Kaempferol promotes primordial follicle activation through the phosphatidylinositol 3‐kinase/protein kinase B signaling pathway and reduces DNA fragmentation of sheep preantral follicles cultured in vitro

The aim of this study was to evaluate the effects of kaempferol on the morphology, follicular activation, growth, and DNA fragmentation of ovine preantral follicles cultured in situ, and the effects of a phosphatidylinositol 3 kinase (PI3K) inhibitor and the expression of phosphorylated protein kinase B (pAKT) after culture. Ovine ovarian fragments were fixed for histological and terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) analyses (fresh control) or cultured in α‐MEM+ alone (control) or with different concentrations of kaempferol (0.1, 1, 10, or 100 μM) for 7 days. Follicles were classified as normal or atretic, primordial or growing, and the oocyte and follicle diameters were measured. Proliferating cells were analyzed and DNA fragmentation was evaluated by the TUNEL assay. Inhibition of PI3K activity was performed through pretreatment in media added with 50 µM LY294002 for 1 hr and pAKT immunohistochemistry was performed after culture in the absence or presence of LY294002. After culture, the percentage of normal follicles was similar among the treatments (p > 0.05), except for 100 µM kaempferol, which had less normal follicles (p < 0.05). Moreover, kaempferol at 10 μM showed a higher percentage of follicular activation and cell proliferation than the other treatments (p < 0.05) and a percentage of TUNEL‐positive cells similar to that in the fresh control and lower than other treatments (p < 0.05). LY294002 significantly inhibited primordial follicle activation stimulated by α‐MEM+ and 10 μM kaempferol and reduced pAKT expression in those follicles. In conclusion, 10 μM kaempferol promotes primordial follicle activation and cell proliferation through the PI3K/AKT pathway and reduces DNA fragmentation of ovine preantral follicles cultured in vitro.     

DDX Profiles in Agricultural Fields Used for Cucurbit Production in Sakarya,Turkey

DDT (2,2-bis(chlorophenyl)-1,1,1- trichloroethane) and its metabolites DDD (2,2-bis(chlorophenyl)-1,1 -dichloroethane) and DDE (2,2-bis(chloraphenyl)-1,1 –dichloroethylene) have half-lives in soil measured in years or decades and are classified as Persistent Organic Pollutants (POPs). In this study, p,p′-DDT, p,p′-DDD, and p,p′-DDE residues were investigated in select agricultural fields of Sakarya Province, Turkey, where Cucurbitaceae have been grown for many years. Total squash and pumpkin production in Sakarya is approximately 3% of total cucurbit production of Turkey but little is known about the concentrations of DDT, DDD, and DDE in these agricultural soils. Thirty-three soil samples were collected from agricultural fields in different counties of Sakarya. p,p′-DDT was detected in all soil samples, with concentrations ranging from 0.23 ng/g to 123 ng/g soil (dry weight). The concentrations of p,p′-DDT metabolites ranged from nondetectable (<0.06 ng/g) to 120 ng/g for p,p′-DDD and from nondetectable (<0.03 ng/g) to 294 ng/g for p,p′-DDE. The highest total DDX (sum of p,p′-DDT, p,p′-DDD, and p,p′-DDE) concentrations among the soil samples was 428 ng/g in a sample collected from Karasu County. Further research in this field was conducted to measure p,p′-DDT, p,p′-DDE, and p,p′-DDD concentrations at multiple locations as a function of soil depth. p,p′-DDT concentrations were measured from 52 ng/g to 1935 ng/g at 0–60 cm depth. The highest DDX concentration was observed at a location where plants have been actively grown since 1987. The lowest DDX concentrations were observed where crops have not been grown since 1987. Our data proved that soil DDX levels at the field gradually increased as a function of how extensively the field has been used for cucurbits production. However, it is not certain whether the application of p,p′-DDT was terminated or if there may still be illegal usage in agricultural soils.     

