Steroids in porcine follicular fluid: analysis by HPLC, capillary CG and capillary CG/MS after purification on SEP-PAK C18 and ion exchange chromatography.

PubMed

Khalil, M W; Lawson, V

1983-04-01

Steroids in porcine follicular fluid have been concentrated by reverse phase chromatography in SEP-PAK C18 and purified further on the cation exchanger SP-Sephadex C-25. Fractionation into unconjugated neutral and phenolic steroids, glucuronides and sulfates was carried out on triethylaminohydroxypropyl Sephadex LH-20 (TEAP-LH-20). The unconjugated neutral fraction was analysed by high pressure liquid chromatography (HPLC) on a C18 radial cartridge 5 mm I.D.; 10 mu, or on a C18 5 mu RESOLVE column, and by capillary gas chromatography (GC) on a 12 M OV-1 cross linked fused silica column. Testosterone, progesterone and androstenedione were the major steroids detected by HPLC monitored at 254 nm, although 17- hydroxy-, 20 alpha-dihydro- and 20 beta-dihydroprogesterone were also present. Pregnenolone, pregnanediol, dehydroepiandrosterone, 17-hydroxypregnenolone and androsterone were detected by capillary CG as their 0-methyloxime trimethylsilyether derivatives. Further confirmation of structure was provided by complete mass spectral data or by selective ion monitoring (SIM).

Ipsilateral versus Contralateral Placement of the Pancreas Allograft in Pancreas after Kidney Transplant Recipients.

PubMed

Yin, Hang; Arpali, Emre; Leverson, Glen E; Sollinger, Hans W; Kaufman, Dixon B; Odorico, Jon S

2018-06-28

In a diabetic, uremic kidney transplant recipient that may receive a future pancreas after kidney (PAK) transplant, the kidney is typically implanted on the left side in anticipation of the subsequent pancreas transplant on the right side. In this study, we sought to determine if ipsilateral PAK (iPAK) is as safe as contralateral PAK (cPAK). 115 PAK transplants (iPAK n=57, cPAK n=58) were performed from 1997-2010 and results were compared between the groups. Kidney graft survival and pancreas graft survival was similar between the two groups. Kidney graft function according to serum creatinine and eGFR was not different between the cPAK and iPAK groups and there were no episodes of kidney graft thrombosis in either group. Subgroup analyses focusing on donor source, also did not show worse outcomes for graft survivals in iPAK group when compared to cPAK group. Pancreas and kidney graft survival in PAK transplants is unaffected by the surgical procedure and iPAK is safe. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Simultaneous determination of bisphenol A and estrogens in hair samples by liquid chromatography-electrospray tandem mass spectrometry.

PubMed

Lee, Chaelin; Kim, Chong Hyeak; Kim, Sunghwan; Cho, Sung-Hee

2017-07-15

Bisphenol A (BPA), an endocrine disrupter, is widely used to make chemicals for polycarbonate, plastics, beverage containers, epoxy resins, and cash register receipts. BPA is one of the known xenoestrogens, which have weak estrogenic activity and cause obesity, diabetes, breast cancer, and reproductive disorders. Even though the concentration level of metabolomes in hair is usually lower than that in urine and blood, there are several reasons why we chose to use hair samples. First, the sampling procedure of hairs is simple. Second, it is also easy to preserve the sample for long term and track the drug-exposure record of a given sample. Third, deformation and contamination of samples rarely occur. In this study, an improved analytical method to determine the levels of BPA and estrogens in hair samples was developed by liquid chromatography-electrospray tandem mass spectrometry (LC-ESI/MS/MS). Hair samples were extracted by an Oasis HLB extraction cartridge after incubation with 1N HCl and derivatized with dansyl chloride to increase sensitivity. BPA and estrogens (estrone, 17β-estradiol, and estriol) were separated using Shiseido CAPCELL PAK C 18 column (2.0×100mm, 3μm) and a mobile phase consisting of 10mM ammonium acetate in water and acetonitrile with a gradient program at a flow rate of 0.3mL/min and were monitored with electrospray tandem mass spectrometry (ESI-MS/MS). The linearity of this method was over 0.995. The limits of detection (LOD) at a signal-to-noise (S/N) ratio of 3 were 0.25-6.0ng/g. The alteration of estrogens levels induced by BPA may play important role to understanding probable endocrine disruptive exposure, and the described methods could be used to evaluate and monitor exposure of endocrine disruptor. Copyright © 2017 Elsevier B.V. All rights reserved.

Reproducibility of corneal astigmatism measurements with a hand held keratometer in preschool children.

PubMed Central

Harvey, E M; Miller, J M; Dobson, V

1995-01-01

AIMS--To evaluate the overall accuracy and reproducibility of the Alcon portable autokeratometer (PAK) measurements in infants and young children. METHODS--The accuracy of the Alcon PAK in measuring toric reference surfaces (1, 3, 5, and 7 D) under various suboptimal measurement conditions was assessed, and the reproducibility of PAK measurements of corneal astigmatism in newborn infants (n = 5), children (n = 19, age 3-5 years), and adults (n = 14) was evaluated. RESULTS--Measurements of toric reference surfaces indicated (a) no significant effect of distance (17-30 mm) on accuracy of measurements, (b) no systematic relation between amount of toricity and accuracy of measurements, (c) no systematic relation between angle of measurement and accuracy, (d) no difference in accuracy of measurements when the PAK is hand held in comparison with when it is mounted, (e) no difference in accuracy of measurements when axis of toricity is oriented obliquely than when it is oriented horizontally, with respect to the PAK, and (f) a small positive bias (+0.16 D) in measurement of spherical equivalent. The PAK did not prove useful for screening newborns. However, measurements were successfully obtained from 18/19 children and 14/14 adults. There was no significant difference in median measurement deviation (deviation of a subject's five measurements from his/her mean) between children (0.21 D) and adults (0.13 D). CONCLUSIONS--The PAK produces accurate measurements of surface curvature under a variety of suboptimal conditions. Variability of PAK measurements in preschool children is small enough to suggest that it would be useful for screening for corneal astigmatism in young children. PMID:8534668

[Studies for analyzing prohibited ingredients such as tetracaine hydrochloride in cosmetics].

PubMed

Tokunaga, Hiroshi; Takeuchi, Orie; Uchino, Tadashi; Ando, Masanori

2004-01-01

Tetracaine hydrochloride (TH) is nominated as the prohibited ingredients in cosmetics in Japanese Pharmaceutical Affairs Act. So the analytical method for TH was investigated by HPLC. After adding 5 ml of TH solution at 10 microg/ml and 2 ml of salicylic acid solution at 75 microg/ml as the internal standard to 0.5 g of the lotion, the mixture was made up to 10 ml with a mixture of water and methanol (1:1) as the testing solution. Milky lotion was procedured as follows: After adding 5 ml of TH solution at 10 microg/ml and 2 ml of internal standard solution to 0.5 g of the milky lotion, the mixture was made up to 10 ml with a mixture of water and methanol (1:1). Two milliliter of this mixture was placed into a centrifuging tube with a cap and 2 ml of hexane was added. After shaking vigorously and centrifuging, the lower layer was used as the testing solution. In the case of the cream, the other procedures were used: 0.5 g of cream was placed into a 10-ml volumetric flask and 1 ml of tetrahydrofuran was added. After dissolving, the mixture of methanol and water (1:1) was added to make up 10.0 ml. Two milliliter of this mixture was placed into a centrifuging tube with a cap and 2.0 ml of hexane was added. After shaking vigorously and centrifuging, the lower layer was used as the testing solution. The testing solution of 20 microl was analyzed by HPLC using the ODS column (CAPCELL PAK C18 column, 4.6 x 250 mm), the mixture of acetonitrile and 50 mmol/l phosphate buffer(pH 2.0)(7:3) and the detection wavelength of 303 nm. The working curves from 0.5 to 6.0 microg/ml showed a linear line between the concentrations of TH and the peak area ratio. There was no interference of peak of TH from the lotion, milky lotion and cream.

PAK2 is cleaved and activated during hyperosmotic shock-induced apoptosis via a caspase-dependent mechanism: evidence for the involvement of oxidative stress.

PubMed

Chan, W H; Yu, J S; Yang, S D

1999-03-01

Hyperosmotic shock elicits a stress response in mammalian cells and can lead to apoptotic cell death. In the present study, we report that hyperosmotic shock can induce activation of a 36 kDa kinase detected by an in-gel kinase assay in several cell types, including mouse Balb/c 3T3 fibroblasts, and human Hep 3B and A431 cells. This 36 kDa kinase can be recognized by an antibody against the C-terminal region of a family of p21Cdc42/Rac-activated kinases (PAKs) on immunoblot. Further studies with this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 demonstrate that hyperosmotic shock can induce cleavage of PAK2 to generate a 36 kDa C-terminal catalytic fragment in cells. The cleavage and activation of PAK2 was found to be closely associated with both DNA fragmentation and activation of an ICE/CED-3 family cysteine protease termed caspase-3 in hyperosmotically shocked cells. Furthermore, pretreating the cells with two caspase inhibitors (Ac-DEVD-cho and Ac-YVAD-cmk) could inhibit both cleavage/activation of PAK2 and DNA fragmentation induced by hyperosmotic shock. Moreover, all these hyperosmotic shock-induced changes (i.e., activation of caspase-3, cleavage/activation of PAK2, and DNA fragmentation) in cells could be blocked by antioxidants such as ascorbic acid (vitamine C), alpha-tocopherol (vitamine E), dithiothreitol, beta-mercaptoethanol, and glutathione. Taken together, our results show that PAK2 is cleaved and activated via a caspase-dependent mechanism during hyperosmotic shock-induced apoptosis and suggest the involvement of antioxidant-preventable oxidative stress in inducing this process.

Novel radiosynthesis of PET HSV-tk gene reporter probes [18F]FHPG and [18F]FHBG employing dual Sep-Pak SPE techniques.

PubMed

Wang, Ji-Quan; Zheng, Qi-Huang; Fei, Xiangshu; Mock, Bruce H; Hutchins, Gary D

2003-11-17

Positron emission tomography (PET) herpes simplex virus thymidine kinase (HSV-tk) gene reporter probes 9-[(3-[(18)F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([(18)F]FHPG) and 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine ([(18)F]FHBG) were prepared by nucleophilic substitution of the appropriate tosylated precursors with [(18)F]KF/Kryptofix 2.2.2 followed by a quick deprotection reaction and purification with a simplified dual Silica Sep-Pak solid-phase extraction (SPE) method in 15-30% radiochemical yield.

Group I Paks support muscle regeneration and counteract cancer-associated muscle atrophy.

PubMed

Cerquone Perpetuini, Andrea; Re Cecconi, Andrea David; Chiappa, Michela; Martinelli, Giulia Benedetta; Fuoco, Claudia; Desiderio, Giovanni; Castagnoli, Luisa; Gargioli, Cesare; Piccirillo, Rosanna; Cesareni, Gianni

2018-05-21

Skeletal muscle is characterized by an efficient regeneration potential that is often impaired during myopathies. Understanding the molecular players involved in muscle homeostasis and regeneration could help to find new therapies against muscle degenerative disorders. Previous studies revealed that the Ser/Thr kinase p21 protein-activated kinase 1 (Pak1) was specifically down-regulated in the atrophying gastrocnemius of Yoshida hepatoma-bearing rats. In this study, we evaluated the role of group I Paks during cancer-related atrophy and muscle regeneration. We examined Pak1 expression levels in the mouse Tibialis Anterior muscles during cancer cachexia induced by grafting colon adenocarcinoma C26 cells and in vitro by dexamethasone treatment. We investigated whether the overexpression of Pak1 counteracts muscle wasting in C26-bearing mice and in vitro also during interleukin-6 (IL6)-induced or dexamethasone-induced C2C12 atrophy. Moreover, we analysed the involvement of group I Paks on myogenic differentiation in vivo and in vitro using the group I chemical inhibitor IPA-3. We found that Pak1 expression levels are reduced during cancer-induced cachexia in the Tibialis Anterior muscles of colon adenocarcinoma C26-bearing mice and in vitro during dexamethasone-induced myotube atrophy. Electroporation of muscles of C26-bearing mice with plasmids directing the synthesis of PAK1 preserves fiber size in cachectic muscles by restraining the expression of atrogin-1 and MuRF1 and possibly by inducing myogenin expression. Consistently, the overexpression of PAK1 reduces the dexamethasone-induced expression of MuRF1 in myotubes and increases the phospho-FOXO3/FOXO3 ratio. Interestingly, the ectopic expression of PAK1 counteracts atrophy in vitro by restraining the IL6-Stat3 signalling pathway measured in luciferase-based assays and by reducing rates of protein degradation in atrophying myotubes exposed to IL6. On the other hand, we observed that the inhibition of group I Paks has no effect on myotube atrophy in vitro and is associated with impaired muscle regeneration in vivo and in vitro. In fact, we found that mice treated with the group I inhibitor IPA-3 display a delayed recovery from cardiotoxin-induced muscle injury. This is consistent with in vitro experiments showing that IPA-3 impairs myogenin expression and myotube formation in vessel-associated myogenic progenitors, C2C12 myoblasts, and satellite cells. Finally, we observed that IPA-3 reduces p38α/β phosphorylation that is required to proceed through various stages of satellite cells differentiation: activation, asymmetric division, and ultimately myotube formation. Our data provide novel evidence that is consistent with group I Paks playing a central role in the regulation of muscle homeostasis, atrophy and myogenesis. © 2018 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.

Inhibition of Tulane Virus replication via exposure to lowbush blueberry (Vaccinium angustifolium) fractional components

USDA-ARS?s Scientific Manuscript database

Tulane Virus (TV) is a common viral surrogate for human norovirus in lab studies. In the present study, the phenotypic response of TV when exposed to fractional components extracted from lowbush blueberries was investigated. Lowbush blueberry extract (F1) was separated using a C-18 Sep-Pak cartridge...

Involvement of Alpha-PAK-Interacting Exchange Factor in the PAK1–c-Jun NH2-Terminal Kinase 1 Activation and Apoptosis Induced by Benzo[a]pyrene

PubMed Central

Yoshii, Shigeto; Tanaka, Masamitsu; Otsuki, Yoshiro; Fujiyama, Toshiharu; Kataoka, Hideki; Arai, Hajime; Hanai, Hiroyuki; Sugimura, Haruhiko

2001-01-01

Benzo[a]pyrene [B(a)P], a potent procarcinogen found in combustion products such as diesel exhaust and cigarette smoke, has been recently shown to activate the c-Jun NH2-terminal kinase 1 (JNK1) and induce caspase-3-mediated apoptosis in Hepa1c1c7 cells. However, the molecules of the signaling pathway that control the mitogen-activated protein kinase cascades induced by B(a)P and the interaction between those and apoptosis by B(a)P have not been well defined. We report here that B(a)P promoted Cdc42/Rac1, p21-activated kinase 1 (PAK1), and JNK1 activities in 293T and HeLa cells. Moreover, alpha-PAK-interacting exchange factor (α PIX) mRNA and its protein expression were upregulated by B(a)P. While overexpression of an active mutant of α PIX (ΔCH) facilitated B(a)P-induced activation of Cdc42/Rac1, PAK1, and JNK1, overexpression of mutated αPIX (L383R, L384S), which lacks guanine nucleotide exchange factor activity, SH3 domain-deleted αPIX (Δ SH3), which lacks the ability to bind PAK, kinase-negative PAK1 (K299R), and kinase-negative SEK1 (K220A, K224L) inhibited B(a)P-triggered JNK1 activation. Interestingly, overexpression of αPIX (Δ CH) and a catalytically active mutant PAK1 (T423E) accelerated B(a)P-induced apoptosis in HeLa cells, whereas αPIX (Δ SH3), PAK1 (K299R), and SEK 1 (K220A, K224L) inhibited B(a)P-initiated apoptosis. Finally, a preferential caspase inhibitor, Z-Asp-CH2-DCB, strongly blocked the αPIX (Δ CH)-enhanced apoptosis in cells treated with B(a)P but did not block PAK1/JNK1 activation. Taken together, these results indicate that αPIX plays a crucial role in B(a)P-induced apoptosis through activation of the JNK1 pathway kinases. PMID:11564864

Study of Turbulence by Photon Correlation Spectroscopy

DTIC Science & Technology

1991-10-23

Hepp, and H . A. Weidenmuller (Springer Lecture Notes in Physics, Vol 12, 1972) p. 402. [17] A. Onuki , Phys. Lett. 127, 143 (1988). [18) K. R...of Turbulence", (W. I. Goldburg, P. Tong, and H . K. Pak), Physica D 38 (1989) 134-140. 2. "Scaling Laws in Weak Turbulence", (with H . K. Pak, W. I...Goldburg and P. Tong), (in press). 3. "An Experimental Study of Weak Turbulence", (with H . K. Pak and W. I. Goldburg), "Fluid Dynamics Research" (in

Biological effects of Trichoderma harzianum peptaibols on mammalian cells.

PubMed

Peltola, Joanna; Ritieni, Alberto; Mikkola, Raimo; Grigoriev, Pavel A; Pócsfalvi, Gabriella; Andersson, Maria A; Salkinoja-Salonen, Mirja S

2004-08-01

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 microg [dry weight] ml of extended boar semen(-1)). The same exposure concentration depleted the boar sperm cells of NADH(2). Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Deltapsi(m)) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.

Biological Effects of Trichoderma harzianum Peptaibols on Mammalian Cells

PubMed Central

Peltola, Joanna; Ritieni, Alberto; Mikkola, Raimo; Grigoriev, Pavel A.; Pócsfalvi, Gabriella; Andersson, Maria A.; Salkinoja-Salonen, Mirja S.

2004-01-01

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 μg [dry weight] ml of extended boar semen−1). The same exposure concentration depleted the boar sperm cells of NADH2. Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C18 cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Δψm) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C18 cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS. PMID:15294840

Rapid synthesis of maleimide functionalized fluorine-18 labeled prosthetic group using "radio-fluorination on the Sep-Pak" method.

PubMed

Basuli, Falguni; Zhang, Xiang; Jagoda, Elaine M; Choyke, Peter L; Swenson, Rolf E

2018-06-30

Following our recently published fluorine-18 labeling method, "Radio-fluorination on the Sep-Pak", we have successfully synthesized 6-[ 18 F]fluoronicotinaldehyde by passing a solution (1:4 acetonitrile: t-butanol) of its quaternary ammonium salt precursor, 6-(N,N,N-trimethylamino)nicotinaldehyde trifluoromethanesulfonate (2), through a fluorine-18 containing anion exchange cartridge (PS-HCO 3 ). Over 80% radiochemical conversion was observed using 10 mg of precursor within 1 minute. The [ 18 F]fluoronicotinaldehyde ([ 18 F]5) was then conjugated with 1-(6-(aminooxy)hexyl)-1H-pyrrole-2,5-dione to prepare the fluorine-18 labeled maleimide functionalized prosthetic group, 6-[ 18 F]fluoronicotinaldehyde O-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl) oxime, 6-[ 18 F]FPyMHO ([ 18 F]6). The current Sep-Pak method not only improves the overall radiochemical yield (50 ± 9%, decay-corrected, n = 9) but also significantly reduces the synthesis time (from 60-90 minutes to 30 minutes) when compared with literature methods for the synthesis of similar prosthetic groups. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Proteolytic cleavage and activation of PAK2 during UV irradiation-induced apoptosis in A431 cells.

PubMed

Tang, T K; Chang, W C; Chan, W H; Yang, S D; Ni, M H; Yu, J S

1998-09-15

Exposure of mammalian cells to ultraviolet (UV) light elicits a cellular response and can also lead to apoptotic cell death. In this report, we show that a 36-kDa myelin basic protein (MBP) kinase detected by an in-gel kinase assay can be dramatically activated during the early stages of UV irradiation-triggered apoptosis of A431 cells. Immunoblot analysis revealed that this 36-kDa MBP kinase could be recognized by an antibody against the C-terminal regions of a family of p21Cdc42/Rac-activated kinases (PAKs). By using this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 as studying tools, we further demonstrated that UV irradiation caused cleavage of PAK2 to generate a 36-kDa C-terminal catalytic fragment and a 30-kDa N-terminal fragment in A431 cells. The appearance of the 36-kDa C-terminal catalytic fragment of PAK2 matched exactly with the activation of the 36-kDa MBP kinase in A431 cells upon UV irradiation. In addition, UV irradiation also led to activation of CPP32/caspase-3, but not ICH-1L/caspase-2 and ICE/caspase-1, in A431 cells and the kinetics of activation of CPP32/caspase-3 appeared to correlate well with that of DNA fragmentation and of cleavage/activation of PAK2, respectively. Moreover, blockage of activation of CPP32/caspase-3 by pretreating the cells with two specific tetrapeptidic inhibitors for caspases (Ac-DEVD-cho and Ac-YVAD-cmk) could significantly attenuate the extent of cleavage/activation of PAK2 induced by UV irradiation. Collectively, the results demonstrate that cleavage and activation of PAK2 can be induced during the early stages of UV irradiation-triggered apoptosis and indicate the involvement of CPP32/caspase-3 in this process.

Dermoscopic clues to differentiate facial lentigo maligna from pigmented actinic keratosis.

PubMed

Lallas, A; Tschandl, P; Kyrgidis, A; Stolz, W; Rabinovitz, H; Cameron, A; Gourhant, J Y; Giacomel, J; Kittler, H; Muir, J; Argenziano, G; Hofmann-Wellenhof, R; Zalaudek, I

2016-05-01

Dermoscopy is limited in differentiating accurately between pigmented lentigo maligna (LM) and pigmented actinic keratosis (PAK). This might be related to the fact that most studies have focused on pigmented criteria only, without considering additional recognizable features. To investigate the diagnostic accuracy of established dermoscopic criteria for pigmented LM and PAK, but including in the evaluation features previously associated with nonpigmented facial actinic keratosis. Retrospectively enrolled cases of histopathologically diagnosed LM, PAK and solar lentigo/early seborrhoeic keratosis (SL/SK) were dermoscopically evaluated for the presence of predefined criteria. Univariate and multivariate regression analyses were performed and receiver operating characteristic curves were used. The study sample consisted of 70 LMs, 56 PAKs and 18 SL/SKs. In a multivariate analysis, the most potent predictors of LM were grey rhomboids (sixfold increased probability of LM), nonevident follicles (fourfold) and intense pigmentation (twofold). In contrast, white circles, scales and red colour were significantly correlated with PAK, posing a 14-fold, eightfold and fourfold probability for PAK, respectively. The absence of evident follicles also represented a frequent LM criterion, characterizing 71% of LMs. White and evident follicles, scales and red colour represent significant diagnostic clues for PAK. Conversely, intense pigmentation and grey rhomboidal lines appear highly suggestive of LM. © 2015 British Association of Dermatologists.

Metagenomes of complex microbial consortia derived from different soils as sources for novel genes conferring formation of carbonyls from short-chain polyols on Escherichia coli.

PubMed

Knietsch, Anja; Waschkowitz, Tanja; Bowien, Susanne; Henne, Anke; Daniel, Rolf

2003-01-01

Metagenomic DNA libraries from three different soil samples (meadow, sugar beet field, cropland) were constructed. The three unamplified libraries comprised approximately 1267000 independent clones and harbored approximately 4.05 Gbp of environmental DNA. Approximately 300000 recombinant Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from short-chain (C2 to C4) polyols such as 1,2-ethanediol, 2,3-butanediol, and a mixture of glycerol and 1,2-propanediol on indicator agar. Twenty-four positive E. COLI clones were obtained during the initial screen. Fifteen of them contained recombinant plasmids, designated pAK201-215, which conferred a stable carbonyl-forming phenotype on E. coli Sequencing revealed that the inserts of pAK201-215 encoded 26 complete and 14 incomplete predicted protein-encoding genes. Most of these genes were similar to genes with unknown functions from other microorganisms or unrelated to any other known gene. The further analysis was focused on the 7 plasmids (pAK204, pAK206, pAK208, and pAK210-213) recovered from the positive clones, which exhibited an NAD(H)-dependent alcohol oxidoreductase activity with polyols or the correlating carbonyls as substrates in crude extracts. Three genes (ORF6, ORF24, and ORF25) conferring this activity were identified during subcloning of the inserts of pAK204, pAK211, and pAK212. The sequences of the three deduced gene products revealed no significant similarities to known alcohol oxidoreductases, but contained putative glycine-rich regions, which are characteristic for binding of nicotinamide cofactors. Copyright 2003 S. Karger AG, Basel

Defense Against Threat. Threat Recognition and Analysis Project

DTIC Science & Technology

1975-09-01

but clustering around it ( land affecting it) are other action structures of different character, now entering strongly on the world scene...seeks .o cap ur ^^’^^try to gestalts of cognition and affect , ^[Jj^ f c!anrPr or country, that put the signature of...TRAMSPORT 8, PETROL PRODUCTS 7.*, USR 27, JAP 18, IND Ik, - FRUITS +NUTS Ik, NATURAL GAS 17, LAMBSKINS 12, USR 30, PAK

Routine production of [(18)f]flumazenil from iodonium tosylate using a sample pretreatment method: a 2.5-year production report.

PubMed

Moon, Byung Seok; Park, Jun Hyung; Lee, Hong Jin; Lee, Byung Chul; Kim, Sang Eun

2014-10-01

[(18)F]Flumazenil, which has the advantage of a longer half-life than [(11)C]flumazenil, is well known for determining of the central benzodiazepine receptor concentrations. However, [(18)F]flumazenil has not been widely used because fluctuating and relatively low yields render automatic production insufficient for routine and multicenter clinical trials. Here, we describe the results of a 2.5-year production study of [(18)F]flumazenil using an iodonium tosylate precursor, which allowed us to overcome the limitations of low and fluctuating radiochemical yields. We developed a clinically applicable production system by modifying a commercial synthesizer for the reliable and reproducible production of [(18)F]flumazenil for routine clinical studies. [(18)F]Flumazenil was prepared at 150 °C for 5 min in the presence of 4-methylphenyl-mazenil iodonium tosylate (4 mg), a radical scavenger (TEMPO, 1 mg), and [(18)F]KF/kryptofix 2.2.2 complex in N,N-dimethylformamide (1 ml). In the purification step, the final mixture was pretreated using different cartridges before performing high-performance liquid chromatography (HPLC) separation. Finally, we measured the radiochemical yield and performed quality-control assays on 94 batches. After carrying out additional purification before HPLC separation using a C18 plus Sep-Pak cartridge, the radiochemical yield of [(18)F]flumazenil increased from 34.4 ± 9.7 % (without the pretreatment, n = 24) to 53.4 ± 9.0 % (n = 94), and the lifetime of the semi-preparative column was five times that of the column without the C18 plus Sep-Pak cartridge. The mean-specific activity of [(18)F]flumazenil was 572 ± 116 GBq/μmol at the end of synthesis, and the radiochemical purity was more than 99 %, as determined by analytical HPLC and radio-TLC. [(18)F]Flumazenil prepared using this method satisfied all quality-control test standards and was highly stable for up to 6 h after preparation. The results of the 2.5-year production study using an iodonium tosylate precursor indicate that [(18)F]flumazenil has commercial and routine clinical applicability.

Flagellin inhibits Myoviridae phage phiCTX infection of Pseudomonas aeruginosa strain GuA18: purification and mapping of binding site.

PubMed

Geiben-Lynn, R; Sauber, K; Lutz, F

2001-11-01

PhiCTX is a double-stranded DNA phage of the Myoviridae family that converts Pseudomonas aeruginosa into a cytotoxin producer. A 42-kDa phiCTX-inhibiting protein was purified from the outer membrane fraction of P. aeruginosa strain GuA18 by octyl-beta-glucoside extraction, DEAE-chromatography, and mono-Q HPLC. This protein had an isoelectric point of 5.4 and bound specifically [125I]-labeled phiCTX. The N-terminal amino acid sequence of six out of seven Lys-C fragments was highly similar (87%) to that of the entire of type-a flagellin of P. aeruginosa strain PAK. At a concentration of 14 nM, purified flagellin protein caused a 50% decrease in the phage titer after a 20-min incubation at 37 degrees C (PhI50). The presence of ethanol was necessary to reconstitute the inhibitory activity. In contrast, no ethanol treatment was necessary for the inhibitory activity of the sheared flagellin filaments from P. aeruginosa strain GuA18, which consists of the 42-kDa flagellin subunits and the synthesized 17-mer phage-binding-peptide NGSNSDSERTALNGEAK, representing flagellin residues 100-116 of P. aeruginosa strain PAK. The PhI50 was 10 nM and 200 nM, respectively. Antisera against the flagellin filament protein as well as against the 17-mer peptide neutralized phage infection. These results indicated that the amino acid region 100-116 of the flagellin subunit of strain GuA18 is involved in phiCTX binding. This region might play a role in phage attachment.

Comparison of the SidePak personal monitor with the Aerosol Particle Sizer (APS).

PubMed

Sánchez Jiménez, Araceli; van Tongeren, Martie; Galea, Karen S; Steinsvåg, Kjersti; MacCalman, Laura; Cherrie, John W

2011-06-01

The aim of this study was to compare the performance of the TSI Aerodynamic Particle Sizer (APS) and the TSI portable photometer SidePak to measure airborne oil mist particulate matter (PM) with aerodynamic diameters below 10 μm, 2.5 μm and 1 μm (PM(10), PM(2.5) and PM(1)). Three SidePaks each fitted with either a PM(10), PM(2.5) or a PM(1) impactor and an APS were run side by side in a controlled chamber. Oil mist from two different mineral oils and two different drilling fluid systems commonly used in offshore drilling technologies were generated using a nebulizer. Compared to the APS, the SidePaks overestimated the concentration of PM(10) and PM(2.5) by one order of magnitude and PM(1) concentrations by two orders of magnitude after exposure to oil mist for 3.3-6.5 min at concentrations ranging from 0.003 to 18.1 mg m(-3) for PM(10), 0.002 to 3.96 mg m(-3) for PM(2.5) and 0.001 to 0.418 mg m(-3) for PM(1) (as measured by the APS). In a second experiment a SidePak monitor previously exposed to oil mist overestimated PM(10) concentrations by 27% compared to measurements from another SidePak never exposed to oil mist. This could be a result of condensation of oil mist droplets in the optical system of the SidePak. The SidePak is a very useful instrument for personal monitoring in occupational hygiene due to its light weight and quiet pump. However, it may not be suitable for the measurement of particle concentrations from oil mist.

Transcriptional regulation of ataxia-telangiectasia and Rad3-related protein by activated p21-activated kinase-1 protects keratinocytes in UV-B-induced premalignant skin lesions.

PubMed

Beesetti, S; Mavuluri, J; Surabhi, R P; Oberyszyn, T M; Tober, K; Pitani, R S; Joseph, L D; Venkatraman, G; Rayala, S K

2017-11-02

Sun-induced skin lesions, in particular actinic keratosis, are generally considered as premalignant skin lesions that can progress into squamous cell carcinoma (SCC) and invasive SCC if left untreated. Therefore, understanding the molecular mechanisms by which the ultraviolet-B (UV-B)-exposed cells are being protected and the signaling pathways that promote the progression of certain premalignant skin lesions to malignant lesions will permit us to prevent or cure skin cancers. In the current study, we found that phospho-p21-activated kinase-1 (Pak1) and Pak1 expression was high in clinical samples of sunlight-induced premalignant skin lesions assessed by immunohistochemistry. Further, we observed that phospho-Pak1 and Pak1 levels are high in UV-B-exposed hairless SKH mouse model skin samples as compared with unexposed skin tissue. Our results from cell line and animal models showed that Pak1 is activated in response to UV-B radiation, and this activated Pak1 translocates from the cytoplasm to the nucleus. Inside the nucleus, Pak1 via C-Fos binds to a specific promoter region of DNA repair kinase ATR (ataxia-telangiectasia and Rad3-related protein) and acts as a transcriptional regulator of ATR. Results from our analysis showed that Pak1 overexpression, knockdown and Pak1 knockout cell line models showed that Pak1 confers protection to keratinocytes from UV-B-induced apoptosis and DNA damage via ATR. To our knowledge, this is the first study that evaluates the functional and clinical significance of a signaling molecule, Pak1, in sun-induced premalignant skin lesions and indicates that increased Pak1 activation and expression could serve as an early warning sign of progression toward non-melanoma skin cancer, if ignored.

Increased expression of microRNA-221 inhibits PAK1 in endothelial progenitor cells and impairs its function via c-Raf/MEK/ERK pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Xiaoping; Mao, Haian; Chen, Jin-yuan

2013-02-15

Highlights: ► MicroRNA-221 is upregulated in the endothelial progenitor cells of atherosclerosis patients. ► PAK1 is a direct target of microRNA-221. ► MicroRNA-221 inhibits EPCs proliferation through c-Raf/MEK/ERK pathway. -- Abstract: Coronary artery disease (CAD) is associated with high mortality and occurs via endothelial injury. Endothelial progenitor cells (EPCs) restore the integrity of the endothelium and protect it from atherosclerosis. In this study, we compared the expression of microRNAs (miRNAs) in EPCs in atherosclerosis patients and normal controls. We found that miR-221 expression was significantly up-regulated in patients compared with controls. We predicted and identified p21/Cdc42/Rac1-activated kinase 1 (PAK1) asmore » a novel target of miR-221 in EPCs. We also demonstrated that miR-221 targeted a putative binding site in the 3′UTR of PAK1, and absence of this site was inversely associated with miR-221 expression in EPCs. We confirmed this relationship using a luciferase reporter assay. Furthermore, overexpression of miR-221 in EPCs significantly decreased EPC proliferation, in accordance with the inhibitory effects induced by decreased PAK1. Overall, these findings demonstrate that miR-221 affects the MEK/ERK pathway by targeting PAK1 to inhibit the proliferation of EPCs.« less

[Determination of azoxystrobin in tea by HPLC].

PubMed

Chonan, T

2001-08-01

A determination method has been developed for azoxystrobin in tea by HPLC. Azoxystrobin was extracted from a sample with acetone, and the extract was passed through an alumina column to remove tannin. The eluate was concentrated to ca. 25 mL and passed through a Sep-Pak Vac tC18 to remove pigments. The eluate was cleaned-up by using liquid-liquid partition, and Florisil and silica-gel columns. The HPLC analysis for azoxystrobin was carried out on a C18 column with acetonitrile-water (9:11) as the mobile phase, with ultraviolet detection at 260 nm. The recovery of azoxystrobin fortified at the level of 0.4 microgram/g was 90.2% and the limit of determination was 0.2 microgram/g.

Determination of phosphatidylethanolamine molecular species in various food matrices by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS2).

PubMed

Zhou, Li; Zhao, Minjie; Ennahar, Saïd; Bindler, Françoise; Marchioni, Eric

2012-04-01

A liquid chromatographic-electrospray ionization-tandem mass spectrometric (LC-ESI-MS(2)) method has been developed for determination of the molecular species of phosphatidylethanolamine (PE) in four food matrices (soy, egg yolk, ox liver, and krill oil). The extraction and purification method consisted of a pressurized liquid extraction procedure for total lipid (TL) extraction, purification of phospholipids (PLs) by adsorption on a silica gel column, and separation of PL classes by semi-preparative normal-phase HPLC. Separation and identification of PE molecular species were performed by reversed-phase HPLC coupled with electrospray ionization tandem mass spectrometry (ESI-MS(2)). Methanol containing 5 mmol L(-1) ammonium formate was used as the mobile phase. A variety of PE molecular species were detected in the four food matrices. (C16:0-C18:2)PE, (C18:2-C18:2)PE, and (C16:0-C18:1)PE were the major PE molecular species in soy. Egg yolk PE contained (C16:0-C18:1)PE, (C18:0-C18:1)PE, (C18:0-C18:2)PE, and (C16:0-C18:2)PE as the major molecular species. Ox liver PE was rich in the species (C18:0-C18:1)PE, (C18:0-C20:4)PE, and (C18:0-C18:2)PE. Finally, krill oil which was particularly rich in (C16:0(alkyl)-C22:6(acyl))plasmanylethanolamine (PakE), (C16:0-C22:6)PE, and (C16:0-C20:5)PE, seemed to be an interesting potential source for supplementation of food with eicosapentaenoic acid and docosahexaenoic acid.

Quantitative determination of regorafenib and its two major metabolites in human plasma with high-performance liquid chromatography and ultraviolet detection.

PubMed

Fujita, Kazuma; Miura, Masatomo; Shibata, Hiroyuki

2016-10-01

A simple, highly sensitive and specific high-performance liquid chromatography (HPLC) method was developed for the simultaneous quantitation of regorafenib, N-oxidemetabolite (M-2) and the desmethyl N-oxide metabolite (M-5) in human plasma. Regorafenib, M-2, M-5 and the internal standard sorafenib were separated using a mobile phase of 0.5% KH2 PO4 (pH 3.5)-acetonitrile (30:70, v/v), on a Capcell PAK MG II column at a flow rate of 0.5 mL/min and measurement at UV 260 nm. The lower limits of quantification for regorafenib, M-2 and M-5 were 10 ng/mL for each analyte. A procedure using solid-phase extraction required only a small amount of plasma (100 μL) for one analysis while providing high extraction recovery (>81% for all compounds) and good selectivity. Coefficients of variation for intra- and inter-day assays were <12.2% for regorafenib, <12.3% for M-2 and <15.1% for M-5. Accuracies for intra- and inter-day assays were <9.4% for regorafenib, <8.0% for M-2 and <12.8% for M-5 over a linear range from 10 to 10,000 ng/mL. This HPLC assay is suitable for clinical pharmacokinetic studies of regorafenib. The present HPLC method is currently in use for our observational studies of patients under treatment. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-08-15

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both autophosphorylation/activation and protein phosphatase-mediated dephosphorylation/inactivation processes. Taken together, our results identify Thr402 as the regulatory autophosphorylation site of auto-kinase, which is a C-terminal catalytic fragment of PAK2.

Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

PubMed Central

Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

1998-01-01

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both autophosphorylation/activation and protein phosphatase-mediated dephosphorylation/inactivation processes. Taken together, our results identify Thr402 as the regulatory autophosphorylation site of auto-kinase, which is a C-terminal catalytic fragment of PAK2. PMID:9693111

Determination of bisphenol A in human serum by high-performance liquid chromatography with multi-electrode electrochemical detection.

PubMed

Inoue, K; Kato, K; Yoshimura, Y; Makino, T; Nakazawa, H

2000-11-10

A simple and sensitive method using high-performance liquid chromatography with multi-electrode electrochemical detection (HPLC-ED) including a coulometric array of four electrochemical sensors has been developed for the determination of bisphenol A in water and human serum. For good separation and detection of bisphenol A, a CAPCELL PAK UG 120 C18 reversed-phase column and a mobile phase consisting of 0.3% phosphoric acid-acetonitrile (60:40) were used. The detection limit obtained by the HPLC-ED method was 0.01 ng/ml (0.5 pg), which was more than 3000-times higher than the detection limit obtained by the ultraviolet (UV) method, and more than 200-times higher than the detection limit obtained by the fluorescence (FL) method. Bisphenol A in water and serum samples was pretreated by solid-phase extraction (SPE) after removing possible contamination derived from a plastic SPE cartridges and water used for the pretreatment. A trace amount (ND approximately 0.013 ng/ml) of bisphenol A was detected from the parts of cartridges (filtration column, sorbent bed and frits) by extraction with methanol, and it was completely removed by washing with at least 15 ml of methanol in the operation process. The concentrations of bisphenol A in tap water and Milli-Q-purified water were found to be 0.01 and 0.02 ng/ml, respectively. For that reason, bisphenol A-free water was made to trap bisphenol A in water using an Empore disk. In every pretreatment, SPE methods using bisphenol A-free water and washing with 15 ml of methanol were done in water and serum samples. The yields obtained from the recovery tests using water to which 0.5 or 0.05 ng/ml of bisphenol A was added were 83.8 to 98.2%, and the RSDs were 3.4 to 6.1%, respectively. The yields obtained from the recovery tests by OASIS HLB using serum to which 1.0 ng/ml or 0.1 ng/ml of bisphenol A was added were 79.0% and 87.3%, and the RSDs were 5.1% and 13.5%, respectively. The limits of quantification in water and serum sample were 0.01 ng/ml and 0.05 ng/ml, respectively. The method was applied to the determination of bisphenol A in healthy human serum sample, and the obtained detection was 0.32 ng/ml. From these results, the HPLC-ED method should be the most useful in the determination of bisphenol A at low concentration levels in water and biological samples.

Separation and purification of astaxanthin from Phaffia rhodozyma by preparative high-speed counter-current chromatography.

PubMed

Du, Xiping; Dong, Congcong; Wang, Kai; Jiang, Zedong; Chen, Yanhong; Yang, Yuanfan; Chen, Feng; Ni, Hui

2016-09-01

An effective high-speed counter-current chromatography (HSCCC) method was established for the preparative isolation and purification of astaxanthin from Phaffia rhodozyma. With a two-phase solvent system composed of n-hexane-acetone-ethanol-water (1:1:1:1, v/v/v/v), 100mg crude extract of P. rhodozyma was separated to yield 20.6mg of astaxanthin at 92.0% purity. By further one step silica gel column chromatography, the purity reached 99.0%. The chemical structure of astaxanthin was confirmed by thin layer chromatography (TLC), UV spectroscopy scanning, high performance liquid chromatography with a ZORBAX SB-C18 column and a Waters Nova-pak C18 column, and ESI/MS/MS. Copyright © 2016 Elsevier B.V. All rights reserved.

A new screening method for amphetamine and methamphetamine using dansyl chloride derivatization and cartridge fluorescence.

PubMed

Yamada, H; Ikeda-Wada, S; Oguri, K

1998-07-01

A new screening method for amphetamines was developed. It consists of derivatization with dansyl chloride, extraction of the derivative using a Sep-Pak C18 or a Bond Elut C18, solid phase extraction columns, and visualization of the fluorescence of the cartridge. A control test using drug-free urine showed no fluorescence. Amphetamine, methamphetamine and the methylenedioxy derivatives exhibited strong fluorescence, while related compounds, such as N-ethylamphetamine and fenetylline, were negative or weakly positive. The disadvantage of the present method is that it is a multi-step procedure and 20-30 min is required for screening. However, since it has a different specificity from the widely used immunochemical technique, it is suggested to be a useful screen for amphetamines.

GIT1/βPIX signaling proteins and PAK1 kinase regulate microtubule nucleation.

PubMed

Černohorská, Markéta; Sulimenko, Vadym; Hájková, Zuzana; Sulimenko, Tetyana; Sládková, Vladimíra; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

2016-06-01

Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (βPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, βPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of βPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and βPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and βPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, βPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and βPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of βPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/βPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Simultaneous Determination of Decursin, Decursinol Angelate, Nodakenin, and Decursinol of Angelica gigas Nakai in Human Plasma by UHPLC-MS/MS: Application to Pharmacokinetic Study.

PubMed

Kim, Sook-Jin; Ko, Se-Mi; Choi, Eun-Jeong; Ham, Seong-Ho; Kwon, Young-Dal; Lee, Yong-Bok; Cho, Hea-Young

2018-04-26

Coumarins in Cham-dang-gwi, the dried root of Angelica gigas Nakai (AGN), possess pharmacological effects on anemia, pain, infection, and articular rheumatism. The AGN root containes decursin (D), decursinol angelate (DA), nodakenin, and decursinol (DOH), a major metabolite of D and DA. The aim of this study was to develop a simultaneous determination method for these four coumarins in human plasma using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Chromatographic separation was performed on dual columns (Kinetex ® C 18 column and Capcell core C 18 column) with mobile phase consisting of water and acetonitrile at a flow rate of 0.3 mL/min using gradient elution. Multiple reaction monitoring was operated in positive ion mode with precursors to product ion transition values of m / z 328.9→228.8, 328.9→228.9, 409.4→248.8, and 246.8→212.9 to measure D, DA, nodakenin, and DOH, respectively. Linear calibration curves were fitted over concentration range of 0.05⁻50 ng/mL for these four components, with correlation coefficient greater than 0.995. Inter- and intra-day accuracies were between 90.60% and 108.24%. These precisions were within 11.19% for all components. The established method was then applied to a pharmacokinetic study for the four coumarins after usual dosing in Korean subjects.

PAK1 translocates into nucleus in response to prolactin but not to estrogen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oladimeji, Peter, E-mail: Peter.Oladimeji@rockets.utoledo.edu; Diakonova, Maria, E-mail: mdiakon@utnet.utoledo.edu

2016-04-22

Tyrosyl phosphorylation of the p21-activated serine–threonine kinase 1 (PAK1) has an essential role in regulating PAK1 functions in breast cancer cells. We previously demonstrated that PAK1 serves as a common node for estrogen (E2)- and prolactin (PRL)-dependent pathways. We hypothesize herein that intracellular localization of PAK1 is affected by PRL and E2 treatments differently. We demonstrate by immunocytochemical analysis that PAK1 nuclear translocation is ligand-dependent: only PRL but not E2 stimulated PAK1 nuclear translocation. Tyrosyl phosphorylation of PAK1 is essential for this nuclear translocation because phospho-tyrosyl-deficient PAK1 Y3F mutant is retained in the cytoplasm in response to PRL. We confirmedmore » these data by Western blot analysis of subcellular fractions. In 30 min of PRL treatment, only 48% of pTyr-PAK1 is retained in the cytoplasm of PAK1 WT clone while 52% re-distributes into the nucleus and pTyr-PAK1 shuttles back to the cytoplasm by 60 min of PRL treatment. In contrast, PAK1 Y3F is retained in the cytoplasm. E2 treatment causes nuclear translocation of neither PAK1 WT nor PAK1 Y3F. Finally, we show by an in vitro kinase assay that PRL but not E2 stimulates PAK1 kinase activity in the nuclear fraction. Thus, PAK1 nuclear translocation is ligand-dependent: PRL activates PAK1 and induces translocation of activated pTyr-PAK1 into nucleus while E2 activates pTyr-PAK1 only in the cytoplasm. - Highlights: • Prolactin but not estrogen causes translocation of PAK1 into nucleus. • Tyrosyl phosphorylation of PAK1 is required for nuclear localization. • Prolactin but not estrogen stimulates PAK1 kinase activity in nucleus.« less

Group II p21-activated kinases as therapeutic targets in gastrointestinal cancer.

PubMed

Shao, Yang-Guang; Ning, Ke; Li, Feng

2016-01-21

P21-activated kinases (PAKs) are central players in various oncogenic signaling pathways. The six PAK family members are classified into group I (PAK1-3) and group II (PAK4-6). Focus is currently shifting from group I PAKs to group II PAKs. Group II PAKs play important roles in many fundamental cellular processes, some of which have particular significance in the development and progression of cancer. Because of their important functions, group II PAKs have become popular potential drug target candidates. However, few group II PAKs inhibitors have been reported, and most do not exhibit satisfactory kinase selectivity and "drug-like" properties. Isoform- and kinase-selective PAK inhibitors remain to be developed. This review describes the biological activities of group II PAKs, the importance of group II PAKs in the development and progression of gastrointestinal cancer, and small-molecule inhibitors of group II PAKs for the treatment of cancer.

Functional PAK-2 knockout and replacement with a caspase cleavage-deficient mutant in mice reveals differential requirements of full-length PAK-2 and caspase-activated PAK-2p34.

PubMed

Marlin, Jerry W; Chang, Yu-Wen E; Ober, Margaret; Handy, Amy; Xu, Wenhao; Jakobi, Rolf

2011-06-01

p21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.

P21-activated kinase in inflammatory and cardiovascular disease.

PubMed

Taglieri, Domenico M; Ushio-Fukai, Masuko; Monasky, Michelle M

2014-09-01

P-21 activated kinases, or PAKs, are serine-threonine kinases that serve a role in diverse biological functions and organ system diseases. Although PAK signaling has been the focus of many investigations, still our understanding of the role of PAK in inflammation is incomplete. This review consolidates what is known about PAK1 across several cell types, highlighting the role of PAK1 and PAK2 in inflammation in relation to NADPH oxidase activation. This review explores the physiological functions of PAK during inflammation, the role of PAK in several organ diseases with an emphasis on cardiovascular disease, and the PAK signaling pathway, including activators and targets of PAK. Also, we discuss PAK1 as a pharmacological anti-inflammatory target, explore the potentials and the limitations of the current pharmacological tools to regulate PAK1 activity during inflammation, and provide indications for future research. We conclude that a vast amount of evidence supports the idea that PAK is a central molecule in inflammatory signaling, thus making PAK1 itself a promising prospective pharmacological target. Copyright © 2014 Elsevier Inc. All rights reserved.

Role of Rac1 in Escherichia coli K1 invasion of human brain microvascular endothelial cells.

PubMed

Rudrabhatla, Rajyalakshmi S; Selvaraj, Suresh K; Prasadarao, Nemani V

2006-02-01

Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) requires the reorganization of host cytoskeleton at the sites of bacterial entry. Both actin and myosin constitute the cytoskeletal architecture. We have previously shown that myosin light chain (MLC) phosphorylation by MLC kinase is regulated during E. coli invasion by an upstream kinase, p21-activated kinase 1 (PAK1), which is an effector protein of Rac and Cdc42 GTPases, but not of RhoA. Here, we report that the binding of only Rac1 to PAK1 decreases in HBMEC upon infection with E. coli K1, which resulted in increased phosphorylation of MLC. Overexpression of a constitutively active (cAc) form of Rac1 in HBMEC blocked the E. coli invasion significantly, whereas overexpression of a dominant negative form had no effect. Increased PAK1 phosphorylation was observed in HBMEC expressing cAc-Rac1 with a concomitant reduction in the phosphorylation of MLC. Immunocytochemistry studies demonstrated that the inhibition of E. coli invasion into cAc-Rac1/HBMEC is due to lack of phospho-MLC recruitment to the sites of E. coli entry. Taken together the data suggest that E. coli modulates the binding of Rac1, but not Cdc42, to PAK1 during the invasion of HBMEC.

Simultaneous determination of carboprost methylate and its active metabolite carboprost in dog plasma by liquid chromatography-tandem mass spectrometry with positive/negative ion-switching electrospray ionization and its application to a pharmacokinetic study.

PubMed

Yin, Lei; Meng, Xiangjun; Zhou, Xiaotong; Zhang, Tinglan; Sun, Heping; Yang, Zhichao; Yang, Bo; Xiao, Ning; Fawcett, J Paul; Yang, Yan; Gu, Jingkai

2015-08-15

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method using positive/negative electrospray ionization (ESI) switching for the simultaneous quantitation of carboprost methylate and carboprost in dog plasma has been developed and validated. After screening, the esterase inhibitor, dichlorvos was added to the whole blood at a ratio of 1:99 (v/v) to stabilize carboprost methylate during blood collection, sample storage and LLE. Indomethacin was added to plasma to inhibit prostaglandins synthesis after sampling. After liquid-liquid extraction of 500μL plasma with ethyl ether-dichloromethane (75:25, v/v), analytes and internal standard (IS), alprostadil-d4, were chromatographed on a CAPCELL PAK Phenyl column (150×2.0mm, 5μm) using acetonitrile-5mM ammonium acetate as mobile phase. Carboprost methylate was detected by positive ion electrospray ionization followed by multiple reaction monitoring (MRM) of the transition at m/z 400.5→329.3; the carboprost and IS were detected by negative ion electrospray ionization followed by MRM of the transitions at m/z 367.2→323.2, and 357.1→321.2, respectively. The method was linear for both analytes in the concentration range 0.05-30ng/mL with intra- and inter-day precisions (as relative standard deviation) of ≤6.75% and accuracy (as relative error) of ≤7.21% and limit of detection (LOD) values were 10 and 20pg/mL, respectively. The method was successfully applied to a pharmacokinetic study of the analytes in beagle dogs after intravaginal administration of a suppository containing 0.5mg carboprost methylate. Copyright © 2015 Elsevier B.V. All rights reserved.

Phosphorylation of tyrosine 285 of PAK1 facilitates βPIX/GIT1 binding and adhesion turnover

PubMed Central

Hammer, Alan; Oladimeji, Peter; De Las Casas, Luis E.; Diakonova, Maria

2015-01-01

The p21-activated serine-threonine kinase (PAK1) regulates cell motility and adhesion. We have previously shown that the prolactin (PRL)-activated tyrosine kinase JAK2 phosphorylates PAK1 in vivo and in vitro and identified tyrosines 153, 201, and 285 in PAK1 as sites of JAK2 tyrosyl phosphorylation. Here, we further investigate the role of the tyrosyl phosphorylated PAK1 (pTyr-PAK1) in regulation of cell adhesion. We use human breast cancer T47D cell lines that stably overexpress PAK1 wild type or PAK1 Y3F mutant in which these 3 JAK2 phosphorylation sites were mutated to phenylalanine. We demonstrate that PRL/JAK2-dependent phosphorylation of these tyrosines promotes a motile phenotype in the cells upon adhesion, participates in regulation of cell adhesion on collagen IV, and is required for maximal PAK1 kinase activity. Down-regulation of PAK1 abolishes the effect of PAK1 on cell adhesion. We show that the tyrosyl phosphorylation of PAK1 promotes PAK1 binding to β-PAK1-interacting guanine-nucleotide exchange factor (βPIX) and G protein-coupled receptor kinase-interacting target 1 (GIT1), phosphorylation of paxillin on Ser273, and formation and distribution of adhesion complexes. Using phosphospecific antibodies (Abs) directed to single phosphorylated tyrosines on PAK1, we identified Tyr285 as a site of PRL-dependent phosphorylation of PAK1 by JAK2. Furthermore, using PAK1 Y285F mutant, we provide evidence for a role of pTyr285 in cell adhesion, enhanced βPIX/GIT1 binding, and adhesion turnover. Our immunohistochemistry analysis demonstrates that pTyr285- PAK1 may modulate PAK1 signaling during tumor progression.—Hammer, A., Oladimeji, P., De La Casas, L. E., Diakonova, M. Phosphorylation of tyrosine 285 of PAK1 facilitates bPIX/GIT1 binding and adhesion turnover. PMID:25466889

Rac1-PAK2 pathway is essential for zebrafish heart regeneration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Xiangwen; He, Quanze; Li, Guobao

P-21 activated kinases, or PAKs, are serine–threonine kinases that play important roles in diverse heart functions include heart development, cardiovascular development and function in a range of models; however, the mechanisms by which PAKs mediate heart regeneration are unknown. Here, we demonstrate that PAK2 and PAK4 expression is induced in cardiomyocytes and vessels, respectively, following zebrafish heart injury. Inhibition of PAK2 and PAK4 using a specific small molecule inhibitor impedes cardiomyocyte proliferation/dedifferentiation and cardiovascular regeneration, respectively. Cdc42 is specifically expressed in the ventricle and may function upstream of PAK2 but not PAK4 under normal conditions and that cardiomyocyte proliferentation duringmore » heart regeneration relies on Rac1-mediated activation of Pak2. Our results indicate that PAKs play a key role in heart regeneration.« less

PAK5, a New Brain-Specific Kinase, Promotes Neurite Outgrowth in N1E-115 Cells

PubMed Central

Dan, Chuntao; Nath, Niharika; Liberto, Muriel; Minden, Audrey

2002-01-01

We have characterized a new member of the mammalian PAK family of serine/threonine kinases, PAK5, which is a novel target of the Rho GTPases Cdc42 and Rac. The kinase domain and GTPase-binding domain (GBD) of PAK5 are most closely related in sequence to those of mammalian PAK4. Outside of these domains, however, PAK5 is completely different in sequence from any known mammalian proteins. PAK5 does share considerable sequence homology with the Drosophila MBT protein (for “mushroom body tiny”), however, which is thought to play a role in development of cells in Drosophila brain. Interestingly, PAK5 is highly expressed in mammalian brain and is not expressed in most other tissues. We have found that PAK5, like Cdc42, promotes the induction of filopodia. In N1E-115 neuroblastoma cells, expression of PAK5 also triggered the induction of neurite-like processes, and a dominant-negative PAK5 mutant inhibited neurite outgrowth. Expression of activated PAK1 caused no noticeable changes in these cells. An activated mutant of PAK5 had an even more dramatic effect than wild-type PAK5, indicating that the morphologic changes induced by PAK5 are directly related to its kinase activity. Although PAK5 activates the JNK pathway, dominant-negative JNK did not inhibit neurite outgrowth. In contrast, the induction of neurites by PAK5 was abolished by expression of activated RhoA. Previous work has shown that Cdc42 and Rac promote neurite outgrowth by a pathway that is antagonistic to Rho. Our results suggest, therefore, that PAK5 operates downstream to Cdc42 and Rac and antagonizes Rho in the pathway, leading to neurite development. PMID:11756552

Signaling, Regulation, and Specificity of the Type II p21-activated Kinases.

PubMed

Ha, Byung Hak; Morse, Elizabeth M; Turk, Benjamin E; Boggon, Titus J

2015-05-22

The p21-activated kinases (PAKs) are a family of six serine/threonine kinases that act as key effectors of RHO family GTPases in mammalian cells. PAKs are subdivided into two groups: type I PAKs (PAK1, PAK2, and PAK3) and type II PAKs (PAK4, PAK5, and PAK6). Although these groups are involved in common signaling pathways, recent work indicates that the two groups have distinct modes of regulation and have both unique and common substrates. Here, we review recent insights into the molecular level details that govern regulation of type II PAK signaling. We also consider mechanisms by which signal transduction is regulated at the level of substrate specificity. Finally, we discuss the implications of these studies for clinical targeting of these kinases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Substance P Induces Rapid and Transient Membrane Blebbing in U373MG Cells in a p21-Activated Kinase-Dependent Manner

PubMed Central

Meshki, John; Douglas, Steven D.; Hu, Mingyue; Leeman, Susan E.; Tuluc, Florin

2011-01-01

U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R). Substance P (SP), the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK) signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK) is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells. PMID:21966499

P21 activated kinase signaling in cancer.

PubMed

Rane, Chetan K; Minden, Audrey

2018-01-09

The p21 Activated Kinases (PAKs) are a family of serine threonine kinases, that consist of 6 members, PAKs 1-6, which are positioned at an intersection of multiple signaling pathways implicated in oncogenesis. The PAKs were originally identified as protein kinases that function downstream of the Ras related Rho GTPases Cdc42 and Rac. PAK1 and PAK4, which belong to Group I and Group II PAKs, respectively, are most often associated with tumorigenesis. On account of their well characterized roles in cancer, several small molecule inhibitors are being developed to inhibit the PAKs, and there is interest in investigating their efficacy as either first line or adjuvant treatments for cancer. Studies to delineate PAK regulated signaling pathways as well as the long term effects of PAK overexpression on gene expression are beginning to shed light on the mechanism by which PAK proteins may lead to cancer when they are overexpressed or activated. This review will describe the association between PAK expression in cancer, with a focus on PAK1 and PAK4, which are most often associated with the disease. The current understanding of the molecular mechanisms by which the PAKs operate in cancer will be discussed. We will also review some of the potential drug candidates, and discuss which of them are currently being tested for their efficacy in cancer treatments. Copyright © 2018 Elsevier Ltd. All rights reserved.

NMR binding and crystal structure reveal that intrinsically-unstructured regulatory domain auto-inhibits PAK4 by a mechanism different from that of PAK1.

PubMed

Wang, Wei; Lim, Liangzhong; Baskaran, Yohendran; Manser, Ed; Song, Jianxing

2013-08-16

Six human PAK members are classified into groups I (PAKs 1-3) and II (PAK4-6). Previously, only group I PAKs were thought to be auto-inhibited but very recently PAK4, the prototype of group II PAKs, has also been shown to be auto-inhibited by its N-terminal regulatory domain. However, the complete auto-inhibitory domain (AID) sequence remains undefined and the mechanism underlying its auto-inhibition is largely elusive. Here, the N-terminal regulatory domain of PAK4 sufficient for auto-inhibiting and binding Cdc42/Rac was characterized to be intrinsically unstructured, but nevertheless we identified the entire AID sequence by NMR. Strikingly, an AID peptide was derived by deleting the binding-unnecessary residues, which has a Kd of 320 nM to the PAK4 catalytic domain. Consequently, the PAK4 crystal structure complexed with the entire AID has been determined, which reveals that the complete kinase cleft is occupied by 20 AID residuescomposed of an N-terminal α-helix and a previously-identified pseudosubstrate motif, thus achieving auto-inhibition. Our study reveals that PAK4 is auto-inhibited by a novel mechanism which is completely different from that for PAK1, thus bearing critical implications for design of inhibitors specific for group II PAKs. Copyright © 2013 Elsevier Inc. All rights reserved.

UnPAKing the class differences among p21-activated kinases.

PubMed

Eswaran, Jeyanthy; Soundararajan, Meera; Kumar, Rakesh; Knapp, Stefan

2008-08-01

The p21-activated kinases (PAKs) are signal transducers, central to many vital cellular processes, including cell morphology, motility, survival, gene transcription and hormone signalling. The mammalian PAK family contains six serine/threonine kinases divided into two subgroups, group I (PAK 1-3) and group II (PAK4-6), based on their domain architecture and regulation. PAKs functioning as dynamic signalling nodes present themselves as attractive therapeutic targets in tumours, neurological diseases and infection. The recent findings across all PAKs, including newly reported structures, shed light on the cellular functions of PAKs, highlighting molecular mechanisms of activation, catalysis and substrate specificity. We believe that a comprehensive understanding of the entire PAK family is essential for developing strategies towards PAK-targeted therapeutics.

Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization.

PubMed

Zhao, Miao; Spiess, Matthias; Johansson, Henrik J; Olofsson, Helene; Hu, Jianjiang; Lehtiö, Janne; Strömblad, Staffan

2017-09-29

p21-activated kinase 4 (PAK4) regulates cell proliferation, apoptosis, cell motility and F-actin remodeling, but the PAK4 interactome has not been systematically analyzed. Here, we comprehensively characterized the human PAK4 interactome by iTRAQ quantitative mass spectrometry of PAK4-immunoprecipitations. Consistent with its multiple reported functions, the PAK4 interactome was enriched in diverse protein networks, including the 14-3-3, proteasome, replication fork, CCT and Arp2/3 complexes. Because PAK4 co-immunoprecipitated most subunits of the Arp2/3 complex, we hypothesized that PAK4 may play a role in Arp2/3 dependent actin regulation. Indeed, we found that PAK4 interacts with and phosphorylates the nucleation promoting factor N-WASP at Ser484/Ser485 and promotes Arp2/3-dependent actin polymerization in vitro. Also, PAK4 ablation in vivo reduced N-WASP Ser484/Ser485 phosphorylation and altered the cellular balance between G- and F-actin as well as the actin organization. By presenting the PAK4 interactome, we here provide a powerful resource for further investigations and as proof of principle, we also indicate a novel mechanism by which PAK4 regulates actin cytoskeleton remodeling.

Determination of 17-oxosteroid glucuronides and sulfates in urine and serum by fluorescence high-performance liquid chromatography using dansyl hydrazine as a prelabeling reagent.

PubMed

Kawasaki, T; Maeda, M; Tsuji, A

1982-12-10

A fluorescence high-performance liquid chromatographic method is described for the direct determination of conjugated 17-oxosteroids in biological fluids without hydrolysis. Conjugated 17-oxosteroids are extracted with Sep-Pak C18 cartridge, labeled with dansyl hydrazine in trichloroacetic acid--benzene solution and then separated by high-performance liquid chromatography on reversed-phase muBondapak C18 column using 0.01 M sodium acetate in methanol-water-acetic acid (65:35:1, v/v) as the mobile phase. The eluate is monitored by a fluorophotometer at 365 nm (excitation) and 520 nm (emission). Linearities of fluorescence intensities (peak heights) with the amounts of various conjugated 17-oxosteroids were obtained between 10 pmol and 100 pmol. This method is sensitive, reliable and useful for the simultaneous determination of conjugated 17-oxosteroids in urine and serum.

Volatile organic compound measurements in the California/Mexico border region during SCOS97

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zielinska, B.; Sagebiel, J.; Uberna, E.

1999-07-01

Measurements of volatile organic compounds (VOC) were carried out in the California/Mexico border region during the Southern California Ozone study in the Summer of 1997 (SCOS97). Integrated 3-hr samples were collected in Rosarito (south of Tijuana, Mexico) and in Mexicali during Intensive Operational Periods (IOP), twice per IOP day. VOC were collected using stainless-steel 6 L canisters; carbonyl compounds were collected using 2,4-dinitrophenylhydrazine (DNPH) impregnated C{sub 18} SepPak cartridges. The canister samples were analyzed for speciated volatile hydrocarbons (C{sub 2}-C{sub 12}), CO, CO{sub 2}, CH{sub 4}, MTBE, and halogenated hydrocarbons. DNPH-impregnated cartridges were analyzed for fourteen C{sub 1}-C{sub 7} carbonylmore » compounds. The results of these measurements will be discussed.« less

PAK4 promotes kinase-independent stabilization of RhoU to modulate cell adhesion

PubMed Central

Dart, Anna E.; Box, Gary M.; Court, William; Gale, Madeline E.; Brown, John P.; Pinder, Sarah E.; Eccles, Suzanne A.

