the production of proinflammatory cytokines in RAW264.7 cells.

The aim of the present study was to examine the anti-inflammatory effect of solanesol and to elucidate the underlying mechanisms. Heme oxygenase-1 (HO-1) plays an important role in cytoprotection against oxidative stress and inflammation. Solanesol induced HO-1 expression both at the level of mRNA and proteins, resulting in increased HO-1 activity. Solanesol treatment enhanced the level of the phosphorylated form, nuclear translocation, ARE-binding, and transcriptional activity of Nrf2. p38 and Akt contributed to ARE-driven HO-1 expression. Solanesol activated both p38 and Akt, and treatments with SB203580 (a p38 kinase inhibitor), LY294002 (an Akt inhibitor), specific p38 siRNA and Akt siRNA suppressed the solanesol-induced activation of Nrf2, resulting in a decrease in HO-1 expression. Solanesol also elevated the autophagic protein LC3B-II level. SnPP (a HO-1 inhibitor) and HO-1 siRNA markedly abolished the anti-inflammatory effect of solanesol against LPS-induced cell damage. Likewise, SB203580, LY294002, 3-MA and Baf-A1 inhibited the solanesol-induced anti-inflammatory effect. These studies demonstrate that solanesol attenuates inflammation by HO-1 induction via p38 and Akt signaling. Thus, it is quite plausible that HO-1 induction by solanesol could trigger anti-inflammatory pathways including limiting LPS-stimulated cytokine production through autophagic signaling via p38 and Akt.

The effects of low-intensity ultrasound on growing bone after sciatic neurectomy.

The purpose of this study was to investigate the therapeutic effects of low-intensity ultrasound (US) on the sciatic-neurectomy-induced bone mass decrement in growing rats. A total of 20 male Sprague-Dawley rats (166.7+/-11.3 g) underwent right leg sciatic neurectomy. They were randomly assigned into two groups, US treatment group (UST) and control group (CON). The rats receiving US treatment were treated with a 125 mW/cm2 continuous low-intensity US stimulation for 15 min/day on the lateral site of the right leg. The control rats did not receive any US treatment. All the animals were euthanized after 4-week US treatment. Both the original data and bilateral difference ratio of femur or tibia weight, histomorphometry data and bone densitometry data showed that the sciatic neurectomy obviously reduced bone mass in the operated limbs of both groups. However, the continuous low-intensity US treatment did not ameliorate the neurectomy-induced loss of bone mass. Thus, the low-intensity US generated micromechanical strains might not induce enough bone formation activity to reverse the bone loss in this model of intact bones.

Distribution and fate of synthetic musks in the Songhua River, Northeastern China: influence of environmental variables.

Contamination levels and spatial and temporal distributions of six typical synthetic musks (SMs) in water and sediment of the Songhua River in Northeastern China were investigated. Experimental data for 72 water and 52 sediment samples collected at 29 sampling sites over 12 months spanning 2011-2012 showed that the Songhua River had been contaminated to different degrees at various sites separately from the river's source. The polycyclic musks 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-(g)-2-benzopyran (HHCB) (Galaxolide) and 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (AHTN) (Tonalide) were found most frequently and at the highest levels. Concentrations of HHCB were <2-37 ng/L in water and <0.5-17.5 ng/g dry weight (dw) in sediment. AHTN was <1-8 ng/L in water and <0.5-5.7 ng/g dw in sediment. Statistical relationships between SM concentrations and four environmental variables (temperature, illumination, runoff, and population density) in the Songhua River Basin were formulated. Concentration levels varied proportionately with the size of the city along the river, while the distribution patterns showed clear seasonal variations. HHCB/AHTN ratios mirrored the transfer and transmitting process of SMs. Concentrations of target compounds were correlated with each other, suggesting similar exposure sources. Environmental risk assessment of SMs presented seasonal variations and provided baseline information on SM exposure in the Songhua River Basin.

Health impact of bioaccessible metal in lip cosmetics to female college students and career women, northeast of China.

Actual measure-based studies have estimated ingestion rate of moderate and high daily use to female college students and career women in northeast of China. Sequential extraction analyses showed that total bioaccessible metals concentration in lipstick ranged from 2.103 to 31.103 µg/g and in lip balm ranged from 0.100 to 3.716 µg/g. The relationship between total bioaccessible metal concentrations and the cost of lip cosmetics showed a negative correlation. Lead was detected in all 30 products (100%), with an average concentration of 0.346 for lip balm and 0.407 µg/g for lipstick. With the exception of chromium content in three lipsticks, the estimated exposure in female college students and career women to target metals via lipstick and lip balm ingestion (calculated for moderate and high use) were much lower than the acceptable reference limits. The findings strongly emphasize the need to focus on the health risk of lip balm.

Assessment on the occupational exposure of urban public bus drivers to bioaccessible trace metals through resuspended fraction of settled bus dust.

Limited information is available on the bioaccessible fraction of trace metals in the resuspended fraction of settled bus dust in order to estimate bus drivers' occupational exposure. In this study, 45 resuspended fraction of settled dust samples were collected from gasoline and compressed natural gas (CNG) powered buses and analyzed for trace metals and their fraction concentrations using a three-step sequential extraction procedure. Experimental results showed that zinc (Zn) had the greatest bioaccessible fraction, recorded as an average of 608.53 mg/kg, followed in order of decreasing concentration by 129.80 mg/kg lead (Pb), 56.77 mg/kg copper (Cu), 34.03 mg/kg chromium (Cr), 22.05 mg/kg nickel (Ni), 13.17 mg/kg arsenic (As) and 2.77 mg/kg cadmium (Cd). Among the three settled bus dust exposure pathways, ingestion was the main route. Total exposure hazard index (HIt) for non-carcinogenic effect trace metals was lower than the safety level of 1. The incremental lifetime cancer risk (ILCR) for drivers was estimated for trace metal exposure. Pb and Ni presented relatively high potential risks in the non-carcinogenic and potentially carcinogenic health assessment for all drivers. ILCR was in the range of 1.84E-05 to 7.37E-05 and 1.74E-05 to 6.95E-05 for gasoline and CNG buses, respectively.

Concentrations of polycyclic aromatic hydrocarbons in resuspendable fraction of settled bus dust and its implications for human exposure.

This preliminary study measured Polycyclic Aromatic Hydrocarbons (PAHs) concentrations in the resuspendable fraction of settled dust on 39 bus lines, to evaluate the impact of engine type (gasoline and compressed natural gas) on exposure for commuters and drivers. Benzo(b)fluoranthene(BbF) was the predominant PAH in resuspendable fraction of settled bus dust. The concentration of total PAHs was 92.90 ± 116.00 µg/g (range: 0.57-410) in gasoline buses and 3.97 ± 1.81 (range: 2.01-9.47) in compressed natural gas (CNG) buses. Based on Benzo[a]pyrene (BaP) equivalent concentrations for the sum of 16 PAHs, the average daily dose (ADD) via dust ingestion and dermal contact was calculated. The ADD of PAHs was higher for commuters and drivers in gasoline-powered buses than in buses using CNG buses. For both short and long duration journeys, young commuters were exposed to higher levels of PAHs via dust ingestion and dermal contact than adult commuters.