Differential Effect of DDT,DDE, and DDD on COX‐2 Expression in the Human Trophoblast Derived HTR‐8/SVneo Cells

The purpose of this study was to investigate the effect of 1,1,1‐trichloro‐2,2‐bis‐(chlorophenyl)ethane (DDT), 1,1‐bis‐(chlorophenyl)‐2,2‐dichloroethene (DDE), and 1,1‐dichloro‐2,2‐bis(chlorophenyl)ethane (DDD) isomers on COX‐2 expression in a human trophoblast‐derived cell line. Cultured HTR‐8/SVneo trophoblast cells were exposed to DDT isomers and its metabolites for 24 h, and COX‐2 mRNA and protein expression were assessed by RT‐PCR, Western blotting, and ELISA. Prostaglandin E₂ production was also measured by ELISA. Both COX‐2 mRNA and protein were detected under control (unexposed) conditions in the HTR‐8/SVneo cell line. COX‐2 protein expression and prostaglandin E₂ production but not COX‐2 mRNA levels increased only after DDE and DDD isomers exposure. It is concluded that DDE and DDD exposure induce the expression of COX‐2 protein, leading to increased prostaglandin E2 production. Interestingly, the regulation of COX‐2 by these organochlorines pesticides appears to be at the translational level. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:454‐460, 2012; View this article online at wileyonlinelibrary.com . DOI 10:1002/jbt.21444     

Assessment of organochlorine pesticides and metals in ring‐tailed lemurs (Lemur catta) at Beza Mahafaly Special Reserve,Madagascar

Like most of Madagascar's endemic primates, ring‐tailed lemurs (Lemur catta) face a number of threats to their survival. Although habitat loss is of greatest concern, other anthropogenic factors including environmental contamination may also affect lemur health and survival. In this study, we examined ring‐tailed lemurs from the Beza Mahafaly Special Reserve (BMSR), southern Madagascar for exposure to organochlorine (OC) pesticides and metals and examined differences in contaminant concentrations between sexes and among age groups, troops, and habitats. A total of 14 pesticides and 13 metals was detected in lemur blood (24 individuals) and hair (65 individuals) samples, respectively. p,p′‐DDT, heptachlor, aldrin, heptachlor epoxide, endrin aldehyde, and endrin were among the most prevalent pesticides detected. Surprisingly, the persistent metabolite of p,p′‐DDT, p,p′‐DDE, was not detected. The most commonly detected metals were aluminum, zinc, boron, phosphorus, silicon, and copper, whereas metals considered more hazardous to wildlife (e.g. arsenic, cadmium, lead, selenium, vanadium) were not found above detection limits. Overall, concentrations of OC pesticides and metals were low and similar to those considered to be background concentrations in other studies examining the ecotoxicology of wild mammals. Few inter‐sex, ‐age, ‐troop, and ‐habitat differences in contaminant concentrations were observed, suggesting a uniform distribution of contaminants within the reserve. Several statistically significant relationships between lemur body size and contaminant concentrations were observed, but owing to the lack of supportive data regarding contaminant exposure in wild primates, the biological significance of these findings remains uncertain. Results of this study document exposure of ring‐tailed lemurs at BMSR to multiple OC pesticides and metals and provide essential baseline data for future health and toxicological evaluations of lemurs and other wild primates, especially those in regions with expanding agricultural and mining operations. Am. J. Primatol. 71:998–1010, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of the 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o,p′DDT)- and estradiol-mediated induction of rat uterine ornithine decarboxylase by prior treatment with o,p′DDT,estradiol, and tamoxifen