2015-01-01

P21-activated kinase 4 (PAK4) is a Cdc42 effector protein thought to regulate cell adhesion disassembly in a kinase-dependent manner. We found that PAK4 expression is significantly higher in high-grade human breast cancer patient samples, whereas depletion of PAK4 modifies cell adhesion dynamics of breast cancer cells. Surprisingly, systematic analysis of PAK4 functionality revealed that PAK4-driven adhesion turnover is neither dependent on Cdc42 binding nor kinase activity. Rather, reduced expression of PAK4 leads to a concomitant loss of RhoU expression. We report that RhoU is targeted for ubiquitination by the Rab40A–Cullin 5 complex and demonstrate that PAK4 protects RhoU from ubiquitination in a kinase-independent manner. Overexpression of RhoU rescues the PAK4 depletion phenotype, whereas loss of RhoU expression reduces cell adhesion turnover and migration. These data support a new kinase-independent mechanism for PAK4 function, where an important role of PAK4 in cellular adhesions is to stabilize RhoU protein levels. Thus, PAK4 and RhoU cooperate to drive adhesion turnover and promote cell migration. PMID:26598620

Phytoavailability of Cadmium (Cd) to Pak Choi (Brassica chinensis L.) Grown in Chinese Soils: A Model to Evaluate the Impact of Soil Cd Pollution on Potential Dietary Toxicity

PubMed Central

Yang, Xiaoe; Xiao, Wendan; Stoffella, Peter J.; Saghir, Aamir; Azam, Muhammad; Li, Tingqiang

2014-01-01

Food chain contamination by soil cadmium (Cd) through vegetable consumption poses a threat to human health. Therefore, an understanding is needed on the relationship between the phytoavailability of Cd in soils and its uptake in edible tissues of vegetables. The purpose of this study was to establish soil Cd thresholds of representative Chinese soils based on dietary toxicity to humans and develop a model to evaluate the phytoavailability of Cd to Pak choi (Brassica chinensis L.) based on soil properties. Mehlich-3 extractable Cd thresholds were more suitable for Stagnic Anthrosols, Calcareous, Ustic Cambosols, Typic Haplustalfs, Udic Ferrisols and Periudic Argosols with values of 0.30, 0.25, 0.18, 0.16, 0.15 and 0.03 mg kg−1, respectively, while total Cd is adequate threshold for Mollisols with a value of 0.86 mg kg−1. A stepwise regression model indicated that Cd phytoavailability to Pak choi was significantly influenced by soil pH, organic matter, total Zinc and Cd concentrations in soil. Therefore, since Cd accumulation in Pak choi varied with soil characteristics, they should be considered while assessing the environmental quality of soils to ensure the hygienically safe food production. PMID:25386790

Phytoavailability of cadmium (Cd) to Pak choi (Brassica chinensis L.) grown in Chinese soils: a model to evaluate the impact of soil Cd pollution on potential dietary toxicity.

PubMed

Rafiq, Muhammad Tariq; Aziz, Rukhsanda; Yang, Xiaoe; Xiao, Wendan; Stoffella, Peter J; Saghir, Aamir; Azam, Muhammad; Li, Tingqiang

2014-01-01

Food chain contamination by soil cadmium (Cd) through vegetable consumption poses a threat to human health. Therefore, an understanding is needed on the relationship between the phytoavailability of Cd in soils and its uptake in edible tissues of vegetables. The purpose of this study was to establish soil Cd thresholds of representative Chinese soils based on dietary toxicity to humans and develop a model to evaluate the phytoavailability of Cd to Pak choi (Brassica chinensis L.) based on soil properties. Mehlich-3 extractable Cd thresholds were more suitable for Stagnic Anthrosols, Calcareous, Ustic Cambosols, Typic Haplustalfs, Udic Ferrisols and Periudic Argosols with values of 0.30, 0.25, 0.18, 0.16, 0.15 and 0.03 mg kg-1, respectively, while total Cd is adequate threshold for Mollisols with a value of 0.86 mg kg-1. A stepwise regression model indicated that Cd phytoavailability to Pak choi was significantly influenced by soil pH, organic matter, total Zinc and Cd concentrations in soil. Therefore, since Cd accumulation in Pak choi varied with soil characteristics, they should be considered while assessing the environmental quality of soils to ensure the hygienically safe food production.

[Pesticide determination in water after separation on reverse-phase minicolumns].

PubMed

Molto, J C; Font, G; Farre, R; Mañes, J

1989-01-01

The lindane, methoxichlor, parathion and methylparathion content of water is measured in a gas-chromatography fitted with a capture electron detector. Prior extraction and concentration are done by using Sep-Pak C18 cartridges with ethyl acetate as eluent, which is offers certain advantages over hexane and methanol. With solutions of 0.5 ppb recovery values of 102.7% (lindane), 85% (methoxychlor), 94.7% (parathion) and 93.1% (methylparathion) are obtained. The method proposed is less time-consuming and less expensive than the usual consisting of extraction with dichloromethane: hexane (15:75 v/V) and concentration in a Kuderna-Danish.

Differential sensitivity of Pak5, Pak6, and Pak5/Pak6 double-knockout mice to the stimulant effects of amphetamine and exercise-induced alterations in body weight.

PubMed

Furnari, Melody A; Jobes, Michelle L; Nekrasova, Tanya; Minden, Audrey; Wagner, George C

2014-04-01

PAK5 and PAK6 are protein kinases highly expressed in the brain. Previously, we observed that Pak6 knockout mice gained significantly more weight during development than Pak5 knockout mice as well as wild-type controls and double-knockout mice lacking both Pak5 and Pak6. In this study, we assessed the effects of exercise on food intake and weight gain of these mice as well as their sensitivity to the stimulant effects of amphetamine. Mice of each genotype were placed in cages with free access to run wheel exercise or in cages without run wheels for a total of 74 days. Food and fluid intake as well as body weight of each mouse were measured on a weekly basis. Finally, mice were given a high dose of amphetamine and activity levels were observed immediately thereafter for 90 minutes. Brains and testes of mice were assayed for protein levels of the estrogen alpha and progesterone receptors. While run wheel mice consumed significantly more food, they weighed less than non-run wheel mice. In addition, although Pak6 knockout mice consumed the same amount of food as wild-type mice, they were significantly heavier regardless of run wheel condition. Pak5 knockout mice were found to be more active than other genotypes after amphetamine treatment. Finally, protein levels of the progesterone and estrogen alpha receptors were altered in brain and testes of the Pak6 knockout mice. Collectively, these data suggest that PAK6 play a role in weight gain unrelated to exercise and caloric intake and that Pak5 knockout mice are more sensitive to the stimulant effects of amphetamine.

p21-activated kinase 1: PAK'ed with potential.

PubMed

Ong, Christy C; Jubb, Adrian M; Zhou, Wei; Haverty, Peter M; Harris, Adrian L; Belvin, Marcia; Friedman, Lori S; Koeppen, Hartmut; Hoeflich, Klaus P

2011-06-01

The p21-activated kinases (PAKs) are central players in growth factor signaling networks and morphogenetic processes that control proliferation, cell polarity, invasion and actin cytoskeleton organization. This raises the possibility that interfering with PAK activity may produce significant anti-tumor activity. In this perspective, we summarize recent data concerning the contribution of the PAK family member, PAK1, in growth factor signaling and tumorigenesis. We further discuss mechanisms by which inhibition of PAK1 can arrest tumor growth and promote cell apoptosis, and the types of cancers in which PAK1 inhibition may hold promise.

Therapeutic Potential of Targeting PAK Signaling.

PubMed

Senapedis, William; Crochiere, Marsha; Baloglu, Erkan; Landesman, Yosef

2016-01-01

The therapeutic potential of targeting p21-Activated Kinases (PAK1 - 6) for the treatment of cancer has recently gained traction in the biotech industry. Many pharmaceutically-viable ATP competitive inhibitors have been through different stages of pre-clinical development with only a single compound evaluated in human trails (PF-3758309). The best studied functional roles of PAK proteins are control of cell adhesion and migration. PAK proteins are known downstream effectors of Ras signaling with PAK expression elevated in cancer (pancreatic, colon, breast, lung and other solid tumors). In addition altered PAK expression is a confirmed driver of this disease, especially in tumors harboring oncogenic Ras. However, there are very few examples of gain-of-function PAK mutations, as a majority of the cancer types have elevated PAK expression due to gene amplification or transcriptional modifications. There is a substantial number of known substrates affected by this aberrant PAK activity. One particular substrate, β-catenin, has garnered interest given its importance in both normal and cancer cell development. These data place PAK proteins between two major signaling pathways in cancer (Ras and β -catenin), making therapeutic targeting of PAKs an intriguing approach for the treatment of a broad array of oncological malignancies.

P21-activated kinase 2 (PAK2) regulates glucose uptake and insulin sensitivity in neuronal cells.

PubMed

Varshney, Pallavi; Dey, Chinmoy Sankar

2016-07-05

P21-activated kinases (PAKs) are recently reported as important players of insulin signaling and glucose homeostasis in tissues like muscle, pancreas and liver. However, their role in neuronal insulin signaling is still unknown. Present study reports the involvement of PAK2 in neuronal insulin signaling, glucose uptake and insulin resistance. Irrespective of insulin sensitivity, insulin stimulation decreased PAK2 activity. PAK2 downregulation displayed marked enhancement of GLUT4 translocation with increase in glucose uptake whereas PAK2 over-expression showed its reduction. Treatment with Akti-1/2 and wortmannin suggested that Akt and PI3K are mediators of insulin effect on PAK2 and glucose uptake. Rac1 inhibition demonstrated decreased PAK2 activity while inhibition of PP2A resulted in increased PAK2 activity, with corresponding changes in glucose uptake. Taken together, present study demonstrates an inhibitory role of insulin signaling (via PI3K-Akt) and PP2A on PAK2 activity and establishes PAK2 as a Rac1-dependent negative regulator of neuronal glucose uptake and insulin sensitivity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The PAKs come of age

PubMed Central

2012-01-01

Protein kinases are versatile signaling molecules that are involved in the regulation most physiological responses. The p21-activated kinases (PAKs) can be activated directly by the small GTPases Rac and Cdc42 and are among the best characterized downstream effectors of these Rho proteins. The structure, substrate specificity and functional role of PAKS are evolutionarily conserved from protozoa to mammals. Vertebrate PAKs are particularly important for cytoskeletal remodeling and focal adhesion assembly, thereby contributing to dynamic processes such as cell migration and synaptic plasticity. This issue of Cellular Logistics focuses on the PAK family of kinases, with ten reviews written by researchers currently working in the field. Here in this introductory overview we highlight some of the most interesting recent discoveries regarding PAK biochemistry and biology. The reviews in this issue cover a range of topics including the atomic structures of PAK1 and PAK4, their role in animals as assessed by knockout studies, and how PAKs are likely to contribute to cancer and neurodegenerative diseases. The promise remains that PAK inhibitors will emerge that validate current pre-clinical studies suggesting that blocking PAK activity will positively contribute to human health. PMID:23125949

The neural cell adhesion molecule (NCAM) associates with and signals through p21-activated kinase 1 (Pak1).

PubMed

Li, Shen; Leshchyns'ka, Iryna; Chernyshova, Yana; Schachner, Melitta; Sytnyk, Vladimir

2013-01-09

The Neural cell adhesion molecule (NCAM) plays an important role in regulation of nervous system development. To expand our understanding of the molecular mechanisms via which NCAM influences differentiation of neurons, we used a yeast two-hybrid screening to search for new binding partners of NCAM and identified p21-activated kinase 1 (Pak1). We show that NCAM interacts with Pak1 in growth cones of neurons. The autophosphorylation and activity of Pak1 were enhanced when isolated growth cones were incubated with NCAM function triggering antibodies, which mimic the interaction between NCAM and its extracellular ligands. The association of Pak1 with cell membranes, the efficiency of Pak1 binding to its activators, and Pak1 activity were inhibited in brains of NCAM-deficient mice. NCAM-dependent Pak1 activation was abolished after lipid raft disruption, suggesting that NCAM promotes Pak1 activation in the lipid raft environment. Phosphorylation of the downstream Pak1 effectors LIMK1 and cofilin was reduced in growth cones from NCAM-deficient neurons, which was accompanied by decreased levels of filamentous actin and inhibited filopodium mobility in the growth cones. Dominant-negative Pak1 inhibited and constitutively active Pak1 enhanced the ability of neurons to increase neurite outgrowth in response to the extracellular ligands of NCAM. Our combined observations thus indicate that NCAM activates Pak1 to drive actin polymerization to promote neuronal differentiation.

Accelerated Evolution of PAK3- and PIM1-like Kinase Gene Families in the Zebra Finch, Taeniopygia guttata

PubMed Central

Kong, Lesheng; Lovell, Peter V.; Heger, Andreas; Mello, Claudio V.; Ponting, Chris P.

2010-01-01

Genes encoding protein kinases tend to evolve slowly over evolutionary time, and only rarely do they appear as recent duplications in sequenced vertebrate genomes. Consequently, it was a surprise to find two families of kinase genes that have greatly and recently expanded in the zebra finch (Taeniopygia guttata) lineage. In contrast to other amniotic genomes (including chicken) that harbor only single copies of p21-activated serine/threonine kinase 3 (PAK3) and proviral integration site 1 (PIM1) genes, the zebra finch genome appeared at first to additionally contain 67 PAK3-like (PAK3L) and 51 PIM1-like (PIM1L) protein kinase genes. An exhaustive analysis of these gene models, however, revealed most to be incomplete, owing to the absence of terminal exons. After reprediction, 31 PAK3L genes and 10 PIM1L genes remain, and all but three are predicted, from the retention of functional sites and open reading frames, to be enzymatically active. PAK3L, but not PIM1L, gene sequences show evidence of recurrent episodes of positive selection, concentrated within structures spatially adjacent to N- and C-terminal protein regions that have been discarded from zebra finch PAK3L genes. At least seven zebra finch PAK3L genes were observed to be expressed in testis, whereas two sequences were found transcribed in the brain, one broadly including the song nuclei and the other in the ventricular zone and in cells resembling Bergmann's glia in the cerebellar Purkinje cell layer. Two PIM1L sequences were also observed to be expressed with broad distributions in the zebra finch brain, one in both the ventricular zone and the cerebellum and apparently associated with glial cells and the other showing neuronal cell expression and marked enrichment in midbrain/thalamic nuclei. These expression patterns do not correlate with zebra finch-specific features such as vocal learning. Nevertheless, our results show how ancient and conserved intracellular signaling molecules can be co-opted, following duplication, thereby resulting in lineage-specific functions, presumably affecting the zebra finch testis and brain. PMID:20237222

P21-activated kinase 4--not just one of the PAK.

PubMed

Dart, Anna E; Wells, Claire M

2013-01-01

P21-activated kinase 4 (PAK4) is a member of the p21-activated kinase (PAK) family. Historically much of the attention has been directed towards founding family member PAK1 but the focus is now shifting towards PAK4. It is a pluripotent serine/threonine kinase traditionally recognised as a downstream effector of the Rho-family GTPases. However, emerging research over the last few years has revealed that this kinase is much more than that. New findings have shed light on the molecular mechanism of PAK4 activation and how this kinase is critical for early development. Moreover, the number of PAK4 substrates and binding partners is rapidly expanding highlighting the increasing amount of cellular functions controlled by PAK4. We propose that PAK4 should be considered a signalling integrator regulating numerous fundamental cellular processes, including actin cytoskeletal dynamics, cell morphology and motility, cell survival, embryonic development, immune defence and oncogenic transformation. This review will outline our current understanding of PAK4 biology. Copyright © 2013 Elsevier GmbH. All rights reserved.

Fusobacterium nucleatum Potentiates Intestinal Tumorigenesis in Mice via a Toll-Like Receptor 4/p21-Activated Kinase 1 Cascade.

PubMed

Wu, Yaxin; Wu, Jiao; Chen, Ting; Li, Qing; Peng, Wei; Li, Huan; Tang, Xiaowei; Fu, Xiangsheng

2018-05-01

The underlying pathogenic mechanism of Fusobacterium nucleatum in the carcinogenesis of colorectal cancer has been poorly understood. Using C57BL/6-Apc Min/+ mice, we investigated gut microbial structures with F. nucleatum, antibiotics, and Toll-like receptor 4 (TLR4) antagonist TAK-242 treatment. In addition, we measured intestinal tumor formation and the expression of TLR4, p21-activated kinase 1 (PAK1), phosphorylated-PAK1 (p-PAK1), phosphorylated-β-catenin S675 (p-β-catenin S675), and cyclin D1 in mice with different treatments. Fusobacterium nucleatum and antibiotics treatment altered gut microbial structures in mice. In addition, F. nucleatum invaded into the intestinal mucosa in large amounts but were less abundant in the feces of F. nucleatum-fed mice. The average number and size of intestinal tumors in F. nucleatum groups was significantly increased compared to control groups in Apc Min/+ mice (P < 0.05). The expression of TLR4, PAK1, p-PAK1, p-β-catenin S675, and cyclin D1 was significantly increased in F. nucleatum groups compared to the control groups (P < 0.05). Moreover, TAK-242 significantly decreased the average number and size of intestinal tumors compared to F. nucleatum groups (P < 0.05). The expression of p-PAK1, p-β-catenin S675, and cyclin D1 was also significantly decreased in the TAK-242-treated group compared to F. nucleatum groups (P < 0.05). Fusobacterium nucleatum potentiates intestinal tumorigenesis in Apc Min/+ mice via a TLR4/p-PAK1/p-β-catenin S675 cascade. Fusobacterium nucleatum-induced intestinal tumorigenesis can be inhibited by TAK-242, implicating TLR4 as a potential target for the prevention and therapy of F. nucleatum-related colorectal cancer.

p21-Activated protein kinases and their emerging roles in glucose homeostasis.

PubMed

Chiang, Yu-ting Alex; Jin, Tianru

2014-04-01

p21-Activated protein kinases (PAKs) are centrally involved in a plethora of cellular processes and functions. Their function as effectors of small GTPases Rac1 and Cdc42 has been extensively studied during the past two decades, particularly in the realms of cell proliferation, apoptosis, and hence tumorigenesis, as well as cytoskeletal remodeling and related cellular events in health and disease. In recent years, a large number of studies have shed light onto the fundamental role of group I PAKs, most notably PAK1, in metabolic homeostasis. In skeletal muscle, PAK1 was shown to mediate the function of insulin on stimulating GLUT4 translocation and glucose uptake, while in pancreatic β-cells, PAK1 participates in insulin granule localization and vesicle release. Furthermore, we demonstrated that PAK1 mediates the cross talk between insulin and Wnt/β-catenin signaling pathways and hence regulates gut proglucagon gene expression and the production of the incretin hormone glucagon-like peptide-1 (GLP-1). The utilization of chemical inhibitors of PAK and the characterization of Pak1(-/-) mice enabled us to gain mechanistic insights as well as to assess the overall contribution of PAKs in metabolic homeostasis. This review summarizes our current understanding of PAKs, with an emphasis on the emerging roles of PAK1 in glucose homeostasis.

PAK4 kinase is essential for embryonic viability and for proper neuronal development.

PubMed

Qu, Jian; Li, Xiaofan; Novitch, Bennet G; Zheng, Ye; Kohn, Matthew; Xie, Jian-Ming; Kozinn, Spencer; Bronson, Roderick; Beg, Amer A; Minden, Audrey

2003-10-01

The serine/threonine kinase PAK4 is a target for the Rho GTPase Cdc42 and has been shown to regulate cell morphology and cytoskeletal organization in mammalian cells. To examine the physiological and developmental functions of PAK4, we have disrupted the PAK4 gene in mice. The absence of PAK4 led to lethality by embryonic day 11.5, a result most likely due to a defect in the fetal heart. Striking abnormalities were also evident in the nervous systems of PAK4-deficient embryos. These embryos had dramatic defects in neuronal development and axonal outgrowth. In particular, spinal cord motor neurons and interneurons failed to differentiate and migrate to their proper positions. This is probably related to the role for PAK4 in the regulation of cytoskeletal organization and cell and/or extracellular matrix adhesion. PAK4-null embryos also had defects in proper folding of the caudal portion of the neural tube, suggesting an important role for PAK4 in neural tube development.

p21-activated kinases and gastrointestinal cancer.

PubMed

He, Hong; Baldwin, Graham S

2013-01-01

p21-activated kinases (PAKs) were initially identified as effector proteins downstream from GTPases of the Rho family. To date, six members of the PAK family have been discovered in mammalian cells. PAKs play important roles in growth factor signalling, cytoskeletal remodelling, gene transcription, cell proliferation and oncogenic transformation. A large body of research has demonstrated that PAKs are up-regulated in several human cancers, and that their overexpression is linked to tumour progression and resistance to therapy. Structural and biochemical studies have revealed the mechanisms involved in PAK signalling, and opened the way to the development of PAK-targeted therapies for cancer treatment. Here we summarise recent findings from biological and clinical research on the role of PAKs in gastrointestinal cancer, and discuss the current status of PAK-targeted anticancer therapies. Copyright © 2012 Elsevier B.V. All rights reserved.

p21-activated kinase inhibitors.

PubMed

Rudolph, Joachim; Crawford, James J; Hoeflich, Klaus P; Chernoff, Jonathan

2013-01-01

The p21-activated kinases (PAKs) are Ser/Thr kinases in the STE20 kinase family with important roles in regulating cytoskeletal organization, cell migration, and signaling. The PAK enzyme family comprises six members subdivided into two groups: Group I, represented by PAK1, 2, and 3, and Group II, represented by PAK 4, 5, and 6, based on sequence and structural homology. Individual PAK isoforms were found to be overexpressed and amplified in a variety of human cancers, and in vitro and in vivo studies using genetically engineered systems as well as small-molecule tool compounds have suggested therapeutic utility of PAKs as oncology targets. The identification of potent and kinome-selective ATP-competitive PAK inhibitors has proven challenging, likely caused by the openness and unique plasticity of the ATP-binding site of PAK enzymes. Progress in achieving increased kinase selectivity has been achieved with certain inhibitors but at the expense of increased molecular weight. Allosteric inhibitors, such as IPA-3, leverage the unique Group I PAK autoregulatory domain for selective inhibition, and this approach might provide an outlet to evade the kinase selectivity challenges observed with ATP-competitive PAK inhibitors. © 2013 Elsevier Inc. All rights reserved.

p21-Activated kinase (Pak) regulates airway smooth muscle contraction by regulating paxillin complexes that mediate actin polymerization.

PubMed

Zhang, Wenwu; Huang, Youliang; Gunst, Susan J

2016-09-01

In airway smooth muscle, tension development caused by a contractile stimulus requires phosphorylation of the 20 kDa myosin light chain (MLC), which activates crossbridge cycling and the polymerization of a pool of submembraneous actin. The p21-activated kinases (Paks) can regulate the contractility of smooth muscle and non-muscle cells, and there is evidence that this occurs through the regulation of MLC phosphorylation. We show that Pak has no effect on MLC phosphorylation during the contraction of airway smooth muscle, and that it regulates contraction by mediating actin polymerization. We find that Pak phosphorylates the adhesion junction protein, paxillin, on Ser273, which promotes the formation of a signalling complex that activates the small GTPase, cdc42, and the actin polymerization catalyst, neuronal Wiskott-Aldrich syndrome protein (N-WASP). These studies demonstrate a novel role for Pak in regulating the contractility of smooth muscle by regulating actin polymerization. The p21-activated kinases (Pak) can regulate contractility in smooth muscle and other cell and tissue types, but the mechanisms by which Paks regulate cell contractility are unclear. In airway smooth muscle, stimulus-induced contraction requires phosphorylation of the 20 kDa light chain of myosin, which activates crossbridge cycling, as well as the polymerization of a small pool of actin. The role of Pak in airway smooth muscle contraction was evaluated by inhibiting acetylcholine (ACh)-induced Pak activation through the expression of a kinase inactive mutant, Pak1 K299R, or by treating tissues with the Pak inhibitor, IPA3. Pak inhibition suppressed actin polymerization and contraction in response to ACh, but it did not affect myosin light chain phosphorylation. Pak activation induced paxillin phosphorylation on Ser273; the paxillin mutant, paxillin S273A, inhibited paxillin Ser273 phosphorylation and inhibited actin polymerization and contraction. Immunoprecipitation analysis of tissue extracts and proximity ligation assays in dissociated cells showed that Pak activation and paxillin Ser273 phosphorylation triggered the formation of an adhesion junction signalling complex with paxillin that included G-protein-coupled receptor kinase-interacting protein (GIT1) and the cdc42 guanine exchange factor, βPIX (Pak interactive exchange factor). Assembly of the Pak-GIT1-βPIX-paxillin complex was necessary for cdc42 and neuronal Wiskott-Aldrich syndrome protein (N-WASP) activation, actin polymerization and contraction in response to ACh. RhoA activation was also required for the recruitment of Pak to adhesion junctions, Pak activation, paxillin Ser273 phosphorylation and paxillin complex assembly. These studies demonstrate a novel role for Pak in the regulation of N-WASP activation, actin dynamics and cell contractility. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Pak functions downstream of Dock to regulate photoreceptor axon guidance in Drosophila.

PubMed

Hing, H; Xiao, J; Harden, N; Lim, L; Zipursky, S L

1999-06-25

The SH2/SH3 adaptor protein Dock has been proposed to transduce signals from guidance receptors to the actin cytoskeleton in Drosophila photoreceptor (R cell) growth cones. Here, we demonstrate that Drosophila p21-activated kinase (Pak) is required in a Dock pathway regulating R cell axon guidance and targeting. Dock and Pak colocalize to R cell axons and growth cones, physically interact, and their loss-of-function phenotypes are indistinguishable. Normal patterns of R cell connectivity require Pak's kinase activity and binding sites for both Dock and Cdc42/Rac. A membrane-tethered form of Pak (Pak(myr) acts as a dominant gain-of-function protein. Retinal expression of Pak(myr) rescues the R cell connectivity phenotype in dock mutants. These data establish Pak as a critical regulator of axon guidance and a downstream effector of Dock in vivo.

A simple and sensitive quantitation of N,N-dimethyltryptamine by gas chromatography with surface ionization detection.

PubMed

Ishii, A; Seno, H; Suzuki, O; Hattori, H; Kumazawa, T

1997-01-01

A simple and sensitive method for determination of N,N-dimethyltryptamine (DMT) by gas chromatography (GC) with surface ionization detection (SID) is presented. Whole blood or urine, containing DMT and gramine (internal standard), was subjected to solid-phase extraction with a Sep-Pak C18 cartridge before analysis by GC-SID. The calibration curve was linear in the DMT range of 1.25-20 ng/mL blood or urine. The detection limit of DMT was about 0.5 ng/mL (10 pg on-column). The recovery of both DMT and gramine spiked in biological fluids was above 86%.

Unmasking Hb Paksé (codon 142, TAA>TAT, α2) and its combinations in patients also carrying Hb Constant Spring (codon 142, TAA>CAA, α2) in northern Thailand.

PubMed

Pornprasert, Sakorn; Panyasai, Sitthichai; Treesuwan, Kallayanee

2012-01-01

The incidence of Hb Paksé (codon 142, TAA>TAT, α2) might have been underestimated due to misidentifying some cases as Hb Constant Spring (Hb CS, codon 142, TAA>CAA, α2) since both abnormal hemoglobins (Hbs) migrate to the same position on Hb electrophoresis or chromatography. Multiplex asymmetric allele-specific polymerase chain reaction (PCR) for identification of Hb CS and Hb Paksé, and a real-time PCR (ReTi-PCR) with SYBR Green1 high resolution melting (HRM) analysis, for detection of the α-thalassemia-1 (α-thal-1) Southeast Asian (- -(SEA)/) type deletion, were performed on 114 blood samples collected from subjects who lived in northern Thailand. These samples were previously identified as carrying Hb CS by capillary electrophoresis (CE) or high performance liquid chromatography (HPLC). Five out of 114 (4.4%) samples were found to carry Hb Paksé with four different genotypes including Hb Paksé trait, compound Hb CS/Hb Paksé, Hb H-Hb Paksé disease and Hb H-Hb Paksé-Hb E disease. These results suggested that Hb Paksé and its various combinations can be misidentified as Hb CS. Although the clinical symptoms of Hb Paksé and Hb CS are similar, to prevent erroneous epidemiological data on Hb CS as well as underestimating the prevalence of Hb Paksé in northern Thailand, DNA analysis is recommended to be performed in all cases when peaks of Hb CS/Hb Paksé are detected on CE or HPLC.

The p21-activated kinase 4-Slug transcription factor axis promotes epithelial-mesenchymal transition and worsens prognosis in prostate cancer.

PubMed

Park, Jung-Jin; Park, Mee-Hee; Oh, Eun Hye; Soung, Nak-Kyun; Lee, Soo Jae; Jung, Jae-Kyung; Lee, Ok-Jun; Yun, Seok Joong; Kim, Wun-Jae; Shin, Eun-Young; Kim, Eung-Gook

2018-05-30

Epithelial-mesenchymal transition (EMT) facilitates cancer invasion and metastasis and thus accelerates cancer progression. p21-activated kinase 4 (PAK4) is a critical regulator of prostate cancer (PC) progression. Here, we report that PAK4 activation promotes PC progression through the EMT regulator Slug. We find that phosphorylated PAK4 S474 (pPAK4) levels, an index of PAK4 activation, were tightly associated with Gleason score (p < 0.001), a clinical indicator of PC progression, but not with prostate serum antigen levels or tumor stage. Stable silencing of PAK4 in PC cells reduced their potential for EMT, cellular invasion, and metastasis in vivo. PAK4 bound and directly phosphorylated Slug at two previously unknown sites, S158 and S254, which resulted in its stabilization. The non-phosphorylatable form Slug S158A/S254A upregulated transcription of CDH1, which encodes E-cadherin, and thus suppressed EMT and invasion, to a greater extent than did wild-type Slug. The strong EMT inducer TGF-β elevated pPAK4 and pSlug S158 levels; PAK4 knockdown or introduction of a dominant-negative form of PAK4 inhibited both TGF-β-stimulated EMT and an increase in pSlug S158 levels. Finally, immunohistochemistry revealed a positive correlation between pPAK4 and pSlug S158 but an inverse correlation between pSlug S158 and E-cadherin. The results suggest that the PAK4-Slug axis represents a novel pathway that promotes PC progression.

Matrix-specific protein kinase A signaling regulates p21 activated kinase activation by flow in endothelial cells

PubMed Central

Funk, Steven Daniel; Yurdagul, Arif; Green, Jonette M.; Jhaveri, Krishna A.; Schwartz, Martin Alexander; Orr, A. Wayne

2010-01-01

Rationale Atherosclerosis is initiated by blood flow patterns that activate inflammatory pathways in endothelial cells. Activation of inflammatory signaling by fluid shear stress is highly dependent on the composition of the subendothelial extracellular matrix. The basement membrane proteins laminin and collagen found in normal vessels suppress flow-induced p21 activated kinase (PAK) and NF-κB activation. By contrast, the provisional matrix proteins fibronectin and fibrinogen found in wounded or inflamed vessels support flow-induced PAK and NF-κB activation. PAK mediates both flow-induced permeability and matrix-specific activation of NF-κB. Objective To elucidate the mechanisms regulating matrix-specific PAK activation. Methods and Results We now show that matrix composition does not affect the upstream pathway by which flow activates PAK (integrin activation, Rac). Instead basement membrane proteins enhance flow-induced protein kinase A (PKA) activation, which suppresses PAK. Inhibiting PKA restored flow-induced PAK and NF-κB activation in cells on basement membrane proteins, whereas stimulating PKA inhibited flow-induced activation of inflammatory signaling in cells on fibronectin. PKA suppressed inflammatory signaling through PAK inhibition. Activating PKA by injection of the PGI2 analog iloprost reduced PAK activation and inflammatory gene expression at sites of disturbed flow in vivo, whereas inhibiting PKA by PKI injection enhanced PAK activation and inflammatory gene expression. Inhibiting PAK prevented the enhancement of inflammatory gene expression by PKI. Conclusions Basement membrane proteins inhibit inflammatory signaling in endothelial cells via PKA-dependent inhibition of PAK. PMID:20224042

P21 activated kinases: structure, regulation, and functions.

PubMed

Rane, Chetan K; Minden, Audrey

2014-01-01

The p21 activated kinases (Paks) are well known effector proteins for the Rho GTPases Cdc42 and Rac. The Paks contain 6 members, which fall into 2 families of proteins. The first family consists of Paks 1, 2, and 3, and the second consists of Paks 4, 5, and 6. While some of the Paks are ubiquitously expressed, others have more restrictive tissue specificity. All of them are found in the nervous system. Studies using cell culture, transgenic mice, and knockout mice, have revealed important roles for the Paks in cytoskeletal organization and in many aspects of cell growth and development. This review discusses the basic structures of the Paks, and their roles in cell growth, development, and in cancer.

p21 Activated Kinase 5 Activates Raf-1 and Targets it to Mitochondria

PubMed Central

Wu, Xiaochong; Carr, Heather S.; Dan, Ippeita; Ruvolo, Peter P.; Frost, Jeffrey A.

2008-01-01

Raf-1 is an important effector of Ras mediated signaling and is a critical regulator of the ERK/MAPK pathway. Raf-1 activation is controlled in part by phosphorylation on multiple residues, including an obligate phosphorylation site at serine 338. Previously PAK1 and casein kinase II have been implicated as serine 338 kinases. To identify novel kinases that phosphorylate this site, we tested the ability of group II PAKs (PAKs 4-6) to control serine 338 phosphorylation. We observed that all group II PAKs were efficient serine 338 kinases, although only PAK1 and PAK5 significantly stimulated Raf-1 kinase activity. We also showed that PAK5 forms a tight complex with Raf-1 in the cell, but not A-Raf or B-Raf. Importantly, we also demonstrated that the association of Raf-1 with PAK5 targets a subpopulation of Raf-1 to mitochondria. These data indicate that PAK5 is a potent regulator of Raf-1 activity and may control Raf-1 dependent signaling at the mitochondria. PMID:18465753

p21‐Activated kinase (Pak) regulates airway smooth muscle contraction by regulating paxillin complexes that mediate actin polymerization

PubMed Central

Zhang, Wenwu; Huang, Youliang

2016-01-01

Key points In airway smooth muscle, tension development caused by a contractile stimulus requires phosphorylation of the 20 kDa myosin light chain (MLC), which activates crossbridge cycling and the polymerization of a pool of submembraneous actin.The p21‐activated kinases (Paks) can regulate the contractility of smooth muscle and non‐muscle cells, and there is evidence that this occurs through the regulation of MLC phosphorylation.We show that Pak has no effect on MLC phosphorylation during the contraction of airway smooth muscle, and that it regulates contraction by mediating actin polymerization.We find that Pak phosphorylates the adhesion junction protein, paxillin, on Ser273, which promotes the formation of a signalling complex that activates the small GTPase, cdc42, and the actin polymerization catalyst, neuronal Wiskott–Aldrich syndrome protein (N‐WASP).These studies demonstrate a novel role for Pak in regulating the contractility of smooth muscle by regulating actin polymerization. Abstract The p21‐activated kinases (Pak) can regulate contractility in smooth muscle and other cell and tissue types, but the mechanisms by which Paks regulate cell contractility are unclear. In airway smooth muscle, stimulus‐induced contraction requires phosphorylation of the 20 kDa light chain of myosin, which activates crossbridge cycling, as well as the polymerization of a small pool of actin. The role of Pak in airway smooth muscle contraction was evaluated by inhibiting acetylcholine (ACh)‐induced Pak activation through the expression of a kinase inactive mutant, Pak1 K299R, or by treating tissues with the Pak inhibitor, IPA3. Pak inhibition suppressed actin polymerization and contraction in response to ACh, but it did not affect myosin light chain phosphorylation. Pak activation induced paxillin phosphorylation on Ser273; the paxillin mutant, paxillin S273A, inhibited paxillin Ser273 phosphorylation and inhibited actin polymerization and contraction. Immunoprecipitation analysis of tissue extracts and proximity ligation assays in dissociated cells showed that Pak activation and paxillin Ser273 phosphorylation triggered the formation of an adhesion junction signalling complex with paxillin that included G‐protein‐coupled receptor kinase‐interacting protein (GIT1) and the cdc42 guanine exchange factor, βPIX (Pak interactive exchange factor). Assembly of the Pak–GIT1–βPIX–paxillin complex was necessary for cdc42 and neuronal Wiskott–Aldrich syndrome protein (N‐WASP) activation, actin polymerization and contraction in response to ACh. RhoA activation was also required for the recruitment of Pak to adhesion junctions, Pak activation, paxillin Ser273 phosphorylation and paxillin complex assembly. These studies demonstrate a novel role for Pak in the regulation of N‐WASP activation, actin dynamics and cell contractility. PMID:27038336

Synapses of Amphids Defective (SAD-A) Kinase Promotes Glucose-stimulated Insulin Secretion through Activation of p21-activated Kinase (PAK1) in Pancreatic β-Cells*

PubMed Central

Nie, Jia; Sun, Chao; Faruque, Omar; Ye, Guangming; Li, Jia; Liang, Qiangrong; Chang, Zhijie; Yang, Wannian; Han, Xiao; Shi, Yuguang

2012-01-01

The p21-activated kinase-1 (PAK1) is implicated in regulation of insulin exocytosis as an effector of Rho GTPases. PAK1 is activated by the onset of glucose-stimulated insulin secretion (GSIS) through phosphorylation of Thr-423, a major activation site by Cdc42 and Rac1. However, the kinase(s) that phosphorylates PAK1 at Thr-423 in islet β-cells remains elusive. The present studies identified SAD-A (synapses of amphids defective), a member of AMP-activated protein kinase-related kinases exclusively expressed in brain and pancreas, as a key regulator of GSIS through activation of PAK1. We show that SAD-A directly binds to PAK1 through its kinase domain. The interaction is mediated by the p21-binding domain (PBD) of PAK1 and requires both kinases in an active conformation. The binding leads to direct phosphorylation of PAK1 at Thr-423 by SAD-A, triggering the onset of GSIS from islet β-cells. Consequently, ablation of PAK1 kinase activity or depletion of PAK1 expression completely abolishes the potentiating effect of SAD-A on GSIS. Consistent with its role in regulating GSIS, overexpression of SAD-A in MIN6 islet β-cells significantly stimulated cytoskeletal remodeling, which is required for insulin exocytosis. Together, the present studies identified a critical role of SAD-A in the activation of PAK1 during the onset of insulin exocytosis. PMID:22669945

Synapses of amphids defective (SAD-A) kinase promotes glucose-stimulated insulin secretion through activation of p21-activated kinase (PAK1) in pancreatic β-Cells.

PubMed

Nie, Jia; Sun, Chao; Faruque, Omar; Ye, Guangming; Li, Jia; Liang, Qiangrong; Chang, Zhijie; Yang, Wannian; Han, Xiao; Shi, Yuguang

2012-07-27

The p21-activated kinase-1 (PAK1) is implicated in regulation of insulin exocytosis as an effector of Rho GTPases. PAK1 is activated by the onset of glucose-stimulated insulin secretion (GSIS) through phosphorylation of Thr-423, a major activation site by Cdc42 and Rac1. However, the kinase(s) that phosphorylates PAK1 at Thr-423 in islet β-cells remains elusive. The present studies identified SAD-A (synapses of amphids defective), a member of AMP-activated protein kinase-related kinases exclusively expressed in brain and pancreas, as a key regulator of GSIS through activation of PAK1. We show that SAD-A directly binds to PAK1 through its kinase domain. The interaction is mediated by the p21-binding domain (PBD) of PAK1 and requires both kinases in an active conformation. The binding leads to direct phosphorylation of PAK1 at Thr-423 by SAD-A, triggering the onset of GSIS from islet β-cells. Consequently, ablation of PAK1 kinase activity or depletion of PAK1 expression completely abolishes the potentiating effect of SAD-A on GSIS. Consistent with its role in regulating GSIS, overexpression of SAD-A in MIN6 islet β-cells significantly stimulated cytoskeletal remodeling, which is required for insulin exocytosis. Together, the present studies identified a critical role of SAD-A in the activation of PAK1 during the onset of insulin exocytosis.

Role of p21-activated kinases in cardiovascular development and function.

PubMed

Kelly, Mollie L; Astsaturov, Artyom; Chernoff, Jonathan

2013-11-01

p21-activated kinases (Paks) are a group of six serine/threonine kinases (Pak1-6) that are involved in a variety of biological processes. Recently, Paks, more specifically Pak1, -2, and -4, have been shown to play important roles in cardiovascular development and function in a range of model organisms including zebrafish and mice. These functions include proper morphogenesis and conductance of the heart, cardiac contractility, and development and integrity of the vasculature. The mechanisms underlying these effects are not fully known, but they likely differ among the various Pak isoforms and include both kinase-dependent and -independent functions. In this review, we discuss aspects of Pak function relevant to cardiovascular biology as well as potential therapeutic implications of small-molecule Pak inhibitors in cardiovascular disease.

Cytotaxonomy of Eurypyga helias (Gruiformes, Eurypygidae): First Karyotypic Description and Phylogenetic Proximity with Rynochetidae

PubMed Central

dos Santos, Michelly da Silva; Tagliarini, Marcella Mergulhão; O´Brien, Patricia C. M.; Ferguson-Smith, Malcolm A.; de Oliveira, Edivaldo H. C.

2015-01-01

The sunbittern (Eurypyga helias) is a South American Gruiformes, the only member of Family Eurypigidae. In most phylogenetic proposals, it is placed in a more distant position than other families of the so-called “core Gruiformes”. Different studies based on molecular, morphological and biogeographical data suggest that the Eurypigidae is closely related to the kagu (Rhynochetos jubatus), the only species in Rynochetidae, another family not included in the core Gruiformes. Here, the karyotype of the sunbittern is described for the first time, by classical and molecular cytogenetics, using whole chromosome probes derived from Gallus gallus and Leucopternis albicollis. We found a diploid number of 80, with only one pair of biarmed autosomal macrochromosomes, similar to that observed in the kagu. Chromosome painting revealed that most syntenies found in the avian putative ancestral karyotype (PAK) were conserved in the sunbittern. However, PAK1, PAK2, and PAK5 corresponded to two chromosome pairs each. Probes derived from L. albicollis confirm that fissions in PAK1 and PAK2 were centric, whereas in PAK5 the fission is interstitial. In addition, there is fusion of segments homologous to PAK2q and PAK5. From a phylogenetic point of view, comparisons of our results with two other Gruiformes belonging to family Rallidae suggest that the PAK5q fission might be a synapomorphy for Gruiformes. Fissions in PAK1 and PAK2 are found only in Eurypigidae, and might also occur in Rynochetidae, in view of the similar chromosomal morphology between the sunbittern and the kagu. This suggests a close phylogenetic relationship between Eurypigidae and Rynochetidae, whose common ancestor was separated by the Gondwana vicariancy in South America and New Caledonia, respectively. PMID:26624624

PAK Inactivation Impairs Social Recognition in 3xTg-AD Mice without Increasing Brain Deposition of Tau and Aβ

PubMed Central

Arsenault, Dany; Dal-Pan, Alexandre; Tremblay, Cyntia; Bennett, David A.; Guitton, Matthieu J.; De Koninck, Yves; Tonegawa, Susumu

2013-01-01

Defects in p21-activated kinase (PAK) are suspected to play a role in cognitive symptoms of Alzheimer's disease (AD). Dysfunction in PAK leads to cofilin activation, drebrin displacement from its actin-binding site, actin depolymerization/severing, and, ultimately, defects in spine dynamics and cognitive impairment in mice. To determine the role of PAK in AD, we first quantified PAK by immunoblotting in homogenates from the parietal neocortex of subjects with a clinical diagnosis of no cognitive impairment (n = 12), mild cognitive impairment (n = 12), or AD (n = 12). A loss of total PAK, detected in the cortex of AD patients (−39% versus controls), was correlated with cognitive impairment (r2 = 0.148, p = 0.027) and deposition of total and phosphorylated tau (r2 = 0.235 and r2 = 0.206, respectively), but not with Aβ42 (r2 = 0.056). Accordingly, we found a decrease of total PAK in the cortex of 12- and 20-month-old 3xTg-AD mice, an animal model of AD-like Aβ and tau neuropathologies. To determine whether PAK dysfunction aggravates AD phenotype, 3xTg-AD mice were crossed with dominant-negative PAK mice. PAK inactivation led to obliteration of social recognition in old 3xTg-AD mice, which was associated with a decrease in cortical drebrin (−25%), but without enhancement of Aβ/tau pathology or any clear electrophysiological signature. Overall, our data suggest that PAK decrease is a consequence of AD neuropathology and that therapeutic activation of PAK may exert symptomatic benefits on high brain function. PMID:23804095

The Multisubstrate Adapter Gab1 Regulates Hepatocyte Growth Factor (Scatter Factor)–c-Met Signaling for Cell Survival and DNA Repair

PubMed Central

Fan, Saijun; Ma, Yong Xian; Gao, Min; Yuan, Ren-Qi; Meng, Qinghui; Goldberg, Itzhak D.; Rosen, Eliot M.

2001-01-01

Hepatocyte growth factor (scatter factor) (HGF/SF) is a pleiotrophic mediator of epithelial cell motility, morphogenesis, angiogenesis, and tumorigenesis. HGF/SF protects cells against DNA damage by a pathway from its receptor c-Met to phosphatidylinositol 3-kinase (PI3K) to c-Akt, resulting in enhanced DNA repair and decreased apoptosis. We now show that protection against the DNA-damaging agent adriamycin (ADR; topoisomerase IIα inhibitor) requires the Grb2-binding site of c-Met, and overexpression of the Grb2-associated binder Gab1 (a multisubstrate adapter required for epithelial morphogenesis) inhibits the ability of HGF/SF to protect MDCK epithelial cells against ADR. In contrast to Gab1 and its homolog Gab2, overexpression of c-Cb1, another multisubstrate adapter that associates with c-Met, did not affect protection. Gab1 blocked the ability of HGF/SF to cause the sustained activation of c-Akt and c-Akt signaling (FKHR phosphorylation). The Gab1 inhibition of sustained c-Akt activation and of cell protection did not require the Gab1 pleckstrin homology or SHP2 phosphatase-binding domain but did require the PI3K-binding domain. HGF/SF protection of parental MDCK cells was blocked by wortmannin, expression of PTEN, and dominant negative mutants of p85 (regulatory subunit of PI3K), Akt, and Pak1; the protection of cells overexpressing Gab1 was restored by wild-type or activated mutants of p85, Akt, and Pak1. These findings suggest that the adapter Gab1 may redirect c-Met signaling through PI3K away from a c-Akt/Pak1 cell survival pathway. PMID:11438654

Molecular pathways: targeting p21-activated kinase 1 signaling in cancer--opportunities, challenges, and limitations.

PubMed

Eswaran, Jeyanthy; Li, Da-Qiang; Shah, Anil; Kumar, Rakesh

2012-07-15

The evolution of cancer cells involves deregulation of highly regulated fundamental pathways that are central to normal cellular architecture and functions. p21-activated kinase 1 (PAK1) was initially identified as a downstream effector of the GTPases Rac and Cdc42. Subsequent studies uncovered a variety of new functions for this kinase in growth factor and steroid receptor signaling, cytoskeleton remodeling, cell survival, oncogenic transformation, and gene transcription, largely through systematic discovery of its direct, physiologically relevant substrates. PAK1 is widely upregulated in several human cancers, such as hormone-dependent cancer, and is intimately linked to tumor progression and therapeutic resistance. These exciting developments combined with the kinase-independent role of PAK1-centered phenotypic signaling in cancer cells elevated PAK1 as an attractive drug target. Structural and biochemical studies revealed the precise mechanism of PAK1 activation, offering the possibility to develop PAK1-targeted cancer therapeutic approaches. In addition, emerging reports suggest the potential of PAK1 and its specific phosphorylated substrates as cancer prognostic markers. Here, we summarize recent findings about the PAK1 molecular pathways in human cancer and discuss the current status of PAK1-targeted anticancer therapies.

Development and validation of a LC-MS/MS method for the determination of clebopride and its application to a pharmacokinetics study in healthy Chinese volunteers.

PubMed

Tan, Zhirong; Ouyang, Dongsheng; Chen, Yao; Zhou, Gan; Cao, Shan; Wang, Yicheng; Peng, Xiujuan; Zhou, Honghao

2010-08-01

A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the identification and quantification of clebopride in human plasma using itopride as an internal standard. The method involves a simple liquid-liquid extraction. The analytes were separated by isocratic gradient elution on a CAPCELL MG-III C(18) (5 microm, 150 mm x 2.1 mm i.d.) column and analyzed in multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI) interface using the respective [M+H](+) ions, m/z 373.9-->m/z184.0 for clebopride, m/z 359.9-->m/z71.5 for itopride. The method was validated over the concentration range of 69.530-4450.0 pg/ml for clebopride. Within- and between-batch precision (RSD%) was all within 6.83% and accuracy ranged from -8.16 to 1.88%. The LLOQ was 69.530 pg/ml. The extraction recovery was on an average 77% for clebopride. The validated method was used to study the pharmacokinetics profile of clebopride in human plasma after oral administration of clebopride. Copyright 2010. Published by Elsevier B.V.

Trio combines with dock to regulate Pak activity during photoreceptor axon pathfinding in Drosophila.

PubMed

Newsome, T P; Schmidt, S; Dietzl, G; Keleman, K; Asling, B; Debant, A; Dickson, B J

2000-04-28

Correct pathfinding by Drosophila photoreceptor axons requires recruitment of p21-activated kinase (Pak) to the membrane by the SH2-SH3 adaptor Dock. Here, we identify the guanine nucleotide exchange factor (GEF) Trio as another essential component in photoreceptor axon guidance. Regulated exchange activity of one of the two Trio GEF domains is critical for accurate pathfinding. This GEF domain activates Rac, which in turn activates Pak. Mutations in trio result in projection defects similar to those observed in both Pak and dock mutants, and trio interacts genetically with Rac, Pak, and dock. These data define a signaling pathway from Trio to Rac to Pak that links guidance receptors to the growth cone cytoskeleton. We propose that distinct signals transduced via Trio and Dock act combinatorially to activate Pak in spatially restricted domains within the growth cone, thereby controlling the direction of axon extension.

The Role of the Pleckstrin Homology Domain-containing Protein CKIP-1 in Activation of p21-activated Kinase 1 (PAK1)*

PubMed Central

Kim, Yong-Bae; Shin, Yong Jae; Roy, Adhiraj; Kim, Jeong-Ho

2015-01-01

Upon growth factor stimulation, PAK1 is recruited to the plasma membrane and activated by a mechanism that requires its phosphorylation at Ser-223 by the protein kinase CK2. However, the upstream signaling molecules that regulate this phosphorylation event are not clearly defined. Here, we demonstrate a major role of the CK2α-interacting protein CKIP-1 in activation of PAK1. CK2α, CKIP-1, and PAK1 are translocated to membrane ruffles in response to the epidermal growth factor (EGF), where CKIP-1 mediates the interaction between CK2α and PAK1 in a PI3K-dependent manner. Consistently, PAK1 mediates phosphorylation and modulation of the activity of p41-Arc, one of its plasma membrane substrate, in a fashion that requires PI3K and CKIP-1. Moreover, CKIP-1 knockdown or PI3K inhibition suppresses PAK1-mediated cell migration and invasion, demonstrating the physiological significance of the PI3K-CKIP-1-CK2-PAK1 signaling pathway. Taken together, these findings identify a novel mechanism for the activation of PAK1 at the plasma membrane, which is critical for cell migration and invasion. PMID:26160174

The discovery and the structural basis of an imidazo[4,5-b]pyridine-based p21-activated kinase 4 inhibitor.

PubMed

Park, Jeung Kuk; Kim, Sunmin; Han, Yu Jin; Kim, Seong Hwan; Kang, Nam Sook; Lee, Hyuk; Park, SangYoun

2016-06-01

p21-Activated kinases (PAKs) which belong to the family of ste20 serine/threonine protein kinases regulate cytoskeletal reorganization, cell motility, cell proliferation, and oncogenic transformation which are all related to the cellular functions during cancer induction and metastasis. The fact that PAK mutations are detected in multiple tumor tissues makes PAKs a novel therapeutic drug target. In this study, an imidazo[4,5-b]pyridine-based PAK4 inhibitor, KY-04045 (6-Bromo-2-(3-isopropyl-1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-b]pyridine), was discovered using a virtual site-directed fragment-based drug design and was validated using an inhibition assay. Although PAK4 affinity to KY-04045 seems much weaker than that of the reported PAK4 inhibitors, the location of KY-04045 is clearly defined in the structure of PAK4 co-crystallized with KY-04045. The crystal structure illustrates that the pyrazole and imidazopyridine rings of KY-04045 are sufficient for mediating PAK4 hinge loop interaction. Hence, we believe that KY-04045 can be exploited as a basic building block in designing novel imidazo[4,5-b]pyridine-based PAK4 inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

PAK1 is a breast cancer oncogene that coordinately activates MAPK and MET signaling

PubMed Central

Shrestha, Yashaswi; Schafer, Eric J.; Boehm, Jesse S.; Thomas, Sapana R.; He, Frank; Du, Jinyan; Wang, Shumei; Barretina, Jordi; Weir, Barbara A.; Zhao, Jean J.; Polyak, Kornelia; Golub, Todd R.; Beroukhim, Rameen; Hahn, William C.

2011-01-01

Activating mutations in the RAS family or BRAF frequently occur in many types of human cancers but are rarely detected in breast tumors. However, activation of the RAS-RAF-MEK-ERK Mitogen-Activated Protein Kinase (MAPK) pathway is commonly observed in human breast cancers, suggesting that other genetic alterations lead to activation of this signaling pathway. To identify breast cancer oncogenes that activate the MAPK pathway, we screened a library of human kinases for their ability to induce anchorage-independent growth in a derivative of immortalized human mammary epithelial cells (HMLE). We identified PAK1 as a kinase that permitted HMLE cells to form anchorage-independent colonies. PAK1 is amplified in several human cancer types, including 33% of breast tumor samples and cancer cell lines. The kinase activity of PAK1 is necessary for PAK1-induced transformation. Moreover, we show that PAK1 simultaneously activates MAPK and MET signaling; the latter via inhibition of Merlin. Disruption of these activities inhibits PAK1-driven anchorage-independent growth. These observations establish PAK1 amplification as an alternative mechanism for MAPK activation in human breast cancer and credential PAK1 as a breast cancer oncogene that coordinately regulates multiple signaling pathways, the cooperation of which leads to malignant transformation. PMID:22105362

PAK1 is a breast cancer oncogene that coordinately activates MAPK and MET signaling.

PubMed

Shrestha, Y; Schafer, E J; Boehm, J S; Thomas, S R; He, F; Du, J; Wang, S; Barretina, J; Weir, B A; Zhao, J J; Polyak, K; Golub, T R; Beroukhim, R; Hahn, W C

2012-07-19

Activating mutations in the RAS family or BRAF frequently occur in many types of human cancers but are rarely detected in breast tumors. However, activation of the RAS-RAF-MEK-ERK MAPK pathway is commonly observed in human breast cancers, suggesting that other genetic alterations lead to activation of this signaling pathway. To identify breast cancer oncogenes that activate the MAPK pathway, we screened a library of human kinases for their ability to induce anchorage-independent growth in a derivative of immortalized human mammary epithelial cells (HMLE). We identified p21-activated kinase 1 (PAK1) as a kinase that permitted HMLE cells to form anchorage-independent colonies. PAK1 is amplified in several human cancer types, including 30--33% of breast tumor samples and cancer cell lines. The kinase activity of PAK1 is necessary for PAK1-induced transformation. Moreover, we show that PAK1 simultaneously activates MAPK and MET signaling; the latter via inhibition of merlin. Disruption of these activities inhibits PAK1-driven anchorage-independent growth. These observations establish PAK1 amplification as an alternative mechanism for MAPK activation in human breast cancer and credential PAK1 as a breast cancer oncogene that coordinately regulates multiple signaling pathways, the cooperation of which leads to malignant transformation.

Potential role of p21 Activated Kinase 1 (PAK1) in the invasion and motility of oral cancer cells.

PubMed

Parvathy, Muraleedharan; Sreeja, Sreeharshan; Kumar, Rakesh; Pillai, Madhavan Radhakrishna

2016-05-16

Oral cancer malignancy consists of uncontrolled division of cells primarily in and around the floor of the oral cavity, gingiva, oropharynx, lower lip and base of the tongue. According to GLOBOCAN 2012 report, oral cancer is one of the most common cancers among males and females in India. Even though significant advancements have been made in the field of oral cancer treatment modalities, the overall prognosis for the patients has not improved in the past few decades and hence, this demands a new thrust for the identification of novel therapeutic targets in oral cancer. p21 Activated Kinases (PAKs) are potential therapeutic targets that are involved in numerous physiological functions. PAKs are serine-threonine kinases and they serve as important regulators of cytoskeletal dynamics and cell motility, transcription through MAP kinase cascades, death and survival signalling, and cell-cycle progression. Although PAKs are known to play crucial roles in cancer progression, the role and clinical significance of PAKs in oral cancer remains poorly understood. Our results suggest that PAK1 is over-expressed in oral cancer cell lines. Stimulation of Oral Squamous Cell Carcinoma (OSCC) cells with serum growth factors leads to PAK1 re-localization and might cause a profound cytoskeletal remodelling. PAK1 was also found to be involved in the invasion, migration and cytoskeletal remodelling of OSCC cells. Our study revealed that PAK1 may play a crucial role in the progression of OSCC. Studying the role of PAK1 and its substrates is likely to enhance our understanding of oral carcinogenesis and potential therapeutic value of PAKs in oral cancer.

PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway.

PubMed

Shu, Xiang-Rong; Wu, Jing; Sun, He; Chi, Li-Qun; Wang, Jin-Huan

2015-09-28

Multiple protein or microRNA markers have been recognized to contribute to the progression and recurrence of cervical cancers. Particular those, which are associated with the chemo- or radio-resistance of cervical cancers, have been proposed to be promising and to facilitate the definition for cervical cancer treatment options. This study was designed to explore the potential prognosis value of p21-activated kinase (PAK)-4 in cervical cancer, via the Kaplan-Meier analysis, log-rank test and Cox regression analysis, and then to investigate the regulatory role of PAK4 in the cisplatin resistance in cervical cancer cells, via the strategies of both PAK4 overexpression and PAK4 knockout. It was demonstrated that PAK4 was upregulated in cervical cancer tissues, in an association with the cancer's malignance variables such as FIGO stage, lymph node or distant metastasis and the poor histological grade. The high PAK4 expression was also independently associated with poor prognosis to cervical cancer patients. Moreover, PAK4 confers cisplatin resistance in cervical cancer Hela or Caski cells. In addition, the PI3K/Akt pathway has been implicated in the PAK4-confered cisplatin resistance. And the PI3K/Akt inhibitor, LY294002, markedly deteriorated the cisplatin-mediated viability reduction of Hela or Caski cells, indicating the involvement of PI3K/Akt pathway in the cisplatin resistance in cervical cancer cells. This study has confirmed the significant prognostic role of PAK4 level in cervical cancer patients and has recognized the regulatory role in cervical cancer progression. Moreover, our study has indicated that PAK4 also confers the chemoresistance of cervical cancer cells in a PI3K/Akt-dependent way. Thus, our study indicates PAK4 as a promising marker for cervical cancer treatment.

Regulation of tamoxifen sensitivity by a PAK1–EBP1 signalling pathway in breast cancer

PubMed Central

Ghosh, A; Awasthi, S; Peterson, J R; Hamburger, A W

2013-01-01

Background: EBP1, an ErbB3-binding protein, sensitises breast cancer cells to tamoxifen in part by decreasing ErbB2 protein levels. The p21-regulated serine/threonine kinase PAK1, implicated in tamoxifen resistance, phosphorylates EBP1 in vitro and in vivo at T261. Phosphorylation of EBP1 at this site induces tamoxifen resistance. We thus postulated that inhibition of PAK1 activity, by restoring EBP1 function, could ameliorate the hormone refractory phenotype of ErbB2-overexpressing breast cancer cells. Methods: Effects of EBP1 on ErbB2 levels were measured by western blotting. Effects of EBP1 and IPA-3 on tamoxifen sensitivity were measured using a tetrazolium based cell viability assay. Results: Transient transfection studies indicated that an EBP1 T261E mutant, which mimics EPB1 phosphorylated by PAK1, increased ErbB2 protein levels. An EBP1 T261A mutant, unable to be phosphorylated by PAK1, ameliorated PAK1-induced tamoxifen resistance, suggesting that phosphorylation of EBP1 by PAK1 contributes to tamoxifen resistance. We then tested if pharmacological inhibition of PAK1 activity might render hormone resistant cells, which endogenously overexpress PAK1, tamoxifen sensitive. IPA-3, a specific small MW PAK1 inhibitor, sensitised cells to tamoxifen only when EBP1 was ectopically expressed. IPA had no effect on tamoxifen resistance in T47D cells in which EBP1 protein had been ablated by shRNA. The IPA-induced increase in tamoxifen sensitivity was accompanied by a decrease in ErbB2 levels only in EBP1-overexpressing cells. Conclusion: These studies suggest that phosphorylation of EBP1 may be one mechanism of PAK1-induced hormone resistance and that PAK1 inhibitors may be useful in cells in which EBP1 is overexpressed. PMID:23361053

Labeling of SR 46349B, a potent and selective 5-HT{sub 2} receptor antagonist

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, P.; Fowler, J.S.; Ding, Y.S.

1995-05-01

SR 46349B is a potent and selective 5-HT{sub 2} receptor antagonist (Kd =1.2 nM) which is currently being evaluated as an antidepressant. We labeled SR46349B with F-18 for PET studies via the nitro-for-fluorine exchange reaction. Among the five nitro-precursors (o-nitroacetophenone) examined for nucleophilic aromatic substitution ({sup 18}F{sup {minus}}, K{sub 2}CO{sub 3}, kryptofix-222, 120{degrees}C, 6 min), only phenol protected ether proceeded well and gave 36.4 {plus_minus} 14.3%(n=19) yield of which was directly hydrolyzed (Hcl, 90{degrees}C, 10 min) to afford. Removal of the nitro-precursor, which was generated in situ during hydrolysis was critical in the purification of the final product and wasmore » accomplished using a combination of C-18 Sep-Pak and silica gel column chromatography. The condensation of {sup 18}F- ketone with Me{sub 2}NCH{sub 2}CH{sub 2}ONH{sub 2}HCl in 2-(2{prime}-methoxyethoxy)ethanol (p-TsOH, 165{degrees}C, 10 min) gave a mixture of [{sup 18}F]SR 46349B and its geometric isomer with ca 1:1 ratio in quantitative yield. [{sup 18}F]SR 46349B was separated from its geometric isomer and other by-products by HPLC [Econosil C-18 semi-prep column, MeOH:MeCN:0.1 MK{sub 2}HPO{sub 4}(27.5:27.5:45), 5 ml/min]. The three step hot synthesis required 170 min and gave a specific activity of 1.14 Ci/{mu}mol, 5% radiochemical yield (EOB) and 96% radiochemical purity.« less

An emerging role for p21-activated kinases (Paks) in viral infections.

PubMed

Van den Broeke, Celine; Radu, Maria; Chernoff, Jonathan; Favoreel, Herman W

2010-03-01

p21-activated protein kinases (Paks) are cytosolic serine/threonine protein kinases that act as effectors for small (p21) GTPases of the Cdc42 and Rac families. It has long been established that Paks play a major role in a host of vital cellular functions such as proliferation, survival and motility, and abnormal Pak function is associated with a number of human diseases. Here, we discuss emerging evidence that these enzymes also play a major role in the entry, replication and spread of many important pathogenic human viruses, including HIV. Careful assessment of the potential role of Paks in antiviral immunity will be pivotal to evaluate thoroughly the potential of agents that inhibit Pak as a new class of anti-viral therapeutics.

Regulation of cardiac excitation and contraction by p21 activated kinase-1.

PubMed

Ke, Yunbo; Lei, Ming; Solaro, R John

2008-01-01

Cardiac excitation and contraction are regulated by a variety of signaling molecules. Central to the regulatory scheme are protein kinases and phosphatases that carry out reversible phosphorylation of different effectors. The process of beta-adrenergic stimulation mediated by cAMP dependent protein kinase (PKA) forms a well-known pathway considered as the most significant control mechanism in excitation and contraction as well as many other regulatory mechanisms in cardiac function. However, although dephosphorylation pathways are critical to these regulatory processes, signaling to phosphatases is relatively poorly understood. Emerging evidence indicates that regulation of phosphatases, which dampen the effect of beta-adrenergic stimulation, is also important. We review here functional studies of p21 activated kinase-1 (Pak1) and its potential role as an upstream signal for protein phosphatase PP2A in the heart. Pak1 is a serine/threonine protein kinase directly activated by the small GTPases Cdc42 and Rac1. Pak1 is highly expressed in different regions of the heart and modulates the activities of ion channels, sarcomeric proteins, and other phosphoproteins through up-regulation of PP2A activity. Coordination of Pak1 and PP2A activities is not only potentially involved in regulation of normal cardiac function, but is likely to be important in patho-physiological conditions.

A fully-automated one-pot synthesis of [18F]fluoromethylcholine with reduced dimethylaminoethanol contamination via [18F]fluoromethyl tosylate.

PubMed

Rodnick, Melissa E; Brooks, Allen F; Hockley, Brian G; Henderson, Bradford D; Scott, Peter J H

2013-08-01

A novel one-pot method for preparing [(18)F]fluoromethylcholine ([(18)F]FCH) via in situ generation of [(18)F]fluoromethyl tosylate ([(18)F]FCH2OTs), and subsequent [(18)F]fluoromethylation of dimethylaminoethanol (DMAE), has been developed. [(18)F]FCH was prepared using a GE TRACERlab FXFN, although the method should be readily adaptable to any other fluorine-(18) synthesis module. Initially ditosylmethane was fluorinated to generate [(18)F]FCH2OTs. DMAE was then added and the reaction was heated at 120 °C for 10 min to generate [(18)F]FCH. After this time, reaction solvent was evaporated, and the crude reaction mixture was purified by solid-phase extraction using C(18)-Plus and CM-Light Sep-Pak cartridges to provide [(18)F]FCH formulated in USP saline. The formulated product was passed through a 0.22 µm filter into a sterile dose vial, and submitted for quality control testing. Total synthesis time was 1.25 h from end-of-bombardment. Typical non-decay-corrected yields of [(18)F]FCH prepared using this method were 91 mCi (7% non-decay corrected based upon ~1.3 Ci [(18)F]fluoride), and doses passed all other quality control (QC) tests. A one-pot liquid-phase synthesis of [(18)F]FCH has been developed. Doses contain extremely low levels of residual DMAE (31.6 µg/10 mL dose or ~3 ppm) and passed all other requisite QC testing, confirming their suitability for use in clinical imaging studies. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Fully-automated One-pot Synthesis of [18F]Fluoromethylcholine with Reduced Dimethylaminoethanol Contamination via [18F]Fluoromethyl Tosylate

PubMed Central

Rodnick, Melissa E.; Brooks, Allen F.; Hockley, Brian G.; Henderson, Bradford D.; Scott, Peter J. H.

2013-01-01

Introduction A novel one-pot method for preparing [18F]fluoromethylcholine ([18F]FCH) via in situ generation of [18F]fluoromethyl tosylate ([18F]FCH2OTs), and subsequent [18F]fluoromethylation of dimethylaminoethanol (DMAE), has been developed. Methods [18F]FCH was prepared using a GE TRACERlab FXFN, although the method should be readily adaptable to any other fluorine-18 synthesis module. Initially ditosylmethane was fluorinated to generate [18F]FCH2OTs. DMAE was then added and the reaction was heated at 120°C for 10 min to generate [18F]FCH. After this time, reaction solvent was evaporated, and the crude reaction mixture was purified by solid-phase extraction using C18-Plus and CM-Light Sep-Pak cartridges to provide [18F]FCH formulated in USP saline. The formulated product was passed through a 0.22 μm filter into a sterile dose vial, and submitted for quality control testing. Total synthesis time was 1.25 hours from end-of-bombardment. Results Typical non-decay-corrected yields of [18F]FCH prepared using this method were 91 mCi (7% non-decay corrected based upon ~1.3 Ci [18F]fluoride), and doses passed all other quality control (QC) tests. Conclusion A one-pot liquid-phase synthesis of [18F]FCH has been developed. Doses contain extremely low levels of residual DMAE (31.6 μg / 10 mL dose or ~3 ppm) and passed all other requisite QC testing, confirming their suitability for use in clinical imaging studies. PMID:23665261

Lentiviral Nef Proteins Utilize PAK2-Mediated Deregulation of Cofilin as a General Strategy To Interfere with Actin Remodeling▿ †

PubMed Central

Stolp, Bettina; Abraham, Libin; Rudolph, Jochen M.; Fackler, Oliver T.

2010-01-01

Nef is an accessory protein and pathogenicity factor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) which elevates virus replication in vivo. We recently described for HIV type 1SF2 (HIV-1SF2) the potent interference of Nef with T-lymphocyte chemotaxis via its association with the cellular kinase PAK2. Mechanistic analysis revealed that this interaction results in deregulation of the actin-severing factor cofilin and thus blocks the chemokine-mediated actin remodeling required for cell motility. However, the efficiency of PAK2 association is highly variable among Nef proteins from different lentiviruses, prompting us to evaluate the conservation of this actin-remodeling/cofilin-deregulating mechanism. Based on the analysis of a total of 17 HIV-1, HIV-2, and SIV Nef proteins, we report here that inhibition of chemokine-induced actin remodeling as well as inactivation of cofilin are strongly conserved activities of lentiviral Nef proteins. Of note, even for Nef variants that display only marginal PAK2 association in vitro, these activities require the integrity of a PAK2 recruitment motif and the presence of endogenous PAK2. Thus, reduced in vitro affinity to PAK2 does not indicate limited functionality of Nef-PAK2 complexes in intact HIV-1 host cells. These results establish hijacking of PAK2 for deregulation of cofilin and inhibition of triggered actin remodeling as a highly conserved function of lentiviral Nef proteins, supporting the notion that PAK2 association may be critical for Nef's activity in vivo. PMID:20147394

Lentiviral Nef proteins utilize PAK2-mediated deregulation of cofilin as a general strategy to interfere with actin remodeling.

PubMed

Stolp, Bettina; Abraham, Libin; Rudolph, Jochen M; Fackler, Oliver T

2010-04-01

Nef is an accessory protein and pathogenicity factor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) which elevates virus replication in vivo. We recently described for HIV type 1(SF2) (HIV-1(SF2)) the potent interference of Nef with T-lymphocyte chemotaxis via its association with the cellular kinase PAK2. Mechanistic analysis revealed that this interaction results in deregulation of the actin-severing factor cofilin and thus blocks the chemokine-mediated actin remodeling required for cell motility. However, the efficiency of PAK2 association is highly variable among Nef proteins from different lentiviruses, prompting us to evaluate the conservation of this actin-remodeling/cofilin-deregulating mechanism. Based on the analysis of a total of 17 HIV-1, HIV-2, and SIV Nef proteins, we report here that inhibition of chemokine-induced actin remodeling as well as inactivation of cofilin are strongly conserved activities of lentiviral Nef proteins. Of note, even for Nef variants that display only marginal PAK2 association in vitro, these activities require the integrity of a PAK2 recruitment motif and the presence of endogenous PAK2. Thus, reduced in vitro affinity to PAK2 does not indicate limited functionality of Nef-PAK2 complexes in intact HIV-1 host cells. These results establish hijacking of PAK2 for deregulation of cofilin and inhibition of triggered actin remodeling as a highly conserved function of lentiviral Nef proteins, supporting the notion that PAK2 association may be critical for Nef's activity in vivo.

p21-Activated kinase inhibitors: a patent review.

PubMed

Crawford, James J; Hoeflich, Klaus P; Rudolph, Joachim

2012-03-01

The p21-activated kinase (PAK) family of serine/threonine protein kinases is activated by binding to the small (p21) GTP-binding proteins Cdc42 and Rac. The PAK family plays important roles in cytoskeletal organisation, cellular morphogenesis and survival, and members of this family have been implicated in a wide range of diseases including cancer, infectious diseases, neurological disorders and arthritis. The present review seeks to summarise recent (up to 2011) reports of small-molecule inhibitors of p21-activated kinases. Where patent applications describe activity against a broad range of kinases and no information was provided specifically on PAK inhibition, these are excluded from this review. In patents considered to be relevant, exemplary compounds were selected and highlighted based on their representation of the chemical matter claimed, potencies, structural features and subsequent disclosure of their properties. Selected information from non-patent literature was also included. A considerable amount of research has been devoted over the past 15 years to exploring the role of PAKs in a wide range of diseases, with a focus on oncology. Published PAK inhibitors are still comparatively rare and few exhibit satisfactory kinase selectivity and 'drug-like' properties. A key question is which profile, pan-PAK, group selective or isoform selective, holds the most promise from both therapeutic and safety standpoints. To investigate this question, isoform-selective, as well as kinome-selective, PAK inhibitor tool compounds will be needed. Pfizer was the first company to progress a PAK inhibitor (pan-PAK) to clinical development; it is expected that, despite the difficulties, other PAK inhibitors will soon follow.

Anti-cancer effect of novel PAK1 inhibitor via induction of PUMA-mediated cell death and p21-mediated cell cycle arrest.

PubMed

Woo, Tae-Gyun; Yoon, Min-Ho; Hong, Shin-Deok; Choi, Jiyun; Ha, Nam-Chul; Sun, Hokeun; Park, Bum-Joon

2017-04-04

Hyper-activation of PAK1 (p21-activated kinase 1) is frequently observed in human cancer and speculated as a target of novel anti-tumor drug. In previous, we also showed that PAK1 is highly activated in the Smad4-deficient condition and suppresses PUMA (p53 upregulated modulator of apoptosis) through direct binding and phosphorylation. On the basis of this result, we have tried to find novel PAK1-PUMA binding inhibitors. Through ELISA-based blind chemical library screening, we isolated single compound, IPP-14 (IPP; Inhibitor of PAK1-PUMA), which selectively blocks the PAK1-PUMA binding and also suppresses cell proliferation via PUMA-dependent manner. Indeed, in PUMA-deficient cells, this chemical did not show anti-proliferating effect. This chemical possessed very strong PAK1 inhibition activity that it suppressed BAD (Bcl-2-asoociated death promoter) phosphorylation and meta-phase arrest via Aurora kinase inactivation in lower concentration than that of previous PAK1 kinase, FRAX486 and AG879. Moreover, our chemical obviously induced p21/WAF1/CIP1 (Cyclin-dependent kinase inhibitor 1A) expression by releasing from Bcl-2 (B-cell lymphoma-2) and by inhibition of AKT-mediated p21 suppression. Considering our result, IPP-14 and its derivatives would be possible candidates for PAK1 and p21 induction targeted anti-cancer drug.

Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

PubMed Central

Phee, Hyewon; Au-Yeung, Byron B; Pryshchep, Olga; O'Hagan, Kyle Leonard; Fairbairn, Stephanie Grace; Radu, Maria; Kosoff, Rachelle; Mollenauer, Marianne; Cheng, Debra; Chernoff, Jonathan; Weiss, Arthur

2014-01-01

The molecular mechanisms that govern thymocyte development and maturation are incompletely understood. The P21-activated kinase 2 (Pak2) is an effector for the Rho family GTPases Rac and Cdc42 that regulate actin cytoskeletal remodeling, but its role in the immune system remains poorly understood. In this study, we show that T-cell specific deletion of Pak2 gene in mice resulted in severe T cell lymphopenia accompanied by marked defects in development, maturation, and egress of thymocytes. Pak2 was required for pre-TCR β-selection and positive selection. Surprisingly, Pak2 deficiency in CD4 single positive thymocytes prevented functional maturation and reduced expression of S1P1 and KLF2. Mechanistically, Pak2 is required for actin cytoskeletal remodeling triggered by TCR. Failure to induce proper actin cytoskeletal remodeling impaired PLCγ1 and Erk1/2 signaling in the absence of Pak2, uncovering the critical function of Pak2 as an essential regulator that governs the actin cytoskeleton-dependent signaling to ensure normal thymocyte development and maturation. DOI: http://dx.doi.org/10.7554/eLife.02270.001 PMID:24843022

ArhGAP15, a Rac-specific GTPase-activating Protein, Plays a Dual Role in Inhibiting Small GTPase Signaling*

PubMed Central

Radu, Maria; Rawat, Sonali J.; Beeser, Alexander; Iliuk, Anton; Tao, Weiguo Andy; Chernoff, Jonathan

2013-01-01

Signaling from small GTPases is a tightly regulated process. In this work we used a protein microarray screen to identify the Rac-specific GAP, ArhGAP15, as a substrate of the Rac effectors Pak1 and Pak2. In addition to serving as a substrate of Pak1/2, we found that ArhGAP15, via its PH domain, bound to these kinases. The association of ArhGAP15 to Pak1/2 resulted in mutual inhibition of GAP and kinase catalytic activity, respectively. Knock-down of ArhGAP15 resulted in activation of Pak1/2, both indirectly, as a result of Rac activation, and directly, as a result of disruption of the ArhGAP15/Pak complex. Our data suggest that ArhGAP15 plays a dual negative role in regulating small GTPase signaling, by acting at the level of the GTPase itself, as well interacting with its effector, Pak kinase. PMID:23760270

Unique catalytic activities and scaffolding of p21 activated kinase-1 in cardiovascular signaling.

PubMed

Ke, Yunbo; Lei, Ming; Wang, Xin; Solaro, R John

2013-09-27

P21 activated kinase-1 (Pak1) has diverse functions in mammalian cells. Although a large number of phosphoproteins have been designated as Pak1 substrates from in vitro studies, emerging evidence has indicated that Pak1 may function as a signaling molecule through a unique molecular mechanism - scaffolding. By scaffolding, Pak1 delivers signals through an auto-phosphorylation-induced conformational change without transfer of a phosphate group to its immediate downstream effector(s). Here we review evidence for this regulatory mechanism based on structural and functional studies of Pak1 in different cell types and research models as well as in vitro biochemical assays. We also discuss the implications of Pak1 scaffolding in disease-related signaling processes and the potential in cardiovascular drug development.

[P21-activated kinases and their role in the nervous system].

PubMed

Qin, Yuan; Ding, Yue-Min; Xia, Qiang

2012-12-25

P21-activated kinases (PAK) participate in a variety of important cellular activities, such as cytoskeleton remodeling, cell migration, cell cycle regulation, and apoptosis or survival. PAK also has an important impact on brain development, neuronal differentiation, and regulation of synaptic plasticity in the nervous system. PAK abnormalities result in diseases including cancer, Parkinson's disease (PD), Alzheimer's disease (AD) and neural retardation. Therefore, it is of vital physiological significance to investigate the neuronal function of PAK. In this paper we review the advancement of research on the neuronal biological function and the underlying mechanisms of PAK.

A p21-activated kinase (PAK1) signaling cascade coordinately regulates F-actin remodeling and insulin granule exocytosis in pancreatic β cells

PubMed Central

Kalwat, Michael A.; Yoder, Stephanie M.; Wang, Zhanxiang; Thurmond, Debbie C.

2012-01-01

Human islet studies implicate an important signaling role for the Cdc42 effector protein p21-activated kinase (PAK1) in the sustained/second-phase of insulin secretion. Because human islets from type 2 diabetic donors lack ~80% of normal PAK1 protein levels, the mechanistic requirement for PAK1 signaling in islet function was interrogated. Similar to MIN6 β cells, human islets elicited glucose-stimulated PAK1 activation that was sensitive to the PAK1 inhibitor, IPA3. Given that sustained insulin secretion has been correlated with glucose-induced filamentous actin (F-actin) remodeling, we tested the hypothesis that a Cdc42-activated PAK1 signaling cascade is required to elicit F-actin remodeling to mobilize granules to the cell surface. Live-cell imaging captured the glucose-induced cortical F-actin remodeling in MIN6 β cells; IPA3-mediated inhibition of PAK1 abolished this remodeling. IPA3 also ablated glucose-stimulated insulin granule accumulation at the plasma membrane, consistent with its role in sustained/second-phase insulin release. Both IPA3 and a selective inhibitor of the Cdc42 GTPase, ML-141, blunted the glucose-stimulated activation of Raf-1, suggesting Raf-1 to be downstream of Cdc42→PAK1. IPA3 also inhibited MEK1/2 activation, implicating the MEK1/2→ERK1/2 cascade to occur downstream of PAK1. Importantly, PD0325901, a new selective inhibitor of MEK1/2→ERK1/2 activation, impaired F-actin remodeling and the sustained/amplification pathway of insulin release. Taken together, these data suggest that glucose-mediated activation of Cdc42 leads to activation of PAK1 and prompts activation of its downstream targets Raf-1, MEK1/2 and ERK1/2 to elicit F-actin remodeling and recruitment of insulin granules to the plasma membrane to support the sustained phase of insulin release. PMID:23246867

High-level expression of P21-Cdc/Rac-activated kinase 7 is closely related to metastatic potential and poor prognosis of colon carcinoma

PubMed Central

Song, Guohe; Xiao, Chao; Yan, Dongwang; Zhong, Lin; Sun, Xing; Wang, Xiaoliang; Yu, Fudong; Yu, Yang; Tang, Huamei; Peng, Zhihai

2016-01-01

P21 protein (Cdc42/Rac)-activated kinase 7 (PAK7) can promote neurite outgrowth, induce microtubule stabilization, and activate cell survival signaling pathways. PAK7 expression was found to increase with colon carcinoma progression, but the prognostic value, clinical significance, and underlying mechanisms have not been explored. In my study, the expression of PAK7 was up-related at both the transcriptional and the translational levels in colon tumors compared to that in adjacent normal colon tissue. Patients with PAK7-positive tumors had a lower rate of overall survival (OS) and metastasis-free survival (MFS) (log-rank test, P < 0.001). A Cox proportional hazards model showed that PAK7 expression was an independent prognostic factor for OS (hazard ration [HR], 2.08; 95% confidence interval [CI], 1.16-3.73; P = 0.004) and MFS (HR, 2.88; 95% CI, 1.53-5.42; P < 0.001) in patients with colon cancer. Patients with tumors that were over-expressing PAK7 experienced metastasis, and died within a significantly shorter time after surgery (P < 0.001). Knockdown of PAK7 by a specific short hairpin RNA (shRNA) significantly suppressed the progression of epithelial to mesechymal transition (EMT), migration, and invasion of colon cancer cells in vitro and tumor growth in vivo. However, overexpression of PAK7 significantly promoted these processes. These findings indicate that aberrant PAK7 expression is associated with the occurrence of metastasis and poor clinical outcomes of human colon cancer by promoting the EMT, and the assessment of PAK7 expression might be helpful in predicting metastasis and prognostication for patients with colon cancer. PMID:27323857

A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

PubMed

Kesanakurti, D; Maddirela, D; Banasavadi-Siddegowda, Y K; Lai, T-H; Qamri, Z; Jacob, N K; Sampath, D; Mohanam, S; Kaur, B; Puduvalli, V K

2017-09-14

Tumor recurrence in glioblastoma (GBM) is, in part, attributed to increased epithelial-to-mesenchymal transition (EMT) and enhanced tumor cell dissemination in adjacent brain parenchyma after ionizing radiation (IR). EMT is associated with aggressive behavior, increased stem-like characteristics and treatment resistance in malignancies; however, the underlying signaling mechanisms that regulate EMT are poorly understood. We identified grade-dependent p21-activated kinases 4 (PAK4) upregulation in gliomas and further determined its role in mesenchymal transition and radioresistance. IR treatment significantly elevated expression and nuclear localization of PAK4 in correlation with induction of reactive oxygen species (ROS) and mesenchymal transition in GBM cells. Stable PAK4 overexpression promoted mesenchymal transition by elevating EMT marker expression in these cells. Of note, transcription factor-DNA-binding arrays and chromatin immunoprecipitation experiments identified the formation of a novel nuclear PAK4/PPARγ complex which was recruited to the promoter of Nox1, a peroxisome proliferator-activated receptor gamma (PPARγ) target gene. In addition, IR further elevated PAK4/PPARγ complex co-recruitment to Nox1 promoter, and increased Nox1 expression and ROS levels associated with mesenchymal transition in these cells. Conversely, specific PAK4 downregulation decreased PPARγ-mediated Nox1 expression and suppressed EMT in IR-treated cells. In vivo orthotopic tumor experiments showed inhibition of growth and suppression of IR-induced PPARγ and Nox1 expression by PAK4 downregulation in tumors. Our results provide the first evidence of a novel role for PAK4 in IR-induced EMT and suggest potential therapeutic efficacy of targeting PAK4 to overcome radioresistance in gliomas.

Coordinating structural and functional synapse development: postsynaptic p21-activated kinase independently specifies glutamate receptor abundance and postsynaptic morphology.

PubMed

Albin, Stephanie D; Davis, Graeme W

2004-08-04

Here, we show that postsynaptic p21-activated kinase (Pak) signaling diverges into two genetically separable pathways at the Drosophila neuromuscular junction. One pathway controls glutamate receptor abundance. Pak signaling within this pathway is specified by a required interaction with the adaptor protein Dreadlocks (Dock). We demonstrate that Dock is localized to the synapse via an Src homology 2-mediated protein interaction. Dock is not necessary for Pak localization but is necessary to restrict Pak signaling to control glutamate receptor abundance. A second genetically separable function of Pak kinase signaling controls muscle membrane specialization through the regulation of synaptic Discs-large. In this pathway, Dock is dispensable. We present a model in which divergent Pak signaling is able to coordinate two different features of postsynaptic maturation, receptor abundance, and muscle membrane specialization.

P21-activated kinase 4 (PAK4) is required for metaphase spindle positioning and anchoring.

PubMed

Bompard, G; Rabeharivelo, G; Cau, J; Abrieu, A; Delsert, C; Morin, N

2013-02-14

The oncogenic kinase PAK4 was recently found to be involved in the regulation of the G1 phase and the G2/M transition of the cell cycle. We have also identified that PAK4 regulates Ran GTPase activity during mitosis. Here, we show that after entering mitosis, PAK4-depleted cells maintain a prolonged metaphase-like state. In these cells, chromosome congression to the metaphase plate occurs with normal kinetics but is followed by an extended period during which membrane blebbing and spindle rotation are observed. These bipolar PAK4-depleted metaphase-like spindles have a defective astral microtubule (MT) network and are not centered in the cell but are in close contact with the cell cortex. As the metaphase-like state persists, centrosome fragmentation occurs, chromosomes scatter from the metaphase plate and move toward the spindle poles with an active spindle assembly checkpoint, a phenotype that is reminiscent of cohesion fatigue. PAK4 also regulates the acto-myosin cytoskeleton and we report that PAK4 depletion results in the induction of cortical membrane blebbing during prometaphase arrest. However, we show that membrane blebs, which are strongly enriched in phospho-cofilin, are not responsible for the poor anchoring of the spindle. As PAK4 depletion interferes with the localization of components of the dynein/dynactin complexes at the kinetochores and on the astral MTs, we propose that loss of PAK4 could induce a change in the activities of motor proteins.

Pak3 promotes cell cycle exit and differentiation of β-cells in the embryonic pancreas and is necessary to maintain glucose homeostasis in adult mice.

PubMed

Piccand, Julie; Meunier, Aline; Merle, Carole; Jia, Zhengping; Barnier, Jean-Vianney; Gradwohl, Gérard

2014-01-01

The transcription factor neurogenin3 (Ngn3) triggers islet cell differentiation in the developing pancreas. However, little is known about the molecular mechanisms coupling cell cycle exit and differentiation in Ngn3(+) islet progenitors. We identified a novel effector of Ngn3 endocrinogenic function, the p21 protein-activated kinase Pak3, known to control neuronal differentiation and implicated in X-linked intellectual disability in humans. We show that Pak3 expression is initiated in Ngn3(+) endocrine progenitor cells and next maintained in maturing hormone-expressing cells during pancreas development as well as in adult islet cells. In Pak3-deficient embryos, the proliferation of Ngn3(+) progenitors and β-cells is transiently increased concomitantly with an upregulation of Ccnd1. β-Cell differentiation is impaired at E15.5 but resumes at later stages. Pak3-deficient mice do not develop overt diabetes but are glucose intolerant under high-fat diet (HFD). In the intestine, Pak3 is expressed in enteroendocrine cells but is not necessary for their differentiation. Our results indicate that Pak3 is a novel regulator of β-cell differentiation and function. Pak3 acts downstream of Ngn3 to promote cell cycle exit and differentiation in the embryo by a mechanism that might involve repression of Ccnd1. In the adult, Pak3 is required for the proper control of glucose homeostasis under challenging HFD.

Anti-cancer effect of novel PAK1 inhibitor via induction of PUMA-mediated cell death and p21-mediated cell cycle arrest

PubMed Central

Woo, Tae-Gyun; Yoon, Min-Ho; Hong, Shin-Deok; Choi, Jiyun; Ha, Nam-Chul; Sun, Hokeun; Park, Bum-Joon

2017-01-01

Hyper-activation of PAK1 (p21-activated kinase 1) is frequently observed in human cancer and speculated as a target of novel anti-tumor drug. In previous, we also showed that PAK1 is highly activated in the Smad4-deficient condition and suppresses PUMA (p53 upregulated modulator of apoptosis) through direct binding and phosphorylation. On the basis of this result, we have tried to find novel PAK1-PUMA binding inhibitors. Through ELISA-based blind chemical library screening, we isolated single compound, IPP-14 (IPP; Inhibitor of PAK1-PUMA), which selectively blocks the PAK1-PUMA binding and also suppresses cell proliferation via PUMA-dependent manner. Indeed, in PUMA-deficient cells, this chemical did not show anti-proliferating effect. This chemical possessed very strong PAK1 inhibition activity that it suppressed BAD (Bcl-2-asoociated death promoter) phosphorylation and meta-phase arrest via Aurora kinase inactivation in lower concentration than that of previous PAK1 kinase, FRAX486 and AG879. Moreover, our chemical obviously induced p21/WAF1/CIP1 (Cyclin-dependent kinase inhibitor 1A) expression by releasing from Bcl-2 (B-cell lymphoma-2) and by inhibition of AKT-mediated p21 suppression. Considering our result, IPP-14 and its derivatives would be possible candidates for PAK1 and p21 induction targeted anti-cancer drug. PMID:28423593

Dynamics of Flagellum- and Pilus-Mediated Association of Pseudomonas aeruginosa with Contact Lens Surfaces▿

PubMed Central

Tran, Victoria B.; Fleiszig, Suzanne M. J.; Evans, David J.; Radke, Clayton J.

2011-01-01

Flagella and pili are appendages that modulate attachment of Pseudomonas aeruginosa to solid surfaces. However, previous studies have mostly reported absolute attachment. Neither the dynamic roles of these appendages in surface association nor those of attachment phenotypes have been quantified. We used video microscopy to address this issue. Unworn, sterile, soft contact lenses were placed in a laminar-flow optical chamber. Initial lens association kinetics for P. aeruginosa strain PAK were assessed in addition to lens-surface association phenotypes. Comparisons were made to strains with mutations in flagellin (fliC) or pilin (pilA) or those in flagellum (motAB) or pilus (pilU) function. PAK and its mutants associated with the contact lens surface at a constant rate according to first-order kinetics. Nonswimming mutants associated ∼30 to 40 times slower than the wild type. PAK and its pilA mutant associated at similar rates, but each ∼4 times faster than the pilU mutant. Lens attachment by wild-type PAK induced multiple phenotypes (static, lateral, and rotational surface movement), each showing only minor detachment. Flagellin (fliC) and flagellar-motility (motAB) mutants did not exhibit surface rotation. Conversely, strains with mutations in pilin (pilA) and pilus retraction (pilU) lacked lateral-surface movement but displayed enhanced surface rotation. Slower surface association of swimming-incapable P. aeruginosa mutants was ascribed to lower convective-diffusion-arrival rates, not to an inability to adhere. Flagellum function (swimming) enhanced lens association, attachment, and rotation; hyperpiliation hindered lens association. P. aeruginosa bound through three different adhesion sites: flagellum, pili, and body. Reduction of bacterial attachment to contact lenses thus requires blockage of multiple adhesion phenotypes. PMID:21498762

The role of p21-activated kinases in hepatocellular carcinoma metastasis.

PubMed

Tse, Edith Yuk Ting; Ching, Yick Pang

2014-01-01

The p21-activated kinases (PAKs) are downstream effectors of the Rho family small GTPases as well as a wide variety of mitogenic factors and have been implicated in cancer formation, development and metastasis. PAKs phosphorylate a wide spectrum of substrates to mediate extracellular signals and regulate cytoskeletal remodeling, cell motility and survival. In this review, we aim to summarize the findings regarding the oncogenic role and the underlying mechanisms of PAKs signaling in various cancers, and in particular highlight the prime importance of PAKs in hepatocellular carcinoma (HCC) progression and metastasis. Recent studies exploring the potential therapeutic application of PAK inhibitors will also be discussed.

p21-activated kinase 1 restricts tonic endocannabinoid signaling in the hippocampus

PubMed Central

Xia, Shuting; Zhou, Zikai; Leung, Celeste; Zhu, Yuehua; Pan, Xingxiu; Qi, Junxia; Morena, Maria; Hill, Matthew N; Xie, Wei; Jia, Zhengping

2016-01-01

PAK1 inhibitors are known to markedly improve social and cognitive function in several animal models of brain disorders, including autism, but the underlying mechanisms remain elusive. We show here that disruption of PAK1 in mice suppresses inhibitory neurotransmission through an increase in tonic, but not phasic, secretion of endocannabinoids (eCB). Consistently, we found elevated levels of anandamide (AEA), but not 2-arachidonoylglycerol (2-AG) following PAK1 disruption. This increased tonic AEA signaling is mediated by reduced cyclooxygenase-2 (COX-2), and COX-2 inhibitors recapitulate the effect of PAK1 deletion on GABAergic transmission in a CB1 receptor-dependent manner. These results establish a novel signaling process whereby PAK1 upregulates COX-2, reduces AEA and restricts tonic eCB-mediated processes. Because PAK1 and eCB are both critically involved in many other organ systems in addition to the brain, our findings may provide a unified mechanism by which PAK1 regulates these systems and their dysfunctions including cancers, inflammations and allergies. DOI: http://dx.doi.org/10.7554/eLife.14653.001 PMID:27296803

Sliding p21-activated kinase 1 to nucleus impacts tamoxifen sensitivity.

PubMed

Rayala, Suresh K; Kumar, Rakesh

2007-08-01

The anti-estrogen, tamoxifen is the most commonly used treatment for patients with estrogen receptor (ER)-alpha-positive breast cancer. Recent data suggest that levels of ER coregulatory proteins as well as extra- and intracellular signaling in response to growth factor stimulation of breast cancer cells play an important role in acquiring resistance to anti-estrogen action. P21-activated kinase 1 (PAK1), a major target of the small GTPases, growth factors and lipid signaling, regulates cell motility, hormone action, invasiveness, and survival, all of which are required for both tumor development and normal mammary gland development. Over the years, the PAK1 has been regarded as cytosolic serine-threonine kinase with regulatory function in cytoskeleton reorganization and motility. However, emerging data now provide evidence of PAK1 function in the nucleus of breast cancer cells. Elevated PAK1 expression in premenopausal breast cancer patients correlates well with the lack of tamoxifen response despite the presence of ER-alpha expression, and such relationship was even distinctly stronger in breast tumors with nuclear PAK1. These typical effects of PAK1 are mechanistically linked with the ability of PAK1 to phosphorylate ER-alpha on serine 305, accompanied by secondary activation of serine 118, and such structural modifications may participate in the development of tamoxifen resistance. These findings suggest that the levels, subcellular localization, and activation status of PAK1 are likely to be important determinants of tamoxifen resistance, and that raising the possibility that tamoxifen resistance might be prevented or reversed by PAK1 inhibition.

Knockout of p21-activated kinase-1 attenuates exercise-induced cardiac remodelling through altered calcineurin signalling

PubMed Central

Davis, Robert T.; Simon, Jillian N.; Utter, Megan; Mungai, Paul; Alvarez, Manuel G.; Chowdhury, Shamim A.K.; Heydemann, Ahlke; Ke, Yunbo; Wolska, Beata M.; Solaro, R. John

2015-01-01

Aims Despite its known cardiovascular benefits, the intracellular signalling mechanisms underlying physiological cardiac growth remain poorly understood. Therefore, the purpose of this study was to investigate a novel role of p21-activated kinase-1 (Pak1) in the regulation of exercise-induced cardiac hypertrophy. Methods and results Wild-type (WT) and Pak1 KO mice were subjected to 6 weeks of treadmill endurance exercise training (ex-training). Cardiac function was assessed via echocardiography, in situ haemodynamics, and the pCa–force relations in skinned fibre preparations at baseline and at the end of the training regimen. Post-translational modifications to the sarcomeric proteins and expression levels of calcium-regulating proteins were also assessed following ex-training. Heart weight/tibia length and echocardiography data revealed that there was marked hypertrophy following ex-training in the WT mice, which was not evident in the KO mice. Additionally, following ex-training, WT mice demonstrated an increase in cardiac contractility, myofilament calcium sensitivity, and phosphorylation of cardiac myosin-binding protein C, cardiac TnT, and tropomyosin compared with KO mice. With ex-training in WT mice, there were also increased protein levels of calcineurin and increased phosphorylation of phospholamban. Conclusions Our data suggest that Pak1 is essential for adaptive physiological cardiac remodelling and support previous evidence that demonstrates Pak1 signalling is important for cardiac growth and survival. PMID:26464331

p21-Activated kinase 5: a pleiotropic kinase.

PubMed

Wen, Yi-Yang; Wang, Xiao-Xia; Pei, Dong-Sheng; Zheng, Jun-Nian

2013-12-15

The PAKs (p21-activated kinases) are highly conserved serine/threonine protein kinases which comprise six mammalian PAKs. PAK5 (p21-activated kinase 5) is the least understood member of PAKs that regulate many intracellular processes when they are stimulated by activated forms of the small GTPases Cdc42 and Rac. PAK5 takes an important part in multiple signal pathways in mammalian cells and controls a variety of cellular functions including cytoskeleton organization, cell motility and apoptosis. The main goal of this review is to describe the structure, mechanisms underlying its activity regulation, its role in apoptosis and the likely directions of further research. Copyright © 2013 Elsevier Ltd. All rights reserved.

Heat sinking for printed circuitry

DOEpatents

Wilson, S.K.; Richardson, G.; Pinkerton, A.L.

1984-09-11

A flat pak or other solid-state device mounted on a printed circuit board directly over a hole extends therethrough so that the bottom of the pak or device extends beyond the bottom of the circuit board. A heat sink disposed beneath the circuit board contacts the bottom of the pak or device and provides direct heat sinking thereto. Pressure may be applied to the top of the pak or device to assure good mechanical and thermal contact with the heat sink.

Correlation between chemotherapy resistance in osteosarcoma patients and PAK5 and Ezrin gene expression

PubMed Central

Liu, Qian; Xu, Bo; Zhou, Wanshan

2018-01-01

The correlation between PAK5 (P21-activated kinase 5) and Ezrin gene expression and chemotherapy resistance of osteosarcoma patients was investigated. The cisplatin (CDDP)-resistance model of osteosarcoma cells SOSP-9607/CDDP was established to detect the cell growth curve. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the drug resistance of cells to chemotherapy drugs. Transwell assay was used to detect the invasive capacity of cells. Semi-quantitative PCR (qPCR) was used to detect the mRNA expression levels in the drug resistance-related genes PAK5 and Ezrin. Western blot analysis was used to detect the protein expression levels in PAK5 and Ezrin. Tumor tissues were taken from the osteosarcoma patients with chemotherapy resistance to detect the expression levels of PAK5 and Ezrin via immunohistochemical detection, and the correlation between PAK5 and Ezrin expressions was studied. The results of MTT assay showed that the growth rate of SOSP-9607 was similar to that of SOSP-9607/CDDP, and the difference was not statistically significant (P>0.05). The sensitivity of SOSP-9607 to CDDP was significantly higher than that of SOSP-9607/CDDP, and the difference was statistically significant (P<0.01). Transwell assay showed that the migration capacity of SOSP-9607/CDDP was significantly better than that of SOSP-9607 (P<0.01), indicating that the drug resistance cell lines of osteosarcoma were constructed successfully. Semi-qPCR and western blot analysis showed that the protein expression levels in PAK5 and Ezrin in SOSP-9607/CDDP were significantly higher than those in SOSP-9607 (P<0.01). The results of immunohistochemistry showed that the expression quantities of PAK5 and Ezrin in osteosarcoma tissues were significantly higher than those in para-tumor tissues (P<0.01). Pearson's correlation analysis showed that expression of PAK5 and Ezrin was positively correlated (r=0.197, P=0.023). The osteosarcoma resistance is closely related to the expression levels of PAK5 and Ezrin genes. Thus, PAK5 and Ezrin genes may affect the tolerance of osteosarcoma patients to chemotherapy drugs during treatment via the synergistic effect. PMID:29391894

Differences in phosphatidic acid signalling and metabolism between ABA and GA treatments of barley aleurone cells.

PubMed

Villasuso, Ana Laura; Di Palma, Maria A; Aveldaño, Marta; Pasquaré, Susana J; Racagni, Graciela; Giusto, Norma M; Machado, Estela E

2013-04-01

Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (GA) response. In this work we investigated the lipid metabolism in response to both hormones. We could detect an in vivo phospholipase D activity (PLD, EC 3.1.4.4). This PLD produced [(32)P]PA (phosphatidic acid) rapidly (minutes) in the presence of ABA, confirming PA involvement in signal transduction, and transiently, indicating rapid PA removal after generation. The presence of PA removal by phosphatidate phosphatase 1 and 2 isoforms (E.C. 3.1.3.4) was verified in isolated aleurone membranes in vitro, the former but not the latter being specifically responsive to the presence of GA or ABA. The in vitro DGPP phosphatase activity was not modified by short time incubation with GA or ABA while the in vitro PA kinase - that allows the production of 18:2-DGPP from 18:2-PA - is stimulated by ABA. The long term effects (24 h) of ABA or GA on lipid and fatty acid composition of aleurone layer cells were then investigated. An increase in PC and, to a lesser extent, in PE levels is the consequence of both hormone treatments. ABA, in aleurone layer cells, specifically activates a PLD whose product, PA, could be the substrate of PAP1 and/or PAK activities. Neither PLD nor PAK activation can be monitored by GA treatment. The increase in PAP1 activity monitored after ABA or GA treatment might participate in the increase in PC level observed after 24 h hormone incubation. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents.

PubMed

Ong, Christy C; Gierke, Sarah; Pitt, Cameron; Sagolla, Meredith; Cheng, Christine K; Zhou, Wei; Jubb, Adrian M; Strickland, Laura; Schmidt, Maike; Duron, Sergio G; Campbell, David A; Zheng, Wei; Dehdashti, Seameen; Shen, Min; Yang, Nora; Behnke, Mark L; Huang, Wenwei; McKew, John C; Chernoff, Jonathan; Forrest, William F; Haverty, Peter M; Chin, Suet-Feung; Rakha, Emad A; Green, Andrew R; Ellis, Ian O; Caldas, Carlos; O'Brien, Thomas; Friedman, Lori S; Koeppen, Hartmut; Rudolph, Joachim; Hoeflich, Klaus P

2015-04-23

Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis. PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n=980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting. We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P=1.29×10(-4) and P=0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis. Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.

The Dynamic Growth Exhibition and Accumulation of Cadmium of Pak Choi (Brassica campestris L. ssp. chinensis) Grown in Contaminated Soils

PubMed Central

Lai, Hung-Yu; Chen, Bo-Ching

2013-01-01

The accumulation of heavy metals, especially cadmium (Cd), in leafy vegetables was compared with other vegetables. Pak choi (Brassica campestris L. ssp. chinensis) is a leafy vegetable consumed in Taiwan and its safety for consumption after growing in contaminated soils is a public concern. A pot experiment (50 days) was conducted to understand the dynamic accumulation of Cd by pak choi grown in artificially contaminated soils. The edible parts of pak choi were sampled and analyzed every 2–3 days. The dry weight (DW) of pak choi was an exponential function of leaf length, leaf width, and chlorophyll content. The accumulation of Cd increased when the soil Cd concentration was raised, but was kept at a constant level during different growth stages. Pak choi had a high bioconcentration factor (BCF = ratio of the concentration in the edible parts to that in the soils), at values of 3.5–4.0. The consumption of pak choi grown in soils contaminated at levels used in this study would result in the ingestion of impermissible amounts of Cd and could possibly have harmful effects on health. PMID:24284350

p21-Activated kinase-1 and its role in integrated regulation of cardiac contractility.

PubMed

Sheehan, Katherine A; Ke, Yunbo; Solaro, R John

2007-09-01

We review here a novel concept in the regulation of cardiac contractility involving variations in the activity of the multifunctional enzyme, p21-activated kinase 1 (Pak1), a member of a family of proteins in the small G protein-signaling pathway that is activated by Cdc42 and Rac1. There is a large body of evidence from studies in noncardiac tissue that Pak1 activity is key in regulation of a number of cellular functions, including cytoskeletal dynamics, cell motility, growth, and proliferation. Although of significant potential impact, the role of Pak1 in regulation of the heart has been investigated in only a few laboratories. In this review, we discuss the structure of Pak1 and its sites of posttranslational modification and molecular interactions. We assemble an overview of the current data on Pak1 signaling in noncardiac tissues relative to similar signaling pathways in the heart, and we identify potential roles of Pak1 in cardiac regulation. Finally, we discuss the current state of Pak1 research in the heart in regard to regulation of contractility through functional myofilament and Ca(2+)-flux modification. An important aspect of this regulation is the modulation of kinase and phosphatase activity. We have focused on Pak1 regulation of protein phosphatase 2A (PP2A), which is abundant in cardiac muscle, thereby mediating dephosphorylation of sarcomeric proteins and sensitizing the myofilaments to Ca(2+). We present a model for Pak1 signaling that provides a mechanism for specifically affecting cardiac cellular processes in which regulation of protein phosphorylation states by PP2A dephosphorylation predominates.

Nuclear p21-activated kinase 1 in breast cancer packs off tamoxifen sensitivity.

PubMed

Rayala, Suresh K; Molli, Poonam R; Kumar, Rakesh

2006-06-15

There is significant clinical interest in the factors that influence the development of tamoxifen resistance in estrogen receptor-alpha (ER-alpha)-positive breast cancers. Recent studies suggest that in ER-positive breast tumor cells, elevated protein levels, and in particular, nuclear localization of p21-activated kinase 1 (PAK1), is associated with the progressive limitation of tamoxifen sensitivity. These phenotypic effects of PAK1 in model systems are mechanistically linked with the ability of PAK1 to phosphorylate ER-alpha on serine 305 and subsequent secondary activation of serine 118. These findings prompt further investigation of how nuclear signaling by PAK1 may affect estrogen's action and whether tamoxifen resistance might be prevented or reversed by PAK1 inhibition.

The p21-activated kinase 1 (Pak1) signalling pathway in cardiac disease: from mechanistic study to therapeutic exploration.

PubMed

Wang, Yanwen; Wang, Shunyao; Lei, Ming; Boyett, Mark; Tsui, Hoyee; Liu, Wei; Wang, Xin

2018-04-01

p21-activated kinase 1 (Pak1) is a member of the highly conserved family of serine/threonine protein kinases regulated by Ras-related small G-proteins, Cdc42/Rac1. It has been previously demonstrated to be involved in cardiac protection. Based on recent studies, this review provides an overview of the role of Pak1 in cardiac diseases including disrupted Ca 2+ homoeostasis-related cardiac arrhythmias, adrenergic stress- and pressure overload-induced hypertrophy, and ischaemia/reperfusion injury. These findings demonstrate the important role of Pak1 mediated through the phosphorylation and transcriptional modification of hypertrophy and/or arrhythmia-related genes. This review also discusses the anti-arrhythmic and anti-hypertrophic, protective function of Pak1 and the beneficial effects of fingolimod (an FDA-approved sphingolipid drug), a Pak1 activator, and its ability to prevent arrhythmias and cardiac hypertrophy. These findings also highlight the therapeutic potential of Pak1 signalling in the treatment and prevention of cardiac diseases. This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc. © 2017 The British Pharmacological Society.

Dermoscopy of Pigmented Actinic Keratosis of the Face: A Study of 232 Cases.

PubMed

Kelati, A; Baybay, H; Moscarella, E; Argenziano, G; Gallouj, S; Mernissi, F Z

2017-11-01

The diagnosis of pigmented actinic keratosis (PAK) is often challenging because of overlapping features with lentigo maligna. To investigate dermoscopic patterns of PAK according to their different evolutionary stages, and to correlate the pattern with clinical characteristics of the patients. Descriptive and analytical study of 232 PAK. Dermoscopic patterns were divided into two categories: the follicule surroundings' abnormalities (FSA) and follicular keratosis' abnormalities (FKA). FSA and FKA dermoscopic patterns were related to male gender, except for star-like appearance, double white clods and dermoscopic horn (p≤0.04). Rhomboidal, annular granular pattern, gray halo, white circle and double clods were dermoscopic pattern significantly related to xeroderma pigmentosum's type of skin. Based on the evolutionary stages of PAK, the jelly sign was significantly related to thin patches of PAK. Central crusts and scales were related to thick plaques and the star-like appearance to hypertrophic PAK. The presence of 2 or more dermoscopic signs in both FSA and FKA was noticed in 99.1% of lesions. The dermoscopic diagnosis of PAK vary according to the evolutionary stages of the disease, this will increase the diagnosis accuracy, with therapeutic implications. Copyright © 2017 AEDV. Publicado por Elsevier España, S.L.U. All rights reserved.

Mapping of the antigenic determinants of Pseudomonas aeruginosa PAK polar pili.

PubMed Central

Watts, T H; Sastry, P A; Hodges, R S; Paranchych, W

1983-01-01

The polar pili of Pseudomonas aeruginosa are flexible filaments 5.2 nm in diameter and 2.5 microns in average length. They consist of a single subunit, pilin, which is a 144-residue polypeptide containing a hydrophobic N-terminal region (residues 1 to 30) and eight hydrophilic regions distributed throughout the remainder of the molecule. To delineate the antigenic regions of pilin, we cleaved the protein at Arg30, Arg53, and Arg120 to produce peptides TCI (residues 1 to 30), TCII (31 to 53), TCIII (54 to 120), and TCIV (121 to 144). TCIII and TCIV were further cleaved into several subfragments. The purified peptides were coupled to bovine serum albumin by using the N-hydroxysuccinimide ester of 4-azidobenzoic acid and were then subjected to immunological analysis, using the enzyme-linked immunosorbent assay and immunoblot procedures with polyclonal antiserum. Four antigenic regions were identified; one in TCI was found to be common to both PAK and PAO pilin. The remaining three were found to be specific to PAK pilin. Two of these were subfragments of TCIII, whereas the third was located close to the C-terminus of the molecule, most likely between Cys129 and Cys142. Modification of these cysteines by reduction and carboxymethylation of the disulfide linkage did not abolish the antigenicity of the C-terminal type-specific antigenic determinant. Images PMID:6194112

p21-activated kinase 1 (PAK1) expression correlates with prognosis in solid tumors: A systematic review and meta-analysis.

PubMed

Fang, Fang; Pan, Jian; Li, Yi-Ping; Li, Gang; Xu, Li-Xiao; Su, Guang-Hao; Li, Zhi-Heng; Feng, Xing; Wang, Jian

2016-05-10

p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) expression appears to be predictive of prognosis in various solid tumors, though the evidence is not yet conclusive. We therefore performed a meta-analysis to explore the relationship between PAK1 and prognosis in patients with solid tumors. Relevant publications were searched in several widely used databases, and 15 studies (3068 patients) were included in the meta-analysis. Pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the association between PAK1 and prognosis. Associations between PAK1 expression and prognosis were observed for overall survival (HR = 2.81, 95% CI = 1.07-7.39) and disease-specific survival (HR = 2.15, 95% CI = 1.47-3.16). No such association was detected for time to tumor progression (HR = 1.78, 95% CI = 0.99-3.21).Our meta-analysis thus indicates that PAK1 expression may be a predictive marker of overall survival and disease-specific survival in patients with solid tumors.

The actin-related p41ARC subunit contributes to p21-activated kinase-1 (PAK1)-mediated glucose uptake into skeletal muscle cells.

PubMed

Tunduguru, Ragadeepthi; Zhang, Jing; Aslamy, Arianne; Salunkhe, Vishal A; Brozinick, Joseph T; Elmendorf, Jeffrey S; Thurmond, Debbie C

2017-11-17

Defects in translocation of the glucose transporter GLUT4 are associated with peripheral insulin resistance, preclinical diabetes, and progression to type 2 diabetes. GLUT4 recruitment to the plasma membrane of skeletal muscle cells requires F-actin remodeling. Insulin signaling in muscle requires p21-activated kinase-1 (PAK1), whose downstream signaling triggers actin remodeling, which promotes GLUT4 vesicle translocation and glucose uptake into skeletal muscle cells. Actin remodeling is a cyclic process, and although PAK1 is known to initiate changes to the cortical actin-binding protein cofilin to stimulate the depolymerizing arm of the cycle, how PAK1 might trigger the polymerizing arm of the cycle remains unresolved. Toward this, we investigated whether PAK1 contributes to the mechanisms involving the actin-binding and -polymerizing proteins neural Wiskott-Aldrich syndrome protein (N-WASP), cortactin, and ARP2/3 subunits. We found that the actin-polymerizing ARP2/3 subunit p41ARC is a PAK1 substrate in skeletal muscle cells. Moreover, co-immunoprecipitation experiments revealed that insulin stimulates p41ARC phosphorylation and increases its association with N-WASP coordinately with the associations of N-WASP with cortactin and actin. Importantly, all of these associations were ablated by the PAK inhibitor IPA3, suggesting that PAK1 activation lies upstream of these actin-polymerizing complexes. Using the N-WASP inhibitor wiskostatin, we further demonstrated that N-WASP is required for localized F-actin polymerization, GLUT4 vesicle translocation, and glucose uptake. These results expand the model of insulin-stimulated glucose uptake in skeletal muscle cells by implicating p41ARC as a new component of the insulin-signaling cascade and connecting PAK1 signaling to N-WASP-cortactin-mediated actin polymerization and GLUT4 vesicle translocation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Simultaneous determination of dextrose, sucrose, maltose, and lactose in sausage products by liquid chromatography.

PubMed

Ali, M S

1988-01-01

A liquid chromatographic (LC) method for the simultaneous determination of dextrose, sucrose, maltose, and lactose in sausage products has been developed. Dextrose, sucrose, maltose, and lactose are extracted from comminuted meat products with 52% ethanol. After filtration, the extracts are purified by passing them through a C18 Sep-Pak cartridge and 2 ion exchange resin Econo-columns in series. After concentration and filtration, extracts are analyzed by LC using a normal phase amino column and a differential refractometer detector. Homogeneously ground samples of cooked and fresh sausages are fortified with dextrose, sucrose, maltose, and lactose at 4 different concentrations. Average recovery for dextrose, sucrose, maltose, and lactose at all 4 levels of fortification was greater than 80% with a coefficient of variation less than 10%.

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection.

PubMed

Ono, I; Matsuda, K; Kanno, S

1996-04-12

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC18, followed by HPLC. The calibration graph for I was linear in the range 0.1-20 micrograms/ml. The limit of quantitation of I, in plasma, was 0.05 microgram/ml. The recovery of spiked I (0.5 microgram/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 microgram/ml) compared to drug-free plasma was 4.3% (n = 8).

Rapid method for measuring rotenone in water at piscicidal concentrations

USGS Publications Warehouse

Dawson, V.K.; Harman, P.D.; Schultz, D.P.; Allen, J.L.

1983-01-01

A high-performance liquid chromatography (HPLC) procedure that is rapid, specific, and sensitive (limit of detection <0.005 mg/liter) was developed for monitoring application and degradation rates of rotenone. For analysis, a water sample is buffered to pH 5 and injected through a Sep Pak(R) C18 disposable cartridge. The cartridge adsorbs and retains the rotenone which then can be eluted quantitatively from the cartridge with a small volume of methanol. This step effectively concentrates the sample and provides sample cleanup. The methanol extract is analyzed directly by HPLC on an MCH 10 reverse-phase column; methanol: water (75:25, volume : volume) is the mobile phase and flow rate is 1.5 ml/minute. The rotenone is detected by ultraviolet spectrophotometry at a wavelength of 295 nm.

Cross-reactive and strain-specific antipeptide antibodies to Pseudomonas aeruginosa PAK and PAO pili.

PubMed Central

Lee, K K; Paranchych, W; Hodges, R S

1990-01-01

Antipeptide antibodies were raised against synthetic peptides corresponding to the amino acid sequences of eight surface predicted regions of the pilin proteins from Pseudomonas aeruginosa PAK and PAO. Four of the anti-PAK peptide antisera cross-reacted with strain PAO pili, while five anti-PAO peptide antisera cross-reacted with strain PAK pili. Only one region of the two pilin proteins (region 88-97) provided strain-specific antibodies when either strain PAK or strain PAO region 88-97 peptides were used to generate antipeptide antibodies. Our results clearly showed that cross-reactive and strain-specific antibodies cannot be based solely on the degree of homology in the aligned protein sequences. The majority of synthetic peptides bound to their homologous antipilus antiserum, suggesting that linear sequences play a significant role in the immunogenic response of native pili. PMID:1974884

The Speech Functions Used by Ibu Muslimah and Pak Harfan in "Laskar Pelangi" Drama

ERIC Educational Resources Information Center

Rahmania

2018-01-01

This research investigates kind of speech function used in utterances created by Ibu Muslimah and Pak Harfan in "Laskar Pelangi" drama. Descriptive qualitative method is conduct for this research. The script of drama Laskar Pelangi was taken as source of data. All utterances produced by bu Muslimah and Pak Harfan as the data in the…

Development and evaluation of the quick anaero-system-a new disposable anaerobic culture system.

PubMed

Yang, Nam Woong; Kim, Jin Man; Choi, Gwang Ju; Jang, Sook Jin

2010-04-01

We developed a new disposable anaerobic culture system, namely, the Quick anaero-system, for easy culturing of obligate anaerobes. Our system consists of 3 components: 1) new disposable anaerobic gas pack, 2) disposable culture-envelope and sealer, and 3) reusable stainless plate rack with mesh containing 10 g of palladium catalyst pellets. To evaluate the efficiency of our system, we used 12 anaerobic bacteria. We prepared 2 sets of ten-fold serial dilutions of the 12 anaerobes, and inoculated these samples on Luria-Bertani (LB) broth and LB blood agar plate (LB-BAP) (BD Diagnostic Systems, USA). Each set was incubated in the Quick anaero-system (DAS Tech, Korea) and BBL GasPak jar with BD GasPak EZ Anaerobe Container System (BD Diagnostic Systems) at 35-37 degrees C for 48 hr. The minimal inoculum size showing visible growth of 12 anaerobes when incubated in both the systems was compared. The minimal inoculum size showing visible growth for 2 out of the 12 anaerobes in the LB broth and 9 out of the 12 anaerobes on LB-BAP was lower for the Quick anaero-system than in the BD GasPak EZ Anaerobe Container System. The mean time (+/-SD) required to achieve absolute anaerobic conditions of the Quick anaero-system was 17 min and 56 sec (+/-3 min and 25 sec). The Quick anaero-system is a simple and effective method of culturing obligate anaerobes, and its performance is superior to that of the BD GasPak EZ Anaerobe Container System.

Identifying Breast Cancer Oncogenes

DTIC Science & Technology

2011-10-01

antibodies . Analytes considered positive (>10) and showing significant (1.5) fold increase in signal are shown as black diamonds. All labeled analytes...Curto et al., 2007). Using a general phospho-tyrosine antibody (4G10) we determined that overexpression of PAK1 indeed upregulates phospho-tyrosine...PAK1. Remarkably, three distinct antibodies identified MET as highly phosphorylated specifically in HMLE-PAK1 cells strongly suggesting that MET could

Recent advances in the development of p21-activated kinase inhibitors.

PubMed

Coleman, Natalia; Kissil, Joseph

2012-04-01

The p21-activated kinases (PAKs) are downstream effectors of the small G-proteins of the Rac and cdc42 family and have been implicated as essential for cell proliferation and survival. Recent studies have also demonstrated the promise of PAKs as therapeutic targets in various types of cancers. The PAKs are divided into two major groups (group I and II) based on sequence similarities. Although the different roles the PAK groups might play are not well understood, recent efforts have focused on the identification of kinase inhibitors that can discriminate between the two groups. In this review these efforts and newly identified inhibitors will be described and future directions discussed.

Cdk1 phosphorylates the Rac activator Tiam1 to activate centrosomal Pak and promote mitotic spindle formation

PubMed Central

Whalley, Helen J.; Porter, Andrew P.; Diamantopoulou, Zoi; White, Gavin R. M.; Castañeda-Saucedo, Eduardo; Malliri, Angeliki

2015-01-01

Centrosome separation is critical for bipolar spindle formation and the accurate segregation of chromosomes during mammalian cell mitosis. Kinesin-5 (Eg5) is a microtubule motor essential for centrosome separation, and Tiam1 and its substrate Rac antagonize Eg5-dependent centrosome separation in early mitosis promoting efficient chromosome congression. Here we identify S1466 of Tiam1 as a novel Cdk1 site whose phosphorylation is required for the mitotic function of Tiam1. We find that this phosphorylation of Tiam1 is required for the activation of group I p21-activated kinases (Paks) on centrosomes in prophase. Further, we show that both Pak1 and Pak2 counteract centrosome separation in a kinase-dependent manner and demonstrate that they act downstream of Tiam1. We also show that depletion of Pak1/2 allows cells to escape monopolar arrest by Eg5 inhibition, highlighting the potential importance of this signalling pathway for the development of Eg5 inhibitors as cancer therapeutics. PMID:26078008

Effect of plant species on nitrogen recovery in aquaponics.

PubMed

Hu, Zhen; Lee, Jae Woo; Chandran, Kartik; Kim, Sungpyo; Brotto, Ariane Coelho; Khanal, Samir Kumar

2015-01-01

Nitrogen transformations in aquaponics with different edible plant species, i.e., tomato (Lycopersicon esculentum) and pak choi (Brassica campestris L. subsp. chinensis) were systematically examined and compared. Results showed that nitrogen utilization efficiencies (NUE) of tomato- and pak choi-based aquaponic systems were 41.3% and 34.4%, respectively. The abundance of nitrifying bacteria in tomato-based aquaponics was 4.2-folds higher than that in pak choi-based aquaponics, primarily due to its higher root surface area. In addition, tomato-based aquaponics had better water quality than that of pak choi-based aquaponics. About 1.5-1.9% of nitrogen input were emitted to atmosphere as nitrous oxide (N2O) in tomato- and pak choi-based aquaponic systems, respectively, suggesting that aquaponics is a potential anthropogenic source of N2O emission. Overall, this is the first intensive study that examined the role plant species played in aquaponics, which could provide new strategy in designing and operating an aquaponic system. Copyright © 2015 Elsevier Ltd. All rights reserved.

miR-133 is a key negative regulator of CDC42-PAK pathway in gastric cancer.

PubMed

Cheng, Zhenguo; Liu, Funan; Wang, Guanqiao; Li, Yanshu; Zhang, Hongyan; Li, Feng

2014-12-01

Cell division cycle 42 (CDC42), an important member of the Ras homolog (Rho) family, plays a key role in regulating multiple cellular processes such as cell cycle progression, migration, cell cytoskeleton organization, cell fate determination and differentiation. Among the downstream effectors of CDC42, P21-activated kinases (PAKs) obtain the most attention. Although a large body of evidence indicates that CDC42/PAKs pathway plays important role in tumor growth, invasion and metastasis, the mechanism of their negative regulation remains unclear. Here, we identified CDC42, a PAKs activating factor, was a target of miR-133. Ectopic overexpression of miRNAs not only downregulated CDC42 expression and PAKs activation, but also inhibited cancer cell proliferation and migration. We also found that miR-133 was down-regulated in 180 pairs gastric cancer tissues. miR-133 expression was negatively associated with tumor size, invasion depth and peripheral organ metastasis. Besides, dysfunction of miR-133 was an independent prognosis factor for overall survival. Our findings could provide new insights into the molecular mechanisms of gastric carcinogenesis, and may help facilitating development of CDC42/PAK-based therapies for human cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

N1, N14-diferuloylspermine as an antioxidative phytochemical contained in leaves of Cardamine fauriei.

PubMed

Abe, Keima; Matsuura, Hideyuki; Ukai, Mitsuko; Shimura, Hanako; Koshino, Hiroyuki; Suzuki, Takashi

2017-10-01

Most Brassicaceae vegetables are ideal dietary sources of antioxidants beneficial for human health. Cardamine fauriei (Ezo-wasabi in Japanese) is a wild, edible Brassicaceae herb native to Hokkaido, Japan. To clarify the main antioxidative phytochemical, an 80% methanol extraction from the leaves was fractionated with Diaion® HP-20, Sephadex® LH-20, and Sep-Pak® C18 cartridges, and the fraction with strong antioxidant activity depending on DPPH method was purified by HPLC. Based on the analyses using HRESIMS and MS/MS, the compound might be N 1 , N 14 -diferuloylspermine. This rare phenol compound was chemically synthesized, whose data on HPLC, MS and 1 H NMR were compared with those of naturally derived compound from C. fauriei. All results indicated they were the same compound. The radical-scavenging properties of diferuloylspermine were evaluated by ORAC and ESR spin trapping methods, with the diferuloylspermine showing high scavenging activities of the ROO · , O 2 ·- , and HO · radicals as was those of conventional antioxidants.

Essential oil of Pinus koraiensis inhibits cell proliferation and migration via inhibition of p21-activated kinase 1 pathway in HCT116 colorectal cancer cells.

PubMed

Cho, Sun-Mi; Lee, Eun-Ok; Kim, Sung-Hoon; Lee, Hyo-Jeong

2014-07-30

The essential oil of Pinus koraiensis (EOPK) is biologically active compound obtained from the leaves of P. koraiensis. The goal of this study was to investigate the anti-cancer mechanism of EOPK in HCT116 colorectal cancer cells. HCT116 cell proliferation was assessed by conducting crystal violet and BrdU assays. To assess the effects of EOPK on cell migration, we performed a wound-healing assay. Further, the contribution of PAK1 to EOPK-induced AKT and extracellular signal-regulated kinase (ERK) suppression was assessed by siRNA-mediated PAK1 knockdown. Changes to the expression and phosphorylation of PAK1 and its effectors were determined by western blotting, and changes to the actin cytoskeleton were determined by performing an immunofluorescence assay. EOPK significantly decreased HCT116 cell proliferation and migration, and induced G1 arrest without affecting normal cells. Additionally, EOPK suppressed the expression of PAK1, and decreased ERK and AKT phosphorylation in HCT116 cells. Finally, EOPK suppressed β-catenin, cyclin D1, and CDK4/6 expression. Our studies indicate that EOPK significantly reduced proliferation and migration of colorectal cancer cells. Furthermore, EOPK suppressed PAK1 expression in a dose-dependent manner, and this suppression of PAK1 led to inhibition of ERK, AKT, and β-catenin activities. Our findings suggest that EOPK exerts its anticancer activity via the inhibition of PAK1 expression, suggesting it may be a potent chemotherapeutic agent for colorectal cancer.

RIT1 controls actin dynamics via complex formation with RAC1/CDC42 and PAK1.

PubMed

Meyer Zum Büschenfelde, Uta; Brandenstein, Laura Isabel; von Elsner, Leonie; Flato, Kristina; Holling, Tess; Zenker, Martin; Rosenberger, Georg; Kutsche, Kerstin

2018-05-01

RIT1 belongs to the RAS family of small GTPases. Germline and somatic RIT1 mutations have been identified in Noonan syndrome (NS) and cancer, respectively. By using heterologous expression systems and purified recombinant proteins, we identified the p21-activated kinase 1 (PAK1) as novel direct effector of RIT1. We found RIT1 also to directly interact with the RHO GTPases CDC42 and RAC1, both of which are crucial regulators of actin dynamics upstream of PAK1. These interactions are independent of the guanine nucleotide bound to RIT1. Disease-causing RIT1 mutations enhance protein-protein interaction between RIT1 and PAK1, CDC42 or RAC1 and uncouple complex formation from serum and growth factors. We show that the RIT1-PAK1 complex regulates cytoskeletal rearrangements as expression of wild-type RIT1 and its mutant forms resulted in dissolution of stress fibers and reduction of mature paxillin-containing focal adhesions in COS7 cells. This effect was prevented by co-expression of RIT1 with dominant-negative CDC42 or RAC1 and kinase-dead PAK1. By using a transwell migration assay, we show that RIT1 wildtype and the disease-associated variants enhance cell motility. Our work demonstrates a new function for RIT1 in controlling actin dynamics via acting in a signaling module containing PAK1 and RAC1/CDC42, and highlights defects in cell adhesion and migration as possible disease mechanism underlying NS.