This study was designed to determine whether prior administration of inducers of rat uterine ornithine decarboxylase (ODC), such as 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o,p′DDT), estradiol-17β (E₂), or tamoxifen, inhibits the elevation of ODC by subsequently administered o,p′ DDT or estradiol-17β. o,p′ DDT (10 mg/day) was injected for 2 days to ovariectomized rats. One or two days later, when the levels of ODC returned to basal levels, o,p′ DDT (10 mg) and E₂ (0.05 μg) were administered intraperitoneally and, 6 or 5 h after these injections, uterine ODC was analyzed. The pretreatment with o,p′ DDT almost entirely blocked the induction of ODC by E₂ or o,p′ DDT. In another experiment, pretreatment with o,p′ DDT for 5 or 6 days eliminated the induction of ODC after injection of o,p′ DDT on Day 7. Similarly, the treatment of rats with the antiestrogen-tamoxifen (0.1 mg/day) for 4 days completely inhibited the subsequent elevation of ODC by either E₂ or o,p′ DDT administered on the fifth day. However, attempts to block the E₂-mediated elevation of ODC by prior treatment with E₂ yielded variable results. Two possibilities were considered to attempt to explain the mechanism of inhibition of induction of ODC by o,p′ DDT and tamoxifen: (a) induction of hepatic monooxygenase by these compounds, resulting in increased metabolism of the subsequently administered o,p′ DDT and E₂ into biologically less active components; (b) involvement of putrescine, the product of ODC action, in inhibiting ODC formation at the pretranslational level or at the post-translational level. It appears unlikely that the o,p′ DDT- and tamoxifen-mediated inhibition of ODC induction was due to an increase in hepatic biotransformation of o,p′ DDT and E₂. Pretreatment with tamoxifen or E₂ did not appear to induce the formation of hepatic microsomal cytochrome P-450, a component of the monooxygenase system. Furthermore, pretreatment with 2,2-bis-(p-chlorophenyl)-1,1-dichloroethylene (a compound with a structure similar to o,p′ DDT), which is not estrogenic but like o,p′ DDT elevates hepatic monooxygenase activity, did not inhibit E₂-or o,p′ DDT-mediated induction of uterine ODC. Concerning the possibility of putrescine inhibitory action, our observations that uterine diamine oxidase activity is negligible and that o,p′ DDT administration has no effect on this enzymatic activity suggested that elevation of ODC may result in higher levels of uterine putrescine or spermidine and spermine. The finding that the administration of putrescine to ovariectomized rats inhibited uterine ODC induction by o, p′ DDT supports the treatise that inhibition of ODC elevation after initial induction of ODC by antiestrogens and o,p′ DDT is due to putrescine- or polyamine-mediated inhibition of ODC. The possible mechanism of such product inhibition of ODC is disucssed.     

Enhancement of insulin-induced PI3K/Akt/GSK-3beta and ERK signaling by neuronal nicotinic receptor/PKC-alpha/ERK pathway: up-regulation of IRS-1/-2 mRNA and protein in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells treated with nicotine (10 µm for 24 h), phosphorylation of Akt, glycogen synthase kinase‐3β (GSK‐3β) and extracellular signal‐regulated kinase (ERK)1/2 induced by insulin (100 nm for 10 min) was enhanced by ～ 62%, without altering levels of these protein kinases. Nicotine produced time (> 12 h)‐ and concentration (EC₅₀ 3.6 and 13 µm )‐dependent increases in insulin receptor substrate (IRS)‐1 and IRS‐2 levels by ～ 125 and 105%, without altering cell surface density of insulin receptors. In these cells, insulin‐induced tyrosine phosphorylation of IRS‐1/IRS‐2 and recruitment of phosphoinositide 3‐kinase (PI3K) to IRS‐1/IRS‐2 were augmented by ～ 63%. The increase in IRS‐1/IRS‐2 levels induced by nicotine was prevented by nicotinic acetylcholine receptor (nAChR) antagonists, the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetra‐acetic acid tetrakis‐acetoxymethyl ester, cycloheximide or actinomycin D. Nicotine increased IRS‐1 and IRS‐2 mRNA levels by ～ 57 and ～ 50%, and this was prevented by conventional protein kinase C (cPKC) inhibitor Gö6976, or ERK kinase inhibitors PD98059 and U0126. Nicotine phosphorylated cPKC‐α, thereby increasing phosphorylation of ERK1/ERK2, as demonstrated by using Gö6976, PD98059 or U0126. Selective activation of cPKC‐α by thymeleatoxin mimicked these effects of nicotine. Thus, stimulation of nAChRs up‐regulated expression of IRS‐1/IRS‐2 via Ca²⁺‐dependent sequential activation of cPKC‐α and ERK, and enhanced insulin‐induced PI3K/Akt/GSK‐3β and ERK signaling pathways.     