RIT1 controls actin dynamics via complex formation with RAC1/CDC42 and PAK1

PubMed Central

von Elsner, Leonie; Flato, Kristina; Holling, Tess; Zenker, Martin; Rosenberger, Georg

2018-01-01

RIT1 belongs to the RAS family of small GTPases. Germline and somatic RIT1 mutations have been identified in Noonan syndrome (NS) and cancer, respectively. By using heterologous expression systems and purified recombinant proteins, we identified the p21-activated kinase 1 (PAK1) as novel direct effector of RIT1. We found RIT1 also to directly interact with the RHO GTPases CDC42 and RAC1, both of which are crucial regulators of actin dynamics upstream of PAK1. These interactions are independent of the guanine nucleotide bound to RIT1. Disease-causing RIT1 mutations enhance protein-protein interaction between RIT1 and PAK1, CDC42 or RAC1 and uncouple complex formation from serum and growth factors. We show that the RIT1-PAK1 complex regulates cytoskeletal rearrangements as expression of wild-type RIT1 and its mutant forms resulted in dissolution of stress fibers and reduction of mature paxillin-containing focal adhesions in COS7 cells. This effect was prevented by co-expression of RIT1 with dominant-negative CDC42 or RAC1 and kinase-dead PAK1. By using a transwell migration assay, we show that RIT1 wildtype and the disease-associated variants enhance cell motility. Our work demonstrates a new function for RIT1 in controlling actin dynamics via acting in a signaling module containing PAK1 and RAC1/CDC42, and highlights defects in cell adhesion and migration as possible disease mechanism underlying NS. PMID:29734338

[Determination of ferulic acid in chuanxiong and in animal serum and cerebrospinal fluid by reversed-phase high performance liquid chromatography].

PubMed

Lü, K; Ding, M Y; Li, H X; Liu, D L

2000-11-01

An easy, rapid and sensitive method for the determination of ferulic acid(FA) in Chuanxiong extracts, animal (mouse) serum and cerebrospinal fluid by RP-HPLC has been developed. The FA was separated on an ODS column, Nova-Pak C18(3.9 mm i.d. x 150 mm) and detected at the wavelength of 320 nm. The mobile phase was methanol-water-acetic acid (35:65:0.5, V/V), with a flow rate of 0.8 mL/min. The detection limit of FA was 1.7 micrograms/L(S/N = 3) and the calibration curve was linear within the range of 0.85 mg/L-4.00 mg/L(r = 0.99904, n = 6). The mean recovery from animal serum and cerebrospinal was 95%-102%.

A novel cold-inducible gene from Pak-choi (Brassica campestris ssp. chinensis), BcWRKY46, enhances the cold, salt and dehydration stress tolerance in transgenic tobacco.

PubMed

Wang, Feng; Hou, Xilin; Tang, Jun; Wang, Zhen; Wang, Shuming; Jiang, Fangling; Li, Ying

2012-04-01

WRKY TFs belong to one of the largest families of transcriptional regulators in plants and form integral parts of signaling webs that modulate many plant processes. BcWRKY46, a cDNA clone encoding a polypeptide of 284 amino acids and exhibited the structural features of group III of WRKY protein family, was isolated from the cold-treated leaves of Pak-choi (Brassica campestris ssp. chinensis Makino, syn. B. rapa ssp. chinensis) using the cDNA-AFLP technique. Expression of this gene was induced quickly and strongly in response to various environmental stresses, including low temperatures, ABA, salt and dehydration. Constitutive expression of BcWRKY46 in tobacco under the control of the CaMV35S promoter reduced the susceptibility of transgenic tobacco to freezing, ABA, salt and dehydration stresses. Our studies suggest that BcWRKY46 plays an important role in responding to ABA and abiotic stress.

Adhesion inhibition of F1C-fimbriated Escherichia coli and Pseudomonas aeruginosa PAK and PAO by multivalent carbohydrate ligands.

PubMed

Autar, Reshma; Khan, A Salam; Schad, Matthias; Hacker, Jörg; Liskamp, Rob M J; Pieters, Roland J

2003-12-05

In order to evaluate their inhibition of bacterial adhesion, the carbohydrate sequences GalNAcbeta1-->4Gal and GalNAcbeta1-->4Galbeta1-->4Glc were synthesized. The disaccharide was conjugated to dendrons based on the 3,5-di-(2-aminoethoxy)-benzoic acid branching unit to yield di- and tetravalent versions of these compounds. A divalent compound was also prepared that had significantly longer spacer arms. Relevant monovalent compounds were prepared for comparison. Their anti-adhesion properties against F1C-fimbriated uropathogenic Escherichia coli were evaluated in an ELISA-type assay by using a recombinant strain and also by using Pseudomonas aeruginosa strains PAO and PAK. Adhesion inhibition was observed in all cases, and multivalency effects of up to one order of magnitude were observed. The combination of spacer and multivalency effects led to a 38-fold increase in the potency of a divalent inhibitor with long spacer arms towards the PAO strain when compared with the free carbohydrate.

Light exposure before learning improves memory consolidation at night

PubMed Central

Shan, Li-Li; Guo, Hao; Song, Ning-Ning; Jia, Zheng-Ping; Hu, Xin-Tian; Huang, Jing-Fei; Ding, Yu-Qiang; Richter-Levine, Gal; Zhou, Qi-Xin; Xu, Lin

2015-01-01

Light is recently recognized as a modulator able to activate the hippocampus and modulate memory processing, but little is known about the molecular mechanisms. Here, we report that in mice, a short pulse of white light before learning dramatically improves consolidation of contextual fear memory during the night. The light exposure increases hippocampal active p21-activated kinase 1 (PAK1) and CA1 long-term potentiation (LTP). These light effects are abolished in PAK1 knockout and dominant-negative transgenic mice, but preserved by expression of constitutively active PAK1 in the hippocampus. Our results indicate that light can act as a switch of PAK1 activity that modulate CA1 LTP and thereby memory consolidation without affecting learning and short-term memory. PMID:26493375

The Search for Therapeutic Bacteriophages Uncovers One New Subfamily and Two New Genera of Pseudomonas-Infecting Myoviridae

PubMed Central

Henry, Marine; Bobay, Louis-Marie; Chevallereau, Anne; Saussereau, Emilie; Ceyssens, Pieter-Jan; Debarbieux, Laurent

2015-01-01

In a previous study, six virulent bacteriophages PAK_P1, PAK_P2, PAK_P3, PAK_P4, PAK_P5 and CHA_P1 were evaluated for their in vivo efficacy in treating Pseudomonas aeruginosa infections using a mouse model of lung infection. Here, we show that their genomes are closely related to five other Pseudomonas phages and allow a subdivision into two clades, PAK_P1-like and KPP10-like viruses, based on differences in genome size, %GC and genomic contents, as well as number of tRNAs. These two clades are well delineated, with a mean of 86% and 92% of proteins considered homologous within individual clades, and 25% proteins considered homologous between the two clades. By ESI-MS/MS analysis we determined that their virions are composed of at least 25 different proteins and electron microscopy revealed a morphology identical to the hallmark Salmonella phage Felix O1. A search for additional bacteriophage homologs, using profiles of protein families defined from the analysis of the 11 genomes, identified 10 additional candidates infecting hosts from different species. By carrying out a phylogenetic analysis using these 21 genomes we were able to define a new subfamily of viruses, the Felixounavirinae within the Myoviridae family. The new Felixounavirinae subfamily includes three genera: Felixounalikevirus, PAK_P1likevirus and KPP10likevirus. Sequencing genomes of bacteriophages with therapeutic potential increases the quantity of genomic data on closely related bacteriophages, leading to establishment of new taxonomic clades and the development of strategies for analyzing viral genomes as presented in this article. PMID:25629728

MiR-145 regulates PAK4 via the MAPK pathway and exhibits an antitumor effect in human colon cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zhigang; Zhang, Xiaoping; Yang, Zhili

2012-10-26

Highlights: Black-Right-Pointing-Pointer MiR-145 targets a putative binding site in the 3 Prime UTR of PAK4. Black-Right-Pointing-Pointer MiR-145 played an important role in inhibiting cell growth by directly targeting PAK4. Black-Right-Pointing-Pointer MiR-145 may function as tumor suppressors. -- Abstract: MicroRNAs (miRNAs) are regulators of numerous cellular events; accumulating evidence indicates that miRNAs play a key role in a wide range of biological functions, such as cellular proliferation, differentiation, and apoptosis in cancer. Down-regulated expression of miR-145 has been reported in colon cancer tissues and cell lines. The molecular mechanisms underlying miR-145 and the regulation of colon carcinogenesis remain unclear. In thismore » study, we investigated the levels of miR-145 in human colon cancer cells using qRT-PCR and found markedly decreased levels compared to normal epithelial cells. We identified PAK4 as a novel target of miR-145 using informatics screening. Additionally, we demonstrated that miR-145 targets a putative binding site in the 3 Prime UTR of PAK4 and that its abundance is inversely associated with miR-145 expression in colon cancer cells; we confirmed this relationship using the luciferase reporter assay. Furthermore, restoration of miR-145 by mimics in SW620 cells significantly attenuated cell growth in vitro, in accordance with the inhibitory effects induced by siRNA mediated knockdown of PAK4. Taken together, these findings demonstrate that miR-145 downregulates P-ERK expression by targeting PAK4 and leads to inhibition of tumor growth.« less

Glucosinolates from pak choi and broccoli induce enzymes and inhibit inflammation and colon cancer differently.

PubMed

Lippmann, Doris; Lehmann, Carsten; Florian, Simone; Barknowitz, Gitte; Haack, Michael; Mewis, Inga; Wiesner, Melanie; Schreiner, Monika; Glatt, Hansruedi; Brigelius-Flohé, Regina; Kipp, Anna P

2014-06-01

High consumption of Brassica vegetables is considered to prevent especially colon carcinogenesis. The content and pattern of glucosinolates (GSLs) can highly vary among different Brassica vegetables and may, thus, affect the outcome of Brassica intervention studies. Therefore, we aimed to feed mice with diets containing plant materials of the Brassica vegetables broccoli and pak choi. Further enrichment of the diets by adding GSL extracts allowed us to analyze the impact of different amounts (GSL-poor versus GSL-rich) and different patterns (broccoli versus pak choi) of GSLs on inflammation and tumor development in a model of inflammation-triggered colon carcinogenesis (AOM/DSS model). Serum albumin adducts were analyzed to confirm the up-take and bioactivation of GSLs after feeding the Brassica diets for four weeks. In agreement with their high glucoraphanin content, broccoli diets induced the formation of sulforaphane-lysine adducts. Levels of 1-methoxyindolyl-3-methyl-histidine adducts derived from neoglucobrassicin were the highest in the GSL-rich pak choi group. In the colon, the GSL-rich broccoli and the GSL-rich pak choi diet up-regulated the expression of different sets of typical Nrf2 target genes like Nqo1, Gstm1, Srxn1, and GPx2. GSL-rich pak choi induced the AhR target gene Cyp1a1 but did not affect Ugt1a1 expression. Both colitis and tumor number were drastically reduced after feeding the GSL-rich pak choi diet while the other three diets had no effect. GSLs can act anti-inflammatory and anti-carcinogenic but both effects depend on the specific amount and pattern of GSLs within a vegetable. Thus, a high Brassica consumption cannot be generally considered to be cancer-preventive.

Mesalamine modulates intercellular adhesion through inhibition of p-21 activated kinase-1

PubMed Central

Khare, Vineeta; Lyakhovich, Alex; Dammann, Kyle; Lang, Michaela; Borgmann, Melanie; Tichy, Boris; Pospisilova, Sarka; Luciani, Gloria; Campregher, Christoph; Evstatiev, Rayko; Pflueger, Maren; Hundsberger, Harald; Gasche, Christoph

2013-01-01

Mesalamine (5-ASA) is widely used for the treatment of ulcerative colitis, a remitting condition characterized by chronic inflammation of the colon. Knowledge about the molecular and cellular targets of 5-ASA is limited and a clear understanding of its activity in intestinal homeostasis and interference with neoplastic progression is lacking. We sought to identify molecular pathways interfered by 5-ASA, using CRC cell lines with different genetic background. Microarray was performed for gene expression profile of 5-ASA-treated and untreated cells (HCT116 and HT29). Filtering and analysis of data identified three oncogenic pathways interfered by 5-ASA: MAPK/ERK pathway, cell adhesion and β-catenin/Wnt signaling. PAK1 emerged as a consensus target of 5-ASA, orchestrating these pathways. We further investigated the effect of 5-ASA on cell adhesion. 5-ASA increased cell adhesion which was measured by cell adhesion assay and transcellular-resistance measurement. Moreover, 5-ASA treatment restored membranous expression of adhesion molecules E-cadherin and β-catenin. Role of PAK1 as a mediator of mesalamine activity was validated in vitro and in vivo. Inhibition of PAK1 by RNA interference also increased cell adhesion. PAK1 expression was elevated in APCmin polyps and 5-ASA treatment reduced its expression. Our data demonstrates novel pharmacological mechanism of mesalamine in modulation of cell adhesion and role of PAK1 in APCmin polyposis. We propose that inhibition of PAK1 expression by 5-ASA can impede with neoplastic progression in colorectal carcinogenesis. The mechanism of PAK1 inhibition and induction of membranous translocation of adhesion proteins by 5-ASA might be independent of its known anti-inflammatory action. PMID:23146664

Mesalamine modulates intercellular adhesion through inhibition of p-21 activated kinase-1.

PubMed

Khare, Vineeta; Lyakhovich, Alex; Dammann, Kyle; Lang, Michaela; Borgmann, Melanie; Tichy, Boris; Pospisilova, Sarka; Luciani, Gloria; Campregher, Christoph; Evstatiev, Rayko; Pflueger, Maren; Hundsberger, Harald; Gasche, Christoph

2013-01-15

Mesalamine (5-ASA) is widely used for the treatment of ulcerative colitis, a remitting condition characterized by chronic inflammation of the colon. Knowledge about the molecular and cellular targets of 5-ASA is limited and a clear understanding of its activity in intestinal homeostasis and interference with neoplastic progression is lacking. We sought to identify molecular pathways interfered by 5-ASA, using CRC cell lines with different genetic background. Microarray was performed for gene expression profile of 5-ASA-treated and untreated cells (HCT116 and HT29). Filtering and analysis of data identified three oncogenic pathways interfered by 5-ASA: MAPK/ERK pathway, cell adhesion and β-catenin/Wnt signaling. PAK1 emerged as a consensus target of 5-ASA, orchestrating these pathways. We further investigated the effect of 5-ASA on cell adhesion. 5-ASA increased cell adhesion which was measured by cell adhesion assay and transcellular-resistance measurement. Moreover, 5-ASA treatment restored membranous expression of adhesion molecules E-cadherin and β-catenin. Role of PAK1 as a mediator of mesalamine activity was validated in vitro and in vivo. Inhibition of PAK1 by RNA interference also increased cell adhesion. PAK1 expression was elevated in APC(min) polyps and 5-ASA treatment reduced its expression. Our data demonstrates novel pharmacological mechanism of mesalamine in modulation of cell adhesion and role of PAK1 in APC(min) polyposis. We propose that inhibition of PAK1 expression by 5-ASA can impede with neoplastic progression in colorectal carcinogenesis. The mechanism of PAK1 inhibition and induction of membranous translocation of adhesion proteins by 5-ASA might be independent of its known anti-inflammatory action. Copyright © 2012 Elsevier Inc. All rights reserved.

Translations on North Korea No. 514

DTIC Science & Technology

1977-03-11

Yong -sok, Kim Ch’i-ku, Chi Chae-ryong, 0...Ch’ongjin were the following: Yim Ch’un-ch’u comrade Pak Yong -sok functionary of the sector concerned Kim Ch’i-ku " Chi Chae- yong 0 Hyon-chu Pak...electric railroad on 22 January were the following: Yim Ch’un-ch’u comrade Pak Yong -sok functionary of the sector concerned Kim Ch’i-ku " Chi Chae- yong

Participation of group I p21-activated kinases in neuroplasticity.

PubMed

Koth, André P; Oliveira, Bruno R; Parfitt, Gustavo M; Buonocore, Juliana de Quadros; Barros, Daniela M

2014-01-01

PAKs are a family of serine/threonine protein kinases activated by small GTPases of the Rho family, including Rac and Cdc42, and are categorized into group I (isoforms 1, 2 and 3) and group II (isoforms 4, 5 and 6). PAK1 and PAK3 are critically involved in biological mechanisms associated with neurodevelopment, neuroplasticity and maturation of the nervous system, and changes in their activity have been detected in pathological disorders, such as Alzheimer's disease, Huntington's disease and mental retardation. The group I PAKs have been associated with neurological processes due to their involvement in intracellular mechanisms that result in molecular and cellular morphological alterations that promote cytoskeletal outgrowth, increasing the efficiency of synaptic transmission. Their substrates in these processes include other intracellular signaling molecules, such as Raf, Mek and LIMK, as well as other components of the cytoskeleton, such as MLC and FLNa. In this review, we describe the characteristics of group I PAKs, such as their molecular structure, mechanisms of activation and importance in the neurobiological processes involved in synaptic plasticity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Pak1 Kinase Maintains Apical Membrane Identity in Epithelia.

PubMed

Aguilar-Aragon, Mario; Elbediwy, Ahmed; Foglizzo, Valentina; Fletcher, Georgina C; Li, Vivian S W; Thompson, Barry J

2018-02-13

Epithelial cells are polarized along their apical-basal axis by the action of the small GTPase Cdc42, which is known to activate the aPKC kinase at the apical domain. However, loss of aPKC kinase activity was reported to have only mild effects on epithelial cell polarity. Here, we show that Cdc42 also activates a second kinase, Pak1, to specify apical domain identity in Drosophila and mammalian epithelia. aPKC and Pak1 phosphorylate an overlapping set of polarity substrates in kinase assays. Inactivating both aPKC kinase activity and the Pak1 kinase leads to a complete loss of epithelial polarity and morphology, with cells losing markers of apical polarization such as Crumbs, Par3/Bazooka, or ZO-1. This function of Pak1 downstream of Cdc42 is distinct from its role in regulating integrins or E-cadherin. Our results define a conserved dual-kinase mechanism for the control of apical membrane identity in epithelia. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Nutritional performance and activity of some digestive enzymes of the cotton bollworm, Helicoverpa armigera , in response to seven tested bean cultivars

PubMed Central

Namin, Foroogh Rahimi; Naseri, Bahram; Razmjou, Jabraeil; Cohen, Allen

2014-01-01

Abstract Nutritional performance and activity of some digestive enzymes (protease and α -amylase) of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) in response to feeding on bean ( Phaseolus vulgaris L. (Fabales: Fabaceae)) cultivars (Shokufa, Akhtar, Sayyad, Naz, Pak, Daneshkadeh, and Talash) were evaluated under laboratory conditions (25 ± 1°C, 65 ± 5% RH, and a 16:8 L:D photoperiod). The highest and lowest respective values of approximate digestibility were observed when fourth, fifth, and sixth larval instar H. armigera were fed red kidney bean Akhtar and white kidney bean Daneshkadeh. The efficiency of conversion of ingested and digested food was highest when H. armigera was fed red kidney beans Akhtar and Naz and lowest when they were fed white kidney bean Pak. The highest protease activity of fifth instars was observed when they were fed red kidney bean Naz, and the highest amylase activity of fifth instars was observed when they were fed red kidney bean Sayyad. Sixth instar larvae that fed on red kidney bean Sayyad showed the highest protease activity. Larvae reared on common bean Talash and white kidney bean Pak showed the highest amylase activity. Among bean cultivars tested, red kidney bean Sayyad was the most unsuitable host for feeding H. armigera . PMID:25368049

Validation of the BrockTB Stat-Pak Assay for Detection of Tuberculosis in Eurasian Badgers (Meles meles) and Influence of Disease Severity on Diagnostic Accuracy▿

PubMed Central

Chambers, Mark A.; Crawshaw, Tim; Waterhouse, Sue; Delahay, Richard; Hewinson, R. Glyn; Lyashchenko, Konstantin P.

2008-01-01

A lateral-flow immunoassay (BrockTB Stat-Pak) for detecting tuberculosis in Eurasian badgers was 49% sensitive and 93% specific against culture for M. bovis (n = 1,464) at necropsy. However, the sensitivity was significantly higher (66 to 78%) in animals with more severe tuberculosis, indicating that the BrockTB Stat-Pak may be useful for the detection of badgers with the greatest risk of transmitting disease. PMID:18272706

Testing the Role of p21-Activated Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely Phenocopies Human NF2 Disease

DTIC Science & Technology

2016-06-01

control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. The major goal of this research project was to genetically and pharmacologically ...with three different pharmacologic PAK inhibitors to determine if targeted PAK inhibition in a preclinical model of schwannoma genesis rescues tumor...this research project was to genetically and pharmacologically test the requirement of Group A PAK signaling in Nf2 deficient schwannoma genesis. We

1,2,3-Triazolyl ester of Ketorolac: A "Click Chemistry"-based highly potent PAK1-blocking cancer-killer.

PubMed

Nguyen, Binh Cao Quan; Takahashi, Hideaki; Uto, Yoshihiro; Shahinozzaman, M D; Tawata, Shinkichi; Maruta, Hiroshi

2017-01-27

An old anti-inflammatory/analgesic drug called Toradol is a racemic form of Ketorolac (50% R-form and 50% S-form) that blocks the oncogenic RAC-PAK1-COX-2 (cyclooxygenase-2) signaling, through the direct inhibition of RAC by the R-form and of COX-2 by the S-form, eventually down-regulating the production of prostaglandins. However, due to its COOH moiety which is clearly repulsive to negatively-charged phospholipid-based plasma membrane, its cell-permeability is rather poor (the IC 50 against the growth of human cancer cells such as A549 is around 13 μM). In an attempt to boost its anti-cancer activity, hopefully by increasing its cell-permeability through abolishing the negative charge, yet keeping its water-solubility, here we synthesized a 1,2,3-triazolyl ester of Toradol through "Click Chemistry". The resultant water-soluble "azo" derivative called "15K" was found to be over 500 times more potent than Toradol with the IC 50 around 24 nM against the PAK1-dependent growth of A549 cancer cells, inactivating PAK1 in cell culture with the apparent IC 50 around 65 nM, and inhibiting COX-2 in vitro with the IC 50 around 6 nM. Furthermore, the Click Chemistry boosts the anti-cancer activity of Ketorolac by 5000 times against the PAK1-independent growth of B16F10 melanoma cells. Using a multi-drug-resistant (MDR) cancer cell line (EMT6), we found that the esterization of Ketorolac boosts its cell-permeability by at least 10 folds. Thus, the Click Chemistry dramatically boosts the anti-cancer activity of Ketorolac, at least in three ways: increasing its cell-permeability, the anti-PAK1 activity of R-form and anti-COX-2 activity of S-form. The resultant "15K" is so far among the most potent PAK1-blockers, and therefore would be potentially useful for the therapy of many different PAK1-dependent diseases/disorders such as cancers. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Trace analysis of carbonyl compounds by liquid chromatography-mass spectrometry after collection as 2,4-dinitrophenylhydrazine derivatives.

PubMed

Sakuragawa, A; Yoneno, T; Inoue, K; Okutani, T

1999-06-04

This study describes the utilization of carbonyl- 2,4-dinitrophenylhydrazine (DNPH) derivatives for the determination of a micro amount of carbonyl compounds in air by liquid chromatography-mass spectrometry (LC-MS). After the carbonyl compounds are collected using a Waters Sep-Pak C18 cartridge column with-impregnated DNPH on octadecylsilica, they are eluted by acetonitrile as carbonyl-DNPH derivatives. A 20-mm3 aliquot of eluent is injected into the LC-MS system. The four derivatives (formaldehyde-, acetaldehyde-, acrolein- and acetone-DNPH) were eluted within 7 min with acetonitrile-water (60:40, v/v) as the mobile phase. The proposed method offers sub-ppb sensitivity and good reproducibility and was applied to the determination of these carbonyl compounds in actual air samples from store rooms, laboratories and offices. The relative standard deviations for these samples (n = 6) were 1 to 3%.

Immunoreactive prohormone atrial natriuretic peptides 1-30 and 31-67 - Existence of a single circulating amino-terminal peptide

NASA Technical Reports Server (NTRS)

Chen, Yu-Ming; Whitson, Peggy A.; Cintron, Nitza M.

1990-01-01

Sep-Pak C18 extraction of human plasma and radioimmunoassay using antibodies which recognize atrial natriuretic peptide (99-128) and the prohormone sequences 1-30 and 31-67 resulted in mean values from 20 normal subjects of 26.2 (+/- 9.2), 362 (+/- 173) and 368 (+/- 160) pg/ml, respectively. A high correlation coefficient between values obtained using antibodies recognizing prohormone sequences 1-30 and 31-67 was observed (R = 0.84). Extracted plasma immunoreactivity of 1-30 and 31-67 both eluted at 46 percent acetonitrile. In contrast, chromatographic elution of synthetic peptides 1-30 and 31-67 was observed at 48 and 39 percent acetonitrile, respectively. Data suggest that the radioimmunoassay of plasma using antibodies recognizing prohormone sequences 1-30 and 31-67 may represent the measurement of a unique larger amino-terminal peptide fragment containing antigenic sites recognized by both antisera.

Molecular screening of the Hbs Constant Spring (codon 142, TAA>CAA, α2) and Paksé (codon 142, TAA>TAT, α2) mutations in Thailand.

PubMed

Pichanun, Dalad; Munkongdee, Thongperm; Klamchuen, Sumonmaln; Butthep, Punnee; Winichagoon, Pranee; Fucharoen, Suthat; Svasti, Saovaros

2010-01-01

Hb Constant Spring [Hb CS, α142(H19)Term] and Hb Paksé [α142(H19)Term] occur from the mutation in the termination codon of the α2-globin gene, TAA>CAA (→Gln) and TAA>TAT (→Tyr), respectively. They are the most common nondeletional α-thalassemia (α-thal) variants causing Hb H disease in Southeast Asia. In this study, 587 cord blood samples were screened for the Hb CS and Hb Paksé mutations by a dot-blot hybridization technique using oligonucleotide probes specific for each mutation. The results showed that the prevalence of Hb CS and Hb Paksé in Central Thailand are 5.80 and 0.51%, respectively, which is in concordance with the results from previous studies.

The role of p21-activated kinases in pancreatic cancer.

PubMed

Yeo, Dannel; He, Hong; Baldwin, Graham S; Nikfarjam, Mehrdad

2015-04-01

Pancreatic cancer is an aggressive cancer with a poor prognosis and an overall 5-year survival rate of less than 5%. Management has not improved significantly over the last 30 years, and a better understanding of the genetic and molecular changes that occur is urgently required. Many of these changes appear to involve the p21-activated kinases (PAKs). The PAK family consists of 6 isoforms, 2 of which, PAK1 and PAK4, are up-regulated and/or hyperactivated in pancreatic cancer. p21-Activated kinases can mediate many different cellular processes especially those contributing to cancer development and progression. These processes include the regulation of cytoskeletal dynamics and cell adhesion, the evasion of apoptosis, and the promotion of cell survival, proliferation, migration, and invasion. p21-Activated kinases may also be involved in characteristics unique to pancreatic tumors, such as interplay with the pancreatic stroma, the re-emergence of embryonic pathways, and the involvement of a subset of microRNAs and heat shock proteins. This review highlights the potential role of PAKs in pancreatic cancer and provides a foundation for more effective therapeutics to improve our current treatment of pancreatic cancer.

FER mediated HGF-independent regulation of HGFR/MET activates RAC1-PAK1 pathway to potentiate metastasis in ovarian cancer.

PubMed

Fan, Gaofeng

2017-11-03

Uncontrolled metastasis significantly contributes to high lethality of patients suffering from ovarian cancer. To date, the detailed molecular mechanisms which account for ovarian tumor cell spreading and metastasis remain largely unknown. In a recent study, we have demonstrated that aberrantly high expression of the non-receptor tyrosine kinase FER is responsible for ovarian tumor cell metastasis both in vitro and in vivo. Mechanistically, we indentified Hepatocyte Growth Factor Receptor HGFR/MET as a novel substrate of FER, and through which the kinase FER modulates ovarian cancer cell motility and invasiveness in a ligand-independent manner. We also observed aberrantly high expression of PAK1 kinase in cancer cells, and RNAi-mediated knockdown of FER kinase inactivated the RAC1-PAK1 signaling pathway and decreased metastatic potential of CAOV4 ovarian cancer cells. Overall, our study revealed a previously uncharacterized, pro-metastatic role of the kinase FER in ovarian cancer through the MET-RAC1-PAK1 pathway. Further efforts are essential to investigating beneficial outcomes towards targeting the RAC1-PAK1 signaling pathway in reducing metastatic burden of this deadly disease.

Dock and Pak regulate olfactory axon pathfinding in Drosophila.

PubMed

Ang, Lay-Hong; Kim, Jenny; Stepensky, Vitaly; Hing, Huey

2003-04-01

The convergence of olfactory axons expressing particular odorant receptor (Or) genes on spatially invariant glomeruli in the brain is one of the most dramatic examples of precise axon targeting in developmental neurobiology. The cellular and molecular mechanisms by which olfactory axons pathfind to their targets are poorly understood. We report here that the SH2/SH3 adapter Dock and the serine/threonine kinase Pak are necessary for the precise guidance of olfactory axons. Using antibody localization, mosaic analyses and cell-type specific rescue, we observed that Dock and Pak are expressed in olfactory axons and function autonomously in olfactory neurons to regulate the precise wiring of the olfactory map. Detailed analyses of the mutant phenotypes in whole mutants and in small multicellular clones indicate that Dock and Pak do not control olfactory neuron (ON) differentiation, but specifically regulate multiple aspects of axon trajectories to guide them to their cognate glomeruli. Structure/function studies show that Dock and Pak form a signaling pathway that mediates the response of olfactory axons to guidance cues in the developing antennal lobe (AL). Our findings therefore identify a central signaling module that is used by ONs to project to their cognate glomeruli.

Improvement of Self-Injury With Dopamine and Serotonin Replacement Therapy in a Patient With a Hemizygous PAK3 Mutation: A New Therapeutic Strategy for Neuropsychiatric Features of an Intellectual Disability Syndrome.

PubMed

Horvath, Gabriella A; Tarailo-Graovac, Maja; Bartel, Tanja; Race, Simone; Van Allen, Margot I; Blydt-Hansen, Ingrid; Ross, Colin J; Wasserman, Wyeth W; Connolly, Mary B; van Karnebeek, Clara D M

2018-01-01

PAK3-related intellectual disability is caused by mutations in the gene encoding the p21-activated kinase (PAK) protein. It is characterized by mild to moderate cognitive impairment, micro/normocephaly, and a neurobehavioral phenotype characterized by short attention span, anxiety, restlessness, aggression, and self-abusive behaviors. The authors report a patient with a novel PAK3 mutation, who presented with intellectual disability, severe automutilation, and epilepsy. His magnetic resonance imaging changes were most likely secondary to lacerations from parenchymal contusions. His behavior was difficult to manage with behavior interventions or multiple medications. After finding low levels of dopamine and borderline low serotonin metabolites in the spinal fluid, treatment with low dose L-dopa/carbidopa and 5-hydroxytryptophan significantly improved his self-injurious behavior. This is the first case of PAK3-related intellectual disability presenting with severe self-injury with improvement following treatment. The patient's response to neurotransmitter replacement therapy raises the question if this treatment intervention might help other individuals suffering genetic syndromes and self-injurious behaviors.

p21-activated kinases in cancer.

PubMed

Kumar, Rakesh; Gururaj, Anupama E; Barnes, Christopher J

2006-06-01

The pivotal role of kinases in signal transduction and cellular regulation has lent them considerable appeal as pharmacological targets across a broad spectrum of cancers. p21-activated kinases (Paks) are serine/threonine kinases that function as downstream nodes for various oncogenic signalling pathways. Paks are well-known regulators of cytoskeletal remodelling and cell motility, but have recently also been shown to promote cell proliferation, regulate apoptosis and accelerate mitotic abnormalities, which results in tumour formation and cell invasiveness. Alterations in Pak expression have been detected in human tumours, which makes them an attractive new therapeutic target.

Measuring Perceptions About the Pashtun People

DTIC Science & Technology

2011-03-01

PakTribune.com, November 22, 2010. BaNo, Sher. “Report from a Pashtun Teen .” New York Times, February 18, 2010. Barnes, Julian E. “Effort to Recruit More...Afghanistan.” http://www.joshuaproject.net/. Kamrany, Nake M. Review of After the Taliban: Nation Building in Afghanistan, by James F. Dobbins. Middle East...Journal 63, no. 5 (Fall 2010). Kamrany, Nake M., and Michael D. Intriligator. “Time to Quit Afghanistan Now.” Huffington Post, November 23, 2010

Issues in Interaction Language Specification and Representation.

DTIC Science & Technology

1983-11-01

of Dialogues for Human-Computer Interfaces," to be submitted for publication (1983). IHEINL75] Heindel, L. and J. Roberto . "LANG-PAK: An Interactive...22043 Bolling Air Force Base Washington, D.C. 20332 Dr. Paul E. Lehner PAR Technology Corp. AFHRL/LRS TDC P.O. Box 2005 Attn: Susan Ewing Reston, VA 22090

Profiling the changes in signaling pathways in ascorbic acid/β-glycerophosphate-induced osteoblastic differentiation.

PubMed

Chaves Neto, Antonio Hernandes; Queiroz, Karla Cristiana; Milani, Renato; Paredes-Gamero, Edgar Julian; Justo, Giselle Zenker; Peppelenbosch, Maikel P; Ferreira, Carmen Veríssima

2011-01-01

Despite numerous reports on the ability of ascorbic acid and β-glycerophosphate (AA/β-GP) to induce osteoblast differentiation, little is known about the molecular mechanisms involved in this phenomenon. In this work, we used a peptide array containing specific consensus sequences (potential substrates) for protein kinases and traditional biochemical techniques to examine the signaling pathways modulated during AA/β-GP-induced osteoblast differentiation. The kinomic profile obtained after 7 days of treatment with AA/β-GP identified 18 kinase substrates with significantly enhanced or reduced phosphorylation. Peptide substrates for Akt, PI3K, PKC, BCR, ABL, PRKG1, PAK1, PAK2, ERK1, ERBB2, and SYK showed a considerable reduction in phosphorylation, whereas enhanced phosphorylation was observed in substrates for CHKB, CHKA, PKA, FAK, ATM, PKA, and VEGFR-1. These findings confirm the potential usefulness of peptide microarrays for identifying kinases known to be involved in bone development in vivo and in vitro and show that this technique can be used to investigate kinases whose function in osteoblastic differentiation is poorly understood.

Postsynaptic density scaffold SAP102 regulates cortical synapse development through EphB and PAK signaling pathway

PubMed Central

Murata, Yasunobu; Constantine-Paton, Martha

2013-01-01

Membrane associated guanylate kinases (MAGUKs), including SAP102, PSD-95, PSD-93 and SAP97, are scaffolding proteins for ionotropic glutamate receptors at excitatory synapses. MAGUKs play critical roles in synaptic plasticity; however, details of signaling roles for each MAGUK remain largely unknown. Here we report that SAP102 regulates cortical synapse development through the EphB and PAK signaling pathways. Using lentivirus-delivered shRNAs, we found that SAP102 and PSD-95, but not PSD-93, are necessary for excitatory synapse formation and synaptic AMPA receptor localization in developing mouse cortical neurons. SAP102 knockdown (KD) increased numbers of elongated dendritic filopodia, which is often observed in mouse models and human patients with mental retardation. Further analysis revealed that SAP102 co-immunoprecipitated the receptor tyrosine kinase EphB2 and RacGEF Kalirin-7 in neonatal cortex, and SAP102 KD reduced surface expression and dendritic localization of EphB. Moreover, SAP102 KD prevented reorganization of actin filaments, synapse formation and synaptic AMPAR trafficking in response to EphB activation triggered by its ligand ephrinB. Lastly, p21-activated kinases (PAKs) were down-regulated in SAP102 KD neurons. These results demonstrate that SAP102 has unique roles in cortical synapse development by mediating EphB and its downstream PAK signaling pathway. Both SAP102 and PAKs are associated with X-linked mental retardation in humans; thus, synapse formation mediated by EphB/SAP102/PAK signaling in the early postnatal brain may be crucial for cognitive development. PMID:23486974

Hair Growth Promoting and Anticancer Effects of p21-activated kinase 1 (PAK1) Inhibitors Isolated from Different Parts of Alpinia zerumbet.

PubMed

Taira, Nozomi; Nguyen, Binh Cao Quan; Tawata, Shinkichi

2017-01-14

PAK1 (p21-activated kinase 1) is an emerging target for the treatment of hair loss (alopecia) and cancer; therefore, the search for PAK1 blockers to treat these PAK1-dependent disorders has received much attention. In this study, we evaluated the anti-alopecia and anticancer effects of PAK1 inhibitors isolated from Alpinia zerumbet (alpinia) in cell culture. The bioactive compounds isolated from alpinia were found to markedly promote hair cell growth. Kaempferol-3- O -β-d-glucuronide (KOG) and labdadiene, two of the isolated compounds, increased the proliferation of human follicle dermal papilla cells by approximately 117%-180% and 132%-226%, respectively, at 10-100 μM. MTD (2,5-bis(1 E ,3 E ,5 E )-6-methoxyhexa-1,3,5-trien-1-yl)-2,5-dihydrofuran) and TMOQ (( E )-2,2,3,3-tetramethyl-8-methylene-7-(oct-6-en-1-yl)octahydro-1 H -quinolizine) showed growth-promoting activity around 164% and 139% at 10 μM, respectively. The hair cell proliferation induced by these compounds was significantly higher than that of minoxidil, a commercially available treatment for hair loss. Furthermore, the isolated compounds from alpinia exhibited anticancer activity against A549 lung cancer cells with IC 50 in the range of 67-99 μM. Regarding the mechanism underlying their action, we hypothesized that the anti-alopecia and anticancer activities of these compounds could be attributed to the inhibition of the oncogenic/aging kinase PAK1.

Determination of monosaccharides and sugar alcohols in tissues from diabetic rats by high-performance liquid chromatography with pulsed amperometric detection.

PubMed

Tomiya, N; Suzuki, T; Awaya, J; Mizuno, K; Matsubara, A; Nakano, K; Kurono, M

1992-10-01

A sensitive and simple high-performance liquid chromatographic method has been developed to determine the concentration of monosaccharides and sugar alcohols in animal tissues. Five neutral monosaccharides (D-glucose, D-galactose, D-mannose, D-fructose, and D-ribose) and three neutral sugar alcohols (myo-inositol, glycerol, and D-sorbitol) predominate in the renal cortices and sciatic nerves of rats. These monosaccharides and sugar alcohols were extracted with distilled water, purified by deproteinization with ethanol, a Sep-Pak C18 cartridge, and columns of Dowex 50W-X8 and Amberlite CG-400, then separated on Ca2+ and Pb2+ cation-exchange columns, eluted with deionized distilled water at 80 degrees C, and detected using integrated pulsed amperometry. About 10 pmol of each sugar was detectable with a signal-to-noise ratio of 10:1. D-Glucose, D-fructose, D-sorbitol, and D-mannose were higher in both the renal and sciatic tissues of diabetic rats than in those of normal animals. D-Ribose and glycerol were higher in the renal cortex of diabetic animals.

Predicting In Vivo Efficacy of Therapeutic Bacteriophages Used To Treat Pulmonary Infections

PubMed Central

Henry, Marine; Lavigne, Rob

2013-01-01

The potential of bacteriophage therapy to treat infections caused by antibiotic-resistant bacteria has now been well established using various animal models. While numerous newly isolated bacteriophages have been claimed to be potential therapeutic candidates on the basis of in vitro observations, the parameters used to guide their choice among billions of available bacteriophages are still not clearly defined. We made use of a mouse lung infection model and a bioluminescent strain of Pseudomonas aeruginosa to compare the activities in vitro and in vivo of a set of nine different bacteriophages (PAK_P1, PAK_P2, PAK_P3, PAK_P4, PAK_P5, CHA_P1, LBL3, LUZ19, and PhiKZ). For seven bacteriophages, a good correlation was found between in vitro and in vivo activity. While the remaining two bacteriophages were active in vitro, they were not sufficiently active in vivo under similar conditions to rescue infected animals. Based on the bioluminescence recorded at 2 and 8 h postinfection, we also define for the first time a reliable index to predict treatment efficacy. Our results showed that the bacteriophages isolated directly on the targeted host were the most efficient in vivo, supporting a personalized approach favoring an optimal treatment. PMID:24041900

p21 Activated kinase 1: Nuclear activity and its role during DNA damage repair.

PubMed

Pérez-Yépez, Eloy Andrés; Saldívar-Cerón, Héctor Iván; Villamar-Cruz, Olga; Pérez-Plasencia, Carlos; Arias-Romero, Luis Enrique

2018-05-01

p21-activated kinase 1 (PAK1) is a serine/threonine kinase activated by the small GTPases Rac1 and Cdc42. It is located in the chromosome 11q13 and is amplified and/or overexpressed in several human cancer types including 25-30% of breast tumors. This enzyme plays a pivotal role in the control of a number of fundamental cellular processes by phosphorylating its downstream substrates. In addition to its role in the cytoplasm, it is well documented that PAK1 also plays crucial roles in the nucleus participating in mitotic events and gene expression through its association and/or phosphorylation of several transcription factors, transcriptional co-regulators and cell cycle-related proteins, including Aurora kinase A (AURKA), polo-like kinase 1 (PLK1), the forkhead transcription factor (FKHR), estrogen receptor α (ERα), and Snail. More recently, PAK signaling has emerged as a component of the DNA damage response (DDR) as PAK1 activity influences the cellular sensitivity to ionizing radiation and promotes the expression of several genes involved in the Fanconi Anemia/BRCA pathway. This review will focus on the nuclear functions of PAK1 and its role in the regulation of DNA damage repair. Copyright © 2018 Elsevier B.V. All rights reserved.

Subverting Host Cell P21-Activated Kinase: A Case of Convergent Evolution across Pathogens.

PubMed

John Von Freyend, Simona; Kwok-Schuelein, Terry; Netter, Hans J; Haqshenas, Gholamreza; Semblat, Jean-Philippe; Doerig, Christian

2017-04-21

Intracellular pathogens have evolved a wide range of strategies to not only escape from the immune systems of their hosts, but also to directly exploit a variety of host factors to facilitate the infection process. One such strategy is to subvert host cell signalling pathways to the advantage of the pathogen. Recent research has highlighted that the human serine/threonine kinase PAK, or p21-activated kinase, is a central component of host-pathogen interactions in many infection systems involving viruses, bacteria, and eukaryotic pathogens. PAK paralogues are found in most mammalian tissues, where they play vital roles in a wide range of functions. The role of PAKs in cell proliferation and survival, and their involvement in a number of cancers, is of great interest in the context of drug discovery. In this review we discuss the latest insights into the surprisingly central role human PAK1 plays for the infection by such different infectious disease agents as viruses, bacteria, and parasitic protists. It is our intention to open serious discussion on the applicability of PAK inhibitors for the treatment, not only of neoplastic diseases, which is currently the primary objective of drug discovery research targeting these enzymes, but also of a wide range of infectious diseases.

Pak and Rac GTPases promote oncogenic KIT–induced neoplasms

PubMed Central

Martin, Holly; Mali, Raghuveer Singh; Ma, Peilin; Chatterjee, Anindya; Ramdas, Baskar; Sims, Emily; Munugalavadla, Veerendra; Ghosh, Joydeep; Mattingly, Ray R.; Visconte, Valeria; Tiu, Ramon V.; Vlaar, Cornelis P.; Dharmawardhane, Suranganie; Kapur, Reuben

2013-01-01

An acquired somatic mutation at codon 816 in the KIT receptor tyrosine kinase is associated with poor prognosis in patients with systemic mastocytosis and acute myeloid leukemia (AML). Treatment of leukemic cells bearing this mutation with an allosteric inhibitor of p21–activated kinase (Pak) or its genetic inactivation results in growth repression due to enhanced apoptosis. Inhibition of the upstream effector Rac abrogates the oncogene-induced growth and activity of Pak. Although both Rac1 and Rac2 are constitutively activated via the guanine nucleotide exchange factor (GEF) Vav1, loss of Rac1 or Rac2 alone moderately corrected the growth of KIT-bearing leukemic cells, whereas the combined loss resulted in 75% growth repression. In vivo, the inhibition of Vav or Rac or Pak delayed the onset of myeloproliferative neoplasms (MPNs) and corrected the associated pathology in mice. To assess the role of Rac GEFs in oncogene-induced transformation, we used an inhibitor of Rac, EHop-016, which specifically targets Vav1 and found that EHop-016 was a potent inhibitor of human and murine leukemic cell growth. These studies identify Pak and Rac GTPases, including Vav1, as potential therapeutic targets in MPN and AML involving an oncogenic form of KIT. PMID:24091327

Isolation and Functional Characterization of a Floral Repressor, BcMAF1, From Pak-choi (Brassica rapa ssp. Chinensis).

PubMed

Huang, Feiyi; Liu, Tongkun; Hou, Xilin

2018-01-01

MADS-box genes form a large gene family in plants and are involved in multiple biological processes, such as flowering. However, the regulation mechanism of MADS-box genes in flowering remains unresolved, especially under short-term cold conditions. In the present study, we isolated BcMAF1 , a Pak-choi ( Brassica rapa ssp. Chinensis ) MADS AFFECTING FLOWERING ( MAF ), as a floral repressor and functionally characterized BcMAF1 in Arabidopsis and Pak-choi. Subcellular localization and sequence analysis indicated that BcMAF1 was a nuclear protein and contained a conserved MADS-box domain. Expression analysis revealed that BcMAF1 had higher expression levels in leaves, stems, and petals, and could be induced by short-term cold conditions in Pak-choi. Overexpressing BcMAF1 in Arabidopsis showed that BcMAF1 had a negative function in regulating flowering, which was further confirmed by silencing endogenous BcMAF1 in Pak-choi. In addition, qPCR results showed that AtAP3 expression was reduced and AtMAF2 expression was induced in BcMAF1 -overexpressing Arabidopsis . Meanwhile, BcAP3 transcript was up-regulated and BcMAF2 transcript was down-regulated in BcMAF1 -silencing Pak-choi. Yeast one-hybrid and dual luciferase transient assays showed that BcMAF1 could bind to the promoters of BcAP3 and BcMAF2 . These results indicated that BcAP3 and BcMAF2 might be the targets of BcMAF1. Taken together, our results suggested that BcMAF1 could negatively regulate flowering by directly activating BcMAF2 and repressing BcAP3 .

MicroRNA-26a is a key regulon that inhibits progression and metastasis of c-Myc/EZH2 double high advanced hepatocellular carcinoma.

PubMed

Zhang, Xiang; Zhang, Xiao; Wang, Ting; Wang, Lei; Tan, Zhijun; Wei, Wei; Yan, Bo; Zhao, Jing; Wu, Kaichun; Yang, Angang; Zhang, Rui; Jia, Lintao

2018-07-10

The transcription factor c-Myc is a key driver for hepatocellular carcinomas (HCCs), while the polycombrepressive complex 2 (PRC2) subunit EZH2 is an essential biomarker of HCC. c-Myc epigenetically silences tumor suppressors by recruiting PRC2 and inducing methylation of histone H3 lysine 27. However, it remains elusive how they are regulated in HCC. We found here that microRNA-26a (miR-26a) suppresses c-Myc, a classical Wnt pathway target gene, by targeting the Wnt pathway coactivator, cyclin-dependent kinase 8 (CDK8); miR-26a also directly targets and inhibits EZH2. The expression of MIR26A2, a predominant origin of miR-26a transcripts in hepatic cells, is repressed by c-Myc/PRC2, thereby forming a c-Myc/miR-26a/CDK8 regulatory circuit in HCC. Meanwhile, miR-26a suppresses migration of HCC by targeting p21-activated kinase 2 (PAK2), a critical kinase linking Rho GTPases to cytoskeleton reorganization. Consequently, in vivo delivery of miR-26a remarkably suppressed the development of xenograft HCC and metastasis of orthotopic HCC by downregulating c-Myc, CDK8 and PAK2. These findings unraveled a novel mechanism of c-Myc and Wnt/β-catenin interplay that dictates HCC pathogenesis, and have implications for the potential applicability of miRNA delivery in targeting the newly identified signaling axis and treating metastatic HCCs. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of small-molecule inhibitors of the group I p21-activated kinases, emerging therapeutic targets in cancer.

PubMed

Yi, Chunling; Maksimoska, Jasna; Marmorstein, Ronen; Kissil, Joseph L

2010-09-01

The p21-activated kinases (PAKs), immediate downstream effectors of the small G-proteins of the Rac/cdc42 family, are critical mediators of signaling pathways regulating cellular behaviors and as such, have been implicated in pathological conditions including cancer. Recent studies have validated the requirement for PAKs in promoting tumorigenesis in breast carcinoma and neurofibromatosis. Thus, there has been considerable interest in the development of inhibitors to the PAKs, as biological markers and leads for the development of therapeutics. While initial approaches were based on screening for competitive organic inhibitors, more recent efforts have focused on the identification of allosteric inhibitors, organometallic ATP-competitive inhibitors and the use of PAK1/inhibitor crystal structures for inhibitor optimization. This has led to the identification of highly selective and potent inhibitors, which will serve as a basis for further development of inhibitors for therapeutic applications. Copyright 2010 Elsevier Inc. All rights reserved.

Slit stimulation recruits Dock and Pak to the roundabout receptor and increases Rac activity to regulate axon repulsion at the CNS midline.

PubMed

Fan, Xueping; Labrador, Juan Pablo; Hing, Huey; Bashaw, Greg J

2003-09-25

Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Here we present evidence that the SH3-SH2 adaptor protein Dreadlocks (Dock), the p21-activated serine-threonine kinase (Pak), and the Rac1/Rac2/Mtl small GTPases can function during Robo repulsion. Loss-of-function and genetic interaction experiments suggest that limiting the function of Dock, Pak, or Rac partially disrupts Robo repulsion. In addition, Dock can directly bind to Robo's cytoplasmic domain, and the association of Dock and Robo is enhanced by stimulation with Slit. Furthermore, Slit stimulation can recruit a complex of Dock and Pak to the Robo receptor and trigger an increase in Rac1 activity. These results provide a direct physical link between the Robo receptor and an important cytoskeletal regulatory protein complex and suggest that Rac can function in both attractive and repulsive axon guidance.

Comparing the influence of selenite (Se4+) and selenate (Se6+) on the inhibition of the mercury (Hg) phytotoxicity to pak choi.

PubMed

Tran, Thi Anh Thu; Dinh, Quang Toan; Cui, Zeiwei; Huang, Jie; Wang, Dan; Wei, Tianjiao; Liang, Dongli; Sun, Xin; Ning, Ping

2018-01-01

Selenite (Se (IV)) and selenate (Se (IV)) have recently been demonstrated to be equally effective in inhibiting mercury (Hg) phytotoxicity to plants. This assertion is still unclear. In this study, we aimed to explore the potential effects of Se species (Se 4+ and Se 6+ ) on the inhibition of the mercury (Hg) bioavailability to pak choi in dry land. Pot experiments with exposure to different dosages of mercuric chloride (HgCl 2 ) and selenite (Na 2 SeO 3 ) or selenate (Na 2 SeO 4 ) were treated. To compare the influence of Se (IV) and Se (VI) on the bioaccumulation and bioavailability of Hg, the levels of total Hg in different pak choi (Brassica chinensis L.) tissues (roots and shoots) and the distribution changes of Hg fractions in soil before planting and after harvest were determined as well as the Hg I R values in soils (relative binding intensity) were analyzed. Results showed that application Se (IV) reduced the concentrations of Hg in pak choi roots more than Se (VI). Hg concentrations were also decreased in pak choi shoots in Se (IV) treatments, while which notably increased in Se (VI) treatments. Thus, Se (IV) plays a more important role than Se (VI) in limiting the absorption and bioaccumulation of Hg in pak choi. Moreover, this inhibition may only significantly occur when Se (IV) is at an appropriate level (2.5mg/kg). In addition, the good correlations between the proportions of mobile Hg fractions (soluble and exchangeable fractions), I R values with the Hg concentrations in plants were observed. This affirmed the importance of the Hg fractions transformation and the I R indicator of Hg in the assessment of their bioavailability. Our findings regarding the importance of Se (IV) influence in reducing Hg bioaccumulation not only provided the correct appraisal about the effect of Se species on the inhibition of the Hg phytotoxicity to pak choi in dry land, but also be a good reference for selecting Se fertilizer forms (Se 4+ or Se 6+ ). Copyright © 2017 Elsevier Inc. All rights reserved.

The Cossidae (Lepidoptera) of Afghanistan with description of three new species and special notes on the fauna of Bande-Amir National Park.

PubMed

Yakovlev, Roman V; Pljustch, Igor G; Skrylnik, Yuriy; Pak, Oleg; Witt, Thomas J

2015-07-23

The annotated list of Cossidae of Afghanistan consists of 44 species in 17 genera from the four subfamilies Catoptinae, Cossinae, Zeuzerinae, and Mehariinae. Three new species are described: Cossulus habibae Yakovlev, Pljustch, Skrylnik & Pak, sp. nov., Semagystia bamiani Yakovlev, Pljustch, Skrylnik & Pak, sp. nov., Phragmacossia bandeamiri Yakovlev, Pljustch, Skrylnik & Pak, sp. nov.; all from Band-e-Amir National Park in Bamian Province. Three species (Dervishiya cadambae (Moore, 1865), Semagystia cossoides (Graeser, 1892), Phragmacossia territa (Staudinger, 1879)) are reported for the first time from Afghanistan. A brief biogeographical analysis of the Cossidae of Afghanistan is given.

Quantitation of tacrolimus in whole blood using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS).

PubMed

Donaldson, Keri J; Shaw, Leslie M

2010-01-01

We describe a multiple reaction monitoring positive ion HPLC/tandem mass spectrometric method for quantification of tacrolimus in human whole blood with online extraction and cleanup. Included in this procedure: API 2000 triple quadrupole mass spectrometer with turbo-ion spray source (Applied Biosystems, Foster City, CA); 10-port diverter/switching valve (Valco, Houston, TX); HPLC system (Agilent Technologies series 1100, Wilmington, DE); 10 mm (C(18)) guard cartridge (Perkin Elmer, Norwalk, CT) used as an extraction column; a Nova-Pak C18 analytical column (2.1 x 150 mm I.D., 4 microm, Waters Corp, Milford, MA); washing solution, methanol: 30 mM ammonium acetate pH 5.1 (80:20); eluting solution, methanol:30 mM ammonium acetate pH 5.1 (97:3); flow rate 0.8 mL/min; and a run-time of 2.8 min. The first and third quadrupoles were set to detect the ammonium adduct ion and a high mass fragment of tacrolimus (m/z 821.5-->768.3), and of an internal standard (ascomycin) (m/z 901.8-->834.4). The lower limit of quantification of this method is 3.75 mg/L. The concentration of drug is determined by comparing peak-area ratios for tacrolimus and internal standard to a standard curve constructed using non-weighted linear through zero regression.

A multiresidue method for determination of trace levels of pesticides in air and water.

PubMed

Millet, M; Wortham, H; Sanusi, A; Mirabel, P

1996-11-01

A multiresidue analytical method is described for the analysis of 13 pesticides in fogwater, rainwater, gas, and particles. This method is based upon solid-liquid extraction using Sep-Pak tC18 light cartridges for aqueous samples, soxhlet for gas (adsorbed on XAD-2) and particles (on glass fiber filters), HPLC-based fractionation of the extracted residues using a silica column, and a linear gradient of n-hexane/tert butyl methyl ether followed by GC-ECD and HPLC-UV analyses of each fraction. Prior to analysis with GC-ECD, a methylation procedure using BF3/methanol was developed for the analysis of the fraction which contains chlorophenoxy acid herbicides. The recoveries of the extraction procedure of liquid samples and of the methylation were greater than 92 and 97% with a standard deviation lower than 8 and 5%, respectively. The detection limits varied between 0.1 and 0.01 microgram.ml-1 for the 13 pesticides studied with a standard deviation less than 9%. This method was used for the determination of pesticides in 18 fogwater samples (soluble + insoluble), 31 rainwater samples, and 17 air (gas + particles) samples collected between 1991 and 1993 in Colmar (east of France).

Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity.

PubMed

Xu, Guanlong; Zhang, Xuxiao; Gao, Weihua; Wang, Chenxi; Wang, Jinliang; Sun, Honglei; Sun, Yipeng; Guo, Lu; Zhang, Rui; Chang, Kin-Chow; Liu, Jinhua; Pu, Juan

2016-09-15

Adaptation of the viral polymerase complex comprising PB1, PB2, and PA is necessary for efficient influenza A virus replication in new host species. We found that PA mutation K356R (PA-K356R) has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of the PA-K356R mutation. Avian H9N2 virus with the PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA and increased viral polymerase activity that resulted in elevated levels of viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutation of PA-K356R and PB2-E627K, a well-known mammalian adaptive marker, in the H9N2 virus showed further cooperative increases in virus production and severity of infection in vitro and in vivo In summary, PA-K356R behaves as a novel mammalian tropism mutation, which, along with other mutations such as PB2-E627K, might render avian H9N2 viruses adapted for human infection. Mutations of the polymerase complex (PB1, PB2, and PA) of influenza A virus are necessary for viral adaptation to new hosts. This study reports a novel and predominant mammalian adaptive mutation, PA-K356R, in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 viruses causes significant increases in virus replication and severity of infection in human cells and mice and that PA-K356R cooperates with the PB2-E627K mutation, a well-characterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo Therefore, the PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants toward human infectivity. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity

PubMed Central

Xu, Guanlong; Zhang, Xuxiao; Gao, Weihua; Wang, Chenxi; Wang, Jinliang; Sun, Honglei; Sun, Yipeng; Guo, Lu; Zhang, Rui; Chang, Kin-Chow; Liu, Jinhua

2016-01-01

ABSTRACT Adaptation of the viral polymerase complex comprising PB1, PB2, and PA is necessary for efficient influenza A virus replication in new host species. We found that PA mutation K356R (PA-K356R) has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of the PA-K356R mutation. Avian H9N2 virus with the PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA and increased viral polymerase activity that resulted in elevated levels of viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutation of PA-K356R and PB2-E627K, a well-known mammalian adaptive marker, in the H9N2 virus showed further cooperative increases in virus production and severity of infection in vitro and in vivo. In summary, PA-K356R behaves as a novel mammalian tropism mutation, which, along with other mutations such as PB2-E627K, might render avian H9N2 viruses adapted for human infection. IMPORTANCE Mutations of the polymerase complex (PB1, PB2, and PA) of influenza A virus are necessary for viral adaptation to new hosts. This study reports a novel and predominant mammalian adaptive mutation, PA-K356R, in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 viruses causes significant increases in virus replication and severity of infection in human cells and mice and that PA-K356R cooperates with the PB2-E627K mutation, a well-characterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo. Therefore, the PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants toward human infectivity. PMID:27384648

Evaluation of Serodiagnostic Assays for Mycobacterium bovis Infection in Elk, White-Tailed Deer, and Reindeer in the United States

PubMed Central

Nelson, Jeffrey T.; Orloski, Kathleen A.; Lloyd, Audra L.; Camacho, Mark; Schoenbaum, Mark A.; Robbe-Austerman, Suelee; Thomsen, Bruce V.; Hall, S. Mark

2012-01-01

In 2011, the United States Department of Agriculture conducted a project in which elk (Cervus elaphus spp.), white-tailed deer (WTD) (Odocoileus virginianus), and reindeer (Rangifer tarandus) were evaluated by the single cervical tuberculin test (SCT), comparative cervical tuberculin test (CCT), and serologic tests. The rapid antibody detection tests evaluated were the CervidTB Stat-Pak (Stat-Pak), and the Dual Path Platform VetTB (DPP). Blood was collected from presumably uninfected animals prior to tuberculin injection for the SCT. A total of 1,783 animals were enrolled in the project. Of these, 1,752 (98.3%) were classified as presumably uninfected, based on originating from a captive cervid herd with no history of exposure to TB. Stat-Pak specificity estimates were 92.4% in reindeer, 96.7% in WTD, and 98.3% in elk and were not significantly different from SCT specificity estimates. Using the DPP in series on Stat-Pak antibody-positive samples improved specificity in the three species. Thirty one animals were classified as confirmed infected, based on necropsy and laboratory results, and 27/31 were antibody positive on Stat-Pak for an estimated sensitivity of 87.1%. The study findings indicate that rapid serologic tests used in series are comparable to the SCT and CCT and may have a greater ability to detect TB-infected cervids. PMID:22792512

Diosgenin inhibits superoxide generation in FMLP-activated mouse neutrophils via multiple pathways.

PubMed

Lin, Y; Jia, R; Liu, Y; Gao, Y; Zeng, X; Kou, J; Yu, B

2014-12-01

Diosgenin possesses anti-inflammatory and anticancer properties. Activated neutrophils produce high concentrations of the superoxide anion which is involved in the pathophysiology of inflammation-related diseases and cancer. In the present study, the inhibitory effect and possible mechanisms of diosgenin on superoxide generation were investigated in mouse bone marrow neutrophils. Diosgenin potently and concentration-dependently inhibited the extracellular and intracellular superoxide anion generation in Formyl-Met-Leu-Phe (FMLP)- activated neutrophils, with IC50 values of 0.50 ± 0.08 μM and 0.66 ± 0.13 μM, respectively. Such inhibition was not mediated by scavenging the superoxide anion or by a cytotoxic effect. Diosgenin inhibited the phosphorylation of p47phox and membrane translocation of p47phox and p67phox, and thus blocking the assembly of nicotinamide adenine dinucleotide phosphate oxidase. Moreover, cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were also effectively increased by diosgenin. It attenuated FMLP-induced increase of phosphorylation of cytosolic phospholipase A (cPLA2), p21-activated kinase (PAK), Akt, p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK). Our data indicate that diosgenin exhibits inhibitory effects on superoxide anion production through the blockade of cAMP, PKA, cPLA2, PAK, Akt and MAPKs signaling pathways. The results may explain the clinical implications of diosgenin in the treatment of inflammation-related disorders.

Melanoma Inhibition by Anthocyanins Is Associated with the Reduction of Oxidative Stress Biomarkers and Changes in Mitochondrial Membrane Potential.

PubMed

Diaconeasa, Zoriţa; Ayvaz, Huseyin; Ruginǎ, Dumitriţa; Leopold, Loredana; Stǎnilǎ, Andreea; Socaciu, Carmen; Tăbăran, Flaviu; Luput, Lavinia; Mada, Diana Carla; Pintea, Adela; Jefferson, Andrew

2017-12-01

Anthocyanins are water soluble pigments which have been proved to exhibit health benefits. Several studies have investigated their effects on several types of cancer, but little attention has been given to melanoma. The phytochemical content of nine different berry samples was assessed by liquid chromatography followed by electrospray ionization mass spectrometry (LC-ESI + -MS). Twenty-six anthocyanins were identified, after a previous C 18 Sep-pak clean-up procedure. Chokeberry and red grape anthocyanins rich extracts (C-ARE and RG-ARE) were selected to be tested on normal and melanoma cell lines, due to their different chemical pattern. C-ARE composition consists of cyanidin aglycone glycosylated with different sugars; while RG-ARE contains glucosylated derivatives of five different aglycones. Both C-ARE and RG-ARE anthocyanins reduced proliferation, increased oxidative stress biomarkers and diminished mitochondrial membrane potential in melanoma cells, having no negative influence on normal cells. A synergistic response may be attributed to the five different aglycones present in RG-ARE, which proved to exert greater effects on melanoma cells than the mixture of cyanidin derivatives with different sugars (C-ARE). In conclusion, C-ARE and RG-ARE anthocyanins may inhibit melanoma cell proliferation and increase the level of oxidative stress, with opposite effect on normal cells. Therefore, anthocyanins might be recommended as active ingredients for cosmetic and nutraceutical industry. Graphical Abstract ᅟ.

Tamoxifen Dependent Interaction Between the Estrogen Receptor and a Novel P21 Activated Kinase

DTIC Science & Technology

2002-06-01

binding domain at the C Cv1 cells were cotransfected with ARE4-Luc reporter (100 teric G prti Tse bindin dominat the C ng), pRL-SV40 ( Renilla control...48 h and assayed for luciferase and Renilla Three of the four residues defining a putative het- activity. erotrimeric G protein binding motif were...nases. Taken together, these results demonstrated expression of the control Renilla reporter regulated by that the PAK6 CRIB domain was functional with re

Separation of aliphatic carboxylic acids and benzenecarboxylic acids by ion-exclusion chromatography with various cation-exchange resin columns and sulfuric acid as eluent.

PubMed

Ohta, Kazutoku; Ohashi, Masayoshi; Jin, Ji-Ye; Takeuchi, Toyohide; Fujimoto, Chuzo; Choi, Seong-Ho; Ryoo, Jae-Jeong; Lee, Kwang-Pill

2003-05-16

The application of various hydrophilic cation-exchange resins for high-performance liquid chromatography (sulfonated silica gel: TSKgel SP-2SW, carboxylated silica gel: TSKgel CM-2SW, sulfonated polymethacrylate resin: TSKgel SP-5PW, carboxylated polymethacrylate resins: TSKgel CM-5PW and TSKgel OA-Pak A) as stationary phases in ion-exclusion chromatography for C1-C7 aliphatic carboxylic acids (formic, acetic, propionic, butyric, isovaleric, valeric, isocaproic, caproic, 2-methylhexanoic and heptanoic acids) and benzenecarboxylic acids (pyromellitic, trimellitic, hemimellitic, o-phthalic, m-phthalic, p-phthalic, benzoic, salicylic acids and phenol) was carried out using diluted sulfuric acid as the eluent. Silica-based cation-exchange resins (TSKgel SP-2SW and TSKgel CM-2SW) were very suitable for the ion-exclusion chromatographic separation of these benzenecarboxylic acids. Excellent simultaneous separation of these benzenecarboxylic acids was achieved on a TSKgel SP-2SW column (150 x 6 mm I.D.) in 17 min using a 2.5 mM sulfuric acid at pH 2.4 as the eluent. Polymethacrylate-based cation-exchange resins (TSKgel SP-5PW, TSKgel CM-5PW and TSKgel OA-Pak A) acted as advanced stationary phases for the ion-exclusion chromatographic separation of these C1-C7 aliphatic carboxylic acids. Excellent simultaneous separation of these C1-C7 acids was achieved on a TSKgel CM-5PW column (150 x 6 mm I.D.) in 32 min using a 0.05 mM sulfuric acid at pH 4.0 as the eluent.

Liquid chromatographic determination of L-ascorbic acid in candies and soft drinks.

PubMed

Maeda, Y; Ochi, S; Masui, T; Matubara, S

1988-01-01

The L-ascorbic acid (AsA) contents of candies and soft drinks available in the market were determined by liquid chromatography (LC). Samples are cleaned up on a disposable Sep-Pak C18 cartridge followed by reverse phase separation on an ODS column using a mobile phase of 0.1% phosphoric acid (pH 2.2). The AsA peak is detected on the basis of the UV absorption at 254 nm. The detection limit was 1 microgram/mL final concentration. Recoveries of AsA added at levels of 1-10 mg/g candy and 1-10 mg/10 mL soft drink were 99.2-101.7% with a coefficient of variation of 0.52-1.20% (n = 5). The present method allows rapid and accurate assays because it is a simple procedure compared with the official dye-titration method, and it is suitable for the routine analysis of AsA in selected candies and soft drinks.

Isolation and Characterization of Anthocyanins from Hibiscus sabdariffa Flowers.

PubMed

Grajeda-Iglesias, Claudia; Figueroa-Espinoza, Maria C; Barouh, Nathalie; Baréa, Bruno; Fernandes, Ana; de Freitas, Victor; Salas, Erika

2016-07-22

The intense red-colored Hibiscus sabdariffa flowers are an inexpensive source of anthocyanins with potential to be used as natural, innocuous, and health-beneficial colorants. An anthocyanin-rich extract from hibiscus flowers was obtained by ultrasound-assisted extraction. By a single-step process fractionation using a Sep-Pak C18 cartridge, the main hibiscus anthocyanins, delphinidin-3-O-sambubioside (Dp-samb) and cyanidin-3-O-sambubioside (Cy-samb), were separated and then characterized via NMR and HPLC-ESIMS data. Since Dp-samb was the most abundant anthocyanin identified in the extract, its colorant properties were studied by the pH jumps method, which allowed the calculation of the single acid-base equilibrium (pK'a 2.92), the acidity (pKa 3.70), and the hydration constants (pKh 3.02). Moreover, by using size-exclusion chromatography, new cyanidin-derived anthocyanins (with three or more sugar units) were successfully identified and reported for the first time in the hibiscus extract.

Analysis of p21-Activated Kinase Function in Neurofibromatosis Type 2

DTIC Science & Technology

2009-01-01

SchΔ(39-121) Pak1+/+ mice developed NF2-related pathologies ( schwannomatosis , nerve sheath tumors, sarcomas), whereas 2/34 P0- SchΔ(39-121) Pak1...for analysis (M.G., personal communication), as schwannomatosis develops in all the transgenic mice by three months of age. We have therefore

Analysis of p21-Activated Kinase Function in Neurofibromatosis Type 2

DTIC Science & Technology

2008-01-01

SchΔ(39-121) Pak1+/+ mice developed NF2-related pathologies ( schwannomatosis , nerve sheath tumors, sarcomas), whereas 1/33 P0-SchΔ(39-121) Pak1...much more favorable for analysis (M.G., personal communication), as schwannomatosis develops in all the transgenic mice by three months of age. We

Specification of Dendritogenesis Site in Drosophila aCC Motoneuron by Membrane Enrichment of Pak1 through Dscam1.

PubMed

Kamiyama, Daichi; McGorty, Ryan; Kamiyama, Rie; Kim, Michael D; Chiba, Akira; Huang, Bo

2015-10-12

Precise positioning of dendritic branches is a critical step in the establishment of neuronal circuitry. However, there is limited knowledge on how environmental cues translate into dendrite initiation or branching at a specific position. Here, through a combination of mutation, RNAi, and imaging experiments, we found that a Dscam-Dock-Pak1 hierarchical interaction defines the stereotypical dendrite growth site in the Drosophila aCC motoneuron. This interaction localizes the Cdc42 effector Pak1 to the plasma membrane at the dendrite initiation site before the activation of Cdc42. Ectopic expression of membrane-anchored Pak1 overrides this spatial specification of dendritogenesis, confirming its function in guiding Cdc42 signaling. We further discovered that Dscam1 localization in aCC occurs through an inter-neuronal contact that involves Dscam1 in the partner MP1 neuron. These findings elucidate a mechanism by which Dscam1 controls neuronal morphogenesis through spatial regulation of Cdc42 signaling and, subsequently, cytoskeletal remodeling. Copyright © 2015 Elsevier Inc. All rights reserved.

Dbo/Henji Modulates Synaptic dPAK to Gate Glutamate Receptor Abundance and Postsynaptic Response.

PubMed

Wang, Manyu; Chen, Pei-Yi; Wang, Chien-Hsiang; Lai, Tzu-Ting; Tsai, Pei-I; Cheng, Ying-Ju; Kao, Hsiu-Hua; Chien, Cheng-Ting

2016-10-01

In response to environmental and physiological changes, the synapse manifests plasticity while simultaneously maintains homeostasis. Here, we analyzed mutant synapses of henji, also known as dbo, at the Drosophila neuromuscular junction (NMJ). In henji mutants, NMJ growth is defective with appearance of satellite boutons. Transmission electron microscopy analysis indicates that the synaptic membrane region is expanded. The postsynaptic density (PSD) houses glutamate receptors GluRIIA and GluRIIB, which have distinct transmission properties. In henji mutants, GluRIIA abundance is upregulated but that of GluRIIB is not. Electrophysiological results also support a GluR compositional shift towards a higher IIA/IIB ratio at henji NMJs. Strikingly, dPAK, a positive regulator for GluRIIA synaptic localization, accumulates at the henji PSD. Reducing the dpak gene dosage suppresses satellite boutons and GluRIIA accumulation at henji NMJs. In addition, dPAK associated with Henji through the Kelch repeats which is the domain essential for Henji localization and function at postsynapses. We propose that Henji acts at postsynapses to restrict both presynaptic bouton growth and postsynaptic GluRIIA abundance by modulating dPAK.

Dbo/Henji Modulates Synaptic dPAK to Gate Glutamate Receptor Abundance and Postsynaptic Response

PubMed Central

Wang, Manyu; Chen, Pei-Yi; Wang, Chien-Hsiang; Lai, Tzu-Ting; Tsai, Pei-I; Cheng, Ying-Ju; Kao, Hsiu-Hua; Chien, Cheng-Ting

2016-01-01

In response to environmental and physiological changes, the synapse manifests plasticity while simultaneously maintains homeostasis. Here, we analyzed mutant synapses of henji, also known as dbo, at the Drosophila neuromuscular junction (NMJ). In henji mutants, NMJ growth is defective with appearance of satellite boutons. Transmission electron microscopy analysis indicates that the synaptic membrane region is expanded. The postsynaptic density (PSD) houses glutamate receptors GluRIIA and GluRIIB, which have distinct transmission properties. In henji mutants, GluRIIA abundance is upregulated but that of GluRIIB is not. Electrophysiological results also support a GluR compositional shift towards a higher IIA/IIB ratio at henji NMJs. Strikingly, dPAK, a positive regulator for GluRIIA synaptic localization, accumulates at the henji PSD. Reducing the dpak gene dosage suppresses satellite boutons and GluRIIA accumulation at henji NMJs. In addition, dPAK associated with Henji through the Kelch repeats which is the domain essential for Henji localization and function at postsynapses. We propose that Henji acts at postsynapses to restrict both presynaptic bouton growth and postsynaptic GluRIIA abundance by modulating dPAK. PMID:27736876

Novel Role for p21-activated Kinase 2 in Thrombin-induced Monocyte Migration*

PubMed Central

Gadepalli, Ravisekhar; Kotla, Sivareddy; Heckle, Mark R.; Verma, Shailendra K.; Singh, Nikhlesh K.; Rao, Gadiparthi N.

2013-01-01

To understand the role of thrombin in inflammation, we tested its effects on migration of THP-1 cells, a human monocytic cell line. Thrombin induced THP-1 cell migration in a dose-dependent manner. Thrombin induced tyrosine phosphorylation of Pyk2, Gab1, and p115 RhoGEF, leading to Rac1- and RhoA-dependent Pak2 activation. Downstream to Pyk2, Gab1 formed a complex with p115 RhoGEF involving their pleckstrin homology domains. Furthermore, inhibition or depletion of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, or Pak2 levels substantially attenuated thrombin-induced THP-1 cell F-actin cytoskeletal remodeling and migration. Inhibition or depletion of PAR1 also blocked thrombin-induced activation of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2, resulting in diminished THP-1 cell F-actin cytoskeletal remodeling and migration. Similarly, depletion of Gα12 negated thrombin-induced Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA, and Pak2 activation, leading to attenuation of THP-1 cell F-actin cytoskeletal remodeling and migration. These novel observations reveal that thrombin induces monocyte/macrophage migration via PAR1-Gα12-dependent Pyk2-mediated Gab1 and p115 RhoGEF interactions, leading to Rac1- and RhoA-targeted Pak2 activation. Thus, these findings provide mechanistic evidence for the role of thrombin and its receptor PAR1 in inflammation. PMID:24025335

Novel tumor suppressive function of Smad4 in serum starvation-induced cell death through PAK1–PUMA pathway

PubMed Central

Lee, S-H; Jung, Y-S; Chung, J-Y; Oh, A Y; Lee, S-J; Choi, D H; Jang, S M; Jang, K-S; Paik, S S; Ha, N-C; Park, B-J

2011-01-01

DPC4 (deleted in pancreatic cancer 4)/Smad4 is an essential factor in transforming growth factor (TGF)-β signaling and is also known as a frequently mutated tumor suppressor gene in human pancreatic and colon cancer. However, considering the fact that TGF-β can contribute to cancer progression through transcriptional target genes, such as Snail, MMPs, and epithelial–mesenchymal transition (EMT)-related genes, loss of Smad4 in human cancer would be required for obtaining the TGF-β signaling-independent advantage, which should be essential for cancer cell survival. Here, we provide the evidences about novel role of Smad4, serum-deprivation-induced apoptosis. Elimination of serum can obviously increase the Smad4 expression and induces the cell death by p53-independent PUMA induction. Instead, Smad4-deficient cells show the resistance to serum starvation. Induced Smad4 suppresses the PAK1, which promotes the PUMA destabilization. We also found that Siah-1 and pVHL are involved in PAK1 destabilization and PUMA stabilization. In fact, Smad4-expressed cancer tissues not only show the elevated expression of PAK1, but also support our hypothesis that Smad4 induces PUMA-mediated cell death through PAK1 suppression. Our results strongly suggest that loss of Smad4 renders the resistance to serum-deprivation-induced cell death, which is the TGF-β-independent tumor suppressive role of Smad4. PMID:22130069

Effects of low light on photosynthetic properties, antioxidant enzyme activity, and anthocyanin accumulation in purple pak-choi (Brassica campestris ssp. Chinensis Makino).

PubMed

Zhu, Hongfang; Li, Xiaofeng; Zhai, Wen; Liu, Yang; Gao, Qianqian; Liu, Jinping; Ren, Li; Chen, Huoying; Zhu, Yuying

2017-01-01

Anthocyanins are secondary metabolites that contribute to red, blue, and purple colors in plants and are affected by light, but the effects of low light on the physiological responses of purple pak-choi plant leaves are still unclear. In this study, purple pak-choi seedlings were exposed to low light by shading with white gauze and black shading in a phytotron. The responses in terms of photosynthetic properties, carbohydrate metabolism, antioxidant enzyme activity, anthocyanin biosynthetic enzyme activity, and the relative chlorophyll and anthocyanin content of leaves were measured. The results showed that chlorophyll b, intracellular CO2 content, stomatal conductance and antioxidant activities of guaiacol peroxidase, catalase and superoxide dismutase transiently increased in the shade treatments at 5 d. The malondialdehyde content also increased under low light stress, which damages plant cells. With the extension of shading time (at 15 d), the relative chlorophyll a, anthocyanin and soluble protein contents, net photosynthetic rate, transpiration rate, stomata conductance, antioxidant enzyme activities, and activities of four anthocyanin biosynthetic enzymes decreased significantly. Thus, at the early stage of low light treatment, the chlorophyll b content increased to improve photosynthesis. When the low light treatment was extended, antioxidant enzyme activity and the activity of anthocyanin biosynthesis enzymes were inhibited, causing the purple pak-choi seedlings to fade from purple to green. This study provides valuable information for further deciphering genetic mechanisms and improving agronomic traits in purple pak-choi under optimal light requirements.

P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate

PubMed Central

Wang, Yiming; Gratzke, Christian; Tamalunas, Alexander; Wiemer, Nicolas; Ciotkowska, Anna; Rutz, Beata; Waidelich, Raphaela; Strittmatter, Frank; Liu, Chunxiao; Stief, Christian G.; Hennenberg, Martin

2016-01-01

Prostate smooth muscle tone and hyperplastic growth are involved in the pathophysiology and treatment of male lower urinary tract symptoms (LUTS). Available drugs are characterized by limited efficacy. Patients’ adherence is particularly low to combination therapies of 5α-reductase inhibitors and α1-adrenoceptor antagonists, which are supposed to target contraction and growth simultaneously. Consequently, molecular etiology of benign prostatic hyperplasia (BPH) and new compounds interfering with smooth muscle contraction or growth in the prostate are of high interest. Here, we studied effects of p21-activated kinase (PAK) inhibitors (FRAX486, IPA3) in hyperplastic human prostate tissues, and in stromal cells (WPMY-1). In hyperplastic prostate tissues, PAK1, -2, -4, and -6 may be constitutively expressed in catecholaminergic neurons, while PAK1 was detected in smooth muscle and WPMY-1 cells. Neurogenic contractions of prostate strips by electric field stimulation were significantly inhibited by high concentrations of FRAX486 (30 μM) or IPA3 (300 μM), while noradrenaline- and phenylephrine-induced contractions were not affected. FRAX486 (30 μM) inhibited endothelin-1- and -2-induced contractions. In WPMY-1 cells, FRAX486 or IPA3 (24 h) induced concentration-dependent (1–10 μM) degeneration of actin filaments. This was paralleled by attenuation of proliferation rate, being observed from 1 to 10 μM FRAX486 or IPA3. Cytotoxicity of FRAX486 and IPA3 in WPMY-1 cells was time- and concentration-dependent. Stimulation of WPMY-1 cells with endothelin-1 or dihydrotestosterone, but not noradrenaline induced PAK phosphorylation, indicating PAK activation by endothelin-1. Thus, PAK inhibitors may inhibit neurogenic and endothelin-induced smooth muscle contractions in the hyperplastic human prostate, and growth of stromal cells. Targeting prostate smooth muscle contraction and stromal growth at once by a single compound is principally possible, at least under experimental conditions. PMID:27071060

P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate.

PubMed

Wang, Yiming; Gratzke, Christian; Tamalunas, Alexander; Wiemer, Nicolas; Ciotkowska, Anna; Rutz, Beata; Waidelich, Raphaela; Strittmatter, Frank; Liu, Chunxiao; Stief, Christian G; Hennenberg, Martin

2016-01-01

Prostate smooth muscle tone and hyperplastic growth are involved in the pathophysiology and treatment of male lower urinary tract symptoms (LUTS). Available drugs are characterized by limited efficacy. Patients' adherence is particularly low to combination therapies of 5α-reductase inhibitors and α1-adrenoceptor antagonists, which are supposed to target contraction and growth simultaneously. Consequently, molecular etiology of benign prostatic hyperplasia (BPH) and new compounds interfering with smooth muscle contraction or growth in the prostate are of high interest. Here, we studied effects of p21-activated kinase (PAK) inhibitors (FRAX486, IPA3) in hyperplastic human prostate tissues, and in stromal cells (WPMY-1). In hyperplastic prostate tissues, PAK1, -2, -4, and -6 may be constitutively expressed in catecholaminergic neurons, while PAK1 was detected in smooth muscle and WPMY-1 cells. Neurogenic contractions of prostate strips by electric field stimulation were significantly inhibited by high concentrations of FRAX486 (30 μM) or IPA3 (300 μM), while noradrenaline- and phenylephrine-induced contractions were not affected. FRAX486 (30 μM) inhibited endothelin-1- and -2-induced contractions. In WPMY-1 cells, FRAX486 or IPA3 (24 h) induced concentration-dependent (1-10 μM) degeneration of actin filaments. This was paralleled by attenuation of proliferation rate, being observed from 1 to 10 μM FRAX486 or IPA3. Cytotoxicity of FRAX486 and IPA3 in WPMY-1 cells was time- and concentration-dependent. Stimulation of WPMY-1 cells with endothelin-1 or dihydrotestosterone, but not noradrenaline induced PAK phosphorylation, indicating PAK activation by endothelin-1. Thus, PAK inhibitors may inhibit neurogenic and endothelin-induced smooth muscle contractions in the hyperplastic human prostate, and growth of stromal cells. Targeting prostate smooth muscle contraction and stromal growth at once by a single compound is principally possible, at least under experimental conditions.

The Drip Impact. [Project ECOLogy ELE Pak, Jack Pak].

ERIC Educational Resources Information Center

Staudacher, Jack

This is one of a series of units for environmental education developed by the Highline Public Schools. The unit is designed for senior high science classes. The primary emphasis of the material is on water, water analysis, and possible methods of watershed management; while the materials were designed for use in and around the Highline Public…

Anthropology - Ecology. [Project ECOLogy ELE Pak, Skidmore Pak].

ERIC Educational Resources Information Center

Skidmore, Margaret

This is one of a series of units for environmental education developed by the Highline Public Schools. The unit may be used as an introduction to the study of anthropology, the influence of ecology on the study of anthropology, and an introduction to the physical school environment. For best results, it should be used at the beginning of the…

ETV TEST REPORT OF CONTROL OF BIOAEROSOLS IN HVAC SYSTEMS GLASFLOSS INDUSTRIES Z-PAK SERIES S, MODEL ZPS24241295BO

EPA Science Inventory

The Environmental Technology Verification report discusses the technology and performance of the Z-Pak Series S, Model ZPS24241295B0 air filter for dust and bioaerosol filtration manufactured by Glasfloss Industries, Inc. The pressure drop across the filter was 91 Pa clean and 34...

Integrated analysis of breast cancer cell lines reveals unique signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heiser, Laura M.; Wang, Nicholas J.; Talcott, Carolyn L.

Cancer is a heterogeneous disease resulting from the accumulation of genetic defects that negatively impact control of cell division, motility, adhesion and apoptosis. Deregulation in signaling along the EGFR-MAPK pathway is common in breast cancer, though the manner in which deregulation occurs varies between both individuals and cancer subtypes. We were interested in identifying subnetworks within the EGFR-MAPK pathway that are similarly deregulated across subsets of breast cancers. To that end, we mapped genomic, transcriptional and proteomic profiles for 30 breast cancer cell lines onto a curated Pathway Logic symbolic systems model of EGFR-MEK signaling. This model was comprised ofmore » 539 molecular states and 396 rules governing signaling between active states. We analyzed these models and identified several subtype specific subnetworks, including one that suggested PAK1 is particularly important in regulating the MAPK cascade when it is over-expressed. We hypothesized that PAK1 overexpressing cell lines would have increased sensitivity to MEK inhibitors. We tested this experimentally by measuring quantitative responses of 20 breast cancer cell lines to three MEK inhibitors. We found that PAK1 over-expressing luminal breast cancer cell lines are significantly more sensitive to MEK inhibition as compared to those that express PAK1 at low levels. This indicates that PAK1 over-expression may be a useful clinical marker to identify patient populations that may be sensitive to MEK inhibitors. All together, our results support the utility of symbolic system biology models for identification of therapeutic approaches that will be effective against breast cancer subsets.« less

Integrated analysis of breast cancer cell lines reveals unique signaling pathways.

PubMed

Heiser, Laura M; Wang, Nicholas J; Talcott, Carolyn L; Laderoute, Keith R; Knapp, Merrill; Guan, Yinghui; Hu, Zhi; Ziyad, Safiyyah; Weber, Barbara L; Laquerre, Sylvie; Jackson, Jeffrey R; Wooster, Richard F; Kuo, Wen Lin; Gray, Joe W; Spellman, Paul T

2009-01-01

Cancer is a heterogeneous disease resulting from the accumulation of genetic defects that negatively impact control of cell division, motility, adhesion and apoptosis. Deregulation in signaling along the EgfR-MAPK pathway is common in breast cancer, though the manner in which deregulation occurs varies between both individuals and cancer subtypes. We were interested in identifying subnetworks within the EgfR-MAPK pathway that are similarly deregulated across subsets of breast cancers. To that end, we mapped genomic, transcriptional and proteomic profiles for 30 breast cancer cell lines onto a curated Pathway Logic symbolic systems model of EgfR-MAPK signaling. This model was composed of 539 molecular states and 396 rules governing signaling between active states. We analyzed these models and identified several subtype-specific subnetworks, including one that suggested Pak1 is particularly important in regulating the MAPK cascade when it is over-expressed. We hypothesized that Pak1 over-expressing cell lines would have increased sensitivity to Mek inhibitors. We tested this experimentally by measuring quantitative responses of 20 breast cancer cell lines to three Mek inhibitors. We found that Pak1 over-expressing luminal breast cancer cell lines are significantly more sensitive to Mek inhibition compared to those that express Pak1 at low levels. This indicates that Pak1 over-expression may be a useful clinical marker to identify patient populations that may be sensitive to Mek inhibitors. All together, our results support the utility of symbolic system biology models for identification of therapeutic approaches that will be effective against breast cancer subsets.

Eco-Kids Fly Off to the Forests. [Project ECOLogy ELE Pak, Bell Pak].

ERIC Educational Resources Information Center

Bell, Loretta

This is one of a series of units for environmental education developed by the Highline Public Schools. This unit was designed for use with fourth-grade students; it focuses on three forest biomes. Each of the biomes has characteristics of its own. The unit includes eight lessons, as well as additional activities. The unit, which incorporates…

Food: The Challenge to Manage. [Project ECOLogy ELE Pak, Roush Pak].

ERIC Educational Resources Information Center

Roush, Judy

This is one of a series of units for environmental education developed by the Highline Public Schools. The unit is designed for students at the senior high level who have a basic knowledge of nutrition, some experience in menu planning, and who are ready to put this knowledge of nutrition to work in selecting foods to attain maximum nutrition with…

Natural or Organic Foods? [Project ECOLogy ELE Pak, Schmidt Pak].

ERIC Educational Resources Information Center

Schmidt, Linda

This is one of a series of units for environmental education developed by the Highline Public Schools. The unit is designed for secondary students in home economics classes. The content of the units focuses on natural and organic foods, characteristics of the foods, and uses of the foods. The seven lessons in this unit are designed to last over a…

Zip Pak (Boys) for Third Reader Level.

ERIC Educational Resources Information Center

Scott, Norval C., Comp.

Aim of the Zip Pak is to improve the migrant child's interest in reading. Behavioral objectives are to have the migrant child indicate 10 percent happier faces on the posttest; to have an equal number of blame-self, blame-others on the posttest; and to have 25 percent more praise-self on the posttest. The workbook for boys includes a story about…

Sediment-water partitioning of inorganic mercury in estuaries.

PubMed

Turner, A; Millward, G E; Le Roux, S M

2001-12-01

The sediment-water partitioning and speciation of inorganic mercury have been studied under simulated estuarine conditions by monitoring the hydrophobicity and uptake of dissolved 203Hg(II) in samples from a variety of estuarine environments. A persistent increase in the distribution coefficientwith increasing salinity is inconsistent with inorganic speciation calculations, which predict an increase in the concentration of the soluble HgCl4(2-) complex (or reduction in sediment-water distribution coefficient) with increasing salinity. Partition data are, however, defined by an empirical equation relating to the salting out of nonelectrolytes via electrostriction and are characterized by salting constants between about 1.4 and 2.0 L mol(-1). Salting out of the neutral, covalent chloro-complex, HgCl2(0), is predicted but cannot account for the magnitude of salting out observed. Since Hg(II) strongly complexes with dissolved (and particulate) organic matter in natural environments, of more significance appears to be the salting out of Hg(II)-organic complexes. Operational measurements of the speciation of dissolved Hg(II) using Sep-Pak C18 columns indicate a reduction in the proportion of hydrophobic (C18-retained) dissolved Hg(II) complexes with increasing salinity, both in the presence and absence of suspended particles. Ratios of hydrophobic Hg(ll) before and after particle addition suggest a coupled salting out-sorption mechanism, with the precise nature of Hg(II) species salted out being determined bythe characteristics and concentrations of dissolved and sediment organic matter.

TnBP⁄Triton X-45 Treatment of Plasma for Transfusion Efficiently Inactivates Hepatitis C Virus

PubMed Central

Chou, Ming-Li; Burnouf, Thierry; Chang, Shun-Pang; Hung, Ting-Chun; Lin, Chun-Ching; Richardson, Christopher D.; Lin, Liang-Tzung

2015-01-01

Risk of transmission of hepatitis C virus (HCV) by clinical plasma remains high in countries with a high prevalence of hepatitis C, justifying the implementation of viral inactivation treatments. In this study, we assessed the extent of inactivation of HCV during minipool solvent/detergent (SD; 1% TnBP / 1% Triton X-45) treatment of human plasma. Luciferase-tagged infectious cell culture-derived HCV (HCVcc) particles were used to spike human plasma prior to treatment by SD at 31 ± 0.5°C for 30 min. Samples were taken before and after SD treatment and filtered on a Sep-Pak Plus C18 cartridge to remove the SD agents. Risk of cytotoxicity was assessed by XTT cell viability assay. Viral infectivity was analyzed based on the luciferase signals, 50% tissue culture infectious dose viral titer, and immunofluorescence staining for HCV NS5A protein. Total protein, cholesterol, and triglyceride contents were determined before and after SD treatment and C18 cartridge filtration. Binding analysis, using patient-derived HCV clinical isolates, was also examined to validate the efficacy of the inactivation by SD. SD treatment effectively inactivated HCVcc within 30 min, as demonstrated by the baseline level of reporter signals, total loss of viral infectivity, and absence of viral protein NS5A. SD specifically targeted HCV particles to render them inactive, with essentially no effect on plasma protein content and hemostatic function. More importantly, the efficacy of the SD inactivation method was confirmed against various genotypes of patient-derived HCV clinical isolates and against HCVcc infection of primary human hepatocytes. Therefore, treatment by 1% TnBP / 1% Triton X-45 at 31°C is highly efficient to inactivate HCV in plasma for transfusion, showing its capacity to enhance the safety of therapeutic plasma products. We propose that the methodology used here to study HCV infectivity can be valuable in the validation of viral inactivation and removal processes of human plasma-derived products. PMID:25658612

Eco-Kids: Experiment with Air on Spaceship Earth. [Project ECOLogy ELE Pak, Bell Pak].

ERIC Educational Resources Information Center

Bell, Loretta

This is one of a series of units for environmental education developed by the Highline Public Schools. The unit, designed for intermediate grades in the elementary schools, is concerned with the study of air, air pollution, effects of air pollution, and ways of improving the quality of the air. Six lessons are included in the unit; most of the…

Degradation Behavior and Accelerated Weathering of Composite Boards Produced from Waste Tetra Pak® Packaging Materials

Treesearch

Nural Yilgor; Coskun Kose; Evren Terzi; Aysel Kanturk Figen; Rebecca Ibach; S. Nami Kartal; Sabriye Piskin

2014-01-01

Manufacturing panels from Tetra Pak® (TP) packaging material might be an alternative to conventional wood-based panels. This study evaluated some chemical and physical properties as well as biological, weathering, and fire performance of panels with and without zinc borate (ZnB) by using shredded TP packaging cartons. Such packaging material, a worldwide well-known...

Chemical aspects of lifetime extension at Paks Nuclear Power Plant

NASA Astrophysics Data System (ADS)

Schunk, J.; Patek, G.; Pintér, T.; Tilky, P.; Ősz, J.; Salamon, T.; Varga, K.

2009-02-01

The review of the water regime used for the Units of Paks Nuclear Power Plant (NPP) was carried out in 2005, after 18-23 years of operation. In order to determine the phase composition of the surface oxide layers primary equipment, samples were measured by Conversion Electron Mössbauer Spectroscopy (CEMS). Due to the absorption of the conversion electrons, information can be obtained from the outermost ˜300 nm thick layer of the surface. It was clearly concluded after processing the huge data base of the water regime and CEMS data, that there is nothing to hamper the life time extension of the Units. In 2006, a new water regime was developed that will be applied during the preparation for the life time extension and the extended service life as well. In connection with this work, recommendations were made for some modifications of the previously used water regime. Currently there is no uniform start-up and shutdown water regime for WWER-440 Units. Therefore, special attention was paid to developing of a start-up water regime, which will be applied for the outages as early as 2008. The summarised recommendations for water regime modification will be subject to international expert review in 2008, and the modifications judged to be implemented will be finalised after the review.

Hispidin and related herbal compounds from Alpinia zerumbet inhibit both PAK1-dependent melanogenesis in melanocytes and reactive oxygen species (ROS) production in adipocytes.

PubMed

Be Tu, Pham Thi; Chompoo, Jamnian; Tawata, Shinkichi

2015-06-01

Recently several compounds from Okinawa plants including Alpinia zerumbet (alpinia) were shown to inhibit directly the oncogenic/ageing kinase PAK1 (p21-activated kinase 1). Furthermore, it was recently revealed that both PAK1 and PAK4 (p21-activated kinase 4) are equally essential for the melanogenesis in melanoma cells. Thus, in this study, we tested if several alpinia compounds inhibit the melanogenesis in melanoma (B16F10) cells, as well as the PAK1-dependent up-regulation of both reactive oxygen species (ROS) and nitric oxide (NO) in cultured adipocytes (3T3-L1) without any cytotoxicity. The effect of alpinia compounds on the melanogenesis was measured by both the melanin content and intracellular tyrosinase activity in melanoma cells treated with 3-isobutyl-1-methylxanthine (IBMX), a melanogenesis stimulating hormone. We found that (1E,3E,5E)-6-methoxyhexa-1,3,5-trien-1-yl)-2,5-dihydrofuran (MTD), 5,6-dehydrokawain (DK), labdadiene, hispidin and dihydro-5,6-dehydrokawain (DDK) at 50 μg/mL reduced the melanin content by 63-79%. The MTD, DK and hispidin, at 50 μg/mL, inhibited tyrosinase activity by 70-83% in melanoma cells. Among these compounds, labdadiene, MTD, (E)-2,2,3,3-Tetramethyl8-methylene-7-(oct-6-en-1-yl)octahydro-1H-quinolizine (TMOQ) and hispidin strongly inhibited the ROS production. Hispidin, labdadiene and MTD at 20 μg/mL inhibited NO production by over 70%. These findings altogether suggest that some of these alpinia compounds could be potentially useful for the prevention or treatment of hyperpigmentation and obesity.

Symptom Assessment in Knee Osteoarthritis Needs to Account for Physical Activity Level

PubMed Central

Lo, Grace H.; McAlindon, Timothy E.; Hawker, Gillian A.; Driban, Jeffrey B.; Price, Lori Lyn; Song, Jing; Eaton, Charles B.; Hochberg, Marc C.; Jackson, Rebecca D.; Kwoh, C. Kent; Nevitt, Michael C.; Dunlop, Dorothy D.

2015-01-01

Objective Pain is not always correlated with radiographic osteoarthritis (OA) severity possibly because people modify activities to manage symptoms. Measures of symptoms that consider pain in the context of activity level may therefore provide greater discrimination than pain alone. Our objective was to compare discrimination of a measure of pain alone with combined measures of pain relative to physical activity across radiographic OA levels. Methods This is a cross-sectional study of the Osteoarthritis Initiative accelerometer substudy, including those with and without knee OA. Two composite pain and activity knee symptom (PAKS) scores were calculated as Western Ontario and McMaster (WOMAC) Universities Osteoarthritis Pain Scale plus one divided by physical activity measures (step and activity counts). Symptom score discrimination across Kellgren and Lawrence (KL) grades were evaluated using histograms and quantile regression. Results 1806 participants, mean age 65.1 (9.1) years, mean BMI 28.4 (4.8) kg/m2, and 55.6% female, were included. WOMAC, but not PAKS scores, exhibited a floor effect. Adjusted median WOMAC by KL grades 0 – 4 were 0, 0, 1, 1, and 3 respectively. Median PAKS1 and PAKS2 were 24.9, 26.0, 32.4, 46.1, 97.9, and 7.2, 7.2, 9.2, 12.9, 23.8, respectively. PAKS scores had more statistically significant comparisons between KL grades compared with WOMAC. Conclusions Symptom assessments incorporating pain and physical activity did not exhibit a floor effect and were better able to discriminate radiographic severity than pain alone, particularly in milder disease. Pain in the context of physical activity level should be used to assess knee OA symptoms. PMID:26407008

Sensitivity, specificity, and confounding factors of novel serological tests used for the rapid diagnosis of bovine tuberculosis in farmed red deer (Cervus elaphus).

PubMed

Buddle, Bryce M; Wilson, Tania; Denis, Michel; Greenwald, Rena; Esfandiari, Javan; Lyashchenko, Konstantin P; Liggett, Simon; Mackintosh, Colin G

2010-04-01

In this study, novel serological tests were used to detect tuberculosis (TB) in groups of farmed red deer (Cervus elaphus) varying in disease status or possible confounding factors. Groups of deer naturally or experimentally infected with Mycobacterium bovis and animals vaccinated against paratuberculosis were studied, as were uninfected animals and animals naturally or experimentally infected with Mycobacterium avium subsp. paratuberculosis. Sera were assayed using two rapid lateral-flow tests, Chembio's CervidTB STAT-PAK and DPP VetTB tests, and results were compared to those from tuberculin skin tests. Both serological tests had a high sensitivity, but specificity was adversely affected after animals had received a vaccine against paratuberculosis and were subsequently skin tested. The specificity of the DPP VetTB test was higher than that of the CervidTB STAT-PAK test, with natural infection with M. avium subsp. paratuberculosis adversely affecting the specificity of only the CervidTB STAT-PAK test. The sera from M. avium subsp. paratuberculosis-infected deer that produced false-positive reactions in the CervidTB STAT-PAK test were retested with a multiantigen print immunoassay (MAPIA), and some of these sera were shown to react with the MPB83 antigen. Combining the results from the serological tests and the skin tests showed only a slight increase in the sensitivity of detection of M. bovis-infected animals. It is concluded that both the CervidTB STAT-PAK and DPP VetTB tests offer rapid, convenient, and easy detection of bovine tuberculosis in deer, albeit with significant interference from paratuberculosis vaccination status and subsequent skin testing. The latter finding illustrates one of the limitations of currently available vaccines against paratuberculosis.

Telemetry Attributes Transfer Standard (TMATS) Handbook

DTIC Science & Technology

2013-02-01

interpretations of TMATS files so that all parties will benefit from its usage. The RCC gives special acknowledgement for production of this document to the...denoted with a *R-CH10* shall indicate the minimum required RCC IRIG 106, Chapter 10 TMATS Setup Record attributes. b. Code names denoted with a * RO ...supported data channel dependent. c. Code names denoted with a * RO -PAK shall indicate the RCC IRIG 106, Chapter 10 TMATS Setup Record attributes

Anthropomorphic Interfaces on Automation Trust, Dependence, and Performance inYounger and Older Adults

DTIC Science & Technology

2015-10-26

www.diabetes.org/diabetes-basics/statistics/ ? loc ¼db-slabnav Banaji, M., and C. D. Hardin. 1996. “Automatic Stereotyping.” Psychological Science 7 (3...May 2014 Accepted by: Dr. Richard Pak, Committee Chair Dr. Leo Gugerty Dr. Christopher Pagano DISTRIBUTION A: Distribution...years. My committee members, Dr. Leo Gugerty and Dr. Christopher Pagano, made my committee complete and I am grateful for their willingness to serve

Ever Stop to Think Man's Survival Is Dependent on His Use of Food Resources? [Project ECOLogy ELE Pak, Nelson Pak].

ERIC Educational Resources Information Center

Nelson, Judy

This is one of a series of units for environmental education developed by the Highline Public Schools. This unit is designed for senior high school students who have a basic knowledge of nutrition and some experience in menu planning. The five lessons provide experiences in selecting, preparing, and storing foods to attain maximum nutrition with a…

High adsorption performance for As(III) and As(V) onto novel aluminum-enriched biochar derived from abandoned Tetra Paks.

PubMed

Ding, Zhuhong; Xu, Xuebin; Phan, Thihongnhung; Hu, Xin; Nie, Guangze

2018-06-12

In order to develop promising sorbents for value-added application of solid wastes, low-cost aluminum-enriched biochar was prepared from abandoned Tetra Pak used to hold milks, a paper-polyethylence-Al foil laminated package box, after acid pretreatment and subsequent slow pyrolysis under an oxygen-limited environment at 600 °C. The basic physicochemical properties of the resultant biochar were characterized and the sorption performance of aqueous As(III) and As(V) was investigated via batch and column sorption experiments. Carbon (49.1%), Ca (7.41%) and Al (13.5%) were the most abundant elements in the resultant biochar; and the specific surface area and the pH value at the point of zero charge (pHPZC) were 174 m 2  g -1 and 9.3, respectively. Batch sorption showed excellent sorption performance for both As(III) (24.2 mg g -1 ) and As(V) (33.2 mg g -1 ) and experimental data were fitted well with Langmuir model for the sorption isotherms and pseudo-second order kinetic model for the sorption kinetics. The residual concentrations of As(V) after sorption were below the limited value of arsenic in WHO Guidelines for Drinking water Quality (0.01 mg L -1 ) even if coexistence of PO 4 3- . Column sorption confirmed the high sorption performance for As(III) and As(V). So the slow pyrolysis of abandoned Tetra Paks as low-cost and value-added sorbents is a sustainable strategy for solid waste disposal and wastewater treatment. Copyright © 2018 Elsevier Ltd. All rights reserved.

WAVE2 targeting to phosphatidylinositol 3,4,5-triphosphate mediated by insulin receptor substrate p53 through a complex with WAVE2.

PubMed

Takahashi, Kazuhide; Suzuki, Katsuo

2010-11-01

Membrane targeting of WAVE2 along microtubules to phosphatidylinositol 3,4,5-triphosphate (PIP(3)) in response to an extracellular stimulus requires Rac1, Pak1, stathmin, and EB1. However, whether WAVE2 interacts directly with PIP(3) or not remains unclear. We demonstrate that insulin-like growth factor I (IGF-I) induces WAVE2 membrane targeting, accompanied by phosphorylation of Pak1 at serine 199/204 (Ser199/204) and stathmin at Ser38 in the inner cytoplasmic region. This is spatially independent of the membrane region where the IGF-I receptor (IGF-IR) is locally activated. WAVE2, phosphorylated Pak1, and phosphorylated stathmin located at the microtubule ends began to accumulate at the leading edge of cells in close proximity to PIP(3) that was produced in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. The PIP(3)-beads binding assay revealed that insulin receptor substrate p53 (IRSp53) and actin rather than WAVE2 bound to PIP(3). IRSp53 constitutively associated with WAVE2 and these two proteins colocalized with PIP(3) at the leading edge after IGF-I stimulation. Suppression of IRSp53 expression by two independent small interfering RNAs (siRNAs) completely inhibited IGF-I-induced membrane targeting and local accumulation of WAVE2 at the leading edge of cells. We propose that IRSp53 constitutively forms a complex with WAVE2 and is crucial for membrane targeting followed by local accumulation of WAVE2 at the leading edge of cells through linking WAVE2 to PIP(3) that is produced near locally activated IGF-IR in response to IGF-I. Copyright (c) 2010 Elsevier Inc. All rights reserved.

C18 solid-phase isolation and high-performance liquid chromatography/ultraviolet diode array determination of fully methoxylated flavones in citrus juices.

PubMed

Sendra, J M; Navarro, J L; Izquierdo, L

1988-09-01

A new analytical methodology for the determination of fully methoxylated flavones (FMFs) in citrus juices is described. Isolation of the FMFs is carried out by percolation of 30 mL of clarified citrus juice (to which tetramethyl-o-kaempferol is previously added as internal standard) through a C18 Sep-Pak cartridge, washing with 3 mL of water followed by 5 mL of water/acetonitrile (3:1), and selective elution of the retained FMFs with 5 mL of water/acetonitrile (9:11). Determination of the isolated FMFs is carried out by reversed-phase high-performance liquid chromatography (HPLC) and UV diode array detection (DAD). Signals at wavelengths 320, 335, and 345 nm (bandwidth 4 nm) are simultaneously acquired, stored, plotted, and integrated. The column used is a microbore (200 x 2.1-mm) Hypersil ODS 5 microns. Elution is in gradient mode, using a ternary mobile phase (water/acetonitrile/tetrahydrofuran). Column temperature is 40 degrees C. Recovery yields are nearly 100% for all the FMFs detected and identified: isosinensetin, hexamethyl-o-gossypetin, sinensetin, tetramethyl-o-isoscutellarein, hexamethyl-o-quercetagetin, nobiletin, tetramethyl-o-scutellarein, heptamethoxyflavone, and tangeretin. Chromatographic separation of the FMFs is extremely dependent upon the minor changes of the mobile phase composition and percentages, gradient rate, and temperature. The UV spectra (230 to 400 nm) of the FMFs obtained under chromatographic conditions are given. The FMFs relative response factors at 320, 335, and 345 nm and their concentrations in hand-squeezed and commercial concentrated orange and mandarin juices are tabulated. The FMF concentration differences found among samples are discussed.

Pancreas outcomes between living and deceased kidney donor in pancreas after kidney transplantation patients.

PubMed

Ventura-Aguiar, Pedro; Ferrer, Joana; Revuelta, Ignacio; Paredes, David; de Sousa-Amorim, Erika; Rovira, Jordi; Esmatjes, Enric; Garcia-Valdecasas, Juan Carlos; Campistol, Josep M; Oppenheimer, Federico; Diekmann, Fritz; Ricart, Maria José

2018-06-08

Pancreas outcomes in pancreas after kidney transplantation (PAK) patients have been reported as being inferior to those of patients who receive simultaneous pancreas and kidney transplantation (SPK). The influence of the kidney donor (i.e. living versus deceased) has never been previously addressed. We retrospectively analysed all pancreas transplants performed in a single centre since 2007 and compared the outcomes between those patients who had previously received a living-donor kidney transplant (pancreas transplantation after living-donor kidney transplantation, PAldK; n = 18) or a deceased-donor kidney transplant (pancreas transplantation after deceased-donor kidney transplantation, PAddK; n = 28), using SPK (n = 139) recipients as a reference. Pancreas survival was similar between all groups, but inferior for PAldK when including only those with a functioning graft at day 90 post-transplantation (P = 0.004). Pancreas acute rejection was significantly increased in PAldK (67%; 1.8 ± 1.4 episodes/graft) when compared with PAddK (25%) and SPK (32%) (P < 0.05) patients. In a multivariate Cox regression model including known risk factors for pancreas rejection, PAldK was the only predictor of acute rejection (hazard ratio 6.82, 95% confidence interval 1.51-30.70, P < 0.05). No association was found between donor-recipient HLA mismatches and graft rejection. Repeated HLA mismatches between kidney and pancreas donors (0 versus 1-6) did not correlate with pancreas graft rejection or survival in either PAK transplantation group (P > 0.05). Pancreas graft outcomes are worse for PAldK when compared with PAddK and SPK patients.

Development of a fractionation method for the detection and identification of oak ellagitannins in red wines.

PubMed

García-Estévez, Ignacio; Escribano-Bailón, M Teresa; Rivas-Gonzalo, Julián C; Alcalde-Eon, Cristina

2010-02-15

During maturation and ageing in oak barrels wines improve their organoleptic properties. Ellagitannins can be released from wood to the wine and be involved in oxidation reactions and seem to influence the astringency and colour properties of the wine. Nevertheless, the ellagitannins levels are lower than those of other wine constituents and, consequently, they are not easily detected. This study has developed a two-step fractionation method consisting of a solid phase extraction in C-18 Sep-Pak cartridges followed by size exclusion chromatography in hand-packed Sephadex LH-20 minicolumn for the detection of oak ellagitannins in different types of wines. An HPLC method has also been developed which allows the separation of compounds with the same m/z ratios, facilitating the ellagitannin identification by means of the mass spectrometric analyses. The main oak ellagitannins (grandinin, vescalagin, roburin E and castalagin) were isolated, detected separately and identified in a spiked wine and in three real ones, proving the usefulness of the fractionation method. Copyright 2009 Elsevier B.V. All rights reserved.

Rapid purification of staphylococcal enterotoxin B by high-pressure liquid chromatography.

PubMed Central

Strickler, M P; Neill, R J; Stone, M J; Hunt, R E; Brinkley, W; Gemski, P

1989-01-01

The Staphylococcus aureus enterotoxins represent a group of proteins that cause emesis and diarrhea in humans and other primates. We have developed a rapid two-step high-pressure liquid chromatography (HPLC) procedure for purification of staphylococcal enterotoxin B (SEB). Sterile filtrates (2.5 liters) of strain 10-275 were adsorbed directly onto a reversed-phase column (50 mm by 30 cm Delta Pak; 300 A [30 nm], 15 microns, C18). SEB was obtained by using a unique sequential gradient system. First, an aqueous ammonium acetate to acetonitrile gradient followed by an aqueous trifluoroacetic acid (TFA) wash was used to remove contaminants. A subsequent TFA to acetonitrile-TFA gradient eluted the bound SEB. Further purification was obtained by rechromatography on a cation-exchange column. From 35 to 45% of the SEB in starting filtrates was recovered. Analysis by immunoblotting of samples separated on sodium dodecyl sulfate-polyacrylamide gels indicated that HPLC-purified SEB exhibited immunological and biochemical properties similar to those of the SEB standard. Induction of an emetic response in rhesus monkeys showed that the HPLC-purified toxin also retained biological activity. Images PMID:2745678

Pak4 Is Required during Epithelial Polarity Remodeling through Regulating AJ Stability and Bazooka Retention at the ZA

PubMed Central

Walther, Rhian F.; Nunes de Almeida, Francisca; Vlassaks, Evi; Burden, Jemima J.; Pichaud, Franck

2016-01-01

Summary The ability of epithelial cells to assemble into sheets relies on their zonula adherens (ZA), a circumferential belt of adherens junction (AJ) material, which can be remodeled during development to shape organs. Here, we show that during ZA remodeling in a model neuroepithelial cell, the Cdc42 effector P21-activated kinase 4 (Pak4/Mbt) regulates AJ morphogenesis and stability through β-catenin (β-cat/Arm) phosphorylation. We find that β-catenin phosphorylation by Mbt, and associated AJ morphogenesis, is needed for the retention of the apical determinant Par3/Bazooka at the remodeling ZA. Importantly, this retention mechanism functions together with Par1-dependent lateral exclusion of Par3/Bazooka to regulate apical membrane differentiation. Our results reveal an important functional link between Pak4, AJ material morphogenesis, and polarity remodeling during organogenesis downstream of Par3. PMID:27052178

Rho Kinase (ROCK) collaborates with Pak to Regulate Actin Polymerization and Contraction in Airway Smooth Muscle.

PubMed

Zhang, Wenwu; Bhetwal, Bhupal P; Gunst, Susan J

2018-05-10

The mechanisms by which Rho kinase (ROCK) regulates airway smooth muscle contraction were determined in tracheal smooth muscle tissues. ROCK may mediate smooth muscle contraction by inhibiting myosin regulatory light chain (RLC) phosphatase. ROCK can also regulate F-actin dynamics during cell migration, and actin polymerization is critical for airway smooth muscle contraction. Our results show that ROCK does not regulate airway smooth muscle contraction by inhibiting myosin RLC phosphatase or by stimulating myosin RLC phosphorylation. We find that ROCK regulates airway smooth muscle contraction by activating the serine-threonine kinase Pak, which mediates the activation of Cdc42 and Neuronal-Wiskott-Aldrich Syndrome protein (N-WASp). N-WASP transmits signals from cdc42 to the Arp2/3 complex for the nucleation of actin filaments. These results demonstrate a novel molecular function for ROCK in the regulation of Pak and cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F-actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase inactive mutant of ROCK, ROCK-K121G, in the tissues or by treating them with the ROCK inhibitor, H-1152P. Our results show no role for ROCK in the regulation of non-muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue. We find that ROCK regulates airway smooth muscle contraction by mediating activation of the serine-threonine kinase, Pak, to promote actin polymerization. Pak catalyzes paxillin phosphorylation on Ser273 and coupling of the GIT1-βPIX-Pak signaling module to paxillin, which activates the GEF activity βPIX towards cdc42. Cdc42 is required for the activation of Neuronal Wiskott-Aldrich Syndrome protein (N-WASp), which transmits signals from cdc42 to the Arp2/3 complex for the nucleation of actin filaments. Our results demonstrate a novel molecular function for ROCK in the regulation of Pak and cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Korean Affairs Report.

DTIC Science & Technology

1986-10-20

Assails Chon’s Linking Explosion With Chongnyon (KCNA, 26 Sep 86) 31 "" 3i "" VNS on Chon Kyong-hwan Bribery Case (Yun Chong- won ; Voice of...Myong-nam; Pyongyang Domestic Service, 25 Sep 86) .. 42 VNS on People’s Rejection of Asiad, Olympics (Ko Il-chol, Min Hye -kyong; Voice of National...86) 109 INTERNATIONAL COMMENTARY United States Accused of Plotting Chemical Warfare (Pak Won -hae; MINJU CH0S0N, 3 Aug 86) 114 /7310 - c

Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

PubMed

Hough, Chris; Radu, Maria; Doré, Jules J E

2012-01-01

The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-β/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-β-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

A validated HPLC method for the determination of pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine in rat plasma and urine.

PubMed

Abu-Qare, A W; Abou-Donia, M B

2001-12-01

A method was developed for the separation and quantification of the anti-nerve agent pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), the analgesic drugs acetaminophen and acetylsalicylic acid, and the stimulant caffeine (3,7-dihydro-1,3,7-trimethyl-1-H-purine-2,6-dione) in rat plasma and urine. The compounds were extracted using C(18) Sep-Pak(R) cartridges then analyzed by high performance liquid chromatography (HPLC) with reversed phase C18 column, and UV detection at 280 nm. The compounds were separated using gradient of 1-85% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.5 ml/min in a period of 14 min. The retention times ranged from 8.8 to 11.5 min. The limits of detection were ranged between 100 and 200 ng/ml, while limits of quantitation were 150-200 ng/ml. Average percentage recovery of five spiked plasma samples were 70.9+/-9.5, 73.7+/-9.8, 88.6+/-9.3, 83.9+/-7.8, and from urine 69.1+/-8.5, 74.5+/-8.7, 85.9+/-9.8, 83.2+/-9.3, for pyridostigmine bromide, acetaminophen, acetylsalicylic acid and caffeine, respectively. The relationship between peak areas and concentration was linear over range between 100 and 1000 ng/ml. The resulting chromatograms showed no interfering peaks from endogenous plasma or urine components. This method was applied to analyze these compounds following oral administration in rats.

Determination of proenkephalin products in brain tissue by high-performance liquid chromatography and a modified bioassay procedure.

PubMed

Bailey, C; Kitchen, I

1985-06-01

A method is described for the separation of proenkephalin products using gradient high-performance liquid chromatography preceded by Sep-Pak chromatography. Samples can be assayed simply by use of a modified mouse vas deferens bioassay which is sufficiently sensitive for most applications. The preliminary Sep-Pak chromatography method excludes alpha-neoendorphin and the dynorphins and thus provides a suitable procedure for separation of prodynorphin and proenkephalin products.

Genetic and Functional Diversity of Human Immunodeficiency Virus Type 1 Subtype B Nef Primary Isolates

PubMed Central

Foster, John L.; Molina, Rene P.; Luo, Tianci; Arora, Vivek K.; Huang, Yaoxing; Ho, David D.; Garcia, J. Victor

2001-01-01

We have characterized the functional integrity of seven primary Nef isolates: five from a long-term nonprogressing human immunodeficiency virus (HIV)-infected individual and one each from two patients with AIDS. One of the seven Nefs was defective for CD4 downregulation, two others were defective for PAK-2 activation, and one Nef was defective for PAK-2 activation and major histocompatibility complex (MHC) class I downregulation. Five of the Nefs were tested and found to be functional for the enhancement of virus particle infectivity. The structural basis for each of the functional defects has been analyzed by constructing a consensus nef, followed by mutational analysis of the variant amino acid residues. Mutations A29V and F193I were deleterious to CD4 downregulation and PAK-2 activation, respectively, while S189R rendered Nef defective for both MHC class I downregulation and PAK-2 activation. A search of the literature identified HIVs from five patients with Nefs predominantly mutated at F193 and from one patient with Nefs predominantly mutated at A29. A29 is highly conserved in all HIV subtypes except for subtype E. F193 is conserved in subtype B (and possibly in the closely related subtype D), but none of the other HIV group M subtypes. Our results suggest that functional distinctions may exist between HIV subtypes. PMID:11160665

Interaction Between a Novel p21 Activated Kinase (PAK6) and Androgen Receptor in Prostate Cancer

DTIC Science & Technology

2005-02-01

Beest , M., van Es, J., 51. Easwaran, V., Pishvaian, M., Salimuddin, and Byers, S. (1999) Curr. Biol. 9, Loureiro, J., Ypma, A., Hursh, D., Jones, T...antibody was generated and used in the study. 40 Immunofluorescence. Cells were cultured in 8-well Lab Tek chambered cover slides (Nalge Nunc...C19220, Transduction Labs , Lex- To further evaluate the roles of IGF-11 and EGF in ington, KY). Proteins were detected using the ECL kit (Am- AR

The Best kept Secrets In Government.

DTIC Science & Technology

1996-09-01

Ray Jones at SeaPak see in the future? One word: " HACCP ." Ray’s not clearing a fish bone. He’s talking about the Administration’s new scientific way... HACCP instructor so I can teach our suppliers and even our competitors the new techniques." Ray explains why SeaPak welcomes a new regulation that...new system is HACCP , the same scientific methods that are making seafood safe. The government sets the goals, but gives each compa- ny the

Tuning the overlap and the cross-layer correlations in two-layer networks: Application to a susceptible-infectious-recovered model with awareness dissemination

NASA Astrophysics Data System (ADS)

Juher, David; Saldaña, Joan

2018-03-01

We study the properties of the potential overlap between two networks A ,B sharing the same set of N nodes (a two-layer network) whose respective degree distributions pA(k ) ,pB(k ) are given. Defining the overlap coefficient α as the Jaccard index, we prove that α is very close to 0 when A and B are random and independently generated. We derive an upper bound αM for the maximum overlap coefficient permitted in terms of pA(k ) , pB(k ) , and N . Then we present an algorithm based on cross rewiring of links to obtain a two-layer network with any prescribed α inside the range (0 ,αM) . A refined version of the algorithm allows us to minimize the cross-layer correlations that unavoidably appear for values of α beyond a critical overlap αc<αM . Finally, we present a very simple example of a susceptible-infectious-recovered epidemic model with information dissemination and use the algorithms to determine the impact of the overlap on the final outbreak size predicted by the model.

HIV-1 Nef interferes with host cell motility by deregulation of Cofilin.

PubMed

Stolp, Bettina; Reichman-Fried, Michal; Abraham, Libin; Pan, Xiaoyu; Giese, Simone I; Hannemann, Sebastian; Goulimari, Polyxeni; Raz, Erez; Grosse, Robert; Fackler, Oliver T

2009-08-20

HIV-1 Nef is a key factor in AIDS pathogenesis. Here, we report that Nef potently inhibits motility of fibroblasts and chemotaxis of HIV-1-infected primary human T lymphocytes toward the chemokines SDF-1alpha, CCL-19, and CCL-21 ex vivo. Furthermore, Nef inhibits guided motility of zebrafish primordial germ cells toward endogenous SDF-1a in vivo. These migration defects result from Nef-mediated inhibition of the actin remodeling normally triggered by migratory stimuli. Nef strongly induces phosphorylation of cofilin, inactivating this evolutionarily conserved actin-depolymerizing factor that promotes cell motility when unphosphorylated. Nef-dependent cofilin deregulation requires association of Nef with the cellular kinase Pak2. Disruption of Nef-Pak2 association restores the cofilin phosphorylation levels and actin remodeling that facilitate cell motility. We conclude that HIV-1 Nef alters Pak2 function, which directly or indirectly inactivates cofilin, thereby restricting migration of infected T lymphocytes as part of a strategy to optimize immune evasion and HIV-1 replication.

Performance of rapid tests and algorithms for HIV screening in Abidjan, Ivory Coast.

PubMed

Loukou, Y G; Cabran, M A; Yessé, Zinzendorf Nanga; Adouko, B M O; Lathro, S J; Agbessi-Kouassi, K B T

2014-01-01

Seven rapid diagnosis tests (RDTs) of HIV were evaluated by a panel group who collected serum samples from patients in Abidjan (HIV-1 = 203, HIV-2 = 25, HIV-dual = 25, HIV = 305). Kit performances were recorded after the reference techniques (enzyme-linked immunosorbent assay). The following RDTs showed a sensitivity of 100% and a specificity higher than 99%: Determine, Oraquick, SD Bioline, BCP, and Stat-Pak. These kits were used to establish infection screening strategies. The combination with 2 or 3 of these tests in series or parallel algorithms showed that series combinations with 2 tests (Oraquick and Bioline) and 3 tests (Determine, BCP, and Stat-Pak) gave the best performances (sensitivity, specificity, positive predictive value, and negative predictive value of 100%). However, the combination with 2 tests appeared to be more onerous than the combination with 3 tests. The combination with Determine, BCP, and Stat-Pak tests serving as a tiebreaker could be an alternative to the HIV/AIDS serological screening in Abidjan.

Heterogeneous water oxidation: surface activity versus amorphization activation in cobalt phosphate catalysts.

PubMed

González-Flores, Diego; Sánchez, Irene; Zaharieva, Ivelina; Klingan, Katharina; Heidkamp, Jonathan; Chernev, Petko; Menezes, Prashanth W; Driess, Matthias; Dau, Holger; Montero, Mavis L

2015-02-16

Is water oxidation catalyzed at the surface or within the bulk volume of solid oxide materials? This question is addressed for cobalt phosphate catalysts deposited on inert electrodes, namely crystallites of pakhomovskyite (Co3(PO4)2⋅8 H2O, Pak) and phosphate-containing Co oxide (CoCat). X-ray spectroscopy reveals that oxidizing potentials transform the crystalline Pak slowly (5-8 h) but completely into the amorphous CoCat. Electrochemical analysis supports high-TOF surface activity in Pak, whereas its amorphization results in dominating volume activity of the thereby formed CoCat material. In the directly electrodeposited CoCat, volume catalysis prevails, but not at very low levels of the amorphous material, implying high-TOF catalysis at surface sites. A complete picture of heterogeneous water oxidation requires insight in catalysis at the electrolyte-exposed "outer surface", within a hydrated, amorphous volume phase, and modes and kinetics of restructuring upon operation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pak4 Is Required during Epithelial Polarity Remodeling through Regulating AJ Stability and Bazooka Retention at the ZA.

PubMed

Walther, Rhian F; Nunes de Almeida, Francisca; Vlassaks, Evi; Burden, Jemima J; Pichaud, Franck

2016-04-05

The ability of epithelial cells to assemble into sheets relies on their zonula adherens (ZA), a circumferential belt of adherens junction (AJ) material, which can be remodeled during development to shape organs. Here, we show that during ZA remodeling in a model neuroepithelial cell, the Cdc42 effector P21-activated kinase 4 (Pak4/Mbt) regulates AJ morphogenesis and stability through β-catenin (β-cat/Arm) phosphorylation. We find that β-catenin phosphorylation by Mbt, and associated AJ morphogenesis, is needed for the retention of the apical determinant Par3/Bazooka at the remodeling ZA. Importantly, this retention mechanism functions together with Par1-dependent lateral exclusion of Par3/Bazooka to regulate apical membrane differentiation. Our results reveal an important functional link between Pak4, AJ material morphogenesis, and polarity remodeling during organogenesis downstream of Par3. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Regulation of Stat5 by FAK and PAK1 in Oncogenic FLT3 and KIT driven Leukemogenesis

PubMed Central

Chatterjee, Anindya; Ghosh, Joydeep; Ramdas, Baskar; Mali, Raghuveer Singh; Martin, Holly; Kobayashi, Michihiro; Vemula, Sasidhar; Canela, Victor H.; Waskow, Emily R.; Visconte, Valeria; Tiu, Ramon V.; Smith, Catherine C.; Shah, Neil; Bunting, Kevin D.; Boswell, H. Scott; Liu, Yan; Chan, Rebecca J.; Kapur, Reuben

2015-01-01

SUMMARY Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPN) and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK), whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis. PMID:25456130

Glutamine nitrogen and ammonium nitrogen supplied as a nitrogen source is not converted into nitrate nitrogen of plant tissues of hydroponically grown pak-choi (Brassica chinensis L.).

PubMed

Wang, H-J; Wu, L-H; Tao, Q-N; Miller, D D; Welch, R M

2009-03-01

Many vegetables, especially leafy vegetables, accumulate NO(-) (3)-N in their edible portions. High nitrate levels in vegetables constitute a health hazard, such as cancers and blue baby syndrome. The aim of this study was to determine if (1) ammonium nitrogen (NH(+) (4)-N) and glutamine-nitrogen (Gln-N) absorbed by plant roots is converted into nitrate-nitrogen of pak-choi (Brassica chinensis L.) tissues, and (2) if nitrate-nitrogen (NO(-) (3)-N) accumulation and concentration of pak-choi tissues linearly increase with increasing NO(-) (3)-N supply when grown in nutrient solution. In experiment 1, 4 different nitrogen treatments (no nitrogen, NH(+) (4)-N, Gln-N, and NO(-) (3)-N) with equal total N concentrations in treatments with added N were applied under sterile nutrient medium culture conditions. In experiment 2, 5 concentrations of N (from 0 to 48 mM), supplied as NO(-) (3)-N in the nutrient solution, were tested. The results showed that Gln-N and NH(+) (4)-N added to the nutrient media were not converted into nitrate-nitrogen of plant tissues. Also, NO(-) (3)-N accumulation in the pak-choi tissues was the highest when plants were supplied 24 mM NO(-) (3)-N in the media. The NO(-) (3)-N concentration in plant tissues was quadratically correlated to the NO(-) (3)-N concentration supplied in the nutrient solution.

Application of TL dosemeters for dose distribution measurements at high temperatures in nuclear reactors.

PubMed

Osvay, M; Deme, S

2006-01-01

Al2O3:Mg,Y ceramic thermoluminescence dosemeters were developed at the Institute of Isotopes for high dose applications at room temperatures. The glow curve of Al2O3:Mg,Y exhibits two peaks--one at 250 degrees C (I) and another peak at approximately 400 degrees C (II). In order to extend the application of these dosemeters to high temperatures, the effect of irradiation temperature was investigated using temperature controlled heating system during high dose irradiation at various temperatures (20-100 degrees C). The new calibration and measuring method has been successfully applied for dose mapping within the hermetic zone of the Paks Nuclear Power Plant even at high temperature parts of blocks.

Purification of a myotoxin from the toadfish Thalassophryne maculosa (Günter) venom.