Plant-originated glycoprotein (24 kDa) has an inhibitory effect on proliferation of BNL CL.2 cells in response to di(2-ethylhexyl)phthalate

Di(2‐ethylhexyl)phthalate (DEHP) is one of the many environmental chemicals that are widely used in polyvinyl chloride products, vinyl flooring, food packaging and infant toys. They cause cell proliferation or dysfunction of human liver. The purpose of this study is to investigate the inhibitory effect of a glycoprotein (24 kDa) isolated from Zanthoxylum piperitum DC (ZPDC) on proliferation of liver cell in the DEHP‐induced BNL CL. 2 cells. [³H]‐thymidine incorporation, intracellular reactive oxygen species (ROS), intracellular Ca²⁺ mobilization and activity of protein kinase C (PKC) were measured using radioactivity and fluorescence method respectively. The expression of mitogen‐activated protein kinases [extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK)], activator protein (AP)‐1 (c‐Jun and c‐Fos), proliferating cell nuclear antigen (PCNA) and cell cycle‐related factors (cyclin D1/cyclin‐dependent kinase [CDK] 4) were evaluated using Western blotting or electrophoretic mobility shift assay. The results in this study showed that the levels of [³H]‐thymidine incorporation, intracellular ROS, intracellular Ca²⁺ mobilization and activity of PKCα were inhibited by ZPDC glycoprotein (100 µg/ml) in the DEHP‐induced BNL CL. 2 cells. Also, activities of ERK, JNK and AP‐1 were reduced by ZPDC glycoprotein (100 µg/ml). With regard to cell proliferation, activities of PCNA and cyclin D1/CDK4 were significantly suppressed at treatment with ZPDC glycoprotein (100 µg/ml) in the presence of DEHP. Taken together, these findings suggest that ZPDC glycoprotein significantly normalized activities of PCNA and cyclin D1/CDK4, which relate to cell proliferation factors. Thus, ZPDC glycoprotein appears to be one of the compounds derived from natural products that are able to inhibit cell proliferation in the phthalate‐induced BNL CL. 2 cells. Copyright © 2011 John Wiley & Sons, Ltd.     

The Systematic Separation and Identification of Pesticides in the First Division

The systematic identification and determination of many kinds of pesticides were tried by a combination of column, thin-layer and gas chromatography. Pesticides in the first division,¹⁾ e.g., aldrin, DMC ethylene, CPAS, DDDS, o,p′-DDT, p,p′-DDT, heptachlor, quintozene, Telodrin and tetrasul, were separated from pesticides in the second division,¹⁾ e.g., α-BHC, β-BHC, γ-BHC, trifluralin, CPA, CNP, nitrofen, endrin, dieldrin, dicofol, etc. by column chromatography. Further, pesticides in the first division were separated from each other and determined approximately by thin-layer and gas chromatography.

The recoveries of these pesticides, except for aldrin and CPAS, were about 100%. The recoveries of aldrin and CPAS were about 70 %, due to decomposition during the procedures, or to adsorption on the adsorbent during the column chromatography. It was presumed from GC-MS data that CPAS was decomposed to DDDS, tetrasul and other compounds during the procedures.     

Oxygen consumption,ammonia excretion,and filtration rate of the marine bivalve Mytilus edulis exposed to methamidophos and omethoate

In this study, the oxygen consumption, ammonia excretion, and filtration rate were monitored in Mytilus edulis in response to administration of the two pesticides, methamidophos and omethoate. Five sublethal concentrations (1, 10, 50, 100, and 200 µgL^?1) were administered over 96 h. Oxygen consumption rates increased following administration of all concentrations of methamidophos for 96 h and to the lower concentrations of omethoate (1, 10, and 50 µg L^?1) from 6 to 24 h. Over 24 h, oxygen consumption decreased significantly. Ammonia excretion rates were higher than the control after 36 h of exposure to methamidophos, while reduction was observed when M. edulis was exposed to omethoate. The O:N ratios increased when the animals were first exposed to these two pesticides and then decreased at all the concentrations. At the concentrations of 100 and 200 µgL^?1, the O:N ratio was below 30 after 72 h. The filtration rate of M. edulis decreased with the increasing concentration of pesticide exposure.     

Therapeutic effect of curcumin on acrylamide‐induced apoptosis mediated by MAPK signaling pathway in Leydig cells

The aim of this study was to investigate the possible therapeutic effects of curcumin (CUR), against acrylamide (AA)‐induced toxic effects on Leydig cells. The AA and CUR‐treated cells were evaluated for cell viability, lipid peroxidation, reactive oxygen species (hydroxyl radical and hydrogen peroxide), antioxidant levels (glutathione peroxidase, glutathione‐S‐transferase, and catalase), apoptosis/necrosis rates and phosphorylation status of mitogen‐activated protein kinases (MAPKs). Leydig cells were exposed to four concentrations of AA (1, 10, 100, 1000 µM) in the presence and absence of CUR (2.5 µM) for 24 hours. According to the present result, AA concentration‐dependently, increased the oxidative stress parameters and suppressed the antioxidant enzyme levels, meanwhile induced apoptosis and activated the phosphorylation of extracellular signal‐regulated kinase, p38, and c‐Jun NH ₂‐terminal kinase. Moreover, CUR ameliorated the detrimental effects of AA. Thus, AA‐induced apoptosis through activation of the MAPK signaling pathway and CUR has a protective effect against AA‐induced damage in Leydig cells.     

Anti‐inflammatory and anti‐endotoxin properties of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti‐inflammatory and antioxidant activities. Several antimicrobial peptides are known to inhibit lipopolysaccharide (LPS)‐induced inflammation in vitro and to protect animals from sepsis. In this study, the cellular anti‐inflammatory and anti‐endotoxin activities of Os and Os‐C, peptides derived from the carboxy‐terminal of a tick defensin, were investigated. Both Os and Os‐C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin‐binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os‐C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione, a known NO scavenger. In contrast, Os‐C showed no scavenging activity. Os and Os‐C inhibited LPS/IFN‐γ induced NO and TNF‐α production in RAW 264.7 cells in a concentration‐dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF‐α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os‐C and glutathione protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os‐C, both peptides have in addition anti‐inflammatory and anti‐endotoxin properties. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

A redox‐linked novel pathway for arsenic‐mediated RET tyrosine kinase activation

We examined the biochemical effects of arsenic on the activities of RET proto‐oncogene (c‐RET protein tyrosine kinases) and RET oncogene (RET‐MEN2A and RET‐PTC1 protein tyrosine kinases) products. Arsenic activated c‐RET kinase with promotion of disulfide bond‐mediated dimerization of c‐RET protein. Arsenic further activated RET‐MEN2A kinase, which was already 3‐ to 10‐fold augmented by genetic mutation compared with c‐RET kinase activity, with promotion of disulfide bond‐mediated dimerization of RET‐MEN2A protein (superactivation). Arsenic also increased extracellular domain‐deleted RET‐PTC1 kinase activity with promotion of disulfide bond‐mediated dimerization of RET‐PTC1 protein. Arsenic increased RET‐PTC1 kinase activity with cysteine 365 (C365) replaced by alanine with promotion of dimer formation but not with cysteine 376 (C376) replaced by alanine. Our results suggest that arsenic‐mediated regulation of RET kinase activity is dependent on conformational change of RET protein through modulation of a special cysteine sited at the intracellular domain in RET protein (relevant cysteine of C376 in RET‐PTC1 protein). Moreover, arsenic enhanced the activity of immunoprecipitated RET protein with increase in thiol‐dependent dimer formation. As arsenic (14.2 µM) was detected in the cells cultured with arsenic (100 µM), direct association between arsenic and RET in the cells might modulate dimer formation. Thus, we demonstrated a novel redox‐linked mechanism of activation of arsenic‐mediated RET proto‐oncogene and oncogene products. J. Cell. Biochem. 110: 399–407, 2010. © 2010 Wiley‐Liss, Inc.     