PubMed

Sosa-Rosales, J I; D'Suze, G; Salazar, V; Fox, J; Sevcik, C

2005-02-01

Venom was milked by gently pressing the base of the opercular and dorsal fin spines. Three fractions were obtained by molecular exclusion high pressure liquid chromatography (HPLC) (Protein Pak 125SW, Millipore Corporation) column, but only the last one with 22.7 min retention time (rt) was biological active (TmPP-22.7). This fraction was rechromatographed on reversed phase HPLC chlorobutylsilane columns (C4, Vydac) nine fractions were obtained, but only one (TmC4-47.2) with 47.2 min rt was biologically active. MALD-TOF mass analysis was carried out on two samples of TmC-47.2 and the results were 15,161.36 and 15,154.70 a.m.u., respectively. Raw venom (1040 microg/ml) depolarised frog (Hyla crepitans) muscle irreversibly from -85 (-88, -81) mV (n=20, median and its 95% CI) to -18 (-24, -15) mV (n=24). The biological activity in TmPP-22.7 (38 microg/ml), which depolarised muscle fibres from -79 (-82, -76) mV (n=20) to -63 (-69 -57) mV (n=24). The depolarising fraction was TmC4-47.2 (50 microg/ml) which depolarised muscles from -87 (91, -82) mV (n=33) to -63 (-76 -51) mV (n=53); the depolarising effect at this concentration was completely reversed on washing with normal saline for 2 h. Muscles treated with 1 microM tetrodotoxin (TTX) were depolarised from -80 (-85, -72) mV (n=49) to -44 (-56, -31) mV (n=44) when 100 microg/ml TmC4-47.2 were applied with TTX; washing 130 min with 1 microM TTX repolarised to -59 (-69, -50) mV (n=25). We also present evidence that TmC4-47.2 induces myonecrosis in mice.

Simultaneous multiresidue determination of metronidazole and spiramycin in fish muscle using high performance liquid chromatography with UV detection.

PubMed

Maher, Hadir M; Youssef, Rasha M; Khalil, Riad H; El-Bahr, Sabry M

2008-12-15

An efficient multiresidue method for the simultaneous determination of metronidazole (MET) and spiramycin (SPY) in tilapia fish muscle, based on high performance liquid chromatography with UV detection (HPLC-UV), has been developed. The drugs were extracted with 0.2% orthophosphoric acid-methanol (6:4), and the extracts were cleaned up on a solid phase extraction cartridge, C18 Sep-Pak light column. The LC separation was performed on a RP stainless-steel C-18 analytical column (150 mm x 4.6 mm, 5 microm) with a gradient elution system of 0.05 M phosphate buffer adjusted to pH 2.4-acetonitrile as the mobile phase at the flow rate of 1.0 ml min(-1). A wavelength programming was applied for the UV detection of the analytes. The method not only enabled the determination of the parent drugs, MET and SPY, but also permitted the determination of their metabolites, hydroxymetronidazole (HMET) and neospiramycin (NSPY). The calibration graphs for each drug were rectilinear in the range of 0.005-1.000 microg g(-1) for MET and HMET and 0.025-1.000 microg g(-1) for SPY and NSPY. With this method, the cited drugs with their metabolites were determined in fortified fish muscle tissues at levels of 0.025, 0.1 and 1.0 microg g(-1) with good accuracy and precision. LOD and LOQ obtained for each drug were as follows: 0.002 and 0.005 microg g(-1) for MET and HMET and 0.005 and 0.025 microg g(-1) for SPY and NSPY. Utilization of the method to successfully analyze tilapia fish muscle samples incurred with MET and SPY was described.

Quantification of penicillin G during labor and delivery by capillary electrophoresis.

PubMed

Thomas, Andrea; Ukpoma, Omon K; Inman, Jennifer A; Kaul, Anil K; Beeson, James H; Roberts, Kenneth P

2008-04-24

In this study, a capillary electrophoresis (CE) method was developed as a means to measure levels of penicillin G (PCN G) in Group B Streptococcus (GBS) positive pregnant women during labor and delivery. Volunteers for this developmental study were administered five million units of PCN G at the onset of labor. Urine, blood, and amniotic fluid samples were collected during labor and post delivery. Samples were semi-purified by solid-phase extraction (SPE) using Waters tC18 SepPak 3cc cartridges with a sodium phosphate/methanol step gradient for elution. Capillary electrophoresis or reversed-phase high-performance liquid chromatography (RP-HPLC) with diode-array absorbance detection were used to separate the samples in less than 30 min. Quantification was accomplished by establishing a calibration curve with a linear dynamic range. The tC18 SPE methodology provided substantial sample clean-up with high recovery yields of PCN G ( approximately 90%). It was found that SPE was critical for maintaining the integrity of the separation column when using RP-HPLC, but was not necessary for sample analysis by CE where no stationary phase is present. Quantification results ranged from millimolar concentrations of PCN G in maternal urine to micromolar concentrations in amniotic fluid. Serum and cord blood levels of PCN G were below quantification limits, which is likely due to the prolonged delay in sample collection after antibiotic administration. These results show that CE can serve as a simple and effective means to characterize the pharmacokinetic distribution of PCN G from mother to unborn fetus during labor and delivery. It is anticipated that similar methodologies have the potential to provide a quick, simple, and cost-effective means of monitoring the clinical efficacy of PCN G and other drugs during pregnancy.

In vivo biosynthesis of L-(/sup 35/S)Cys-arginine vasopressin, -oxytocin, and -somatostatin: rapid estimation using reversed phase high pressure liquid chromatography. [Rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Franco-Bourland, R.E.; Fernstrom, J.D.

1981-01-01

L(/sup 35/S)Cys-arginine vasopressin, -oxytocin, and -somatostatin were purified from hypothalami and neurohypophyses 4 h after rats received L(/sup 35/S)Cys via the third ventricle. After acetic acid extraction, Sephadex G-25 filtration, and chemoadsorption to C18-silica (Sep-Pak cartridges), the labeled peptides were rapidly separated by gradient elution, reversed phase, high pressure liquid chromatography (HPLC). The identity and isotopic purity of the labeled peptides were determined by several reversed phase HPLC procedures in conjunction with chemical modification. The labeled peptide fractions were at least 50% radiochemically pure. Using this HPLC isolation procedure, incorporation of L-(/sup 35/S)Cys into each peptide was determined in hydratedmore » and dehydrated rats. Label incorporation into arginine vasopressin and oxytocin in the hypothalamus and the neurohypophysis of dehydrated rats was 2-3 times greater than that in hydrated rats. Incorporation of label into hypothalamic and neurohypophyseal somatostatin was unaffected by the hydration state of the animal. This procedure thus provides a very rapid, but sensitive, set of techniques for studying the control of small peptide biosynthesis in the brain.« less

Nontransmission of deoxynivalenol (vomitoxin) to milk following oral administration to dairy cows.

PubMed

Prelusky, D B; Trenholm, H L; Lawrence, G A; Scott, P M

1984-10-01

The absorption of deoxynivalenol (DON; vomitoxin), a trichothecene mycotoxin produced by Fusarium species, was studied in the dairy cow. Serum and milk DON levels were quantitated following a single oral dose of 920 mg DON to each of two lactating cows of similar weight. Maximum blood levels for the two animals following DON administration were 200 and 90 ng/ml serum, occurring at times 4.7 and 3.5 hr, respectively. By 24 hr after dosing only trace levels (less than 2 ng/ml) were still detectable. DON in its conjugated form accounted for 24-46% of the total levels present in serum. Free and conjugated DON were also present in cow's milk, but only extremely low amounts (less than 4 ng/ml) were detected. Detection of DON was carried out utilizing Sep-Pak C18 extraction cartridges for isolation, with additional purification of the sample achieved by passing the extract through a short charcoal/alumina column. The extract was then reacted with N-heptafluorobutyrylimidazole prior to quantitation of the resulting DON-tris-heptafluorobutyrate derivative by combined gas chromatography-quadrupole mass spectrometry, using multiple selected ion monitoring. Detection limits were as low as 1 ng/ml (1 ppb).

Optimized determination method for trans-10-hydroxy-2-decenoic acid content in royal jelly by high-performance liquid chromatography with an internal standard.

PubMed

Zhou, Jinhui; Xue, Xiaofeng; Li, Yi; Zhang, Jinzhen; Zhao, Jing

2007-01-01

An optimized reversed-phase high-performance liquid chromatography method was developed to detect the trans-10-hydroxy-2-decenoic acid (10-HDA) content in royal jelly cream and lyophilized powder. The sample was extracted using absolute ethanol. Chromatographic separation of 10-HDA and methyl 4-hydroxybenzoate as the internal standard was performed on a Nova-pak C18 column. The average recoveries were 95.0-99.2% (n = 5) with relative standard deviation (RSD) values of 1.3-2.1% for royal jelly cream and 98.0-100.0% (n = 5) with RSD values of 1.6-3.0% for lyophilized powder, respectively. The limits of detection and quantitation were 0.5 and 1.5 mg/kg, respectively, for both royal jelly cream and lyophilized powder. The method was validated for the determination of practical royal jelly products. The concentration of 10-HDA ranged from 1.26 to 2.21% for pure royal jelly cream samples and 3.01 to 6.19% for royal jelly lyophilized powder samples. For 30 royal jelly products, the 10-HDA content varied from not detectable to 0.98%.

Molecular pathways: targeting RAC-p21-activated serine-threonine kinase signaling in RAS-driven cancers.

PubMed

Baker, Nicole M; Yee Chow, Hoi; Chernoff, Jonathan; Der, Channing J

2014-09-15

Cancers driven by oncogenic Ras proteins encompass some of the most deadly human cancer types, and there is a pressing need to develop therapies for these diseases. Although recent studies suggest that mutant Ras proteins may yet be druggable, the most promising and advanced efforts involve inhibitors of Ras effector signaling. Most efforts to target Ras signaling have been aimed at the ERK mitogen-activated protein kinase and the phosphoinositide 3-kinase signaling networks. However, to date, no inhibitors of these Ras effector pathways have been effective against RAS-mutant cancers. This ineffectiveness is due, in part, to the involvement of additional effectors in Ras-dependent cancer growth, such as the Rac small GTPase and the p21-activated serine-threonine kinases (PAK). PAK proteins are involved in many survival, cell motility, and proliferative pathways in the cell and may present a viable new target in Ras-driven cancers. In this review, we address the role and therapeutic potential of Rac and group I PAK proteins in driving mutant Ras cancers. ©2014 American Association for Cancer Research.

Inhibition of Pseudomonas aeruginosa biofilm formation by 2,2'-bipyridyl, lipoic, kojic and picolinic acids.

PubMed

Çevik, Kübra; Ulusoy, Seyhan

2015-08-01

The inhibitory effects of iron chelators, and FeCl3 chelation on biofilm formation and swarming motility were investigated against an opportunistic human pathogen Pseudomonas aeruginosa. The inhibitory activity of 2,2'-bipyridyl, lipoic acid, kojic acid and picolinic acid on biofilm formation of P. aeruginosa strain PAO1 and three clinical isolates (P. aeruginosa PAK01, P. aeruginosa PAK02 and P. aeruginosa PAK03) were investigated, based on crystal violet assay, and swarming motility test. The kojic, lipoic and picolinic acid inhibited biofilm formation by 5-33% in all tested P. aeruginosa isolates. When chelated iron was added, biofilm inhibition rates were determined to be 39-57%. Among the tested chelators against P. aeruginosa, lipoic acid (84%) and kojic acid (68%) presented the highest inhibition of swarming motility. This is the first study to report the inhibitory effect of lipoic acid on biofilm formation and swarming motility of P. aeruginosa. It is considered that lipoic and picolinic acids can serve as alternatives for the treatment of the P. aeruginosa infections by inhibiting biofilm formation.

Regulation of Stat5 by FAK and PAK1 in Oncogenic FLT3- and KIT-Driven Leukemogenesis.

PubMed

Chatterjee, Anindya; Ghosh, Joydeep; Ramdas, Baskar; Mali, Raghuveer Singh; Martin, Holly; Kobayashi, Michihiro; Vemula, Sasidhar; Canela, Victor H; Waskow, Emily R; Visconte, Valeria; Tiu, Ramon V; Smith, Catherine C; Shah, Neil; Bunting, Kevin D; Boswell, H Scott; Liu, Yan; Chan, Rebecca J; Kapur, Reuben

2014-11-20

Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs), and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription, is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK) whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Structural and Functional Studies Indicate That the EPEC Effector, EspG, Directly Binds p21-Activated Kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Germane, Katherine L.; Spiller, Benjamin W.

2011-09-20

Bacterial pathogens secrete effectors into their hosts that subvert host defenses and redirect host processes. EspG is a type three secretion effector with a disputed function that is found in enteropathogenic Escherichia coli. Here we show that EspG is structurally similar to VirA, a Shigella virulence factor; EspG has a large, conserved pocket on its surface; EspG binds directly to the amino-terminal inhibitory domain of human p21-activated kinase (PAK); and mutations to conserved residues in the surface pocket disrupt the interaction with PAK.

Identification of pilin pools in the membranes of Pseudomonas aeruginosa.

PubMed Central

Watts, T H; Worobec, E A; Paranchych, W

1982-01-01

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain. Images PMID:6813311

Direct-Acting Antivirals in Chronic Hepatitis C Genotype 4 Infection in Community Care Setting.

PubMed

Gayam, Vijay; Khalid, Mazin; Mandal, Amrendra Kumar; Hussain, Muhammad Rajib; Mukhtar, Osama; Gill, Arshpal; Garlapati, Pavani; Shrestha, Binav; Guss, Debra; Sherigar, Jagannath; Mansour, Mohammed; Mohanty, Smruti

2018-04-01

Limited data exists comparing the safety, tolerability, and efficacy of direct-acting antivirals (DAAs) in patients with chronic hepatitis C genotype 4 (HCV GT-4) in the community practice setting. We aim to evaluate the treatment response of DAAs in these patients. All the HCV GT-4 patients treated with DAAs between January 2014 and October 2017 in a community clinic setting were retrospectively analyzed. Pretreatment baseline patient characteristics, treatment efficacy with sustained virologic response (SVR) at 12 weeks post treatment (SVR12), and adverse reactions were assessed. Fifty-two patients of Middle Eastern (primarily Egyptian) descent were included in the study. Thirty-two patients were treated with ledipasvir/sofosbuvir (Harvoni ® ) ± ribavirin, 12 patients were treated with ombitasvir/paritaprevir/ritonavir/dasabuvir (ViekiraPak ® ) ± ribavirin, and eight patients were treated with sofosbuvir/Velpatasvir (Epclusa ® ). Ten patients (19.2%) had compensated cirrhosis. Overall, SVR at 12 weeks was achieved in 94% in patients who received one of the three DAA regimens (93.8% in Harvoni ® group, 91.7 % in ViekiraPak ® group and 100% in Epclusa ® group). Prior treatment status and type of regimen used in the presence of compensated cirrhosis had no statistical significance on overall SVR achievement (P value = 0.442 and P value = 0.091, respectively). The most common adverse effect was fatigue (27%). In the real-world setting, DAAs are effective and well tolerated in patients with chronic HCV GT-4 infection with a high overall SVR rate of 94%. Large-scale studies are needed to further assess this SVR in these groups.

Effect of selenium-enriched organic material amendment on selenium fraction transformation and bioavailability in soil.

PubMed

Wang, Dan; Dinh, Quang Toan; Anh Thu, Tran Thi; Zhou, Fei; Yang, Wenxiao; Wang, Mengke; Song, Weiwei; Liang, Dongli

2018-05-01

To exploit the plant byproducts from selenium (Se) biofortification and reduce environmental risk of inorganic Se fertilizer, pot experiment was conducted in this study. The effects of Se-enriched wheat (Triticum aestivum L.) straw (WS + Se) and pak choi (Brassica chinensis L.) (P + Se) amendment on organo-selenium speciation transformation in soil and its bioavailability was evaluated by pak choi uptake. The Se contents of the cultivated pak choi in treatments amended with the same amount of Se-enriched wheat straw and pak choi were 1.7 and 9.7 times in the shoots and 2.3 and 6.3 times in the roots compared with control treatment. Soil respiration rate was significantly increased after all organic material amendment in soil (p < 0.05), which accelerated the mineralization of organic materials and thus resulted in soluble Se (SOL-Se), exchangeable Se (EX-Se), and fulvic acid-bound Se (FA-Se) fraction increasing by 25.2-29.2%, 9-13.8%, and 4.92-8.28%, respectively. In addition, both Pearson correlation and cluster analysis showed that EX-Se and FA-Se were better indicators for soil Se availability in organic material amendment soils. The Marquardt-Levenberg Model well described the dynamic kinetics of FA-Se content after Se-enriched organic material amendment in soil mainly because of the mineralization of organic carbon and organo-selenium. The utilization of Se in P + Se treatment was significantly higher than those in WS + Se treatment because of the different mineralization rates and the amount of FA-Se in soil. Se-enriched organic materials amendment can not only increase the availability of selenium in soil but also avoid the waste of valuable Se source. Copyright © 2018 Elsevier Ltd. All rights reserved.

Complex two-component signaling regulates the general stress response in Alphaproteobacteria.

PubMed

Kaczmarczyk, Andreas; Hochstrasser, Ramon; Vorholt, Julia A; Francez-Charlot, Anne

2014-12-02

The general stress response (GSR) in Alphaproteobacteria was recently shown to be controlled by a partner-switching mechanism that is triggered by phosphorylation of the response regulator PhyR. Activation of PhyR ultimately results in release of the alternative extracytoplasmic function sigma factor σ(EcfG), which redirects transcription toward the GSR. Little is known about the signal transduction pathway(s) controlling PhyR phosphorylation. Here, we identified the single-domain response regulator (SDRR) SdrG and seven histidine kinases, PakA to PakG, belonging to the HWE/HisKA2 family as positive modulators of the GSR in Sphingomonas melonis Fr1. Phenotypic analyses, epistasis experiments, and in vitro phosphorylation assays indicate that Paks directly phosphorylate PhyR and SdrG, and that SdrG acts upstream of or in concert with PhyR, modulating its activity in a nonlinear pathway. Furthermore, we found that additional SDRRs negatively affect the GSR in a way that strictly requires PhyR and SdrG. Finally, analysis of GSR activation by thermal, osmotic, and oxidative stress indicates that Paks display different degrees of redundancy and that a specific kinase can sense multiple stresses, suggesting that the GSR senses a particular condition as a combination of, rather than individual, molecular cues. This study thus establishes the alphaproteobacterial GSR as a complex and interlinked network of two-component systems, in which multiple histidine kinases converge to PhyR, the phosphorylation of which is, in addition, subject to regulation by several SDRRs. Our finding that most HWE/HisKA2 kinases contribute to the GSR in S. melonis Fr1 opens the possibility that this notion might also be true for other Alphaproteobacteria.

Inhibition of Pseudomonas aeruginosa biofilm formation by 2,2’-bipyridyl, lipoic, kojic and picolinic acids

PubMed Central

Çevik, Kübra; Ulusoy, Seyhan

2015-01-01

Objective(s): The inhibitory effects of iron chelators, and FeCl3 chelation on biofilm formation and swarming motility were investigated against an opportunistic human pathogen Pseudomonas aeruginosa. Materials and Methods: The inhibitory activity of 2,2’-bipyridyl, lipoic acid, kojic acid and picolinic acid on biofilm formation of P. aeruginosa strain PAO1 and three clinical isolates (P. aeruginosa PAK01, P. aeruginosa PAK02 and P. aeruginosa PAK03) were investigated, based on crystal violet assay, and swarming motility test. Results: The kojic, lipoic and picolinic acid inhibited biofilm formation by 5-33% in all tested P. aeruginosa isolates. When chelated iron was added, biofilm inhibition rates were determined to be 39-57%. Among the tested chelators against P. aeruginosa, lipoic acid (84%) and kojic acid (68%) presented the highest inhibition of swarming motility. This is the first study to report the inhibitory effect of lipoic acid on biofilm formation and swarming motility of P. aeruginosa. Conclusion: It is considered that lipoic and picolinic acids can serve as alternatives for the treatment of the P. aeruginosa infections by inhibiting biofilm formation. PMID:26557964

RhoJ Regulates Melanoma Chemoresistance by Suppressing Pathways that Sense DNA Damage

PubMed Central

Ho, Hsiang; Aruri, Jayavani; Kapadia, Rubina; Mehr, Hootan; White, Michael A.; Ganesan, Anand K.

2012-01-01

Melanomas resist conventional chemotherapeutics in part through intrinsic disrespect of apoptotic checkpoint activation. In this study, using an unbiased genome-wide RNAi screen we identified RhoJ and its effector Pak1, as key modulators of melanoma cell sensitivity to DNA damage. We find that RhoJ activates Pak1 in response to drug-induced DNA damage, which then uncouples ATR from its downstream effectors, ultimately resulting in a blunted DNA damage response (DDR). In addition, ATR suppression leads to the decreased phosphorylation of ATF2, and consequent increased expression of the melanocyte survival gene Sox10 resulting in a higher DDR threshold required to engage melanoma cell death. In the setting of normal melanocyte behavior, this regulatory relationship may facilitate appropriate epidermal melanization in response to UV-induced DNA damage. However, pathological pathway activation during oncogenic transformation produces a tumor that is intrinsically resistant to chemotherapy and has the propensity to accumulate additional mutations. These findings identify DNA damage agents and pharmacological inhibitors of RhoJ/PAK1 as novel synergistic agents that can be used to treat melanomas that are resistant to conventional chemotherapies. PMID:22971344

Determination of blood cyanide by HPLC-MS.

PubMed

Tracqui, A; Raul, J S; Géraut, A; Berthelon, L; Ludes, B

2002-04-01

An original high-performance liquid chromatographic-mass spectrometric (HPLC-MS) procedure was developed for the determination of cyanide (CN) in whole blood. After the addition of K13C15N as internal standard, blood was placed in a microdiffusion device, the inner well of which was filled with a mixture of taurine (50mM in water)/naphthalene-2,3-dicarboxaldehyde (NDA, 10mM in methanol)/methanol/ concentrated (approximately 20%) ammonia solution (25:25:45:5, v/v). Concentrated H2SO4 was added to the blood sample, and the microdiffusion chamber was sealed. After 30 min of gentle agitation, 2 microL of the contents of the inner vial were pipetted and directly injected onto a NovaPak C18 HPLC column. Separation was performed by a gradient of acetonitrile in 2mM NH4COOH, pH 3.0 buffer (35-80% in 10 min). Detection was done with a Perkin-Elmer Sciex API-100 mass analyzer with an ionspray interface, operated in the negative ionization mode. MS data were collected as either TIC or SIM at m/z (299 + 191) and (301 + 193) for the derivatives formed with CN and 13C15N, respectively. Inspired by previous works dealing with the complexation of CN by NDA + taurine to form a 1-cyano [f] benzoisoindole derivative analyzed by HPLC-fluorimetry, this method appears simple, rapid, and extremely specific. Limits of detection and quantitation for blood CN are 5 and 15 ng/mL, respectively. The use of 13C15N as internal standard allows the quantitation of CN with elegance and accuracy in comparison with previously reported methods.

A Stability-Indicating HPLC Method for the Determination of Memantine Hydrochloride in Dosage Forms through Derivatization with 1-Fluoro-2,4-dinitrobenzene

PubMed Central

Jalalizadeh, Hassan; Raei, Mahdi; Tafti, Razieh Fallah; Farsam, Hassan; Kebriaeezadeh, Abbas; Souri, Effat

2014-01-01

Memantine is chemically a tricyclic amine and is used for Parkinson’s disease and movement disorders. Although several HPLC methods with different derivatization reagents have been developed for the determination of memantine in biological fluids, there are some complications which limit the use of these methods in routine analysis of memantine in in vitro tests. We established a simple, sensitive, precise, and accurate HPLC method for the quantification of memantine in dosage forms. Pre-column derivatization of memantine was performed with 1-fluoro-2,4-dinitrobenzene and the reaction product was separated on a Nova-Pak C18 column. A mixture of acetonitrile and sodium dihydrogenphosphate (pH 2.5; 0.05 M) (70: 30, v/v) was used as the mobile phase. UV detection was performed at 360 nm. Forced degradation studies were performed on a powdered tablet sample of memantine hydro-chloride using acidic (0.1 M hydrochloric acid), basic (0.1 M sodium hydroxide), oxidative (10% hydrogen peroxide), thermal (105°C), photolytic, and humidity conditions. Good linearity (r2=0.999) was obtained over the range of 1–12 μg mL−1 of memantine hydrochloride with acceptable within-day and between-day precision values in the range of 0.05–0.95%. The proposed method was used for the assay determination and dissolution rate study of memantine dosage forms with excellent specificity. PMID:24959398

Storing of Extracts in Polypropylene Microcentrifuge Tubes Yields Contaminant Peak During Ultra-flow Liquid Chromatographic Analysis.

PubMed

Kshirsagar, Parthraj R; Hegde, Harsha; Pai, Sandeep R

2016-05-01

This study was designed to understand the effect of storage in polypropylene microcentrifuge tubes and glass vials during ultra-flow liquid chromatographic (UFLC) analysis. One ml of methanol was placed in polypropylene microcentrifuge tubes (PP material, Autoclavable) and glass vials (Borosilicate) separately for 1, 2, 4, 8, 10, 20, 40, and 80 days intervals stored at -4°C. Contaminant peak was detected in methanol stored in polypropylene microcentrifuge tubes using UFLC analysis. The contaminant peak detected was prominent, sharp detectable at 9.176 ± 0.138 min on a Waters 250-4.6 mm, 4 μ, Nova-Pak C18 column with mobile phase consisting of methanol:water (70:30). It was evident from the study that long-term storage of biological samples prepared using methanol in polypropylene microcentrifuge tubes produce contaminant peak. Further, this may mislead in future reporting an unnatural compound by researchers. Long-term storage of biological samples prepared using methanol in polypropylene microcentrifuge tubes produce contaminant peakContamination peak with higher area under the curve (609993) was obtained in ultra-flow liquid chromatographic run for methanol stored in PP microcentrifuge tubesContamination peak was detected at retention time 9.113 min with a lambda max of 220.38 nm and 300 mAU intensity on the given chromatographic conditionsGlass vials serve better option over PP microcentrifuge tubes for storing biological samples. Abbreviations used: UFLC: Ultra Flow Liquid Chromatography; LC: Liquid Chromatography; MS: Mass spectrometry; AUC: Area Under Curve.

Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase

PubMed Central

Han, Shi-Chong; Guo, Hui-Chen; Sun, Shi-Qi; Jin, Ye; Wei, Yan-Quan; Feng, Xia; Yao, Xue-Ping; Cao, Sui-Zhong; Xiang Liu, Ding; Liu, Xiang-Tao

2016-01-01

Virus entry is an attractive target for therapeutic intervention. Here, using a combination of electron microscopy, immunofluorescence assay, siRNA interference, specific pharmacological inhibitors, and dominant negative mutation, we demonstrated that the entry of foot-and-mouth disease virus (FMDV) triggered a substantial amount of plasma membrane ruffling. We also found that the internalization of FMDV induced a robust increase in fluid-phase uptake, and virions internalized within macropinosomes colocalized with phase uptake marker dextran. During this stage, the Rac1-Pak1 signaling pathway was activated. After specific inhibition on actin, Na+/H+ exchanger, receptor tyrosine kinase, Rac1, Pak1, myosin II, and protein kinase C, the entry and infection of FMDV significantly decreased. However, inhibition of phosphatidylinositol 3-kinase (PI3K) did not reduce FMDV internalization but increased the viral entry and infection to a certain extent, implying that FMDV entry did not require PI3K activity. Results showed that internalization of FMDV exhibited the main hallmarks of macropinocytosis. Moreover, intracellular trafficking of FMDV involves EEA1/Rab5-positive vesicles. The present study demonstrated macropinocytosis as another endocytic pathway apart from the clathrin-mediated pathway. The findings greatly expand our understanding of the molecular mechanisms of FMDV entry into cells, as well as provide potential insights into the entry mechanisms of other picornaviruses. PMID:26757826

Enterocin TW21, a novel bacteriocin from dochi-isolated Enterococcus faecium D081821.

PubMed

Chang, S-Y; Chen, Y-S; Pan, S-F; Lee, Y-S; Chang, C-H; Chang, C-H; Yu, B; Wu, H-C

2013-09-01

Purification and characterization of a novel bacteriocin produced by strain Enterococcus faecium D081821. Enterococcus faecium D081821, isolated from the traditional Taiwanese fermented food dochi (fermented black beans), was previously found to produce a bacteriocin against Listeria monocytogenes and some Gram-positive bacteria. This bacteriocin, termed enterocin TW21, was purified from culture supernatant by ammonium sulfate precipitation, Sep-Pak C18 cartridge, ion-exchange and gel filtration chromatography. Mass spectrometry analysis showed the mass of the peptide to be approximately 5300·6 Da. The N-terminal amino acid sequencing yielded a partial sequence NH2 -ATYYGNGVYxNTQK by Edman degradation, and it contains the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The open reading frame (ORF) encoding the bacteriocin was identified from the draft genome sequence of Enterococcus faecium D081821, and sequence analysis of this peptide indicated that enterocin TW21 is a novel bacteriocin. Enterococcus faecium D081821 produced a bacteriocin named enterocin TW21, the molecular weight and amino acid sequence both revealed it to be a novel bacteriocin. A new member of class IIa bacteriocin was identified. This bacteriocin shows great inhibitory ability against L. monocytogenes and could be applied as a natural food preservative. © 2013 The Society for Applied Microbiology.

Selenium speciation methods and application to soil saturation extracts from San Joaquin Valley, California

USGS Publications Warehouse

Fio, John L.; Fujii, Roger

1990-01-01

Methods to determine soluble concentrations of selenite, selenate, and organic Se were evaluated on saturation extracts of soil samples collected from three sites on the Panoche Creek alluvial fan in the western San Joaquin Valley, California. The methods were used in combination with hydride-generation atomic-absorption spectrometry for detection of Se, and included a selective chemical-digestion method and three chromatographic methods using XAD-8 resin, Sep-Pak C18 cartridge, and a combination of XAD-8 resin and activated charcoal. The chromatography methods isolate dissolved organic matter that can inhibit Se detection by hydride-generation atomic-absorption spectrometry. Isolation of hydrophobic organic matter with XAD-8 did not affect concentrations of selenite and selenate, and the isolated organic matter represents a minimal estimation of organic Se. Ninety-eight percent of the Se in the extracts was selenate and about 100% of the isolated organic Se was associated with the humic acid fraction of dissolved organic matter. The depth distribution of Se species in the soil saturation extracts support a hypothesis that the distribution of soluble Se and salinity in these soils is the result of evaporation from a shallow water table and leaching by irrigation water low in Se and salinity.

A methodology for multivariate phenotype-based genome-wide association studies to mine pleiotropic genes.

PubMed

Park, Sung Hee; Lee, Ji Young; Kim, Sangsoo

2011-01-01

Current Genome-Wide Association Studies (GWAS) are performed in a single trait framework without considering genetic correlations between important disease traits. Hence, the GWAS have limitations in discovering genetic risk factors affecting pleiotropic effects. This work reports a novel data mining approach to discover patterns of multiple phenotypic associations over 52 anthropometric and biochemical traits in KARE and a new analytical scheme for GWAS of multivariate phenotypes defined by the discovered patterns. This methodology applied to the GWAS for multivariate phenotype highLDLhighTG derived from the predicted patterns of the phenotypic associations. The patterns of the phenotypic associations were informative to draw relations between plasma lipid levels with bone mineral density and a cluster of common traits (Obesity, hypertension, insulin resistance) related to Metabolic Syndrome (MS). A total of 15 SNPs in six genes (PAK7, C20orf103, NRIP1, BCL2, TRPM3, and NAV1) were identified for significant associations with highLDLhighTG. Noteworthy findings were that the significant associations included a mis-sense mutation (PAK7:R335P), a frame shift mutation (C20orf103) and SNPs in splicing sites (TRPM3). The six genes corresponded to rat and mouse quantitative trait loci (QTLs) that had shown associations with the common traits such as the well characterized MS and even tumor susceptibility. Our findings suggest that the six genes may play important roles in the pleiotropic effects on lipid metabolism and the MS, which increase the risk of Type 2 Diabetes and cardiovascular disease. The use of the multivariate phenotypes can be advantageous in identifying genetic risk factors, accounting for the pleiotropic effects when the multivariate phenotypes have a common etiological pathway.

Direct-Acting Antivirals in Chronic Hepatitis C Genotype 4 Infection in Community Care Setting

PubMed Central

Gayam, Vijay; Khalid, Mazin; Mandal, Amrendra Kumar; Hussain, Muhammad Rajib; Mukhtar, Osama; Gill, Arshpal; Garlapati, Pavani; Shrestha, Binav; Guss, Debra; Sherigar, Jagannath; Mansour, Mohammed; Mohanty, Smruti

2018-01-01

Background Limited data exists comparing the safety, tolerability, and efficacy of direct-acting antivirals (DAAs) in patients with chronic hepatitis C genotype 4 (HCV GT-4) in the community practice setting. We aim to evaluate the treatment response of DAAs in these patients. Methods All the HCV GT-4 patients treated with DAAs between January 2014 and October 2017 in a community clinic setting were retrospectively analyzed. Pretreatment baseline patient characteristics, treatment efficacy with sustained virologic response (SVR) at 12 weeks post treatment (SVR12), and adverse reactions were assessed. Results Fifty-two patients of Middle Eastern (primarily Egyptian) descent were included in the study. Thirty-two patients were treated with ledipasvir/sofosbuvir (Harvoni®) ± ribavirin, 12 patients were treated with ombitasvir/paritaprevir/ritonavir/dasabuvir (ViekiraPak®) ± ribavirin, and eight patients were treated with sofosbuvir/Velpatasvir (Epclusa®). Ten patients (19.2%) had compensated cirrhosis. Overall, SVR at 12 weeks was achieved in 94% in patients who received one of the three DAA regimens (93.8% in Harvoni® group, 91.7 % in ViekiraPak® group and 100% in Epclusa® group). Prior treatment status and type of regimen used in the presence of compensated cirrhosis had no statistical significance on overall SVR achievement (P value = 0.442 and P value = 0.091, respectively). The most common adverse effect was fatigue (27%). Conclusions In the real-world setting, DAAs are effective and well tolerated in patients with chronic HCV GT-4 infection with a high overall SVR rate of 94%. Large-scale studies are needed to further assess this SVR in these groups. PMID:29707080

The Role of Startups

ScienceCinema

Babinec, Sue; Kirkpatrick, Doug

2018-05-30

Many ARPA-E-funded universities and research institutions have created start-up companies to further catalyze their next-generation technologies. Ambri and BlackPak are two examples of ARPA-E projects that were spun out by other institutions—Massachusetts Institute of Technology and SRI International, respectively—in an effort to get their technologies out of the lab and into the market quickly. This video features remarks from ARPA-E Senior Commercialization Advisor Sue Babinec and interviews with technologists at Ambri and BlackPak, who each tell the story of how their new companies spun out of the lab and have become agile startups capable of delivering real products to the marketplace.

The Role of Startups

DOE Office of Scientific and Technical Information (OSTI.GOV)

Babinec, Sue; Kirkpatrick, Doug

2015-02-11

Many ARPA-E-funded universities and research institutions have created start-up companies to further catalyze their next-generation technologies. Ambri and BlackPak are two examples of ARPA-E projects that were spun out by other institutions—Massachusetts Institute of Technology and SRI International, respectively—in an effort to get their technologies out of the lab and into the market quickly. This video features remarks from ARPA-E Senior Commercialization Advisor Sue Babinec and interviews with technologists at Ambri and BlackPak, who each tell the story of how their new companies spun out of the lab and have become agile startups capable of delivering real products to themore » marketplace.« less

Qualitative identification of permitted and non-permitted colour additives in food products.

PubMed

Harp, Bhakti Petigara; Miranda-Bermudez, Enio; Baron, Carolina I; Richard, Gerald I

2012-01-01

Colour additives are dyes, pigments or other substances that can impart colour when added or applied to food, drugs, cosmetics, medical devices, or the human body. The substances must be pre-approved by the US Food and Drug Administration (USFDA) and listed in Title 21 of the US Code of Federal Regulations before they may be used in products marketed in the United States. Some also are required to be batch certified by the USFDA prior to their use. Both domestic and imported products sold in interstate commerce fall under USFDA jurisdiction, and the USFDA's district laboratories use a combination of analytical methods for identifying or confirming the presence of potentially violative colour additives. We have developed a qualitative method for identifying 17 certifiable, certification exempt, and non-permitted colour additives in various food products. The method involves extracting the colour additives from a product and isolating them from non-coloured components with a C(18) Sep-Pak cartridge. The colour additives are then separated and identified by liquid chromatography (LC) with photodiode array detection, using an Xterra RP18 column and gradient elution with aqueous ammonium acetate and methanol. Limits of detection (LODs) ranged from 0.02 to 1.49 mg/l. This qualititative LC method supplements the visible spectrophotometric and thin-layer chromatography methods currently used by the USFDA's district laboratories and is less time-consuming and requires less solvent compared to the other methods. The extraction step in the new LC method is a simple and an efficient process that can be used for most food types.

Potential for false positive HIV test results with the serial rapid HIV testing algorithm.

PubMed

Baveewo, Steven; Kamya, Moses R; Mayanja-Kizza, Harriet; Fatch, Robin; Bangsberg, David R; Coates, Thomas; Hahn, Judith A; Wanyenze, Rhoda K

2012-03-19

Rapid HIV tests provide same-day results and are widely used in HIV testing programs in areas with limited personnel and laboratory infrastructure. The Uganda Ministry of Health currently recommends the serial rapid testing algorithm with Determine, STAT-PAK, and Uni-Gold for diagnosis of HIV infection. Using this algorithm, individuals who test positive on Determine, negative to STAT-PAK and positive to Uni-Gold are reported as HIV positive. We conducted further testing on this subgroup of samples using qualitative DNA PCR to assess the potential for false positive tests in this situation. Of the 3388 individuals who were tested, 984 were HIV positive on two consecutive tests, and 29 were considered positive by a tiebreaker (positive on Determine, negative on STAT-PAK, and positive on Uni-Gold). However, when the 29 samples were further tested using qualitative DNA PCR, 14 (48.2%) were HIV negative. Although this study was not primarily designed to assess the validity of rapid HIV tests and thus only a subset of the samples were retested, the findings show a potential for false positive HIV results in the subset of individuals who test positive when a tiebreaker test is used in serial testing. These findings highlight a need for confirmatory testing for this category of individuals.

Potential for false positive HIV test results with the serial rapid HIV testing algorithm

PubMed Central

2012-01-01

Background Rapid HIV tests provide same-day results and are widely used in HIV testing programs in areas with limited personnel and laboratory infrastructure. The Uganda Ministry of Health currently recommends the serial rapid testing algorithm with Determine, STAT-PAK, and Uni-Gold for diagnosis of HIV infection. Using this algorithm, individuals who test positive on Determine, negative to STAT-PAK and positive to Uni-Gold are reported as HIV positive. We conducted further testing on this subgroup of samples using qualitative DNA PCR to assess the potential for false positive tests in this situation. Results Of the 3388 individuals who were tested, 984 were HIV positive on two consecutive tests, and 29 were considered positive by a tiebreaker (positive on Determine, negative on STAT-PAK, and positive on Uni-Gold). However, when the 29 samples were further tested using qualitative DNA PCR, 14 (48.2%) were HIV negative. Conclusion Although this study was not primarily designed to assess the validity of rapid HIV tests and thus only a subset of the samples were retested, the findings show a potential for false positive HIV results in the subset of individuals who test positive when a tiebreaker test is used in serial testing. These findings highlight a need for confirmatory testing for this category of individuals. PMID:22429706

A developed determination of midazolam and 1'-hydroxymidazolam in plasma by liquid chromatography-mass spectrometry: application of human pharmacokinetic study for measurement of CYP3A activity.

PubMed

Shimizu, Mikiko; Uno, Tsukasa; Tamura, Hiro-omi; Kanazawa, Hideko; Murakami, Isao; Sugawara, Kazunobu; Tateishi, Tomonori

2007-03-01

This paper describes sensitive and reliable determination of midazolam (MDZ) and its major metabolite 1'-hydroxymidazolam (1-OHMDZ) in human plasma by liquid chromatography-mass spectrometry (LC-MS) with a sonic spray ionization (SSI) interface. MDZ, 1-OHMDZ and diazepam as an internal standard were extracted from 1ml of alkalinized plasma using n-hexane-chloroform (70:30, v/v). The extract was injected into an analytical column (YMC-Pak Pro C(18), 50mmx2.0mmi.d.). The mobile phase for separation consisted of 10mM ammonium acetate and methanol (50:50, v/v) and was delivered at a flow-rate of 0.2ml/min. The drift voltage was 100V. The sampling aperture was heated at 120 degrees C and the shield temperature was 260 degrees C. The total time for chromatographic separation was less than 16min. The validated concentration ranges of this method were 0.25-50ng/ml for both MDZ and 1-OHMDZ. Mean recoveries were 93.6% for MDZ and 86.6% for 1-OHMDZ. Intra- and inter-day coefficient variations were less than 6.5 and 5.5% for MDZ, and 6.1 and 5.7% for 1-OHMDZ at 0.3, 4, 20 and 40ng/ml. The limits of quantification were 0.25ng/ml for both MDZ and 1-OHMDZ. This method was sensitive and reliable enough for pharmacokinetic studies on healthy volunteers, and was applied for the measurement of CYP3A activity in humans after an intravenous (1mg) and a single-oral administration (2mg) of subtherapeutic MDZ dose.

PrimaTB STAT-PAK Assay, a Novel, Rapid Lateral-Flow Test for Tuberculosis in Nonhuman Primates▿

PubMed Central

Lyashchenko, Konstantin P.; Greenwald, Rena; Esfandiari, Javan; Greenwald, David; Nacy, Carol A.; Gibson, Susan; Didier, Peter J.; Washington, Marc; Szczerba, Peter; Motzel, Sherri; Handt, Larry; Pollock, John M.; McNair, James; Andersen, Peter; Langermans, Jan A. M.; Verreck, Frank; Ervin, Sean; Ervin, Frank; McCombs, Candace

2007-01-01

Tuberculosis (TB) is the most important zoonotic bacterial disease in nonhuman primates (NHP). The current diagnostic method, the intradermal palpebral tuberculin test, has serious shortcomings. We characterized antibody responses in NHP against Mycobacterium tuberculosis to identify immunodominant antigens and develop a rapid serodiagnostic test for TB. A total of 422 NHP were evaluated, including 243 rhesus (Macaca mulatta), 46 cynomolgus (Macaca fascicularis), and 133 African green (Cercopithecus aethiops sabaeus) monkeys at five collaborative centers. Of those, 50 monkeys of the three species were experimentally inoculated with M. tuberculosis. Antibody responses were monitored every 2 to 4 weeks for up to 8 months postinfection by MultiAntigen Print ImmunoAssay with a panel of 12 recombinant antigens. All of the infected monkeys produced antibodies at various levels and with different antigen recognition patterns. ESAT-6 and MPB83 were the most frequently recognized proteins during infection. A combination of selected antigens which detected antibodies in all of the infected monkeys was designed to develop the PrimaTB STAT-PAK assay by lateral-flow technology. Serological evaluation demonstrated high diagnostic sensitivity (90%) and specificity (99%). The highest rate of TB detection was achieved when the skin test was combined with the PrimaTB STAT-PAK kit. This novel immunoassay provides a simple, rapid, and accurate test for TB in NHP. PMID:17652522

Amiodarone

MedlinePlus

... Kaletra, in Viekira Pak); ledipasvir and sofosbuvir (Harvoni); lithium (Lithobid); loratadine (Claritin); medications for diabetes or seizures; ... rash weight loss or gain restlessness weakness nervousness irritability intolerance to heat or cold thinning hair excessive ...

Fast trace determination of nine odorant and estrogenic chloro- and bromo-phenolic compounds in real water samples through automated solid-phase extraction coupled with liquid chromatography tandem mass spectrometry.

PubMed

Yuan, Su-Fen; Liu, Ze-Hua; Lian, Hai-Xian; Yang, Chuang-Tao; Lin, Qing; Yin, Hua; Lin, Zhang; Dang, Zhi

2018-02-01

A fast and reliable method was developed for simultaneous trace determination of nine odorous and estrogenic chloro- and bromo-phenolic compounds (CPs and BPs) in water samples using solid-phase extraction (SPE) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS). For sample preparation, the extraction efficiencies of two widely applied cartridges Oasis HLB and Sep-Pak C18 were compared, and the Oasis HLB cartridge showed much better extraction performance; pH of water sample also plays important role on extraction, and pH = 2-3 was found to be most appropriate. For separation of the target compounds, small addition of ammonium hydroxide can obviously improve the detection sensitivity, and the optimized addition concentration was determined as 0.2%. The developed efficient method was validated and showed excellent linearity (R 2  > 0.995), low limit of detection (LOD, 1.9-6.2 ng/L), and good recovery efficiencies of 57-95% in surface and tap water with low relative standard deviation (RSD, 1.3-17.4%). The developed method was finally applied to one tap and one surface water samples and most of these nine targets were detected, but all of them were below their odor thresholds, and their estrogen equivalent (EEQ) were also very low.

Determination of neonicotinoid insecticides and their metabolites in honey bee and honey by liquid chromatography tandem mass spectrometry.

PubMed

Gbylik-Sikorska, Malgorzata; Sniegocki, Tomasz; Posyniak, Andrzej

2015-05-15

The original analytical method for the simultaneous determination and confirmation of neonicotinoids insecticides (imidacloprid, clothianidin, acetamiprid, thiametoxam, thiacloprid, nitenpyram, dinotefuran) and some of their metabolites (imidacloprid guanidine, imidacloprid olefin, imidacloprid urea, desnitro-imidacloprid hydrochloride, thiacloprid-amid and acetamiprid-N-desmethyl) in honey bee and honey was developed. Preparation of honey bee samples involves the extraction with mixture of acetonitrile and ethyl acetate followed by cleaned up using the Sep-Pak Alumina N Plus Long cartridges. Honey samples were dissolved in 1% mixture of acetonitrile and ethyl acetate with addition of TEA, then extracts were cleaned up with Strata X-CW cartridges. The identity of analytes was confirmed using liquid chromatography tandem mass spectrometry. All compounds were separated on a Luna C18 column with gradient elution. The whole procedure was validated according to the requirements of SANCO 12571/2013. The average recoveries of the analytes ranged from 85.3% to 112.0%, repeatabilities were in the range of 2.8-11.2%, within-laboratory reproducibility was in the range of 3.3-14.6%, the limits of quantitation were in the range of 0.1-0.5μgkg(-1), depending of analyte and matrices. The validated method was successfully applied for the determination of clothianidin, imidacloprid and imidacloprid urea in real incurred honey bee samples and clothianidin in honey. Copyright © 2015 Elsevier B.V. All rights reserved.

Role of endopeptidase 3.4.24.16 in the catabolism of neurotensin, in vivo, in the vascularly perfused dog ileum.

PubMed Central

Barelli, H.; Fox-Threlkeld, J. E.; Dive, V.; Daniel, E. E.; Vincent, J. P.; Checler, F.

1994-01-01

1. The degradation of tritiated and unlabelled neurotensin (NT) following close intra-arterial infusion of the peptides in ileal segments of anaesthetized dogs was examined. 2. Intact NT and its catabolites recovered in the venous effluents were purified by chromatography on Sep-Pak columns followed by reverse-phase h.p.l.c. and identified by their retention times or by radioimmunoassay. 3. The half-life of neurotensin was estimated to be between 2 and 6 min. Four labelled catabolites, corresponding to free tyrosine, neurotensin (1-8), neurotensin (1-10) and neurotensin (1-11), were detected. 4. Neurotensin (1-11) was mainly generated by a phosphoramidon-sensitive cleavage, probably elicited by endopeptidase 24-11. 5. Two endopeptidase 3.4.24.16 inhibitors, phosphodiepryl 03 and the dipeptide Pro-Ile, dose-dependently potentiated the recovery of intact neurotensin. Furthermore, both agents inhibited the formation of neurotensin (1-10), the product that results from the hydrolysis of neurotensin by purified endopeptidase 3.4.24.16. In contrast, the endopeptidase 3.4.24.15 inhibitor Cpp-AAY-pAB neither protected neurotensin from degradation nor modified the production of neurotensin (1-10). 6. Our study is the first evidence to indicate that endopeptidase 3.4.24.16 contributes to the catabolism of neurotensin, in vivo, in the dog intestine. PMID:8032633

Precision Nanoparticles

ScienceCinema

John Hemminger

2017-12-09

A revolutionary technology that efficiently produces nanoparticles in uniform and prescribed sizes (1-100 nanometers) using supercritical fluids. INL researcher Robert Fox was joined by Idaho State University researchers Rene Rodriquez and Joshua Pak in d

Field evaluation of three blood-based assays for elk (Cervus canadensis) naturally infected with Mycobacterium bovis.

PubMed

Shury, Todd K; Bergeson, Doug; Surujballi, Om; Lyashchenko, Konstantin P; Greenwald, Rena

2014-08-01

Diagnosis of Mycobacterium bovis in wild populations is very challenging due to complications imposed by the use of traditional skin tests, poor sensitivity of gold standard tests which rely on culture of M. bovis from tissues and wide variations in severity of disease. Various combinations of a lymphocyte stimulation test (LST), fluorescence polarization assay (FPA) and the Cervid TB Stat-Pak were evaluated using two different validation approaches: a latent class analysis and classical statistical approach using culture as a gold standard. A validation subsample consisting of animals culled for population control and mortalities from capture provided an unbiased estimate of test performance for comparison. The sensitivity of the LST (0.83, 95% CI: [0.70-0.97] as a single test was similar to existing tuberculin skin tests, but the sensitivity of the FPA (0.40, 95% CI: [0.22-0.58]) and Cervid TB Stat-Pak (0.62, 95% CI: [0.41-0.83]) were lower in this population. Test performance of the LST and Cervid TB Stat-Pak in parallel was similar to the use of all three tests in parallel and inclusion of the FPA did not greatly enhance test performance. Prevalence of M. bovis in elk varied substantially between the high risk area of southern Manitoba (9.1%, 95% CI: [6.09-12.1%]) and lower risk areas outside this zone (0.76%, 95% CI: [0-2.26%]). Bayesian latent class analysis indicated lack of covariance between the two antibody tests (FPA and Cervid TB Stat-Pak) while the classical two-stage analysis indicated there was conditional dependence between the tests. All three tests when used in parallel resulted in 100% NPV using all three validation methods, indicating few elk were misclassified as false negative by post mortem culture. Similar to previous studies, this study found that combinations of blood tests that utilize cell mediated responses along with humoral antibody responses maximize the sensitivity of tests for diagnosis of M. bovis in wild cervid populations. Copyright © 2013 CROWN COPYRIGHT and Elsevier B.V. Published by Elsevier B.V. All rights reserved.

Evaluation of three commercial rapid tests for detecting antibodies to human immunodeficiency virus.

PubMed

Ng, K P; Saw, T L; Baki, A; Kamarudin, R

2003-08-01

Determine HIV-1/2, Chembio HIV-1/2 STAT-PAK and PenTest are simple/rapid tests for the detection of antibodies to HIV-1 and HIV-2 in human whole blood, serum and plasma samples. The assay is one step and the result is read visually within 15 minutes. Using 92 known HIV-1 reactive sera and 108 known HIV-1 negative sera, the 3 HIV tests correctly identified all the known HIV-1 reactive and negative samples. The results indicated that Determine HIV-1/2, Chembio HIV-1/2 STAT-PAK and PenTest HIV are as sensitive and specific (100% concordance) as Microparticle Enzyme Immunoassay. The data indicated that these 3 HIV tests are effective testing systems for diagnosis of HIV infection in a situation when the conventional Enzyme Immunoassay is not suitable.

Optimization of the treatment of wheat samples for the determination of phytic acid by HPLC with refractive index detection.

PubMed

Amaro, Rosa; Murillo, Miguel; González, Zurima; Escalona, Andrés; Hernández, Luís

2009-01-01

The treatment of wheat samples was optimized before the determination of phytic acid by high-performance liquid chromatography with refractive index detection. Drying by lyophilization and oven drying were studied; drying by lyophilization gave better results, confirming that this step is critical in preventing significant loss of analyte. In the extraction step, washing of the residue and collection of this water before retention of the phytates in the NH2 Sep-Pak cartridge were important. The retention of phytates in the NH2 Sep-Pak cartridge and elimination of the HCI did not produce significant loss (P = 0.05) in the phytic acid content of the sample. Recoveries of phytic acid averaged 91%, which is a substantial improvement with respect to values reported by others using this methodology.

The mixed low-level waste problem in BE/NWN capsule

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hensley, D.C.

1999-07-01

The Boh Environmental, LLC (BE) and Northwest Nuclear, LLC (NWN) program addresses the problem of diminishing capacity in the United States to store mixed waste. A lack of an alternative program has caused the US Department of Energy (DOE) to indefinitely store all of its mixed waste in Resource Conservation and Recovery Act (RCRA) compliant storage facilities. Unfortunately, this capacity is fast approaching the administrative control limit. The combination of unique BE encapsulation and NWN waste characterization technologies provides an effective solution to DOE's mixed-waste dilemma. The BE ARROW-PAK technique encapsulates mixed low-level waste (MLLW) in extra-high molecular weight, high-densitymore » polyethylene, pipe-grade resin cylinders. ARROW-PAK applications include waste treatment, disposal, transportation (per 49 CFR 173), vault encasement, and interim/long-term storage for 100 to 300 yr. One of the first demonstrations of this treatment/storage technique successfully treated 880 mixed-waste debris drums at the DOE Hanford Site in 1997. NWN, deploying the APNea neutron assay technology, provides the screening and characterization capability necessary to ensure that radioactive waste is correctly categorized as either transuranic (TRU) or LLW. MLLW resulting from D and D activities conducted at the Oak Ridge East Tennessee Technology Park will be placed into ARROW-PAK containers following comprehensive characterization of the waste by NWN. The characterized and encapsulated waste will then be shipped to a commercial disposal facility, where the shipments meet all waste acceptance criteria of the disposal facility including treatment criteria.« less

Measurements of atmospheric nitrous acid and nitric acid

NASA Astrophysics Data System (ADS)

Huang, Gu; Zhou, Xianliang; Deng, Guohong; Qiao, Huancheng; Civerolo, Kevin

A highly sensitive technique for the measurement of atmospheric HONO and HNO 3 is reported. The technique is based on aqueous scrubbing using two coil samplers, followed by conversion of HNO 3 to nitrite, derivatization of nitrite to a highly light-absorbing azo dye with sulfanilamide (SA) and N-(1-naphthyl) ethylenediamine (NED), and high performance liquid chromatography (HPLC) analysis. HNO 3 concentration was obtained by the difference of the two channels. Two scrubbing solutions were used for sampling the two species: a 1-mM phosphate buffer solution (pH 7) for the measurement of HONO and a 180 mM NH 4Cl/NH 3 buffer solution (pH 8.5) for the measurement of HONO+HNO 3. The scrubbing solution flow rate was 0.24 ml min -1 and the gas sampling flow rate was 2 l min -1. HNO 3 in the NH 4Cl/NH 3 buffer solution was quantitatively reduced to nitrite along an on-line 0.8-cm Cd reductor column. Nitrite in both channels was derivatized with 2 mM SA and 0.2 mM NED in 25 mM HCl. Quantitative derivatization was achieved within 5 min at 55°C. The azo dye derivative was then separated from the SA/NED reagent by reversed-phase HPLC and detected with a UV-vis detector at 540 nm. With an on-line SEP-PAK C-18 cartridge for the reagent purification, the method detection limit is estimated to be better than 1 pptv for HONO and about 20 pptv for HNO 3. The sample integration time was about 2 min and the sampling frequency is every 10 min. Data collected in downtown Albany and Whiteface Mountain, NY, are shown as examples of applications of this technique in both urban and remote clean environments.

OPTN/SRTR 2016 Annual Data Report: Pancreas.

PubMed

Kandaswamy, R; Stock, P G; Gustafson, S K; Skeans, M A; Curry, M A; Prentice, M A; Fox, A; Israni, A K; Snyder, J J; Kasiske, B L

2018-01-01

The number of pancreas transplants performed in the United States increased by 7.0% in 2016 over the previous year, the first such increase in more than a decade, largely attributable to an increase in simultaneous kidney pancreas transplants. Transplant rates increased in 2016, and mortality on the waiting list decreased. The declining enthusiasm for pancreas after kidney (PAK) transplants persisted. The uniform definition of graft failure was approved by the OPTN Board of Directors in 2015 and will be implemented in early 2018. Meanwhile, SRTR continues to refrain from reporting pancreas graft failure data. The OPTN/UNOS Pancreas Transplantation Committee is seeking to broaden allocation of pancreata across compatible ABO blood types in a proposal out for public comment July 31 to October 2, 2017. A new initiative to provide guidance on the benefits of PAK transplants is also out for public comment.  .

Near real time observational data collection for SPRUCE experiment- PakBus protocol for slow satellite connections

NASA Astrophysics Data System (ADS)

Krassovski, Misha; Hanson, Paul; Riggs, Jeff

2017-04-01

Climate change studies are one of the most important aspects of modern science and related experiments are getting bigger and more complex. One such experiment is the Spruce and Peatland Responses Under Climatic and Environmental Change experiment (SPRUCE, http://mnspruce.ornl.gov) conducted in in northern Minnesota, 40 km north of Grand Rapids, in the USDA Forest Service Marcell Experimental Forest (MEF). The SPRUCE experimental mission is to assess ecosystem-level biological responses of vulnerable, high carbon terrestrial ecosystems to a range of climate warming manipulations and an elevated CO2 atmosphere. This manipulation experiment generates a lot of observational data and requires a reliable onsite data collection system, dependable methods to transfer data to a robust scientific facility, and real-time monitoring capabilities. This publication shares our experience of establishing near real time data collection and monitoring system via a satellite link using PakBus protocol.

Impact of different feedstocks derived biochar amendment with cadmium low uptake affinity cultivar of pak choi (Brassica rapa ssb. chinensis L.) on phytoavoidation of Cd to reduce potential dietary toxicity.

PubMed

Yasmin Khan, Kiran; Ali, Barkat; Cui, Xiaoqiang; Feng, Ying; Yang, Xiaoe; Joseph Stoffella, Peter

2017-07-01

Biochar has become eco-friendly amendment used for phytoavoidation with low cadmium (Cd) accumulating cultivars of crops to ensure food safety in Cd contaminated soils. In this study, biochar with different waste feedstock material were evaluated for their effectiveness on essential trace metals mobility, Cd bioavailability and its accumulation in two contrasting Cd accumulating cultivars of pak choi (Brassica rapa ssp. chinensis L.) grown in Cd contaminated Mollisol soil. A greenhouse experiment was conducted with plants grown in Cd contaminated soil that had been amended with biochar derived from barley straw, tomato green waste, chicken manure, duck manure and swine manure at application rate of 0%, 2.5% and 5.0% (w/w). The results showed that soil pH was significantly increased by all treatments. Biochar increased plant dry biomass, micronutrients bioavailability with significant differences in the Cd sorption capacity, with the effectiveness higher with increasing biochar application rate. However, tomato green waste (TGW) and chicken manure (CM) derived biochar were more effective than the other biochar in reducing Cd mobilization in soil by 35-54% and 26-43% and reduced its accumulation in shoots of pak choi cultivars by 34-76% and 33-72% in low Cd accumulator cultivar and 64-85% and 55-80% in high Cd accumulator cultivar than the control. Overall, results indicate that TGW and CM biochar can efficiently immobilize Cd, thereby reducing bioavailability in Cd contaminated Mollisol soil to ensure food safety. Copyright © 2017 Elsevier Inc. All rights reserved.

Metabolomics differentiation of canola genotypes: toward an understanding of canola allelochemicals

PubMed Central

Asaduzzaman, M.; Pratley, James E.; An, Min; Luckett, David J.; Lemerle, Deirdre

2015-01-01

Allelopathy is one crop attribute that could be incorporated in an integrated weed management system as a supplement to synthetic herbicides. However, the underlying principles of crop allelopathy and secondary metabolite production are still poorly understood including in canola. In this study, an allelopathic bioassay and a metabolomic analysis were conducted to compare three non-allelopathic and three allelopathic canola genotypes. Results from the laboratory bioassay showed that there were significant differences among canola genotypes in their ability to inhibit root and shoot growth of the receiver annual ryegrass; impacts ranged from 14% (cv. Atr-409) to 76% (cv. Pak85388-502) and 0% (cv. Atr-409) to 45% (cv. Pak85388-502) inhibition respectively. The root length of canola also differed significantly between genotypes, there being a non-significant negative interaction (r = -0.71; y = 0.303x + 21.33) between the root length of donor canola and of receiver annual ryegrass. Variation in chemical composition was detected between organs (root extracts, shoot extracts) and root exudates and also between canola genotypes. Root extracts contained more secondary metabolites than shoot extracts while fewer compounds were recorded in the root exudates. Individual compound assessments identified a total of 14 secondary metabolites which were identified from the six tested genotypes. However, only Pak85388-502 and Av-opal exuded sinapyl alcohol, p-hydroxybenzoic acid and 3,5,6,7,8-pentahydroxy flavones in agar growth medium, suggesting that the synergistic effect of these compounds playing a role for canola allelopathy against annual ryegrass in vitro. PMID:25620971

Folliculotropism in pigmented facial macules: Differential diagnosis with reflectance confocal microscopy.

PubMed

Persechino, Flavia; De Carvalho, Nathalie; Ciardo, Silvana; De Pace, Barbara; Casari, Alice; Chester, Johanna; Kaleci, Shaniko; Stanganelli, Ignazio; Longo, Caterina; Farnetani, Francesca; Pellacani, Giovanni

2018-03-01

Pigmented facial macules are common on sun damage skin. The diagnosis of early stage lentigo maligna (LM) and lentigo maligna melanoma (LMM) is challenging. Reflectance confocal microscopy (RCM) has been proven to increase diagnostic accuracy of facial lesions. A total of 154 pigmented facial macules, retrospectively collected, were evaluated for the presence of already-described RCM features and new parameters depicting aspects of the follicle. Melanocytic nests, roundish pagetoid cells, follicular infiltration, bulgings from the follicles and many bright dendrites and infiltration of the hair follicle (ie, folliculotropism) were found to be indicative of LM/LMM compared to non-melanocytic skin neoplasms (NMSNs), with an overall sensitivity of 96% and specificity of 83%. Concerning NMSNs, solar lentigo and lichen planus-like keratosis resulted better distinguishable from LM/LMM because usually lacking malignant features and presenting characteristic diagnostic parameters, such as epidermal cobblestone pattern and polycyclic papillary contours. On the other hand, distinction of pigmented actinic keratosis (PAK) resulted more difficult, and needing evaluation of hair follicle infiltration and bulging structures, due to the frequent observation of few bright dendrites in the epidermis, but predominantly not infiltrating the hair follicle (estimated specificity for PAK 53%). A detailed evaluation of the components of the folliculotropism may help to improve the diagnostic accuracy. The classification of the type, distribution and amount of cells, and the presence of bulging around the follicles seem to represent important tools for the differentiation between PAK and LM/LMM at RCM analysis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Spleen tyrosine kinase mediates high glucose-induced transforming growth factor-{beta}1 up-regulation in proximal tubular epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Won Seok; Chang, Jai Won; Han, Nam Jeong

The role of spleen tyrosine kinase (Syk) in high glucose-induced intracellular signal transduction has yet to be elucidated. We investigated whether Syk is implicated in high glucose-induced transforming growth factor-{beta}1 (TGF-{beta}1) up-regulation in cultured human proximal tubular epithelial cells (HK-2 cell). High glucose increased TGF-{beta}1 gene expression through Syk, extracellular signal-regulated kinase (ERK), AP-1 and NF-{kappa}B. High glucose-induced AP-1 DNA binding activity was decreased by Syk inhibitors and U0126 (an ERK inhibitor). Syk inhibitors suppressed high glucose-induced ERK activation, whereas U0126 had no effect on Syk activation. High glucose-induced NF-{kappa}B DNA binding activity was also decreased by Syk inhibitors. Highmore » glucose increased nuclear translocation of p65 without serine phosphorylation of I{kappa}B{alpha} and without degradation of I{kappa}B{alpha}, but with an increase in tyrosine phosphorylation of I{kappa}B{alpha} that may account for the activation of NF-{kappa}B. Both Syk inhibitors and Syk-siRNA attenuated high glucose-induced I{kappa}B{alpha} tyrosine phosphorylation and p65 nuclear translocation. Depletion of p21-activated kinase 2 (Pak2) by transfection of Pak2-siRNA abolished high glucose-induced Syk activation. In summary, high glucose-induced TGF-{beta}1 gene transcription occurred through Pak2, Syk and subsequent ERK/AP-1 and NF-{kappa}B pathways. This suggests that Syk might be implicated in the diabetic kidney disease.« less

High throughput screening of CO2-tolerating microalgae using GasPak bags

PubMed Central

2013-01-01

Background Microalgae are diverse in terms of their speciation and function. More than 35,000 algal strains have been described, and thousands of algal cultures are maintained in different culture collection centers. The ability of CO2 uptake by microalgae varies dramatically among algal species. It becomes challenging to select suitable algal candidates that can proliferate under high CO2 concentration from a large collection of algal cultures. Results Here, we described a high throughput screening method to rapidly identify high CO2 affinity microalgae. The system integrates a CO2 mixer, GasPak bags and microplates. Microalgae on the microplates will be cultivated in GasPak bags charged with different CO2 concentrations. Using this method, we identified 17 algal strains whose growth rates were not influenced when the concentration of CO2 was increased from 2 to 20% (v/v). Most CO2 tolerant strains identified in this study were closely related to the species Scenedesmus and Chlorococcum. One of Scenedesmus strains (E7A) has been successfully tested in in the scale up photo bioreactors (500 L) bubbled with flue gas which contains 10-12% CO2. Conclusion Our high throughput CO2 testing system provides a rapid and reliable way for identifying microalgal candidate strains that can grow under high CO2 condition from a large pool of culture collection species. This high throughput system can also be modified for selecting algal strains that can tolerate other gases, such as NOx, SOx, or flue gas. PMID:24341988

Further Adventures of Dr. Sony's Mad Monster Machine, or Cutting the Studio Umbilical Cord

ERIC Educational Resources Information Center

Steffin, Sherwin

1977-01-01

We must reexamine our often total reliance on the studio as the only source for instructional television production. The porta-pak provides an alternative. Tips are provided for successful field production. (Author/STS)

Sirolimus induces apoptosis and reverses multidrug resistance in human osteosarcoma cells in vitro via increasing microRNA-34b expression.

PubMed

Zhou, Yan; Zhao, Rui-hua; Tseng, Kuo-fu; Li, Kun-peng; Lu, Zhi-gang; Liu, Yuan; Han, Kun; Gan, Zhi-hua; Lin, Shu-chen; Hu, Hai-yan; Min, Da-liu

2016-04-01

Multi-drug resistance poses a critical bottleneck in chemotherapy. Given the up-regulation of mTOR pathway in many chemoresistant cancers, we examined whether sirolimus (rapamycin), a first generation mTOR inhibitor, might induce human osteosarcoma (OS) cell apoptosis and increase the sensitivity of OS cells to anticancer drugs in vitro. Human OS cell line MG63/ADM was treated with sirolimus alone or in combination with doxorubicin (ADM), gemcitabine (GEM) or methotrexate (MTX). Cell proliferation and apoptosis were detected using CCK-8 assay and flow cytometry, respectively. MiRNAs in the cells were analyzed with miRNA microarray. The targets of miR-34b were determined based on TargetScan analysis and luciferase reporter assays. The expression of relevant mRNA and proteins was measured using qRT-PCR and Western blotting. MiR-34, PAK1 and ABCB1 levels in 40 tissue samples of OS patients were analyzed using qRT-PCR and in situ hybridization assays. Sirolimus (1-100 nmol/L) dose-dependently suppressed the cell proliferation (IC50=23.97 nmol/L) and induced apoptosis. Sirolimus (10 nmol/L) significantly sensitized the cells to anticancer drugs, leading to decreased IC50 values of ADM, GEM and MTX (from 25.48, 621.41 and 21.72 μmol/L to 4.93, 73.92 and 6.77 μmol/L, respectively). Treatment of with sirolimus increased miR-34b levels by a factor of 7.5 in the cells. Upregulation of miR-34b also induced apoptosis and increased the sensitivity of the cells to the anticancer drugs, whereas transfection with miR-34b-AMO, an inhibitor of miR-34b, reversed the anti-proliferation effect of sirolimus. Two key regulators of cell cycle, apoptosis and multiple drug resistance, PAK1 and ABCB1, were demonstrated to be the direct targets of miR-34b. In 40 tissue samples of OS patients, significantly higher miR-34 ISH score and lower PAK5 and ABCB1 scores were detected in the chemo-sensitive group. Sirolimus increases the sensitivity of human OS cells to anticancer drugs in vitro by up-regulating miR-34b interacting with PAK1 and ABCB1. A low miR-34 level is an indicator of poor prognosis in OS patients.

Simultaneous determination of pyridostigmine bromide, N,N-diethyl-m-toluamide, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography.

PubMed

Abu-Qare, A W; Abou-Donia, M B

2000-12-01

A rapid and simple method was developed for the separation and quantification of the anti nerve agent drug pyridostignmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), its metabolites m-toluamide and m-toluic acid, the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), and two of its metabolites m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid in rat plasma and urine. The method is based on using C18 Sep-Pak cartridges for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with reversed-phase C18 column, and gradient UV detection ranging between 208 and 230 nm. The compounds were separated using gradient of 1 to 99% acetonitrile in water (pH 3.20) at a flow-rate ranging between 0.5 and 1.7 ml/min in a period of 17 min. The retention times ranged from 5.7 to 14.5 min. The limits of detection were ranged between 20 and 100 ng/ml, while limits of quantitation were 150-200 ng/ml. Average percentage recovery of five spiked plasma samples were 51.4+/-10.6, 71.1+/-11.0, 82.3+/-6.7, 60.4+/-11.8, 63.6+/-10.1, 69.3+/-8.5, 68.3+/-12.0, 82.6+/-8.1, and from urine 55.9+/-9.8, 60.3+/-7.4, 77.9+/-9.1, 61.7+/-13.5, 68.6+/-8.9, 62.0+/-9.5, 72.9+/-9.1, and 72.1+/-8.0, for pyridostigmine bromide, DEET, permethrin, N-methyl-3-hydroxypyridinium bromide, m-toluamide, m-toluic acid, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 5000 ng/ml. This method was applied to analyze the above chemicals and metabolites following their administration in rats.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism.

PubMed

Francis, Andrew J; Resendiz, Marino J E

2017-07-28

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5'-[CUA CGG AAU CAU]-3' containing zero, one, or two modifications located at the C2'-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2'-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Fray, a Drosophila serine/threonine kinase homologous to mammalian PASK, is required for axonal ensheathment

NASA Technical Reports Server (NTRS)

Leiserson, W. M.; Harkins, E. W.; Keshishian, H.

2000-01-01

Fray is a serine/threonine kinase expressed by the peripheral glia of Drosophila, whose function is required for normal axonal ensheathment. Null fray mutants die early in larval development and have nerves with severe swelling and axonal defasciculation. The phenotype is associated with a failure of the ensheathing glia to correctly wrap peripheral axons. When the fray cDNA is expressed in the ensheathing glia of fray mutants, normal nerve morphology is restored. Fray belongs to a novel family of Ser/Thr kinases, the PF kinases, whose closest relatives are the PAK kinases. Rescue of the Drosophila mutant phenotype with PASK, the rat homolog of Fray, demonstrates a functional homology among these proteins and suggests that the Fray signaling pathway is widely conserved.

UnPAKing RUNX3 functions-Both sides of the coin.

PubMed

Kumar, Arun; Sundaram, Sandhya; Rayala, Suresh K; Venkatraman, Ganesh

2017-06-19

Post translational modifications of RUNX3 have been shown to play an important role in directing RUNX3 functions. In this review we highlight the phosphorylation dependent functions of RUNX3 as regulated by PAK1 and its implications on tumorigenesis.

OPTIMIZING THE PAKS METHOD FOR MEASURING AIRBORNE ACROLEIN

EPA Science Inventory

Airborne acrolein is produced from the combustion of fuel and tobacco and is of concern due to its potential for respiratory tract irritation and other adverse health effects. Active sampling with DNPH-coated solid sorbents has been widely used for sampling airborne carbonyls; ...

OPTIMIZING THE PAKS METHOD FOR MEASURING AIRBORNE ACROLEIN

EPA Science Inventory

Airborne acrolein is produced from the combustion of fuel and tobacco and is of concern due to its potential for respiratory tract irritation and other adverse health effects. DNPH active-sampling is a method widely used for sampling airborne aldehydes and ketones (carbonyls); ...

LIM Kinase, a Newly Identified Regulator of Presynaptic Remodeling by Rod Photoreceptors After Injury

PubMed Central

Wang, Weiwei; Townes-Anderson, Ellen

2015-01-01

Purpose Rod photoreceptors retract their axon terminals and develop neuritic sprouts in response to retinal detachment and reattachment, respectively. This study examines the role of LIM kinase (LIMK), a component of RhoA and Rac pathways, in the presynaptic structural remodeling of rod photoreceptors. Methods Phosphorylated LIMK (p-LIMK), the active form of LIMK, was examined in salamander retina with Western blot and confocal microscopy. Axon length within the first 7 hours and process growth after 3 days of culture were assessed in isolated rod photoreceptors treated with inhibitors of upstream regulators ROCK and p21-activated kinase (Pak) (Y27632 and IPA-3) and a direct LIMK inhibitor (BMS-5). Porcine retinal explants were also treated with BMS-5 and analyzed 24 hours after detachment. Because Ca2+ influx contributes to axonal retraction, L-type channels were blocked in some experiments with nicardipine. Results Phosphorylated LIMK is present in rod terminals during retraction and in newly formed processes. Axonal retraction over 7 hours was significantly reduced by inhibition of LIMK or its regulators, ROCK and Pak. Process growth was reduced by LIMK or Pak inhibition especially at the basal (axon-bearing) region of the rod cells. Combining Ca2+ channel and LIMK inhibition had no additional effect on retraction but did further inhibit sprouting after 3 days. In detached porcine retina, LIMK inhibition reduced rod axonal retraction and improved retinal morphology. Conclusions Thus structural remodeling, in the form of either axonal retraction or neuritic growth, requires LIMK activity. LIM kinase inhibition may have therapeutic potential for reducing pathologic rod terminal plasticity after retinal injury. PMID:26658506

Heat shock increases oxidative stress to modulate growth and physico-chemical attributes in diverse maize cultivars

NASA Astrophysics Data System (ADS)

Hussain, Iqbal; Ashraf, Muhammad Arslan; Rasheed, Rizwan; Iqbal, Muhammad; Ibrahim, Muhammad; Ashraf, Shamila

2016-10-01

The present investigation was conducted to appraise the physiochemical adjustments in contrasting maize cultivars, namely, PakAfgoi (tolerant) and EV-5098 (sensitive) subjected to heat shock. Seven-day-old seedlings were exposed to heat shock for different time intervals (1, 3, 6, 24, 48 and 72 h) and data for various physiochemical attributes determined to appraise time course changes in maize. After 72 h of heat shock, the plants were grown under normal conditions for 5 d and data for different growth attributes and photosynthetic pigments recorded. Exposure to heat shock reduced growth and photosynthetic pigments in maize cultivars. The plants exposed to heat shock for up to 3 h recovered growth and photosynthetic pigments when stress was relieved. A time course rise in the relative membrane permeability, hydrogen peroxide (H2O2) and malondialdehyde contents was recorded particularly in the EV-5098 indicating that heat shock-induced oxidative stress. Activities of different enzymatic antioxidants greatly altered due to heat shock. For instance, an increase in superoxide dismutase activity was recorded in both maize cultivars. The activity of ascorbate peroxidase was greater in Pak-Afgoi. However, the peroxidase and catalase activities were higher in plants of EV-5098. Heat shock caused a significant rise in the proline and decline in the total free amino acids. Overall, the performance of Pak-Afgoi was better in terms of having lesser oxidative damage and greater cellular levels of proline. The results suggested that oxidative stress indicators (relative membrane permeability, H2O2 and malondialdehyde) and proline can be used as markers for heat shock tolerant plants.

Effect of fetal exposure to bisphenol A on brain mediated by X-chromosome inactivation.

PubMed

Kumamoto, Takayuki; Oshio, Shigeru

2013-01-01

Recent studies have reported that bisphenol A (BPA) influences brain development in fetal exposure to mice. The X-chromosome codes many neurodevelopment-related genes leading to abnormal development, such as mental retardation and intellectual deficiency. For females, most of expressions of X-linked genes are regulated by X-chromosome inactivation (XCI), which occurs during fetal period, and this mechanism is regulated by Xist and its antisense, Tsix. To clarify the possibility of X-mediated effect as a mechanism of neurodevelopmental disorders by BPA, pregnant ICR mice were orally administered 0.02 or 50 mg/kg of BPA on gestational days 6 and 15. Postnatally at days 2, 4 and weeks 3 and 7, mRNA expression of XCI-regulating factors (Xist and Tsix), X-linked neurodevelopment-related genes (Fmr1, Gdi1, Nlgn3, Pak3 and Ophn1), and sexual differentiation-related genes (ERα, ERβ and AR) were examined in cerebrums of female pups. Anogenital distance (AGD) and serum estradiol were also examined. In the 50 mg/kg exposed-group, reduced Xist, Fmr1, Gdi1, Nlgn3, and Pak3 and increased Tsix were observed simultaneously. Moderately reduced Xist, Gdi1, Nlgn3 and Pak3 were observed at 0.02 mg/kg BPA. ERα, ERβ and AR expression changes, shortened AGDs and reduced estradiol levels were observed in each exposure group. Fetal exposure to BPA changed expression of XCI-regulating factors and may alter the expression levels of X-linked neurodevelopment-related genes disrupting the XCI mechanism and function. This X-mediated effect is considered one of the mechanisms of various BPA-induced neurodevelopmental disorders.

Inter-Rater Agreement of Pressure Ulcer Risk and Prevention Measures in the National Database of Nursing Quality Indicators(®) (NDNQI).

PubMed

Waugh, Shirley Moore; Bergquist-Beringer, Sandra

2016-06-01

In this descriptive multi-site study, we examined inter-rater agreement on 11 National Database of Nursing Quality Indicators(®) (NDNQI(®) ) pressure ulcer (PrU) risk and prevention measures. One hundred twenty raters at 36 hospitals captured data from 1,637 patient records. At each hospital, agreement between the most experienced rater and each other team rater was calculated for each measure. In the ratings studied, 528 patients were rated as "at risk" for PrU and, therefore, were included in calculations of agreement for the prevention measures. Prevalence-adjusted kappa (PAK) was used to interpret inter-rater agreement because prevalence of single responses was high. The PAK values for eight measures indicated "substantial" to "near perfect" agreement between most experienced and other team raters: Skin assessment on admission (.977, 95% CI [.966-.989]), PrU risk assessment on admission (.978, 95% CI [.964-.993]), Time since last risk assessment (.790, 95% CI [.729-.852]), Risk assessment method (.997, 95% CI [.991-1.0]), Risk status (.877, 95% CI [.838-.917]), Any prevention (.856, 95% CI [.76-.943]), Skin assessment (.956, 95% CI [.904-1.0]), and Pressure-redistribution surface use (.839, 95% CI [.763-.916]). For three intervention measures, PAK values fell below the recommended value of ≥.610: Routine repositioning (.577, 95% CI [.494-.661]), Nutritional support (.500, 95% CI [.418-.581]), and Moisture management (.556, 95% CI [.469-.643]). Areas of disagreement were identified. Findings provide support for the reliability of 8 of the 11 measures. Further clarification of data collection procedures is needed to improve reliability for the less reliable measures. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT, PAINT OVERSPRAY ARRESTOR, KOCH FILTER CORPORATION, DUO-PAK 650

EPA Science Inventory

Paint overspray arrestors (POAs) were evaluated by the Air Pollution Control Technology (APCT) pilot of the Environmental Technology Verification (ETV) Program. The performance factor verified was the particle filtration efficiency as a function of size for particles smaller than...

Simple method for determination of patulin production by Penicillium griseofulvum Dierckx.

PubMed Central

Torres, M; Sanchis, V; Riba, M; Canela, R

1986-01-01

Patulin production by Penicillium griseofulvum was monitored with Sep-Pak cartridges and high-pressure liquid chromatography. Determination and quantification of this metabolite proved to be very simple, and our method saved time and a large amount of organic solvents. PMID:3513700

A Robot to Help Make the Rounds

NASA Technical Reports Server (NTRS)

2003-01-01

This paper presents a discussion on the Pyxis HelpMate SecurePak (SP) trackless robotic courier designed by Transitions Research Corporation, to navigate autonomously throughout medical facilities, transporting pharmaceuticals, laboratory specimens, equipment, supplies, meals, medical records, and radiology films between support departments and nursing floors.

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT: PAINT OVERSPRAY ARRESTOR, AAF INTERNATIONAL DRI-PAK 40-45%

EPA Science Inventory

Paint overspray arrestors (POAs) were evaluated by the Air Pollution Control Technology (APCT) pilot of the Environmental Technology Verification (ETV) Program. The performance factor verified was the particle filtration efficiency as a function of size for particles smaller than...

Activation of Entorhinal Cortical Projections to the Dentate Gyrus Underlies Social Memory Retrieval.

PubMed

Leung, Celeste; Cao, Feng; Nguyen, Robin; Joshi, Krutika; Aqrabawi, Afif J; Xia, Shuting; Cortez, Miguel A; Snead, O Carter; Kim, Jun Chul; Jia, Zhengping

2018-05-22

Social interactions are essential to our mental health, and a deficit in social interactions is a hallmark characteristic of numerous brain disorders. Various subregions within the medial temporal lobe have been implicated in social memory, but the underlying mechanisms that tune these neural circuits remain unclear. Here, we demonstrate that optical activation of excitatory entorhinal cortical perforant projections to the dentate gyrus (EC-DG) is necessary and sufficient for social memory retrieval. We further show that inducible disruption of p21-activated kinase (PAK) signaling, a key pathway important for cytoskeletal reorganization, in the EC-DG circuit leads to impairments in synaptic function and social recognition memory, and, importantly, optogenetic activation of the EC-DG terminals reverses the social memory deficits in the transgenic mice. These results provide compelling evidence that activation of the EC-DG pathway underlies social recognition memory recall and that PAK signaling may play a critical role in modulating this process. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Laboratory preparation of DNPH derivatives of carbonyl compounds on Sep-Pak{reg_sign} cartridges for quality assurance purposes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harrell, R.M.

1994-12-31

Aldehydes and ketones are receiving increased attention both as hazardous substances and as promoters in the photochemical formation of ozone in the atmosphere. They enter the atmosphere in the exhaust of motor vehicles and other equipment using hydrocarbon and alcohol fuels. Formaldehyde, the most prevalent aldehyde, is widely used as a preservative, a textile-treatment agent, and an intermediate in the manufacture of urea-formaldehyde and phenol-formaldehyde resins. The formaldehyde concentration ranges for several types of environments are presented. Waters Sep-Pak{reg_sign} DNPH-Silica cartridges are convenient, reproducible sampling devices for quantifying aldehydes and ketones in gases, including air. These cartridges trap the compoundsmore » by reacting them with the DNPH, 2,4-Dinitrophenylhydrazine, on the cartridge to form stable hydrazone derivatives. Derivatives are later eluted and analyzed by HPLC. Cartridges spiked in the laboratory are used for quality assurance and instrument performance verification.« less

Myxoma and vaccinia viruses exploit different mechanisms to enter and infect human cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villa, Nancy Y.; Bartee, Eric; Mohamed, Mohamed R.

2010-06-05

Myxoma (MYXV) and vaccinia (VACV) viruses have recently emerged as potential oncolytic agents that can infect and kill different human cancer cells. Although both are structurally similar, it is unknown whether the pathway(s) used by these poxviruses to enter and cause oncolysis in cancer cells are mechanistically similar. Here, we compared the entry of MYXV and VACV-WR into various human cancer cells and observed significant differences: 1 - low-pH treatment accelerates fusion-mediated entry of VACV but not MYXV, 2 - the tyrosine kinase inhibitor genistein inhibits entry of VACV, but not MYXV, 3 - knockdown of PAK1 revealed that itmore » is required for a late stage event downstream of MYXV entry into cancer cells, whereas PAK1 is required for VACV entry into the same target cells. These results suggest that VACV and MYXV exploit different mechanisms to enter into human cancer cells, thus providing some rationale for their divergent cancer cell tropisms.« less

Separation properties of aluminium-plastic laminates in post-consumer Tetra Pak with mixed organic solvent.

PubMed

Zhang, S F; Zhang, L L; Luo, K; Sun, Z X; Mei, X X

2014-04-01

The separation properties of the aluminium-plastic laminates in postconsumer Tetra Pak structure were studied in this present work. The organic solvent blend of benzene-ethyl alcohol-water was used as the separation reagent. Then triangle coordinate figure analysis was taken to optimize the volume proportion of various components in the separating agent and separation process. And the separation temperature of aluminium-plastic laminates was determined by the separation time, efficiency, and total mass loss of products. The results show that cost-efficient separations perform best with low usage of solvents at certain temperatures, for certain times, and within a certain range of volume proportions of the three components in the solvent agent. It is also found that similar solubility parameters of solvents and polyethylene adhesives (range 26.06-34.85) are a key factor for the separation of the aluminium-plastic laminates. Such multisolvent processes based on the combined-system concept will be vital to applications in the recycling industry.

Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways.

PubMed

Li, Hui; Yang, Duxiao; Ning, Shanglei; Xu, Yinghui; Yang, Fan; Yin, Rusha; Feng, Taihu; Han, Shouqing; Guo, Lu; Zhang, Pengju; Qu, Wenjie; Guo, Renbo; Song, Chen; Xiao, Peng; Zhou, Chengjun; Xu, Zhigang; Sun, Jin-Peng; Yu, Xiao

2018-01-01

The protein tyrosine phosphatase nonreceptor type 12 (PTPN12) is a multifunctional protein and has elicited much research attention because its decreased protein level has been associated with poor prognosis of several types of cancers. Recently, we have solved the crystal structure of the phosphatase domain of PTPN12, which disclosed a specific PTPN12-insert-loop harboring a cyclin-dependent kinase 2 (CDK2) phosphorylation site. However, the functional significance of this phosphorylation is undefined. In the present study, we found that S19 site phosphorylation of PTPN12 by CDK2 discharged its antitumor activity by down-regulation of its inhibitory role in cell migration, but not affecting its other regulatory functions. Phosphorylation of PTPN12 at the S19 site changed its substrate interface, and by doing so, selectively decreased its activity toward the human epidermal growth factor receptor 2 (HER2)- pY 1196 site, but not other HER2 phosphorylation sites or other known PTPN12 substrates. A further in-depth mechanism study revealed that the phosphorylation of PTPN12 by CDK2 impaired recruitment of the serine/threonine-protein kinase 1 (PAK1) to HER2, resulted in the blockade of the HER2-pY 1196 -PAK1-T 423 signaling pathway, thus increased tumor cell motility. Taken together, our results identified a new phosphorylation-based substrate recognition mechanism of PTPN12 by CDK2, which orchestrated signaling crosstalk between the oncogenic CDK2 and HER2 pathways. The newly identified governing mechanism of the substrate selectivity of a particular phosphatase was previously unappreciated and exemplifies how a phospho-network is precisely controlled in different cellular contexts.-Li, H., Yang, D., Ning, S., Xu, Y., Yang, F., Yin, R., Feng, T., Han, S., Guo, L., Zhang, P., Qu, W., Guo, R., Song, C., Xiao, P., Zhou, C., Xu, Z., Sun, J.-P., Yu, X. Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways. © FASEB.

[Mental disturbances in children and adolescents in Germany. Results of a representative study:age,gender and rater effects].

PubMed

Döpfner, M; Plück, J; Berner, W; Fegert, J M; Huss, M; Lenz, K; Schmeck, K; Lehmkuhl, U; Poustka, F; Lehmkuhl, G

1997-12-01

A study on behavioral and emotional problems and competence in children and adolescents in Germany (PAK-KID study) is described. It is the first nationwide representative survey of this kind of children and adolescents aged 4 to 18 years in Germany. For children aged 4 to 10 years the parents completed the German version of Achenbach's Child Behavior Checklist (CBCL 4-18) developed by the Arbeitsgruppe Deutsche Child Behavior Checklist. Children and adolescents aged 11 years and older filled out the German version of the Youth Self-Report that is part of Achenbach's CBCL in addition to the parents completing the German version of the CBCL. A total of 2856 parent questionnaires and 1798 self-report questionnaires completed by children and adolescents were analyzed. The sample was representative with respect to the main sociodemographic variables. On all problem scales children and adolescents aged 11 to 18 years reported significantly more problems than their parents did. The frequency of internalizing problems (social withdrawal, somatic complaints, anxiety/depression) and delinquent behavior of children and adolescents reported by parents increased with the children's age, whereas aggressive behavior and attention problems decreased with age. Girls reported significantly more problems than boys on all internalizing scales of the Youth Self-Report. The effect was not totally replicated in the parent reports. In the parent reports, boys had more attention problems and more aggressive and delinquent behavior than girls.

UTILIZING THE PAKS METHOD FOR MEASURING ACROLEIN AND OTHER ALDEHYDES IN DEARS

EPA Science Inventory

Acrolein is a hazardous air pollutant of high priority due to its high irritation potency and other potential adverse health effects. However, a reliable method is currently unavailable for measuring airborne acrolein at typical environmental levels. In the Detroit Exposure and A...

Pancreas Transplantation of US and Non-US Cases from 2005 to 2014 as Reported to the United Network for Organ Sharing (UNOS) and the International Pancreas Transplant Registry (IPTR)

PubMed Central

Gruessner, Angelika C.; Gruessner, Rainer W.G.

2016-01-01

This report is an update of pancreas and kidney transplant activities in the US and non-US region in two periods, 2005-2009 and 2010-2014. The aim of the report was to analyze transplant progress and success in the US compared to non-US countries, and to compare trends between the two periods. Between 2005-2009 and 2010-2014, the number of US pancreas transplants declined by over 20%, while the overall number of pancreas transplants performed outside the US has increased. The decline in US numbers is predominantly due to the decline in primary and secondary pancreas after kidney transplants (PAK). During the time period studied, the number of PAK transplants dropped by 50%. In contrast, the number of simultaneous pancreas/kidney transplants (SPK) declined by only 10%, and the number of pancreas transplants alone (PTA) by 20%. Over 90% of pancreas transplants worldwide were performed, with a simultaneous kidney transplant and excellent results. Transplant outcomes in SPK improved significantly because of a decrease in the rates of technical and immunologic graft loss. In 2010-2014 vs. 2005-2009, US SPK transplant patient survival at 1 year post-transplant increased from 95.7% to 97.4%, pancreas graft function from 88.3% to 91.3%, and kidney function from 93.6% to 95.5%. A significant improvement was also noted in PAK transplants. One-year patient survival increased from 96.4% to 97.9% and pancreas graft function from 81.0% to 86.0%. PTA 1-year patient survival remained constant at 97%, and pancreas 1-year graft survival improved from 81.0% to 85.7%. With the decline in the number of transplants, a change towards better pancreas donor selection was observed. In solitary transplants, the donors were primarily young trauma victims, and the pancreas preservation time was relatively short. A general tendency towards transplanting older recipients was noted. In 2010-2014 vs. 2005-2009, PTA recipients 50 years of age or older accounted for 32% vs. 22%, PAK for 28% vs. 22%, and SPK for 22% vs. 20%. This may be due to a relatively lower immunologic graft loss rate, especially in solitary transplants, which historically has been high in young recipients. The number of pancreas transplants in patients with type 2 diabetes and end-stage renal disease has increased, and accounted for 9% of all SPK recipients in 2010-2014. PMID:26982345

A Stability-Indicating HPLC Method for the Determination of Fingolimod in Pharmaceutical Dosage Forms.

PubMed

Souri, Effat; Zargarpoor, Mohammad; Mottaghi, Siavash; Ahmadkhaniha, Reza; Kebriaeezadeh, Abbas

2015-01-01

Fingolimod is an immunosuppressive agent which is used for the prophylaxis of organ transplantation rejection or multiple sclerosis treatment. In this study, systematic forced degradation studies on fingolimod bulk powder were performed to develop a stability-indicating HPLC method. Separation of fingolimod and its degradation products was achieved on a Nova-Pak C8 column. The mobile phase was a mixture of potassium dihydrogenphosphate 50 mM (pH 3.0) and acetonitrile (45:55, v/v) at a flow rate of 1 ml/min. The proposed method was linear in the range of 0.125-20 μg mL(-1). The within-day and between-day coefficients of variation were in the range of 0.6-1.2%. The developed method was successfully applied for the determination of the fingolimod amount in pharmaceutical dosage forms.

78 FR 1718 - Approved Tests for Bovine Tuberculosis in Cervids

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-09

.... APHIS-2012-0087] Approved Tests for Bovine Tuberculosis in Cervids AGENCY: Animal and Plant Health... Stat-Pak[supreg] and DPP[supreg] tests as official tuberculosis tests for the following species of... because we have determined that the tests can reliably detect the presence or absence of antibodies to...

The Pak-U.S. Alliance in the Fight Against Terrorism: A Cost-Benefit Analysis

DTIC Science & Technology

2011-12-01

Micro-Alliance, Entrepreneurship , Game Theoretical Approach, Perceptions and Realities, Military-Centric Assistance, Population-Centric Approach...40 4. Entrepreneurship and Micro-Alliances ...........................................41 E. SUMMARY...through educational reforms, trade and employment ( entrepreneurship ). 6 6. Hypothesis-3: Promoting converging interests/common goals and removing

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT: PAINT OVERSPRAY ARRESTOR, AAF INTERNATIONAL DRIPAK 90-95%

EPA Science Inventory

The report gives results of March 26-29, 1999, tests of AAF International's DriPak 90-95% paint overspray arrestor (POA) as part of an evaluation of POAs by EPA's Air Pollution Control Technology (APCT) Environmental Technology Verification (ETV) Program. The basic performance fa...

Chromatographic method for the determination of diazepam, pyridostigmine bromide, and their metabolites in rat plasma and urine.

PubMed

Abu-Qare, A W; Abou-Donia, M B

2001-04-25

This study describes a chromatographic method for the determination of diazepam, an anxiolytic drug that is also used as an antidote against nerve agent seizures, its metabolites N-desmethyldiazepam, and temazepam, the anti-nerve agent drug pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) and its metabolite N-methyl-3-hydroxypyridinium bromide in rat plasma and urine. The compounds were extracted using C18 Sep-Pak Vac 3cc (500 mg) cartridges and separated using isocratic mobile phase of methanol, acetonitrile and water (pH 3.2) (10:40:50) at a flow-rate of 0.5 ml/min in a period of 12 min, and UV detection ranging between 240 and 280 nm. The limits of detection for all analytes ranged between 20 and 50 ng/ml, while limits of quantitation were 100 ng/ml. Average percentage extraction recoveries of five spiked plasma samples were 79.1+/-7.7, 83.5+/-6.4, 83.9+/-5.9, 71.3+/-6.0 and 77.7+/-5.6, and from urine 79.4+/-7.9, 83.1+/-6.9, 73.6+/-7.7, 74.3+/-7.1 and 77.6+/-5.9 for diazepam, N-desmethyldiazepam, temazepam, pyridostigmine bromide, and N-methyl-3-hydroxypyridinium bromide, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 1000 ng/ml. This method was applied to determine the above analytes following a single oral administration in rats as a tool to study the pharmacokinetic profile of each compound, alone and in combination.

Quantitative determination and sampling of azathioprine residues for cleaning validation in production area.

PubMed

Fazio, Tatiana Tatit; Singh, Anil Kumar; Kedor-Hackmann, Erika Rosa Maria; Santoro, Maria Inês Rocha Miritello

2007-03-12

Cleaning validation is an integral part of current good manufacturing practices in any pharmaceutical industry. Nowadays, azathioprine and several other pharmacologically potent pharmaceuticals are manufactured in same production area. Carefully designed cleaning validation and its evaluation can ensure that residues of azathioprine will not carry over and cross contaminate the subsequent product. The aim of this study was to validate simple analytical method for verification of residual azathioprine in equipments used in the production area and to confirm efficiency of cleaning procedure. The HPLC method was validated on a LC system using Nova-Pak C18 (3.9 mm x 150 mm, 4 microm) and methanol-water-acetic acid (20:80:1, v/v/v) as mobile phase at a flow rate of 1.0 mL min(-1). UV detection was made at 280 nm. The calibration curve was linear over a concentration range from 2.0 to 22.0 microg mL(-1) with a correlation coefficient of 0.9998. The detection limit (DL) and quantitation limit (QL) were 0.09 and 0.29 microg mL(-1), respectively. The intra-day and inter-day precision expressed as relative standard deviation (R.S.D.) were below 2.0%. The mean recovery of method was 99.19%. The mean extraction-recovery from manufacturing equipments was 83.5%. The developed UV spectrophotometric method could only be used as limit method to qualify or reject cleaning procedure in production area. Nevertheless, the simplicity of spectrophotometric method makes it useful for routine analysis of azathioprine residues on cleaned surface and as an alternative to proposed HPLC method.

The effect of American cranberry (Vaccinium macrocarpon) constituents on the growth inhibition, membrane integrity, and injury of Escherichia coli O157:H7 and Listeria monocytogenes in comparison to Lactobacillus rhamnosus.

PubMed

Lacombe, Alison; McGivney, Christine; Tadepalli, Shravani; Sun, Xiaohong; Wu, Vivian C H

2013-06-01

The antimicrobial properties of the American cranberry were studied against Escherichia coli O157:H7, Listeria monocytogenes, and Lactobacillus rhamnosus to determine the effects on growth inhibition, membrane permeability, and injury. Cranberry powder was separated using a C-18 Sep-Pak cartridge into sugars plus organic acids (F1), monomeric phenolics (F2), and anthocyanins plus proanthocyanidins (F3). Fraction 3 was further separated into anthocyanins (F4) and proanthocyanidins (F5) using an LH-20 Sephadex column. Each fraction was diluted in the brain heart infusion (BHI) broth to determine the minimum inhibitory/bactericidal concentrations (MIC/MBC). L. monocytogenes was the most susceptible to cranberry fraction treatment with the lowest MIC/MBC for each treatment, followed by E. coli O157:H7 and L. rhamnosus. Membrane permeability and potential was studied using LIVE/DEAD viability assay and using Bis (1, 3-dibutylbarbituric acid) trimethine oxonol (DiBAC4), respectively. L. rhamnosus demonstrated the highest permeability followed by E. coli O157:H7, and L. monocytogenes. L. rhamnosus demonstrated the highest recovery followed by E. coli O157:H7, and L. monocytogenes. Each cranberry fraction demonstrated membrane hyperpolarization at their native pH, while F2, F3, and F5 demonstrated membrane depolarization at neutral pH. With this knowledge cranberry compounds may be used to prevent maladies and potentially substitute for synthetic preservatives and antibiotics. Copyright © 2013 Elsevier Ltd. All rights reserved.

Spectrofluorometry, thin layer chromatography, and column high-performance liquid chromatography determination of rabeprazole sodium in the presence of its acidic and oxidized degradation products.

PubMed

Osman, Afaf Osman; Osman, Afaf; Osman, Mohamed

2009-01-01

The objective of this study is to develop validated stability-indicating spectrofluorometric, TLC-densitometric, and HPLC methods for the determination of rabeprazole sodium and its degradation products. The first method was based on measuring the fluorescence intensity of the drug at 416 and 311 nm for the emission and at 320 and 274 nm for the excitation for acid and oxidized solutions, respectively. The second method was based on the separation of the drug from its acidic and oxidized degradation products followed by densitometric measurement of the intact drug spot at 284 nm. The separation was carried out on Fluka TLC sheets of silica gel 60 F254 using isopropyl alcohol--30% ammonia (80 + 2, v/v) mobile phase. The third method was based on HPLC separation of rabeprazole sodium from its acidic and oxidized degradation products on a reversed-phase Waters Nova-Pak C18 column using 0.05 M potassium dihydrogen phosphate-methanol-acetonitrile (5 + 3 + 2, v/v/v) pH 7 +/- 0.2 mobile phase. The proposed procedures were successfully applied for the determination of rabeprazole sodium in pure form, laboratory-prepared mixtures, tablet, and expired batch. The obtained results were statistically compared with those of a reported method and validated according to United States Pharmacopeia guidelines. Two main acidic degradation products of the drug were separated and subjected to IR spectrometry and MS to confirm their structures, and the schemes for their formation were elucidated.

Micellar liquid chromatographic determination of arbutin and hydroquinone in medicinal plant extracts and commercial cosmetic products.

PubMed

Thogchai, W; Liawruangrath, B

2013-06-01

A simple micellar liquid chromatographic (MLC) procedure for simultaneous determination of arbutin and hydroquinone in medicinal plant extracts and commercial cosmetic products was proposed. This method was developed and validated. The chromatographic conditions were also optimized. All analyses were performed at room temperature in an isocratic mode, using a mixture of 1% (v/v) acetonitrile and 0.006 mol L⁻¹ Brij 35 (pH 6.0) as a mobile phase. The flow rate was set at 1.0 mL min⁻¹. The analytical column was a 150 × 3.9 mm Nova-Pak C-18 column. The effluent from the analytical column was monitored by UV detection at 280 nm. Under the optimum conditions, arbutin and hydroquinone could be determined within a concentration range of 2-50 μg mL⁻¹ of arbutin, and hydroquinone was obtained with the regression equations; y = 0.045x + 0.042 (r² = 0.9923) and y = 0.091x + 0.050 (r² = 0.9930) respectively. The limits of detection were found to be 0.51 μg mL⁻¹ and 0.37 μg mL⁻¹ for arbutin and hydroquinone respectively. The proposed MLC method was applied for the determination of arbutin and hydroquinone contents in medicinal plant extracts and commercial cosmetic products. This proposed MLC method is thus suitable for routine analysis of arbutin and hydroquinone in the pharmaceutical formulations, cosmetic products and raw medicinal plant extracts. ICS © 2013 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Chemical composition and radical scavenging activity of essential oil and methanolic extract of Eremostachys azerbaijanica Rech.f. from Iran.

PubMed

Asnaashari, Solmaz; Afshar, Fariba Heshmati; Ebrahimi, Atefeh; Moghadam, Sedigheh Bamdad; Delazar, Abbas

2016-01-01

In the present study, the chemical composition of the essential oil and methanol (MeOH) extract of aerials of E. azerbaijanica were identified. Furthermore, the free radical scavenging properties of the volatile oil as well as the MeOH extract of the plant were assessed. The essential oil of the air-dried aerial parts was obtained by hydro-distillation using a Clevenger-type apparatus. The oil was then analyzed by gas chromatography-mass spectrometry and gas chromatography with flame ionization detector. Soxhlet extraction was performed on the aerial parts using n-hexane, dichloromethane and MeOH. The MeOH extract was then subjected to solid-phase extraction using a C18 Sep-Pak cartridge. Isolation and structural elucidation of the pure components was accomplished by high-performance liquid chromatography and spectroscopic methods (UV, (1)H-NMR). The free radical scavenging properties were determined by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay. A total of 59 components representing 95.9% of the oil constituents were identified which were primarily characterized as terpenoids or aliphatic skeletons. The major components of the oil were hexahydrofarnesyl acetone (27.1%), 2-methyl-6-propyl-dodecane (16.4%) and tricosane (9.3%). One flavonoid (luteolin-7-O-rutinoside) and one phenylethanoid (verbascoside) were also isolated and identified from the MeOH extract. The results of DPPH assays showed that the essential oil of E. azerbaijanica possessed weak free radical scavenging activity whereas the MeOH extract and its pure constituents showed significant scavenging activities in comparison with positive controls.

Complete genome sequence of a recent panzootic virulent Newcastle disease virus from Pakistan

USDA-ARS?s Scientific Manuscript database

Complete genome sequence of a new strain of Newcastle disease virus (NDV) (chicken/Pak/Lahore-611/2013) is reported. The strain was isolated from a vaccinated chicken flock in Pakistan in 2013 and has panzootic features. The genome is 15192 nucleotides in length and is classified as sub-genotype V...

Working with Children to Protect Our Future

ERIC Educational Resources Information Center

Williams, Nick

2011-01-01

The author has been teaching primary school children for 20 years, and has always been passionate about teaching young people about the environment. In this article, he describes his work with Tetra Pak and WWF-UK to develop a national, school-based competition and teaching programme to help children understand the importance of using renewable…

Field application of serodiagnostics to identify elephants with Tuberculosis prior to case confirmation by culture

USDA-ARS?s Scientific Manuscript database

Three serologic methods for antibody detection in elephant tuberculosis (TB), multiantigen print immunoassay (MAPIA), ElephantTB STAT-PAK kit, and DPP VetTB test, were validated prospectively using serial serum samples from 14 captive elephants in 5 countries which were diagnosed with TB by positive...

ENVIRONMENTAL TECHNOLOGY VERIFICATON: TEST REPORT OF CONTROL OF BIOAEROSOLS IN HVAC SYSTEMS AAF INTERNATIONAL DRIPAK 90/95%

EPA Science Inventory

The Environmental Technology Verification report discusses the technology and performance of the DriPak 90/95% air filter for dust and bioaerosol filtration manufactured by AAF International. The pressure drop across the filter was 104 Pa clean and 348 Pa dust loaded, and the fil...

77 FR 2124 - Notice of Delays in Processing of Special Permits Applications

Federal Register 2010, 2011, 2012, 2013, 2014

2012-01-13

... Alsworth, AK. 10656-M Conference of 4 03-31-2012 Radiation Control Program Directors, Inc., Frankfort, KY... Control Program Directors, Inc., Frankfort, KY. 10898-M Hydac 3 03-31-2012 Corporation, Bethlehem, PA... Berlin Packaging (Former Grantee All-Pak, Inc.), Bridgeville, PA. 8826-M Phoenix Air 4 03-31-2012 Group...

Autism-like Deficits in Shank3-Deficient Mice Are Rescued by Targeting Actin Regulators.

PubMed

Duffney, Lara J; Zhong, Ping; Wei, Jing; Matas, Emmanuel; Cheng, Jia; Qin, Luye; Ma, Kaijie; Dietz, David M; Kajiwara, Yuji; Buxbaum, Joseph D; Yan, Zhen

2015-06-09

Haploinsufficiency of the Shank3 gene, which encodes a scaffolding protein at glutamatergic synapses, is a highly prevalent and penetrant risk factor for autism. Using combined behavioral, electrophysiological, biochemical, imaging, and molecular approaches, we find that Shank3-deficient mice exhibit autism-like social deficits and repetitive behaviors, as well as the significantly diminished NMDA receptor (NMDAR) synaptic function and synaptic distribution in prefrontal cortex. Concomitantly, Shank3-deficient mice have a marked loss of cortical actin filaments, which is associated with the reduced Rac1/PAK activity and increased activity of cofilin, the major actin depolymerizing factor. The social deficits and NMDAR hypofunction are rescued by inhibiting cofilin or activating Rac1 in Shank3-deficient mice and are induced by inhibiting PAK or Rac1 in wild-type mice. These results indicate that the aberrant regulation of synaptic actin filaments and loss of synaptic NMDARs contribute to the manifestation of autism-like phenotypes. Thus, targeting actin regulators provides a strategy for autism treatment. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Rac1/Pak1/p38/MMP-2 axis regulates angiogenesis in ovarian cancer

PubMed Central

Gonzalez-Villasana, Vianey; Fuentes-Mattei, Enrique; Ivan, Cristina; Dalton, Heather J.; Rodriguez-Aguayo, Cristian; Fernandez-de Thomas, Ricardo J.; Aslan, Burcu; Monroig, Paloma del C.; Velazquez-Torres, Guermarie; Previs, Rebecca A.; Pradeep, Sunila; Kahraman, Nermin; Wang, Huamin; Kanlikilicer, Pinar; Ozpolat, Bulent; Calin, George; Sood, Anil K.; Lopez-Berestein, Gabriel

2015-01-01

Purpose Zoledronic acid (ZA) is being increasingly recognized for its anti-tumor properties, but the underlying functions are not well understood. In this study, we hypothesized that ZA inhibits ovarian cancer (OC) angiogenesis preventing Rac1 activation. Experimental Design The biological effects of ZA were examined using a series of in vitro (cell invasion, cytokine production, Rac1 activation, reverse-phase protein array and in vivo (orthotopic mouse models) experiments. Results There was significant inhibition of OC (HeyA8-MDR and OVCAR-5) cell invasion as well as reduced production of pro-angiogenic cytokines in response to ZA treatment. Furthermore, ZA inactivated Rac1 and decreased the levels of Pak1/p-38/matrix metalloproteinase-2 in OC cells. In vivo, ZA reduced tumor growth, angiogenesis and cell proliferation and inactivated Rac1 in both HeyA8-MDR and OVCAR-5 models. These in vivo antitumor effects were enhanced in both models when ZA was combined with nab-paclitaxel. Conclusion ZA has robust anti-tumor and anti-angiogenic activity and merits further clinical development as OC treatment. PMID:25595279

The effects of LED illumination spectra and intensity on carotenoid content in Brassicaceae microgreens.

PubMed

Brazaitytė, Aušra; Sakalauskienė, Sandra; Samuolienė, Giedrė; Jankauskienė, Julė; Viršilė, Akvilė; Novičkovas, Algirdas; Sirtautas, Ramūnas; Miliauskienė, Jurga; Vaštakaitė, Viktorija; Dabašinskas, Laurynas; Duchovskis, Pavelas

2015-04-15

The objective of this study was to evaluate the effects of irradiance levels and spectra produced by solid-state light-emitting diodes (LEDs) on carotenoid content and composition changes in Brassicaceae microgreens. A system of five high-power, solid-state lighting modules with standard 447-, 638-, 665-, and 731-nm LEDs was used in the experiments. Two experiments were performed: (1) evaluation of LED irradiance levels of 545, 440, 330, 220, and 110 μmol m(-2) s(-1) photosynthetically active flux density (PPFD) and (2) evaluation of the effects of 520-, 595-, and 622-nm LEDs supplemental to the standard set of LEDs. Concentrations of various carotenoids in red pak choi and tatsoi were higher under illumination of 330-440 μmol m(-2) s(-1) and at 110-220 μmol m(-2) s(-1) in mustard. All supplemental wavelengths increased total carotenoid content in mustard but decreased it in red pak choi. Carotenoid content increased in tatsoi under supplemental yellow light. Copyright © 2014 Elsevier Ltd. All rights reserved.

Separation and quantitative determination of 6α-hydroxycortisol and 6β-hydroxycortisol in human urine by high-performance liquid chromatography with ultraviolet absorption detection.

PubMed

Shibasaki, Hiromi; Okamoto, Sawako; Inoue, Risako; Okita, Misato; Yokokawa, Akitomo; Furuta, Takashi

2012-03-01

The present study developed an high-performance liquid chromatography (HPLC) method for the simultaneous determination of urinary metabolites of endogenous cortisol, 6α-hydroxycortisol (6α-OHF) and 6β-hydroxycortisol (6β-OHF), in human urine, using 6α-hydroxycorticosterone as internal standard. 6α-OHF and 6β-OHF were extracted from urine with ethyl acetate by using a Sep-Pak C(18) plus cartridge. Separation of the stereoisomers was achieved on a reversed-phase hybrid column by a gradient elution of (A) 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH (pH 3.77) and (B) 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH/acetonitrile (2:3, v/v). 6α-OHF and 6β-OHF were well separated on an XTerra MS C(18) 5 μm column using two types of stepwise gradient elution program (programs 2 and 3). Resolutions of 6α-OHF and 6β-OHF were Rs = 4.41 for program 2 and Rs = 4.60 for program 3. The analysis was performed within 23~26 min, monitored by UV absorbance at 239 nm. The lower limits of detection of 6α-OHF and 6β-OHF were 0.80 ng per injection (s/n = ca. 8), and the lower limits of quantification were 5.02 ng/ml for 6α-OHF and 41.08 ng/ml for 6β-OHF, respectively. The within-day reproducibilities in the amounts of 6α-OHF and 6β-OHF determined were in good agreement with the actual amounts added, the relative errors being -5.37% and -3.73% (gradient 2) and -5.69% and -3.96% (gradient 3) for both 6α-OHF and 6β-OHF, respectively. The inter-assay precisions (RSDs) for 6α-OHF and 6β-OHF were less than 1.99% (gradient 2) and 2.61% (gradient 3), respectively. The present HPLC method was applied to the measurement of 6α-OHF and 6β-OHF in urine to evaluate the time courses of 6α-hydroxylation and 6β-hydroxylation clearances of cortisol during 40 days for phenotyping CYP3A in a healthy subject.

Purification and partial characterization of an early pregnancy factor-induced suppressor factor (EPF-S1).

PubMed

Rolfe, B A; Athanasas-Platsis, S; Hoskin, M J; Morton, H; Cavanagh, A C

1995-06-01

The immunomodulatory properties of early pregnancy factor (EPF) are mediated through induction of at least two lymphokines, designated EPF-S1 and EPF-S2 (previously estimated M(r) 15,000 and 55,000 respectively). The activity of the former is MHC-restricted while the latter is restricted to a locus (or loci) outside the MHC. The present study established further criteria by which EPF-S1 and EPF-S2 might be distinguished from each other and compared with other suppressor factors. In addition, techniques have been developed to purify EPF-S1 to homogeneity. Congenic mouse strains were used to map the genetic restriction of EPF-S2 in the rosette inhibition test and high performance gel permeation chromatography was used to demonstrate that EPF-S1 induces EPF-S2 but not vice versa. Further studies then focused on isolation of this first component of the cascade, EPF-S1, from immune ascites (from growth in athymic mice of the anti-EPF-S1 producing rat-mouse hybridoma R2T gamma, in which EPF-S1 is complexed to antibody). Techniques used were acidification followed by application to Sep-pak C18 cartridges, high performance cation-exchange chromatography and two reversed-phased HPLC steps on a C3 column. Purified material was analyzed by SDS-PAGE and Edman degradation. Approximately 10 micrograms EPF-S1 were isolated fom 60 ml ascitic fluid. Homogeneity of the purified material was demonstrated by SDS-PAGE, where it ran as a single band of approximate M(r) 12,000 coincident with biological activity. Attempts at Edman degradation indicate that the molecule is N-blocked. Definitive primary characterization of EPF-S1 must await the preparation and isolation of proteolytic fragments of the molecule, but the present studies establish conditions which make such structural analysis possible.

Highly efficient enrichment of low-abundance intact proteins by core-shell structured Fe3O4-chitosan@graphene composites.

PubMed

Zhang, Peng; Fang, Xiaoni; Yan, Guoquan; Gao, Mingxia; Zhang, Xiangmin

2017-11-01

In proteomics research, the screening and monitoring of disease biomarkers is still a major challenge, mainly due to their low concentration in biological samples. However, the universal enrichment of intact proteins has not been further studied. In this work, we developed a Fe 3 O 4 -chitosan@graphene (Fe 3 O 4 -CS@G) core-shell composite to enrich low-abundance proteins from biological samples. Fe 3 O 4 -CS@G composite holds chitosan layer decorated Fe 3 O 4 core, which improves the hydrophilicity of materials greatly. Meanwhile, the graphene nanosheets shell formed via electrostatic assembly endows the composite with huge surface area (178m 2 /g). The good water dispersibility ensures the sufficient contact opportunities between graphene composites and proteins, and the large surface area provides enough adsorption sites for the enrichment of proteins. Using Fe 3 O 4 -CS@G, four standard proteins Cyt-c, BSA, Myo and OVA were enriched with better adsorption capacity and recovery rate, compared with previously reported magnetic graphene composites. Additionally, the mechanism of compared to" is corrected into "compared with". proteins adsorption on Fe 3 O 4 -CS@G was further studied, which indicates that hydrophobic and electrostatic interaction work together to facilitate the universal and efficient enrichment of proteins. Human plasma sample was employed to further evaluate the enrichment performance of Fe 3 O 4 -CS@G. Eventually, 123 proteins were identified from one of SAX fractions of human plasma, which is much better than commercial Sep-pak C18 enrichment column (39 proteins). All these outstanding performances suggest that Fe 3 O 4 -CS@G is an ideal platform for the enrichment of low-abundance intact proteins and thus holds great potential to facilitate the identification of biomarkers from biological samples in proteomics research. Copyright © 2017 Elsevier B.V. All rights reserved.

A liquid chromatography with tandem mass spectrometry method for simultaneous determination of UTL-5g and its metabolites in human plasma.

PubMed

Shaw, Jiajiu; Wiegand, Richard; Wu, Jianmei; Bao, Xun; Valeriote, Frederick; Li, Jing

2015-06-01

UTL-5g is a novel small-molecule TNF-α inhibitor under investigation as both a chemoprotective and radioprotective agent. Animal studies showed that pretreatment of UTL-5g protected kidney, liver, and platelets from cisplatin-induced toxicity. In addition, UTL-5g reduced liver and lung injuries induced by radiation in vivo. Although a number of preclinical studies have been conducted, a validated bioanalytical method for UTL-5g in human plasma has not been published. In this work, a sensitive and reproducible reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the determination of UTL-5g and its metabolites, 5-methylisoxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA), in human plasma. The method involves a simple methanol precipitation step followed by injection of the supernatant onto a Waters 2695 HPLC system coupled with a Waters Quattro Micro™ triple quadrupole mass spectrometer. Chromatographic separation was accomplished using a Waters Nova-Pak C18 column maintained at 30°C, running at gradient mode with mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in methanol at a flow rate of 0.2mL/min. The analytes were monitored under positive electrospray ionization (ESI). Quantitation of these compounds in plasma was linear from 0.05 to 10μM. The lower limit of quantitation (LLOQ) was 0.05, 0.1, and 0.2μM for UTL-5g, ISOX and DCA, respectively. The accuracy and intra-and inter-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method provides a practical tool to measure and characterize the plasma concentration-time profiles for UTL-5g and its metabolites, ISOX and DCA. Copyright © 2015 Elsevier B.V. All rights reserved.

Cytoskeletal role in protection of the failing heart by β-adrenergic blockade

PubMed Central

Cheng, Guangmao; Kasiganesan, Harinath; Baicu, Catalin F.; Wallenborn, J. Grace; Kuppuswamy, Dhandapani

2012-01-01

Formation of a dense microtubule network that impedes cardiac contraction and intracellular transport occurs in severe pressure overload hypertrophy. This process is highly dynamic, since microtubule depolymerization causes striking improvement in contractile function. A molecular etiology for this cytoskeletal alteration has been defined in terms of type 1 and type 2A phosphatase-dependent site-specific dephosphorylation of the predominant myocardial microtubule-associated protein (MAP)4, which then decorates and stabilizes microtubules. This persistent phosphatase activation is dependent upon ongoing upstream activity of p21-activated kinase-1, or Pak1. Because cardiac β-adrenergic activity is markedly and continuously increased in decompensated hypertrophy, and because β-adrenergic activation of cardiac Pak1 and phosphatases has been demonstrated, we asked here whether the highly maladaptive cardiac microtubule phenotype seen in pathological hypertrophy is based on β-adrenergic overdrive and thus could be reversed by β-adrenergic blockade. The data in this study, which were designed to answer this question, show that such is the case; that is, β1- (but not β2-) adrenergic input activates this pathway, which consists of Pak1 activation, increased phosphatase activity, MAP4 dephosphorylation, and thus the stabilization of a dense microtubule network. These data were gathered in a feline model of severe right ventricular (RV) pressure overload hypertrophy in response to tight pulmonary artery banding (PAB) in which a stable, twofold increase in RV mass is reached by 2 wk after pressure overloading. After 2 wk of hypertrophy induction, these PAB cats during the following 2 wk either had no further treatment or had β-adrenergic blockade. The pathological microtubule phenotype and the severe RV cellular contractile dysfunction otherwise seen in this model of RV hypertrophy (PAB No Treatment) was reversed in the treated (PAB β-Blockade) cats. Thus these data provide both a specific etiology and a specific remedy for the abnormal microtubule network found in some forms of pathological cardiac hypertrophy. PMID:22081703

A Stability-Indicating HPLC Method for the Determination of Fingolimod in Pharmaceutical Dosage Forms

PubMed Central

Souri, Effat; Zargarpoor, Mohammad; Mottaghi, Siavash; Ahmadkhaniha, Reza; Kebriaeezadeh, Abbas

2015-01-01

Fingolimod is an immunosuppressive agent which is used for the prophylaxis of organ transplantation rejection or multiple sclerosis treatment. In this study, systematic forced degradation studies on fingolimod bulk powder were performed to develop a stability-indicating HPLC method. Separation of fingolimod and its degradation products was achieved on a Nova-Pak C8 column. The mobile phase was a mixture of potassium dihydrogenphosphate 50 mM (pH 3.0) and acetonitrile (45:55, v/v) at a flow rate of 1 ml/min. The proposed method was linear in the range of 0.125–20 μg mL−1. The within-day and between-day coefficients of variation were in the range of 0.6–1.2%. The developed method was successfully applied for the determination of the fingolimod amount in pharmaceutical dosage forms. PMID:26839803

Effect of non-thermal air atmospheric pressure plasma jet treatment on gingival wound healing

NASA Astrophysics Data System (ADS)

Lee, Jung-Hwan; Choi, Eun-Ha; Kim, Kwang-Mahn; Kim, Kyoung-Nam

2016-02-01

Non-thermal atmospheric pressure plasmas have been applied in the biomedical field for the improvement of various cellular activities. In dentistry, the healing of gingival soft tissue plays an important role in health and aesthetic outcomes. While the biomedical application of plasma has been thoroughly studied in dentistry, a detailed investigation of plasma-mediated human gingival fibroblast (HGF) migration for wound healing and its underlying biological mechanism is still pending. Therefore, the aim of this study is to apply a non-thermal air atmospheric pressure plasma jet (NTAAPPJ) to HGF to measure the migration and to reveal the underlying biological mechanisms involved in the migration. After the characterization of NTAAPPJ by optical emission spectroscopy, the adherent HGF was treated with NTAAPPJ or air with a different flow rate. Cell viability, lipid peroxidation, migration, intracellular reactive oxygen species (ROS), and the expression of migration-related genes (EGFR, PAK1, and MAPK3) were investigated. The level of statistical significance was set at 0.05. NTAAPPJ and air treatment with a flow rate of 250-1000 standard cubic centimetres per minute (sccm) for up to 30 s did not induce significant decreases in cell viability or membrane damage. A significant increase in the migration of mitomycin C-treated HGF was observed after 30 s of NTAAPPJ treatment compared to 30 s air-only treatment, which was induced by high levels of intracellular reactive oxygen species (ROS). An increase in migration-related gene expression and EGFR activation was observed following NTAAPPJ treatment in an air flow rate-dependent manner. This is the first report that NTAAPPJ treatment induces an increase in HGF migration without changing cell viability or causing membrane damage. HGF migration was related to an increase in intracellular ROS, changes in the expression of three of the migration-related genes (EGFR, PAK1, and MAPK1), and EGFR activation. Therefore, NTAAPPJ for gingival tissue healing is a promising method for health and aesthetic outcomes.

R-ketorolac Targets Cdc42 and Rac1 and Alters Ovarian Cancer Cell Behaviors Critical for Invasion and Metastasis

PubMed Central

Guo, Yuna; Kenney, Shelby Ray; Muller, Carolyn Y.; Adams, Sarah; Rutledge, Teresa; Romero, Elsa; Murray-Krezan, Cristina; Prekeris, Rytis; Sklar, Larry A.; Hudson, Laurie G.; Wandinger-Ness, Angela

2015-01-01

Cdc42 (cell division control protein 42) and Rac1 (Ras-related C3 botulinum toxin substrate 1) are attractive therapeutic targets in ovarian cancer based on established importance in tumor cell migration, adhesion and invasion. Despite a predicted benefit, targeting GTPases has not yet been translated to clinical practice. We previously established that Cdc42 and constitutively active Rac1b are overexpressed in primary ovarian tumor tissues. Through high throughput screening and computational shape homology approaches we identified R-ketorolac as a Cdc42 and Rac1 inhibitor; distinct from the anti-inflammatory, cyclooxygenase inhibitory activity of S-ketorolac. In the present study, we establish R-ketorolac as an allosteric inhibitor of Cdc42 and Rac1. Cell-based assays validate R-ketorolac activity against Cdc42 and Rac1. Studies on immortalized human ovarian adenocarcinoma cells (SKOV3ip), and primary, patient-derived ovarian cancer cells show R-ketorolac is a robust inhibitor of growth factor or serum dependent Cdc42 and Rac1 activation with a potency and cellular efficacy similar to small molecule inhibitors of Cdc42 (CID2950007/ML141) and Rac1 (NSC23766). Furthermore, GTPase inhibition by R-ketorolac reduces downstream p21-activated kinases (PAK1/PAK2) effector activation by >80%. Multiple assays of cell behavior using SKOV3ip and primary patient-derived ovarian cancer cells show that R-ketorolac significantly inhibits cell adhesion, migration and invasion. In sum, we provide evidence for R-ketorolac as direct inhibitor of Cdc42 and Rac1 that is capable of modulating downstream GTPase-dependent, physiological responses, which are critical to tumor metastasis. Our findings demonstrate the selective inhibition of Cdc42 and Rac1 GTPases by an FDA approved drug-racemic ketorolac that can be used in humans. PMID:26206334

R-Ketorolac Targets Cdc42 and Rac1 and Alters Ovarian Cancer Cell Behaviors Critical for Invasion and Metastasis.

PubMed

Guo, Yuna; Kenney, S Ray; Muller, Carolyn Y; Adams, Sarah; Rutledge, Teresa; Romero, Elsa; Murray-Krezan, Cristina; Prekeris, Rytis; Sklar, Larry A; Hudson, Laurie G; Wandinger-Ness, Angela

2015-10-01

Cdc42 (cell division control protein 42) and Rac1 (Ras-related C3 botulinum toxin substrate 1) are attractive therapeutic targets in ovarian cancer based on established importance in tumor cell migration, adhesion, and invasion. Despite a predicted benefit, targeting GTPases has not yet been translated to clinical practice. We previously established that Cdc42 and constitutively active Rac1b are overexpressed in primary ovarian tumor tissues. Through high-throughput screening and computational shape homology approaches, we identified R-ketorolac as a Cdc42 and Rac1 inhibitor, distinct from the anti-inflammatory, cyclooxygenase inhibitory activity of S-ketorolac. In the present study, we establish R-ketorolac as an allosteric inhibitor of Cdc42 and Rac1. Cell-based assays validate R-ketorolac activity against Cdc42 and Rac1. Studies on immortalized human ovarian adenocarcinoma cells (SKOV3ip) and primary patient-derived ovarian cancer cells show that R-ketorolac is a robust inhibitor of growth factor or serum-dependent Cdc42 and Rac1 activation with a potency and cellular efficacy similar to small-molecule inhibitors of Cdc42 (CID2950007/ML141) and Rac1 (NSC23766). Furthermore, GTPase inhibition by R-ketorolac reduces downstream p21-activated kinases (PAK1/PAK2) effector activation by >80%. Multiple assays of cell behavior using SKOV3ip and primary patient-derived ovarian cancer cells show that R-ketorolac significantly inhibits cell adhesion, migration, and invasion. In summary, we provide evidence for R-ketorolac as a direct inhibitor of Cdc42 and Rac1 that is capable of modulating downstream GTPase-dependent, physiologic responses, which are critical to tumor metastasis. Our findings demonstrate the selective inhibition of Cdc42 and Rac1 GTPases by an FDA-approved drug, racemic ketorolac, that can be used in humans. ©2015 American Association for Cancer Research.

From LAMP to Koha: Case Study of the Pakistan Legislative Assembly Libraries

ERIC Educational Resources Information Center

Shafi-Ullah, Farasat; Qutab, Saima

2012-01-01

Purpose: This paper aims to elaborate the library data migration process from LAMP (Library Automation Management Program) to the open source software Koha's (2.2.8 Windows based) Pakistani flavour PakLAG-Koha in six legislative assembly libraries of Pakistan. Design/methodology/approach: The paper explains different steps of the data migration…

Restoring fire suppressed Texas pak woodlands to historic conditions using prescribed fire

Treesearch

Jeff C. Sparks; Michael C. Stambaugh; Eric L. Keith

2012-01-01

Comparable to many oak ecosystems across the eastern United States, oak woodlands in Texas display characteristics of changing composition and structure due to altered fire regimes. Information describing historic fire regimes suggests woodlands underwent relatively frequent and repeated burning prior to major Euro-American influence in the early 19th century. Oak...

How Does Culture Shape Students' Perceptions of Scientists? Cross-National Comparative Study of American and Chinese Elementary Students

ERIC Educational Resources Information Center

Farland-Smith, Donna

2009-01-01

For decades, researchers have been convinced that one stereotypic image of scientists existed among children worldwide (Chambers, 1983; Chiang & Guo, 1996; Fung, 2002; Maoldomhnaigh & Hunt, 1988; Newton & Newton, 1992, 1998; She, 1998; Song, Pak, & Jang, 1992). This study, however, moves beyond that stereotypic image and examines…

Wetlands systems in southern Thailand: The essential resources for sustainable regional development

Treesearch

Rotchanatch Darnsawasdi; Prassert Chitpong

2000-01-01

Parts of Southern Thailand are inundated by water for months annually resulting in various wetlands including, among others, Tapi River Basin, Pak Panang River Basin, Songkhla Lake Basin, Pangnga Bay, Pattani River Basin, and Narathiwas Peat Swamp. Most wetlands perform functions such as flood retention, water filtration, bird and wildlife habitat,and tree growth....

A portrait of tissue phosphoprotein stability in the clinical tissue procurement process.