Scale‐up and intensification of (S)‐1‐(2‐chlorophenyl)ethanol bioproduction: Economic evaluation of whole cell‐catalyzed reduction of o‐Chloroacetophenone

Escherichia coli cells co‐expressing genes coding for Candida tenuis xylose reductase and Candida boidinii formate dehydrogenase were used for the bioreduction of o‐chloroacetophenone with in situ coenzyme recycling. The product, (S)‐1‐(2‐chlorophenyl)ethanol, is a key chiral intermediate in the synthesis of polo‐like kinase 1 inhibitors, a new class of chemotherapeutic drugs. Production of the alcohol in multi‐gram scale requires intensification and scale‐up of the biocatalyst production, biotransformation, and downstream processing. Cell cultivation in a 6.9‐L bioreactor led to a more than tenfold increase in cell concentration compared to shaken flask cultivation. The resultant cells were used in conversions of 300 mM substrate to (S)‐1‐(2‐chlorophenyl)ethanol (e.e. >99.9%) in high yield (96%). Results obtained in a reaction volume of 500 mL were identical to biotransformations carried out in 1 mL (analytical) and 15 mL (preparative) scale. Optimization of product isolation based on hexane extraction yielded 86% isolated product. Biotransformation and extraction were accomplished in a stirred tank reactor equipped with pH and temperature control. The developed process lowered production costs by 80% and enabled (S)‐1‐(2‐chlorophenyl)ethanol production within previously defined economic boundaries. A simple and efficient way to synthesize (S)‐1‐(2‐chlorophenyl)ethanol in an isolated amount of 20 g product per reaction batch was demonstrated. Biotechnol. Bioeng. 2013; 110: 2311–2315. © 2013 Wiley Periodicals, Inc.     

Resveratrol induces cell‐cycle disruption and apoptosis in chemoresistant B16 melanoma

Resveratrol, a naturally occurring polyphenol, has been shown to possess chemopreventive activities. In this study, we show that resveratrol (0–500 µM) inhibits the growth of a doxorubicin‐resistant B16 melanoma cell subline (B16/DOX) (IC₅₀ = 25 µM after 72 h, P < 0.05). This was accomplished by imposing an artificial checkpoint at the G₁–S phase transition, as demonstrated by cell‐cycle analysis and down‐regulation of cyclin D1/cdk4 and increased of p53 expression level. The G₁‐phase arrest of cell cycle in resveratrol‐treated (10–100 µM) B16/DOX cells was followed by the induction of apoptosis, which was revealed by pyknotic nuclei and fragmented DNA. Resveratrol also potentiated at subtoxic dose (25 µM for 24 h) doxorubicin cytotoxicity in the chemoresistant B16 melanoma (P < 0.01). When administered to mice, resveratrol (12.5 mg/kg) reduced the growth of an established B16/DOX melanoma and prolonged survival (32% compared to untreated mice). All these data support a potential use of resveratrol alone or in combination with other chemotherapeutic agents in the management of chemoresistant tumors. J. Cell. Biochem. 110: 893–902, 2010. © 2010 Wiley‐Liss, Inc.     

Advanced glycation end‐products activate extracellular signal‐regulated kinase via the oxidative stress‐EGF receptor pathway in renal fibroblasts

Advanced glycation end‐products (AGEs), epidermal growth factor receptor (EGFR), reactive oxygen species (ROS), and extracellular signal‐regulated kinases (ERK) are implicated in diabetic nephropathy (DN). Therefore, we asked if AGEs‐induced ERK protein phosphorylation and mitogenesis are dependent on the receptor for AGEs (RAGE)–ROS–EGFR pathway in normal rat kidney interstitial fibroblast (NRK‐49F) cells. We found that AGEs (100 µg/ml) activated EGFR and ERK1/2, which was attenuated by RAGE short‐hairpin RNA (shRNA). AGEs also increased RAGE protein and intracellular ROS levels while RAGE shRNA and N‐acetylcysteine (NAC) attenuated AGEs‐induced intracellular ROS. Hydrogen peroxide (5–25 µM) increased RAGE protein level while activating both EGFR and ERK1/2. Low‐dose hydrogen peroxide (5 µM) increased whereas high‐dose hydrogen peroxide (100 µM) decreased mitogenesis at 3 days. AGEs‐activated EGFR and ERK1/2 were attenuated by an anti‐oxidant (NAC) and an EGFR inhibitor (Iressa). Moreover, AGEs‐induced mitogenesis was attenuated by RAGE shRNA, NAC, Iressa, and an ERK1/2 inhibitor (PD98059). In conclusion, it was found that AGEs‐induced mitogenesis is dependent on the RAGE–ROS–EGFR–ERK1/2 pathway whereas AGEs‐activated ERK1/2 is dependent on the RAGE–ROS–EGFR pathway in NRK‐49F cells. J. Cell. Biochem. 109: 38–48, 2010. © 2009 Wiley‐Liss, Inc.     

Elevated Concentrations of Pesticides and PCBs in Soils at the Southern Caspian Sea (Iran) are Related to Land Use

Soil contamination by organochlorine pesticides or PCBs is almost undocumented for Iran. Here we report a soil survey in Mazandaran and Guilan provinces that hold >30% of the agricultural areas of Iran where pesticide use is widespread. Concentration of DDTs, HCHs, cyclodienes, and PCBs were measured in 45 soil samples from different agricultural land uses and forest land. The average concentrations of ∑DDT (37 μg kg^?1) and ∑HCH (21 μg kg^?1) in agricultural soils are among the largest ever reported and exceed international soil screening standards. All residues were larger in agricultural than in forest soil. Within agricultural land, ∑DDT were largest for tea gardens, lindane was largest in rice fields, and cyclodiens largest in citrus orchards. The ratio of (DDD + DDE)/DDT is an index of the extent of DDT degradation in soil and was lower in tea gardens than in other soils (0.7 versus 2–5), indicating either ongoing DDT input or lower degradation rate in the tea gardens that are more acid than the other soils (pH 4.5 versus 6.5–7.0). The o,p′–DDT/p,p′–DDT ratio was about 3 in forest soils, suggesting that DDT is derived from dicofol application and not from technical DDT as in agricultural soils. The PCB 28, 180, and 138 showed the highest mean concentration compared with other PCB congeners in all land uses. This survey is the first of this kind for Iran and illustrates that concentrations of organochlorine pesticide in soil are relatively large.     

Disruption of LptA oligomerization and affinity of the LptA–LptC interaction

The lipopolysaccharide (LPS)‐rich outer membrane (OM) is a unique feature of Gram‐negative bacteria, and LPS transport across the inner membrane (IM) and through the periplasm is essential to the biogenesis and maintenance of the OM. LPS is transported across the periplasm to the outer leaflet of the OM by the LPS transport (Lpt) system, which in Escherichia coli is comprised of seven recently identified proteins, including LptA, LptC, LptDE, and LptFGB₂. Structures of the periplasmic protein LptA and the soluble portion of the membrane‐associated protein LptC have been solved and show these two proteins to be highly structurally homologous with unique folds. LptA has been shown to form concentration dependent oligomers that stack end‐to‐end. LptA and LptC have been shown to associate in vivo and are expected to form a similar protein–protein interface to that found in the LptA dimer. In these studies, we disrupted LptA oligomerization by introducing two point mutations that removed a lysine and glutamine side chain from the C‐terminal β‐strand of LptA. This loss of oligomerization was characterized using EPR spectroscopy techniques and the affinity of the interaction between the mutant LptA protein and WT LptC was determined using EPR spectroscopy (K_d = 15 µM) and isothermal titration calorimetry (K_d = 14 µM). K_d values were also measured by EPR spectroscopy for the interaction between LptC and WT LptA (4 µM) and for WT LptA oligomerization (29 µM). These data suggest that the affinity between LptA and LptC is stronger than the affinity for LptA oligomerization.