PubMed

Espina, Virginia; Edmiston, Kirsten H; Heiby, Michael; Pierobon, Mariaelena; Sciro, Manuela; Merritt, Barbara; Banks, Stacey; Deng, Jianghong; VanMeter, Amy J; Geho, David H; Pastore, Lucia; Sennesh, Joel; Petricoin, Emanuel F; Liotta, Lance A

2008-10-01

Little is known about the preanalytical fluctuations of phosphoproteins during tissue procurement for molecular profiling. This information is crucial to establish guidelines for the reliable measurement of these analytes. To develop phosphoprotein profiles of tissue subjected to the trauma of excision, we measured the fidelity of 53 signal pathway phosphoproteins over time in tissue specimens procured in a community clinical practice. This information provides strategies for potential surrogate markers of stability and the design of phosphoprotein preservative/fixation solutions. Eleven different specimen collection time course experiments revealed augmentation (+/-20% from the time 0 sample) of signal pathway phosphoprotein levels as well as decreases over time independent of tissue type, post-translational modification, and protein subcellular location (tissues included breast, colon, lung, ovary, and uterus (endometrium/myometrium) and metastatic melanoma). Comparison across tissue specimens showed an >20% decrease of protein kinase B (AKT) Ser-473 (p < 0.002) and myristoylated alanine-rich C-kinase substrate protein Ser-152/156 (p < 0.0001) within the first 90-min postexcision. Proteins in apoptotic (cleaved caspase-3 Asp-175 (p < 0.001)), proliferation/survival/hypoxia (IRS-1 Ser-612 (p < 0.0003), AMP-activated protein kinase beta Ser-108 (p < 0.005), ERK Thr-202/Tyr-204 (p < 0.003), and GSK3alphabeta Ser-21/9 (p < 0.01)), and transcription factor pathways (STAT1 Tyr-701 (p < 0.005) and cAMP response element-binding protein Ser-133 (p < 0.01)) showed >20% increases within 90-min postprocurement. Endothelial nitric-oxide synthase Ser-1177 did not change over the time period evaluated with breast or leiomyoma tissue. Treatment with phosphatase or kinase inhibitors alone revealed that tissue kinase pathways are active ex vivo. Combinations of kinase and phosphatase inhibitors appeared to stabilize proteins that exhibited increases in the presence of phosphatase inhibitors alone (ATF-2 Thr-71, SAPK/JNK Thr-183/Tyr-185, STAT1 Tyr-701, JAK1 Tyr-1022/1023, and PAK1/PAK2 Ser-199/204/192/197). This time course study 1) establishes the dynamic nature of specific phosphoproteins in excised tissue, 2) demonstrates augmented phosphorylation in the presence of phosphatase inhibitors, 3) shows that kinase inhibitors block the upsurge in phosphorylation of phosphoproteins, 4) provides a rational strategy for room temperature preservation of proteins, and 5) constitutes a foundation for developing evidence-based tissue procurement guidelines.

High-performance liquid chromatographic determination of pyridostigmine bromide, nicotine, and their metabolites in rat plasma and urine.

PubMed

Abu-Qare, A W; Abou-Donia, M B

2001-07-01

This study reports on the development of a rapid and simple method for the determination of the antinerve agent drug pyridostigmine bromide (3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) (PB), its metabolite N-methyl-3-hydroxypyridinium bromide, nicotine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidine), and its metabolites nornicotine (2-(3-pyridyl)pyrrolidine) and cotinine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidone) in rat plasma and urine. The compounds are extracted and eluted by methanol and acetonitrile using C18 Sep-Pak cartridges and separated using high-performance liquid chromatography by a gradient of methanol, acetonitrile, and water (pH 3.2) at a flow rate of 0.8 mL/min in a period of 14 min. UV detection was at 260 nm for nicotine and its metabolites and at 280 nm for PB and its metabolite. The limits of detection ranged between 20 and 70 ng/mL, and the limits of quantitation were 50-100 ng/mL. The average percent recovery of five spiked plasma samples were 85.7 +/- 7.3%, 80.4 +/- 5.8%, 78.9 +/- 5.4%, 76.7 +/- 6.4%, and 79.7 +/- 5.7% and for urine were 85.9 +/- 5.9%, 75.5 +/- 6.9%, 82.6 +/- 7.9%, 73.6 +/- 5.9%, and 77.7 +/- 6.3% for nicotine, nornicotine, cotinine, PB, and N-methyl-3-hydroxypyridinium bromide, respectively. The calibration curves for standard solutions of the compounds of peak areas and concentration are linear for a range between 100 and 1,000 ng/mL. This method is applied in order to analyze the previously mentioned chemicals and metabolites following their oral administration in rats.

A rapid and sensitive LC-MS/MS-ESI method for the determination of tolvaptan and its two main metabolites in human plasma.

PubMed

Jiang, Juanjuan; Tian, Lei; Huang, Yiling; Yan, Yan; Li, Yishi

2016-08-01

A liquid chromatography-tandem mass spectrometry (LC-MS) method to quantify tolvaptan and its two main metabolites and applied to human study was first developed and validated as a measure of compliance in clinical research. Because of the structure similarity of tolvaptan and its multiple metabolites, the method was optimized to obtain a chromatographic and MS separation of the endogenous interference and isotope ions as well as high analysis throughput. Tolvaptan, its two main metabolites and the internal standard were extracted from human serum (0.1mL) using solid-phase extraction, separated on a Waters nova-pak C18 column (150×3.9mm, 5μm) using isocratic elution with a mobile phase composed of acetonitrile, water and formic acid (65:35:0.25, v/v/v). The total run-time was shortened to 3.5min. The mass transition ranges under positive electrospray ionisation that were monitored for quantitation included m/z 449-252 for tolvaptan, m/z 479-252 for metabolite DM-4103, m/z 481-252 for metabolite DM-4107 and m/z 463-266 for the internal standard (IS). The limit of quantification in plasma for all three analytes was 1ng/mL. The method was validated over a linear range from 1 to 500ng/mL for all three analytes with acceptable inter- and intra-assay precision and accuracy. The stability of the analytes was determined to be suitable for routine laboratory practices. The method was successfully applied to samples taken from research volunteers who ingested a 15mg tolvaptan tablet. Copyright © 2016. Published by Elsevier B.V.

Circulating miRNAs as Potential Alternative Cell Signaling Associated with Maternal Recognition of Pregnancy in the Mare.

PubMed

Klohonatz, Kristin M; Cameron, Ashely D; Hergenreder, Joanna R; da Silveira, Juliano C; Belk, Aeriel D; Veeramachaneni, D N R; Bouma, Gerrit J; Bruemmer, Jason E

2016-12-01

During early pregnancy, the conceptus and mare communicate to establish pregnancy. Cell-secreted vesicles (e.g., exosomes) have been reported in serum. Exosomes contain bioactive materials, such as miRNA, that can mediate cell responses. We hypothesized that a) exosomes are present in mare circulation and quantity varies with pregnancy status, b) exosomes contain miRNAs unique to pregnancy status, and c) miRNAs target pathways in endometrium based upon pregnancy status of the mare. First, serum samples were obtained from mares in a crossover design, with each mare providing samples from a pregnant and nonmated control cycle (n = 3/sample day) on Days 12, 14, 16, and 18 postovulation. Flow cytometry revealed the presence of serum microvesicles in mares in two different-sized populations (greater than or less than 100 nm), validated by transmission electron microscopy. Second, serum was collected on Days 9, 11, and 13 (n = 4/day), and endometrial biopsies were collected on Days 11 and 13 (n = 3/day) from pregnant and nonmated mares. Total RNA from serum exosomes was evaluated with quantitative RT-PCR using equine-specific miRNA sequences. A total of 12 miRNAs were found in different quantities on the specified days. Pathway analysis suggested that miRNAs targeted focal adhesion molecules (FAMs). Transcripts corresponding to FAMs were evaluated in endometrial biopsies. Protein levels and localization for PAK6 and RAF1 were further evaluated. Our data suggest that serum exosomes contain miRNA that differ based upon pregnancy status, and may affect mRNA expression related to focal adhesion pathway in the endometrium, with a potential role in maternal recognition of pregnancy. © 2016 by the Society for the Study of Reproduction, Inc.

Residue depletion of oxytetracycline (OTC) and 4-epi-oxytetracycline (4-epi-OTC) in broiler chicken's claws by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

PubMed

Cornejo, J; Pokrant, E; Araya, D; Briceño, C; Hidalgo, H; Maddaleno, A; Araya-Jordán, C; San Martin, B

2017-04-01

Antibiotics are widely used in poultry production for the treatment of bacterial diseases. However, residues may remain in products and by-products destined for human consumption or animal feeding. The claws of chickens, which are a by-product of the poultry industry, can directly or indirectly enter the food chain as meals destined to feed other productive animals. Thus, it becomes necessary to determine and quantify antimicrobial residues present in this matrix. The objective of the study was to assess the depletion of oxytetracycline (OTC) and its metabolite 4-epi-OTC in broiler chicken's claws. Claws of 32 broilers treated with a therapeutic dosage of 10% OTC during 7 days were analysed. Samples were taken at days 3, 9, 15 and 19 post-treatment. As for the control group, eight broiler chickens were raised under the same conditions. Extraction was carried out through EDTA-McIlvaine buffer, and clean-up employed a SPE C-18 Sep-Pak®. Instrumental analysis was performed through LC-MS/MS. The concentrations of both analytes were determined in claw samples until day 19 post-treatment. Average concentrations were within the LOD (20 μg kg - 1 ) and LOQ (22 µg kg - 1 ) for OTC and 84 μg kg - 1 for 4-epi-OTC. Withdrawal times (WDTs) of 39 days for OTC and 54 days for 4-epi-OTC were established in claws based on 95% confidence. These findings demonstrate that claws can be a source of antimicrobial residue entry into the food chain, since the results showed that OTC and its metabolite can be found in chicken's claws for long periods, even exceeding the average lifespan of a broiler chicken.

Cost-effectiveness of sofosbuvir-based treatments for chronic hepatitis C in the US.

PubMed

Zhang, Sai; Bastian, Nathaniel D; Griffin, Paul M

2015-08-05

The standard care of treatment of interferon plus ribavirin (plus protease inhibitor for genotype 1) are effective in 50 % to 70 % of patients with CHC. Several new treatments including Harvoni, Olysio + Sovaldi, Viekira Pak, Sofosbuvir-based regimens characterized with potent inhibitors have been approved by the Food and Drug Administration (FDA) providing more options for CHC patients. Trials have shown that the new treatments increased the rate to 80% to 95%, though with a substantial increase in cost. In particular, current market pricing of a 12-week course of sofosbuvir is approximately US$84,000. We determine the cost-effectiveness of new treatments in comparison with the standard care of treatments. A Markov simulation model of CHC disease progression is used to evaluate the cost-effectiveness of different treatment strategies based on genotype. The model calculates the expected lifetime medical costs and quality adjusted life years (QALYs) of hypothetical cohorts of identical patients receiving certain treatments. For genotype 1, we compare: (1) peginterferon + ribavirin + telaprevir for 12 weeks, followed by 12 or 24 weeks treatment of peginterferon + ribavirin dependent on HCV RNA level at week 12; (2) Harvoni treatment, 12 weeks; (3) Olysio + Sovaldi, 12 weeks for patients without cirrhosis, 24 weeks for patients with cirrhosis; (4) Viekira Pak + ribavirin, 12 weeks for patients without cirrhosis, 24 weeks for patients with cirrhosis; (5) sofosbuvir + peginterferon + ribavirin, 12 weeks for patients with or without cirrhosis. For genotypes 2 and 3, treatment strategies include: (1) peginterferon + ribavirin, 24 weeks for treatment-naïve patients; (2) sofosbuvir + ribavirin, 12 weeks for patients with genotype 2, 24 weeks for genotype 3; (3) peginterferon + ribavirin as initial treatment, 24 weeks for patients with genotype 2/3, follow-up treatment with sofosbuvir + ribavirin for 12/16 weeks are performed on non-responders and relapsers. Viekira Pak is cost-effective for genotype 1 patients without cirrhosis, whereas Harvoni is cost-effective for genotype 1 patients with cirrhosis. Sofosbuvir-based treatments for genotype 1 in general are not cost-effective due to its substantial high costs. Two-phase treatments with 12-week and 16-week follow-ups are cost-effective for genotype 3 patients and for genotype 2 patients with cirrhosis. The results were shown to be robust over a broad range of parameter values through sensitivity analysis. For genotype 1, sofosbuvir-based treatments are not cost-effective compared to Viekira Pak and Harvoni, although a 30% reduction in sofosbuvir price would change this result. Sofosbuvir + ribavirin are cost-effective as second-phase treatments following peginterferon + ribavirin initial treatment for genotypes 2 and 3. However, there is limited data on sofosbuvir-involved treatment, and the results obtained in this study must be interpreted within the model assumptions.

Simple synthesis of carbon-11 labeled styryl dyes as new potential PET RNA-specific, living cell imaging probes.

PubMed

Wang, Min; Gao, Mingzhang; Miller, Kathy D; Sledge, George W; Hutchins, Gary D; Zheng, Qi-Huang

2009-05-01

A new type of styryl dyes have been developed as RNA-specific, live cell imaging probes for fluorescent microscopy technology to study nuclear structure and function. This study was designed to develop carbon-11 labeled styryl dyes as new probes for biomedical imaging technique positron emission tomography (PET) imaging of RNA in living cells. Precursors (E)-2-(2-(1-(triisopropylsilyl)-1H-indol-3-yl)vinyl)quinoline (2), (E)-2-(2,4,6-trimethoxystyryl)quinoline (3) and (E)-4-(2-(6-methoxyquinolin-2-yl)vinyl)-N,N-diemthylaniline (4), and standards styryl dyes E36 (6), E144 (7) and F22 (9) were synthesized in multiple steps with moderate to high chemical yields. Precursor 2 was labeled by [(11)C]CH(3)OTf, trapped on a cation-exchange CM Sep-Pak cartridge following a quick deprotecting reaction by addition of (n-Bu)(4)NF in THF, and isolated by solid-phase extraction (SPE) purification to provide target tracer [(11)C]E36 ([(11)C]6) in 40-50% radiochemical yields, decay corrected to end of bombardment (EOB), based on [(11)C]CO(2). The target tracers [(11)C]E144 ([(11)C]7) and [(11)C]F22 ([(11)C]9) were prepared by N-[(11)C]methylation of the precursors 3 and 4, respectively, using [(11)C]CH(3)OTf and isolated by SPE method in 50-70% radiochemical yields at EOB. The specific activity of the target tracers [(11)C]6, [(11)C]7 and [(11)C]9 was in a range of 74-111GBq/mumol at the end of synthesis (EOS).

Postmortem photonic imaging of lux-modified Salmonella typhimurium within the gastrointestinal tract of swine following oral inoculation in vivo

USDA-ARS?s Scientific Manuscript database

The study objective was to monitor Salmonella progression by photonic detection through segments of the gastrointestinal tract following oral inoculation. Pigs (~ 80 kg) were inoculated orally with 3.1 or 4.1×10*10 colony forming units (cfu) of Salmonella typhimurium transformed with plasmid pAK1-lu...

Psychoanalysis and Transformation of Heroes in Mohsin Hamid's Novels "Moth Smoke" and "The Reluctant Fundamentalist"

ERIC Educational Resources Information Center

Awan, Abdul Ghafoor; Andleeb, Shaista; Yasin, Farhat

2016-01-01

Mohsin Hamid is an exponent of postcolonial characterization and possesses a specific touch of today's hero in local Asian context. His pen is fluent on social fiction portraying Indo-Pak culture. Few writers could rise to the height of fame right with only a couple of preliminary works. Hamid secured it through his first novel "Moth…

Initial evaluation of two native egg parasitoids for the control of Bagrada hilaris, an invasive stink bug in western USA

USDA-ARS?s Scientific Manuscript database

Bagrada bug, Bagrada hilaris (Hem.: Pentatomidae), is a major pest of cole crops (Brassicaceae). It was first reported in California in 2008 and has now spread to several U.S. states and Mexico. We studied host phylogeography by sequencing CO1 from 20 bagrada bug populations and determined that Pak...

Requirement of Treg-intrinsic CTLA4/PKCη signaling pathway for suppressing tumor immunity

PubMed Central

Pedros, Christophe; Canonigo-Balancio, Ann J.; Kong, Kok-Fai

2017-01-01

The ability of Tregs to control the development of immune responses is essential for maintaining immune system homeostasis. However, Tregs also inhibit the development of efficient antitumor responses. Here, we explored the characteristics and mechanistic basis of the Treg-intrinsic CTLA4/PKCη signaling pathway that we recently found to be required for contact-dependent Treg-mediated suppression. We show that PKCη is required for the Treg-mediated suppression of tumor immunity in vivo. The presence of PKCη-deficient (Prkch–/–) Tregs in the tumor microenvironment was associated with a significantly increased expression of the costimulatory molecule CD86 on intratumoral CD103+ DCs, enhanced priming of antigen-specific CD8+ T cells, and greater levels of effector cytokines produced by these cells. Similar to mouse Tregs, the GIT/PAK/PIX complex also operated downstream of CTLA4 and PKCη in human Tregs, and GIT2 knockdown in Tregs promoted antitumor immunity. Collectively, our data suggest that targeting the CTLA4/PKCη/GIT/PAK/PIX signaling pathway in Tregs could represent a novel immunotherapeutic strategy to alleviate the negative impact of Tregs on antitumor immune responses. PMID:29212947

An approach to Moho discontinuity recovery from on-orbit GOCE data with application over Indo-Pak region

NASA Astrophysics Data System (ADS)

Eshagh, Mehdi; Hussain, Matloob

2016-10-01

In this research, a modified form of Vening Meinesz-Moritz (VMM) theory of isostasy for the second-order radial derivative of gravitational potential, measured from the Gravity field and steady-state Ocean Circulation Explorer (GOCE), is developed for local Moho depth recovery. An integral equation is organised for inverting the GOCE data to compute a Moho model in combination with topographic/bathymetric heights of SRTM30, sediment and consolidated crystalline basement and the laterally-varying density contrast model of CRUST1.0. A Moho model from EGM2008 to degree and order 180 is also computed based on the same principle for the purpose of comparison. In addition, we compare both of them with the 3 available seismic Moho models; two global and one regional over the Indo-Pak region. Numerical results show that our GOCE-based Moho model is closer to the all seismic models than that of EGM2008. The model is closest to the regional one with a standard deviation of 5.5 km and a root mean squares error of 7.8 km, which is 2.3 km smaller than the corresponding one based on EGM2008.

Rac1/RhoA antagonism defines cell-to-cell heterogeneity during epidermal morphogenesis in nematodes

PubMed Central

Ouellette, Marie-Hélène

2016-01-01

The antagonism between the GTPases Rac1 and RhoA controls cell-to-cell heterogeneity in isogenic populations of cells in vitro and epithelial morphogenesis in vivo. Its involvement in the regulation of cell-to-cell heterogeneity during epidermal morphogenesis has, however, never been addressed. We used a quantitative cell imaging approach to characterize epidermal morphogenesis at a single-cell level during early elongation of Caenorhabditis elegans embryos. This study reveals that a Rac1-like pathway, involving the Rac/Cdc42 guanine-exchange factor β-PIX/PIX-1 and effector PAK1/PAK-1, and a RhoA-like pathway, involving ROCK/LET-502, control the remodeling of apical junctions and the formation of basolateral protrusions in distinct subsets of hypodermal cells. In these contexts, protrusions adopt lamellipodia or an amoeboid morphology. We propose that lamella formation may reduce tension building at cell–cell junctions during morphogenesis. Cell-autonomous antagonism between these pathways enables cells to switch between Rac1- and RhoA-like morphogenetic programs. This study identifies the first case of cell-to-cell heterogeneity controlled by Rac1/RhoA antagonism during epidermal morphogenesis. PMID:27821782

Geochronology of Middle and Upper Pleistocene Loess Sections in Hungary

NASA Astrophysics Data System (ADS)

Frechen, Manfred; Horváth, Erzsébet; Gábris, Gyula

1997-11-01

The application of both thermoluminescence and infrared stimulated luminescence dating to the extensively studied "classical" Hungarian loess/paleosol sequences from Basaharc, Mende, and Paks provides a reliable chronological framework and climatostratigraphic reconstruction for the last interglacial/glacial cycle. Based on this combined luminescence dating study a new chronology is proposed for the "Young Loess" in Hungary. Luminescence dating suggests that the loess below the MF2 horizon formed during the penultimate glaciation. The MF1 horizon probably formed during an interstade within oxygen isotope stage 3. For the youngest loess, overlying MF1, a very high accumulation rate was determined. Large time gaps occur above MF2 and MF1, indicating that most of the record of the last glaciation is missing in the standard sections at Basaharc, Mende, and Paks. Either large discontinuities or a very low accumulation rate occurred in all three type sections during the soil-forming periods. High-resolution studies of climatic proxies using this combined luminescence dating approach provide a reliable chronological framework for loess and loess derivatives of the last glacial cycle in Hungary, although a precise and complete chronostratigraphic reconstruction cannot be achieved from the incomplete records found at these sites.

Antioxidative properties of defatted dabai pulp and peel prepared by solid phase extraction.

PubMed

Khoo, Hock Eng; Azlan, Azrina; Ismail, Amin; Abas, Faridah

2012-08-14

Solid phase extraction (SPE) using Sep-Pak® cartridges is one of the techniques used for fractionation of antioxidant compounds in waste of dabai oil extraction (defatted dabai parts). The aim of this study was to determine the phenolic compounds and antioxidant capacity in crude extracts and several SPE fractions from methanolic extract of defatted dabai pulp and peel. Based on SPE, Sep-Pak® cyanopropyl and C₁₈ cartridges were used to fractionate the antioxidant-rich crude extracts into water and methanolic fractions. Analyzed using LC-MS, flavonoids, anthocyanins, saponin derivatives and other unknown antioxidative compounds were detected in the defatted dabai crude extracts and their SPE fractions. Anthocyanins were the major phenolic compounds identified in the defatted dabai peel and detected in most of the SPE fractions. Methanolic fractions of defatted dabai parts embraced higher total phenolics and antioxidant capacity than water fractions. This finding also revealed the crude extracts of defatted dabai peel have the most significant antioxidant properties compared to the methanolic and water fractions studied. The crude extract of defatted dabai parts remain as the most potent antioxidant as it contains mixture of flavonoids, anthocyanins and other potential antioxidants.

Feasibility of Neurapheresis™ as a Therapy for Multidrug Resistant Gram-negative Bacterial Meningitis

PubMed Central

Ballard, Christi; Ashraf, Bilal; Ejikeme, Tiffany; Hansen, Brenda; Charalambous, Lefko; Pagadala, Promila; Sharma-Kuinkel, Batu K; Giamberardino, Charles; Hedstrom, Blake; Verbick, Laura Zitella; Mccabe, Aaron; Lad, Shivanand P; Fowler, Vance; Perfect, John R

2017-01-01

Abstract Background The World Health Organization has identified Pseudomonas, Acinetobacter and Klebsiella (PAK) as three multidrug resistant (MDR) gram-negative pathogens that pose a threat to human health. The greatest threat lies in hospitals, nursing homes, and patients with devices such as intravenous catheters and ventilators. Gram-negative bacterial meningitis (GBM) manifests when these bacteria invade the central nervous system. Due to the threat of increasing antibiotic resistance and the high mortality associated with MDR GBM, we have tested a closed-loop, extracorporeal cerebrospinal fluid (CSF) filtration system (NeurapheresisTM) for its applicability in this context. Here we demonstrate feasibility of Neurapheresis for MDR GBM and characterize system parameters for bacterial clearance. Methods PAK cultures were grown and diluted to 1 × 107 cells/mL in artificial CSF or Luria-Miller broth. Both single pass and closed loop filtration were performed with various tangential flow filtration (TFF) and dead-end filter paradigms. Samples were taken either immediately post-filter or after every full CSF volume cycle (150 mL) during a long-term closed loop experiment. Bacterial load, endotoxin and cytokines were quantified. Results In single pass tests, 5kDa and 100kDa TFF filters and 0.2µm and 0.45µm dead-end filters excluded all PAK organisms completely. The 100kDa and 5kDa TFF filters significantly reduced endotoxin concentration by >95% and >99% of baseline, respectively. The 5 kDa TFF filters produced a 2-log (>99%) reduction in cytokines (IL-1ra, IL-6, TNF, CRP, and CXCL10). In closed-loop experiments, both TFF filters demonstrated a 1–2 Log CFU (90–99%) reduction of all PAK organisms over 4 filtration cycles. Conclusion Neurapheresis shows potential to be an efficient multi-modal tool for controlling and treating MDR GBM in this in vitro model. Extending closed loop filtration over time demonstrates capability for rapid sterilization of the CSF. Future iterations may include adjunctive intrathecal drug delivery to further accelerate elimination of bacteria. Reduction of both endotoxin and cytokines by Neurapheresis may have significant implications for controlling the damaging neuro-inflammatory response during MDR GBM. Disclosures B. Hedstrom, Minnetronix, Inc.: Employee, Salary; L. Zitella Verbick, Minnetronix, Inc.: Employee, Salary; A. Mccabe, Minnetronix, Inc.: Employee, Salary; S. P. Lad, Minnetronix, Inc.: Collaborator and Scientific Advisor, Licensing agreement or royalty, Research grant and Research support; V. Fowler Jr., Pfizer, Novartis, Galderma, Novadigm, Durata, Debiopharm, Genentech, Achaogen, Affinium, Medicines Co., Cerexa, Tetraphase, Trius, MedImmune, Bayer, Theravance, Cubist, Basilea, Affinergy, Janssen, xBiotech, Contrafect: Consultant, Consulting fee; NIH, MedImmune, Cerexa/Forest/Actavis/Allergan, Pfizer, Advanced Liquid Logics, Theravance, Novartis, Cubist/Merck; Medical Biosurfaces; Locus; Affinergy; Contrafect; Karius: Grant Investigator, Grant recipient; Green Cross, Cubist, Cerexa, Durata, Theravance; Debiopharm: Consultant, Consulting fee; UpToDate: Royalties, Royalties

Postmortem Photonic Imaging of Lux-Modified Salmonella Typhimuium Within the Gastrointestinal Tract of Swine Following Oral Inoculation In Vivo

USDA-ARS?s Scientific Manuscript database

The study objective was to monitor Salmonella progression by photonic detection through segments of the gastrointestinal tract after oral inoculation. Pigs (~80 kg) were inoculated orally with 3.1 or 4.1 x 1010 cfu of Salmonella Typhimurium transformed with plasmid pAK1-lux for a 6-h (n = 6) or 12-h...

Use of GC/MS Analysis to Distinguish Between Vapor Intrusion and Indoor Sources of VOCs - Standardized Protocol for On-Site Evaluation of Vapor Intrusion

DTIC Science & Technology

2014-07-01

Testing a Sealed Crack in a Concrete Floor .................................................................. 14 Figure 5: VOC Responses to...Engineered Fluid Toluene Some paints and adhesives SprayPAK Enamel , Minwax Wood Finish Xylenes Adhesives, paints, gasoline Bonide Tree Sprays and...expansion joints, plumbing penetrations, or cracks . 3 Note that if indoor air concentrations are

The Effect of an Instructional Unit Incorporating Live Animals on Knowledge of Nutrition for Different Age Levels.

ERIC Educational Resources Information Center

Roth, Anne I.; Wunderlich, Kenneth W.

A nutrition education unit, Rat Pak, developed by Dairy Council, Inc., is an attempt to influence students to make wise food choices. It consists of eleven lessons in an instructional sequence which incorporates the use of white rats as a means of illustrating the effect of improper diet while teaching proper diet. The purpose of this…

Super star clusters, their environment, and the formation of galactic winds

NASA Astrophysics Data System (ADS)

Westmoquette, Mark S.

Starbursts and starburst-driven outflows play a central role in the evolution of galaxies. However, the paucity of detailed observations of superwinds limits our current understanding of these complex systems. To this end we have undertaken two intensive ground- and space-based observing campaigns aimed at studying the ionized gas conditions in two nearby starburst galaxies, M82 and NGC 1569. These two systems host starbursts on different scales: M82 contains densely-packed star cluster complexes that drive a large-scale bipolar superwind, whereas NGC 1569 exhibits a set of discrete superbubbles powered by only a handful of young massive clusters. We have used long-slit spectra, obtained with the Hubble Space Telescope (HST), together with HST and ground-based imaging from the WIYN 3.5 m telescope, to observe M82 at optical wavelengths. The high quality HST spectroscopy obtained with the Space Telescope Imaging Spectrograph (STIS), have allowed us to investigate the properties of the gas across the starburst core. By combining high-resolution HST imaging with deep WIYN observations, we have created the most comprehensive image of the M82 superwind to date, and used it to characterise the outflow morphology. We also observed the centre of NGC 1569 with the Integral Field Unit (IFU) of the Gemini Multi-Object Spectrograph (GMOS) on the Gemini-North telescope, and M82 with the WIYN/DensePak and SparsePak IFUs. We decomposed the observed emission-line profile shapes, and identified an underlying broad (>100 kms-1) component across the starburst cores of both galaxies. By mapping the spatial variation of each individual line component, we have developed a new model to explain the broad emission and the state of the interstellar medium (ISM) in the central starbursts. We have also observed the outer-wind environment of NGC 1569 with the WIYN SparsePak instrument. We find that the broad line is only found within 500-700 pc of the centre, and speculate that the boundary of this region may indicate the point at which bulk motions begin to dominate over turbulence.

An overview of triple infection with hepatitis B, C and D viruses.

PubMed

Riaz, Mehwish; Idrees, Muhamad; Kanwal, Hifza; Kabir, Firoz

2011-07-27

Viral hepatitis is one of the major health problems worldwide, particularly in South East Asian countries including Pakistan where hepatitis C virus (HCV) and hepatitis B virus (HBV) infections are highly endemic. Hepatitis delta virus (HDV) is also not uncommon world-wide. HCV, HBV, and HDV share parallel routes of transmission due to which dual or triple viral infection can occur in a proportion of patients at the same time. HBV and HCV are important factors in the development of liver cirrhosis (LC) and hepatocellular carcinoma (HCC). In addition to LC and HCC, chronic HDV infection also plays an important role in liver damage with oncogenic potential.The current article reviews the available literature about the epidemiology, pathogenesis, transmission, symptoms, diagnosis, replication, disease outcome, treatment and preventive measures of triple hepatitis infection by using key words; epidemiology of triple infection, risk factors, awareness status, treatment and replication cycle in PubMed, PakMediNet, Directory of Open Access Journals (DOAJ) and Google Scholar. Total data from 74 different studies published from 1983 to 2010 on triple hepatitis infections were reviewed and included in this study. The present article briefly describes triple infection with HCV, HBV and HDV.

The changing landscape of hepatitis C virus therapy: focus on interferon-free treatment.

PubMed

Lam, Brian P; Jeffers, Thomas; Younoszai, Zahra; Fazel, Yousef; Younossi, Zobair M

2015-09-01

Chronic hepatitis C (CHC) affects over 185 million individuals worldwide, approximately 3% of the world's population. CHC can lead to quality of life impairment, cirrhosis, hepatocellular carcinoma (HCC), liver failure and liver-related death. While CHC has been associated with increases in HCC, liver-related mortality and all-cause mortality, being cured of CHC is associated with improvement in these outcomes. Older interferon-based regimens were complex and toxic and required 6-12 months of therapy, with cure rates averaging around 40-45% for HCV genotype 1. Newer interferon-free regimens are now available in the US, Europe, Japan and in other countries. These regimens have short durations, minimal side effects, low pill burden and efficacy approaching 90-100%. We may eventually see single-tablet regimens lasting no more than 4-6 weeks. This review will summarize the data regarding these interferon-free regimens, including Gilead's Harvoni (sofosbuvir/ledipasvir), AbbVie's Viekira Pak (paritaprevir/ritonavir/ombitasvir with dasabuvir), and Janssen's Olysio (simeprevir) with sofosbuvir. Some practical considerations as we move into an interferon-free era will also be discussed, such as patient adherence and drug-drug interactions.

The changing landscape of hepatitis C virus therapy: focus on interferon-free treatment

PubMed Central

Lam, Brian P.; Jeffers, Thomas; Younoszai, Zahra; Fazel, Yousef

2015-01-01

Chronic hepatitis C (CHC) affects over 185 million individuals worldwide, approximately 3% of the world’s population. CHC can lead to quality of life impairment, cirrhosis, hepatocellular carcinoma (HCC), liver failure and liver-related death. While CHC has been associated with increases in HCC, liver-related mortality and all-cause mortality, being cured of CHC is associated with improvement in these outcomes. Older interferon-based regimens were complex and toxic and required 6–12 months of therapy, with cure rates averaging around 40–45% for HCV genotype 1. Newer interferon-free regimens are now available in the US, Europe, Japan and in other countries. These regimens have short durations, minimal side effects, low pill burden and efficacy approaching 90–100%. We may eventually see single-tablet regimens lasting no more than 4–6 weeks. This review will summarize the data regarding these interferon-free regimens, including Gilead’s Harvoni (sofosbuvir/ledipasvir), AbbVie’s Viekira Pak (paritaprevir/ritonavir/ombitasvir with dasabuvir), and Janssen’s Olysio (simeprevir) with sofosbuvir. Some practical considerations as we move into an interferon-free era will also be discussed, such as patient adherence and drug–drug interactions. PMID:26327920

Tuberculosis in Laos, who is at risk: the mahouts or their elephants?

PubMed

Lassausaie, J; Bret, A; Bouapao, X; Chanthavong, V; Castonguay-Vanier, J; Quet, F; Mikota, S K; Théorêt, C; Buisson, Y; Bouchard, B

2015-04-01

SUMMARY Tuberculosis (TB) in elephants has the potential to infect humans and is an increasing public health concern. Lao PDR is one of the last countries where elephants are still used for timber extraction and where they live in close contact with their mahouts. There are 500 animals at work in the country, some interacting with wild herds. Although human TB prevalence is known to be high in Laos, studies on elephant TB had yet to be undertaken. From January to July 2012, screening was performed using the ElephantTB Stat-Pak assay on 80 elephants working around the Nam Pouy National Park in Sayaboury Province. This represents more than 18% of the total registered national working elephant population. Here we report that 36% of the elephants were seroreactive to the test. Of these, 31% had contacts with wild individuals, which suggests potential transmission of mycobacteria to the local wild herds. Clinical examination, chest X-rays, sputum microscopy and culture were performed on their 142 mahouts or owners. Despite high TB seroreactivity in elephants, no participant was smear- or culture-positive for Mycobacterium tuberculosis or M. bovis, although atypical mycobacteria were isolated from 4% of participants.

Korean Affairs Report.

DTIC Science & Technology

1986-08-14

Plan 86 Libya Receives DPRK Economic Delegation 86 Ethiopian Envoy Received 86 Peruvian Visitor Received 87 - d Egyptian Anniversary 87 Kim Il...in the designing of the hotel is a cinema house with several hundreds of seats. Guest flats will be arranged from the third floor. Each flat will... PERUVIAN VISITOR RECEIVED—Pyongyang, July 22 (KCNA)—Comrade Pak Song-chol, member of the Political Bureau of the Central Committee of the Workers

JPRS Report, East Europe.

DTIC Science & Technology

1991-04-10

adversely influenced by certain inconsistencies and by mourn those who died innocently. Let us place flowers criminal failures to act, but we rejoice...velvet revolution we have pineapple and strawberries, all the while carrying on a been hopefully mouthing the slogan of a return to leisurely conversation...of was made that Paks Nuclear Plant should buy, in accor- our population’s overall income. This is supplemented by dance with a long-term agreement

Annotated Bibliography of Water Optical Properties of Ocean Waters.

DTIC Science & Technology

1982-05-01

24. Barrett, B. B. ( 1971 ). Cooperative Gulf of Mexico Estuarine Inventory and Study, Louisiana, Phase II, Hydrology and Phase III, Sedimentology...Assoc. U.K. (39), 227-238. Ambient light measurements to depths of 400 m. 35. Boulter, Jacques ( 1971 ). Photometric Sous-marine. Measures effectuees...Florida coastal water with a Hydroproducts transmissometer (550 nm). 41. Carder, Kendall L., G. F. Beardsley, Jr., and Hasong Pak ( 1971 ). Particle Size

Korean Affairs Report.

DTIC Science & Technology

1987-01-08

King H4 Mali Ambassador Presents Credentials 114 Greetings to Finland’s Koivisto 114 Greetings to Togolese Congress 114 Pak Song-chol Meets GDR...council’s Standing Committee at the Sejong Cultural Center in Seoul. The resolution then pledged to propagate the national will of constructing a counter...12232 GREETINGS TO THAI RING—Pyongyang 4 December (RCNA)—President Rim Il-song on 3 December sent a message of greetings to Phumiphon Adunyadet, king

Defense Technical Information Center Cataloging, Abstracting and Indexing Guidelines

DTIC Science & Technology

2008-05-01

APPEARS AS: ENTER AS: General Electric Co., Daytona Beach , Florida. Apollo Support Dept. General Electric Co Daytona Beach FL Apollo Support Dept...OMAN OM PAKISTAN PK PALAU PW PANAMA PA PAPUA NEW GUINEA PG PARAGUAY PY PERU PE PHILIPPINES PH PITCAIRN PN POLAND PL PORTUGAL PT PUERTO...OMN PAKISTAN PAK PALAU PLW PANAMA PAN PAPUA NEW GUINEA PNG PARAGUAY PRY PERU PER PHILIPPINES PHL PITCAIRN PCN POLAND POL PORTUGAL PRT

Piper betle L. Modulates Senescence-Associated Genes Expression in Replicative Senescent Human Diploid Fibroblasts

PubMed Central

Durani, Lina Wati; Tan, Jen Kit; Chua, Kien Hui

2017-01-01

Piper betle (PB) is a traditional medicine that is widely used to treat different diseases around Asian region. The leaf extracts contain various bioactive compounds, which were reported to have antidiabetic, antibacterial, anti-inflammatory, antioxidant, and anticancer effects. In this study, the effect of PB aqueous extracts on replicative senescent human diploid fibroblasts (HDFs) was investigated by determining the expressions of senescence-associated genes using quantitative PCR. Our results showed that PB extracts at 0.4 mg/ml can improve cell proliferation of young (143%), presenescent (127.3%), and senescent (157.3%) HDFs. Increased expressions of PRDX6, TP53, CDKN2A, PAK2, and MAPK14 were observed in senescent HDFs compared to young and/or presenescent HDFs. Treatment with PB extracts modulates the transcriptional profile changes in senescent HDFs. By contrast, expressions of SOD1 increased, whereas GPX1, PRDX6, TP53, CDKN2A, PAK2, and MAPK14 were decreased in PB-treated senescent HDFs compared to untreated senescent HDFs. In conclusion, this study indicates the modulation of PB extracts on senescence-associated genes expression of replicative senescent HDFs. Further studies warrant determining the mechanism of PB in modulating replicative senescence of HDFs through these signaling pathways. PMID:28596968

Piper betle L. Modulates Senescence-Associated Genes Expression in Replicative Senescent Human Diploid Fibroblasts.

PubMed

Durani, Lina Wati; Khor, Shy Cian; Tan, Jen Kit; Chua, Kien Hui; Mohd Yusof, Yasmin Anum; Makpol, Suzana

2017-01-01

Piper betle (PB) is a traditional medicine that is widely used to treat different diseases around Asian region. The leaf extracts contain various bioactive compounds, which were reported to have antidiabetic, antibacterial, anti-inflammatory, antioxidant, and anticancer effects. In this study, the effect of PB aqueous extracts on replicative senescent human diploid fibroblasts (HDFs) was investigated by determining the expressions of senescence-associated genes using quantitative PCR. Our results showed that PB extracts at 0.4 mg/ml can improve cell proliferation of young (143%), presenescent (127.3%), and senescent (157.3%) HDFs. Increased expressions of PRDX6 , TP53 , CDKN2A , PAK2 , and MAPK14 were observed in senescent HDFs compared to young and/or presenescent HDFs. Treatment with PB extracts modulates the transcriptional profile changes in senescent HDFs. By contrast, expressions of SOD1 increased, whereas GPX1 , PRDX6 , TP53 , CDKN2A , PAK2 , and MAPK14 were decreased in PB-treated senescent HDFs compared to untreated senescent HDFs. In conclusion, this study indicates the modulation of PB extracts on senescence-associated genes expression of replicative senescent HDFs. Further studies warrant determining the mechanism of PB in modulating replicative senescence of HDFs through these signaling pathways.

Implementation of New Reactivity Measurement System and New Reactor Noise Analysis Equipment in a VVER-440 Nuclear Power Plant

NASA Astrophysics Data System (ADS)

Vegh, János; Kiss, Sándor; Lipcsei, Sándor; Horvath, Csaba; Pos, István; Kiss, Gábor

2010-10-01

The paper deals with two recently developed, high-precision nuclear measurement systems installed at the VVER-440 units of the Hungarian Paks NPP. Both developments were motivated by the reactor power increase to 108%, and by the planned plant service time extension. The first part describes the RMR start-up reactivity measurement system with advanced services. High-precision picoampere meters were installed at each reactor unit and measured ionization chamber current signals are handled by a portable computer providing data acquisition and online reactivity calculation service. Detailed offline evaluation and analysis of reactor start-up measurements can be performed on the portable unit, too. The second part of the paper describes a new reactor noise diagnostics system using state-of-the-art data acquisition hardware and signal processing methods. Details of the new reactor noise measurement evaluation software are also outlined. Noise diagnostics at Paks NPP is a standard tool for core anomaly detection and for long-term noise trend monitoring. Regular application of these systems is illustrated by real plant data, e.g., results of standard reactivity measurements during a reactor startup session are given. Noise applications are also illustrated by real plant measurements; results of core anomaly detection are presented.

Thallium contamination in arable soils and vegetables around a steel plant-A newly-found significant source of Tl pollution in South China.

PubMed

Liu, Juan; Luo, Xuwen; Wang, Jin; Xiao, Tangfu; Chen, Diyun; Sheng, Guodong; Yin, Meiling; Lippold, Holger; Wang, Chunlin; Chen, Yongheng

2017-05-01

Thallium (Tl) is a highly toxic rare element. Severe Tl poisoning can cause neurological brain damage or even death. The present study was designed to investigate contents of Tl and other associated heavy metals in arable soils and twelve common vegetables cultivated around a steel plant in South China, a newly-found initiator of Tl pollution. Potential health risks of these metals to exposed population via consumption of vegetables were examined by calculating hazard quotients (HQ). The soils showed a significant contamination with Tl at a mean concentration of 1.34 mg/kg. The Tl levels in most vegetables (such as leaf lettuce, chard and pak choy) surpassed the maximum permissible level (0.5 mg/kg) according to the environmental quality standards for food in Germany. Vegetables like leaf lettuce, chard, pak choy, romaine lettuce and Indian beans all exhibited bioconcentration factors (BCF) and transfer factors (TF) for Tl higher than 1, indicating a hyperaccumulation of Tl in these plants. Although the elevated Tl levels in the vegetables at present will not immediately pose significant non-carcinogenic health risks to residents, it highlights the necessity of a permanent monitoring of Tl contamination in the steel-making areas. Copyright © 2017 Elsevier Ltd. All rights reserved.

A microarray study of gene and protein regulation in human and rat brain following middle cerebral artery occlusion

PubMed Central

Mitsios, Nick; Saka, Mohamad; Krupinski, Jerzy; Pennucci, Roberta; Sanfeliu, Coral; Wang, Qiuyu; Rubio, Francisco; Gaffney, John; Kumar, Pat; Kumar, Shant; Sullivan, Matthew; Slevin, Mark

2007-01-01

Background Altered gene expression is an important feature of ischemic cerebral injury and affects proteins of many functional classes. We have used microarrays to investigate the changes in gene expression at various times after middle cerebral artery occlusion in human and rat brain. Results Our results demonstrated a significant difference in the number of genes affected and the time-course of expression between the two cases. The total number of deregulated genes in the rat was 335 versus 126 in the human, while, of 393 overlapping genes between the two array sets, 184 were changed only in the rat and 36 in the human with a total of 41 genes deregulated in both cases. Interestingly, the mean fold changes were much higher in the human. The expression of novel genes, including p21-activated kinase 1 (PAK1), matrix metalloproteinase 11 (MMP11) and integrase interactor 1, was further analyzed by RT-PCR, Western blotting and immunohistochemistry. Strong neuronal staining was seen for PAK1 and MMP11. Conclusion Our findings confirmed previous studies reporting that gene expression screening can detect known and unknown transcriptional features of stroke and highlight the importance of research using human brain tissue in the search for novel therapeutic agents. PMID:17997827

The interaction between HIV-1 Nef and adaptor protein-2 reduces Nef-mediated CD4+ T cell apoptosis.

PubMed

Jacob, Rajesh Abraham; Johnson, Aaron L; Pawlak, Emily N; Dirk, Brennan S; Van Nynatten, Logan R; Haeryfar, S M Mansour; Dikeakos, Jimmy D

2017-09-01

Acquired Immune Deficiency Syndrome is characterized by a decline in CD4 + T cells. Here, we elucidated the mechanism underlying apoptosis in Human Immunodeficiency Virus-1 (HIV-1) infection by examining host apoptotic pathways hijacked by the HIV-1 Nef protein in the CD4 + T-cell line Sup-T1. Using a panel of Nef mutants unable to bind specific host proteins we uncovered that Nef generates pro- and anti-apoptotic signals. Apoptosis increased upon mutating the motifs involved in the interaction of Nef:AP-1 (Nef M20A or Nef EEEE62-65AAAA ) or Nef:AP-2 (Nef LL164/165AA ), implying these interactions limit Nef-mediated apoptosis. In contrast, disrupting the Nef:PAK2 interaction motifs (Nef H89A or Nef F191A ) reduced apoptosis. To validate further, apoptosis was measured after short-hairpin RNA knock-down of AP-1, AP-2 and PAK2. AP-2α depletion enhanced apoptosis, demonstrating that disrupting the Nef:AP-2α interaction limits Nef-mediated apoptosis. Collectively, we describe a mechanism by which HIV-1 regulates cell survival and demonstrate the consequence of interfering with Nef:host protein interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

A systematic review of treatment response rates in Pakistani hepatitis C virus patients; current prospects and future challenges

PubMed Central

Ali, Muhammad; Afzal, Samia; Zia, Asad; Hassan, Ahmed; Khalil, Ali Talha; Ovais, Muhammad; Shinwari, Zabta Khan; Idrees, Muhammad

2016-01-01

Abstract Background: The estimated hepatitis C virus (HCV) carriers are approximately 10 million in Pakistan which usually progresses to chronic hepatitis, with rare cases of spontaneous viral eradication. The present article reviews the treatment status of HCV infection in Pakistani population and various factors associated with the treatment response rates. Methods: Literature on anti-HCV therapy was searched in PubMed, Google Scholar and PakMediNet. Thirty three different studies representing different geographic regions of Pakistan published from 2002 to 2016 were included in the present review. Weighted mean, standard error estimates (SE) and standard deviation (SD) were determined for each population group. Results: Mean value for sustained virological response (SVR) for standard IFN plus ribavirin (RBV) combination therapy was 68.38% ± 14.13% (range 33.8%–87.10%; SE 3.08) and pegylated-IFN plus RBV combination therapy 64.38% ± 8.68% (range 55.0%–76.00%; SE 3.88). The lowest value for SVR has been reported to be 24.3% (for genotype 1; administering INF-α 2b 3MU 3 times/week and RBV 1000–1200 mg/day for 48 weeks) while highest of 87.5% (genotype 3a; INF-α 2a 3MU 3 times/week and RBV 1000–1200 mg/day for 24 weeks). The mean value for rapid virological response (RVR) was found to be 48.18% ± 29.20% (SE 9.73). As PEG-interferon and direct acting antivirals (DAAs) are relatively expensive, interferon-alfa (IFN-α) and RBV combination therapy have been used widely to treat HCV infected patients in Pakistan for the last one and half decade. On average, 2.45% of the patients discontinued treatment due to severe side effects. Conclusion: We encourage further studies on understanding host and viral factors associated with specific focus on harder to treat viral variants (relapsers and nonresponders). These variants are currently rising in the country. PMID:27977575

Association of hepatitis C virus with insulin resistance: evidences from animal studies and clinical studies.

PubMed

Badar, Sadaf; Khubaib, Bushra; Idrees, Muhammad; Hussain, Abrar; Awan, Zunaira; Butt, Sadia; Afzal, Samia; Akram, Madeeha; Fatima, Zareen; Aftab, Mahwish; Saleem, Sana; Munir, Sara; Rauff, Bisma; Naudhani, Mahrukh; Ali, Liaquat; Ali, Muhammaad; Rehman, Irshadul

2012-01-01

HCV infection is strongly associated with development of insulin resistance and type-2 diabetes, however molecular mechanism of these associations is not known. The aim of this review was to conduct a comprehensive literature search to understand the nature of the association between hepatitis C virus (HCV) infection and insulin resistance (IR). We also explored the role of HCV core protein and NS5a in modulating the course of the insulin-signaling pathway. We searched Directory of Open Access Journals (DOAJ) Google Scholar, Pubmed (NLM), LISTA (EBSCO), Web of Science (TS and PakMediNet). Emerging evidence suggests an association between HCV infection and carotid/coronary vascular disease. IR appears to be a dominant underlying cause of accelerated atherosclerosis in patients with chronic hepatitis C (CHC). HCV can induce IR directly through the stimulation of SOCS3 and PPA2, and both of these molecules have been shown to inhibit interferon-α signaling. Improvement of insulin sensitivity may increase the response rate to antiviral treatment and prevent IR complications, including vascular diseases. The results of several clinical trials that have used insulin sensitizers (metformin and PPAR-γ agonists) have been inconclusive. Beside the association between HCV and IR, the published data also have showed the possible association of HCV core and NS5A protein with IR.

Assessment of intrinsic small signal parameters of submicron SiC MESFETs

NASA Astrophysics Data System (ADS)

Riaz, Mohammad; Ahmed, Muhammad Mansoor; Rafique, Umair; Ahmed, Umer Farooq

2018-01-01

In this paper, a technique has been developed to estimate intrinsic small signal parameters of submicron SiC MESFETs, designed for high power microwave applications. In the developed technique, small signal parameters are extracted by involving drain-to-source current, Ids instead of Schottky barrier depletion layer expression. It has been demonstrated that in SiC MESFETs, the depletion layer gets modified due to intense transverse electric field and/or self-heating effects, which are conventionally not taken into account. Thus, assessment of AC small signal parameters by employing depletion layer expression loses its accuracy for devices meant for high power applications. A set of expressions for AC small signal elements has been developed using Ids and its dependence on device biasing has been discussed. The validity of the proposed technique has been demonstrated using experimental data. Dr. Ahmed research interests are in Microelectronics, Microwave and RF Engineering and he has supervised numerous MS and PhD research projects. He authored over 100 research papers in the field of microelectronics. Dr. Ahmed is a fellow of the Institution of Engineering and Technology (IET), UK.; a Chartered Engineer (CEng) from the UK Engineering Council and holds the title of European Engineer (Eur Ing) from the European Federation of National Engineering Association (FEANI), Brussels. He is a life member of PEC (Pak); EDS & MTTS (USA).

Ocean Optical Database

DTIC Science & Technology

1992-05-01

ocean color for retrieving ocean k(490) values are examined. The validation of the optical database from the satellite is accessed through comparison...for sharing results of this validation study. We wish to thank J. Mueller for helpful discussions in optics and satellite processing and for sharing his...of these data products are displayable as 512 x 512 8-bit image maps compatible with the PC-SeaPak image format. Valid data ranges are from 1 to 255

Drop Tower Characterization of Army Research Lab (ARL)-Fabricated Thin-Film Lead Zirconate Titanate (PZT) Transducers

DTIC Science & Technology

2012-04-01

TRADE NAME, TRADEMARK, MANUFACTURER , OR OTHERWISE, DOES NOT CONSTITUTE OR IMPLY ITS ENDORSEMENT, RECOMMENDATION, OR FAVORING BY THE U.S. GOVERNMENT...TRADE NAMES USE OF TRADE NAMES OR MANUFACTURERS IN THIS REPORT DOES NOT CONSTITUTE AN OFFICIAL ENDORSEMENT OR APPROVAL OF THE USE OF...on Gel-Pak Ready for Noodle Wire Attachment ..................... 10 13. Waveshaper (on Right) Beside a 0.5-Inch Steel Ball

Korean Affairs Report

DTIC Science & Technology

1985-09-11

president of Pyongyang Music and Dance Univers- ity Kim Tong-sop, president of University of Commerce, and academician, Dr and Prof Pak Si-hyong and...investigation of the building. They decided to demand a meeting with the American Ambassador to Korea and a press conference with reporters from both within and...Chong-chin and Sim Yong-chin who at the underground fountain of the Lotte Department Store at the Ulchiro en- trance collected rocks from the nearby

Bomb blast injuries: an exploration of patient characteristics and outcome using Pakistan National Emergency Departments Surveillance (Pak-NEDS) data

PubMed Central

2015-01-01

Background Bomb blast injuries result in premature deaths and burdening of healthcare systems. The objective of this study was to explore the characteristics and outcome of patients presenting to the emergency departments in Pakistan with bomb blast injuries. Methods Active surveillance was conducted in seven major emergency departments of Pakistan from November 2010-March 2011. All the sites are tertiary care urban centers. All the patients who presented to the hospital's emergency department (ED) following a bomb blast injury as per self-report or the ambulance personnel were included in the study. Frequency of demographics, injury pattern, and outcomes were calculated. Results A total of 103 patients with bomb blast injuries presented to the selected emergency departments. The median age of patients was 30 years. Around three-fourth of the patients were males (n = 74, 74.7%). Most of the bomb blast patients were seen in Peshawar (n = 41, 39.8%) and Karachi city (n = 31, 30.1%) and the most common mode of arrival was non-ambulance transport (n = 71, 76.3%). Upper limb injuries (n = 12, 40%) were common in the under 18 age group and lower limb injuries (n = 31, 39.2%) in the 18 years and above group. There were a total of 8 (7.7%) deaths reported out of these 103 patients. Conclusion Bomb blast injuries in Pakistan generally affect young males. Non-ambulance transport is the most common way to access emergency departments (ED). Overall ED mortality is high and capturing data during a disaster in an emergency department is challenging. PMID:26692453

Bomb blast injuries: an exploration of patient characteristics and outcome using Pakistan National Emergency Departments Surveillance (Pak-NEDS) data.

PubMed

Khan, Irum; Khan, Nadeem; Naeem, Rubaba; Kerai, Salima; Allen, Kate; Zia, Nukhba; Shahbaz, Sana; Afridi, Shiraz; Siddiqui, Emaduddin; Khan, Uzma; Hyder, Adnan A; Razzak, Junaid A

2015-01-01

Bomb blast injuries result in premature deaths and burdening of healthcare systems. The objective of this study was to explore the characteristics and outcome of patients presenting to the emergency departments in Pakistan with bomb blast injuries. Active surveillance was conducted in seven major emergency departments of Pakistan from November 2010-March 2011. All the sites are tertiary care urban centers. All the patients who presented to the hospital's emergency department (ED) following a bomb blast injury as per self-report or the ambulance personnel were included in the study. Frequency of demographics, injury pattern, and outcomes were calculated. A total of 103 patients with bomb blast injuries presented to the selected emergency departments. The median age of patients was 30 years. Around three-fourth of the patients were males (n = 74, 74.7%). Most of the bomb blast patients were seen in Peshawar (n = 41, 39.8%) and Karachi city (n = 31, 30.1%) and the most common mode of arrival was non-ambulance transport (n = 71, 76.3%). Upper limb injuries (n = 12, 40%) were common in the under 18 age group and lower limb injuries (n = 31, 39.2%) in the 18 years and above group. There were a total of 8 (7.7%) deaths reported out of these 103 patients. Bomb blast injuries in Pakistan generally affect young males. Non-ambulance transport is the most common way to access emergency departments (ED). Overall ED mortality is high and capturing data during a disaster in an emergency department is challenging.

Survival Escherichia coli O157:H7 transformed with either the pAK1-lux or pXEN-13 plasmids in in vitro bovine ruminal and fecal microbial fermentations

USDA-ARS?s Scientific Manuscript database

The use of luminescent technology may serve as a viable model for the real-time validation of various pre-harvest interventions on the colonization or shedding of Escherichia coli O157:H7 within cattle. The objective of this study was to determine if the growth of E. coli O157:H7 (ATCC 43888) in ru...

Behavior of Materials at Cold Regions Temperatures. Part 1. Program Rationale and Test Plan

DTIC Science & Technology

1988-07-01

10 15 i" 0% I 0 0 0 2 3 4 5a 0 1 2 3 4 5 6 7 lf CSTRAIN (IN/INbST AI OW N V, Figure B47. Static stress vs strain curves for resilient expanded ... polystyrene foam (Resilo-Pak) , at temperature extremes (0.5, 0. 75 and 1.3 lb Ifft3 densities) (Titus 1967). so ,eSF 50 -- 8 * TEMPERATURE. K 0 so 100 is

World Food Conference: Issues and Preparation for Conference,

DTIC Science & Technology

1974-01-17

what appears to be a serious underestimating of the strength of the derived demand for feedgrains. However, before setting up a new model the FAO...or PCA D 14S In contrast to the developed part of the world where food supplies on the average are substantially above minimum calorie and protein ...1970 base, which incor- porates the probable progress to 1970 of the LDC’s (principally India and Pak- istan) in applying the High Yield Variety

Gfi1b controls integrin signaling-dependent cytoskeleton dynamics and organization in megakaryocytes.

PubMed

Beauchemin, Hugues; Shooshtarizadeh, Peiman; Vadnais, Charles; Vassen, Lothar; Pastore, Yves D; Möröy, Tarik

2017-03-01

Mutations in GFI1B are associated with inherited bleeding disorders called GFI1B -related thrombocytopenias. We show here that mice with a megakaryocyte-specific Gfi1b deletion exhibit a macrothrombocytopenic phenotype along a megakaryocytic dysplasia reminiscent of GFI1B -related thrombocytopenia. GFI1B deficiency increases megakaryocyte proliferation and affects their ploidy, but also abrogates their responsiveness towards integrin signaling and their ability to spread and reorganize their cytoskeleton. Gfi1b -null megakaryocytes are also unable to form proplatelets, a process independent of integrin signaling. GFI1B-deficient megakaryocytes exhibit aberrant expression of several components of both the actin and microtubule cytoskeleton, with a dramatic reduction of α-tubulin. Inhibition of FAK or ROCK, both important for actin cytoskeleton organization and integrin signaling, only partially restored their response to integrin ligands, but the inhibition of PAK, a regulator of the actin cytoskeleton, completely rescued the responsiveness of Gfi1b -null megakaryocytes to ligands, but not their ability to form proplatelets. We conclude that Gfi1b controls major functions of megakaryocytes such as integrin-dependent cytoskeleton organization, spreading and migration through the regulation of PAK activity whereas the proplatelet formation defect in GFI1B-deficient megakaryocytes is due, at least partially, to an insufficient α-tubulin content. Copyright© Ferrata Storti Foundation.

DO COMMERCIAL SEROLOGIC TESTS FOR TRYPANOSOMA CRUZI INFECTION DETECT MEXICAN STRAINS IN WOMEN AND NEWBORNS?

PubMed Central

Gamboa-León, Rubi; Gonzalez-Ramirez, Claudia; Padilla-Raygoza, Nicolas; Sosa-Estani, Sergio; Caamal-Kantun, Alejandra; Buekens, Pierre; Dumonteil, Eric

2012-01-01

We sought to determine the serological test that could be used for Trypanosoma cruzi seroprevalence studies in Mexico, where lineage I predominates. In a previous study among pregnant women and their newborns in the states of Yucatan and Guanajuato, we reported a 0.8–0.9% of prevalence for T. cruzi–specific antibodies by Stat-Pak and Wiener ELISA. We have expanded this study here by performing an additional non-commercial ELISA and confirming the seropositives with Western blot, using whole antigens of a local parasite strain. We found a seroprevalence of 0.6% (3/500) in Merida and 0.4% in Guanajuato (2/488). The 5 seropositive umbilical cord samples reacted to both non-commercial ELISA and Western blot tests, and only 1 of the maternal samples was not reactive to non-commercial ELISA. A follow-up of the newborns at 10 mo was performed in Yucatan to determine the presence of T. cruzi antibodies in children as evidence of congenital infection. None of the children was seropositive. One newborn from an infected mother died at 2 wk of age of cardiac arrest, but T. cruzi infection was not confirmed. The T. cruzi seroprevalence data obtained with both commercial tests (Stat-Pak and ELISA Wiener) are similar to those from non-commercial tests using a local Mexican strain of T. cruzi. PMID:21506787

Chronic Cigarette Smoke Mediated Global Changes in Lung Mucoepidermoid Cells: A Phosphoproteomic Analysis.

PubMed

Solanki, Hitendra S; Advani, Jayshree; Khan, Aafaque Ahmad; Radhakrishnan, Aneesha; Sahasrabuddhe, Nandini A; Pinto, Sneha M; Chang, Xiaofei; Prasad, Thottethodi Subrahmanya Keshava; Mathur, Premendu Prakash; Sidransky, David; Gowda, Harsha; Chatterjee, Aditi

2017-08-01

Proteomics analysis of chronic cigarette smoke exposure is a rapidly emerging postgenomics research field. While smoking is a major cause of lung cancer, functional studies using proteomics approaches could enrich our mechanistic understanding of the elusive lung cancer global molecular signaling and cigarette smoke relationship. We report in this study on a stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analysis of a human lung mucoepidermoid carcinoma cell line, H292 cells, chronically exposed to cigarette smoke. Using high resolution Orbitrap Velos mass spectrometer, we identified the hyperphosphorylation of 493 sites, which corresponds to 341 proteins and 195 hypophosphorylated sites, mapping to 142 proteins upon smoke exposure (2.0-fold change). We report differential phosphorylation of multiple kinases, including PAK6, EPHA4, LYN, mitogen-activated protein kinase, and phosphatases, including TMEM55B, PTPN14, TIGAR, among others, in response to chronic cigarette smoke exposure. Bioinformatics analysis revealed that the molecules differentially phosphorylated upon chronic exposure of cigarette smoke are associated with PI3K/AKT/mTOR and CDC42-PAK signaling pathways. These signaling networks are involved in multiple cellular processes, including cell polarity, cytoskeletal remodeling, cellular migration, protein synthesis, autophagy, and apoptosis. The present study contributes to emerging proteomics insights on cigarette smoke mediated global signaling in lung cells, which in turn may aid in development of precision medicine therapeutics and postgenomics biomarkers.

Systemic stereoselectivity study of flufiprole: Stereoselective bioactivity, acute toxicity and environmental fate.

PubMed

Tian, Mingming; Zhang, Qing; Hua, Xiude; Tang, Bowen; Gao, Beibei; Wang, Minghua

2016-12-15

In this study, the stereoselectivity of flufiprole enantiomers in regards to their bioactivity, acute toxicity and environmental fate is reported for the first time. Four types of representative insects (Plutella xylostella, Nilaparvata lugens, Mythimna separata and Acyrthosiphon pisum) were used to investigate enantioselective bioactivity. Acute toxicities of flufiprole enantiomers toward two non-target organisms were also evaluated. Moreover, stereoselective degradation in four vegetables under field conditions was studied in response to food safety concerns. The bioactivity of (R)-flufiprole was 1.9-5.1 times higher than that of (S)-flufiprole. (R)-flufiprole also showed 3.7-5.7 times higher acute toxicity to Scenedesmus obliquus and Trichogramma japonicum Ashmead than (S)-flufiprole. Opposite stereoselective degradation of the two enantiomers was observed in pak choi, spinach cucumber, and tomato. (S)-flufiprole degraded faster in pak choi and spinach, resulting in an enrichment of (R)-isomer. By contrast, (R)-isomer was preferentially degraded in cucumber and tomato. Molecular simulation technology was used to illuminate the mechanism of enantioselective bioactivity. The Glide Score (-5.82kcal/mol) for (R)-isomer was better than that (-5.11kcal/mol) of (S)-isomer and this calculation showed (R)-flufiprole was more effective in pest control. Consequently, significant stereoselectivity of flufiprole enantiomers should be taken into account when assessing the environmental health risk of the pesticide. Copyright © 2016 Elsevier B.V. All rights reserved.

Preliminary kinetic evaluation of an immobilized polysaccharide sub-2μm column using a low dispersion supercritical fluid chromatograph.

PubMed

Berger, Terry A

2017-08-11

The performance of a 3×50mm, 1.6μm d p column with an immobilized polysaccharide stationary phase (ChiralPak IA-U) was evaluated for efficiency, and pressure drop, with respect to flow rate and modifier concentration using supercritical fluid chromatography (SFC). This appears to be the first such report using such a column in SFC. A unique low dispersion (ultra-high performance) SFC was used for the evaluation. The minimum reduced plate height of 2.78, indicates that the maximum efficiency was similar to or better than coated polysaccharide columns. Selectivity was different from ChiralPak AD, with the same chiral selector, as reported by many others. At high flows and high methanol concentrations, pump pressures sometimes approached 600bar. With 5% methanol, pressure vs. flow rate was non-linear suggesting turbulent flow in the connector tubing. The optimum flow rate (F opt ) at 40% methanol was ≈0.8mL/min, where the column efficiency was highest. At 5% methanol, F opt increased to ≈1.6mL/min, but efficiency degraded noticeably. The differences in F opt suggests that the solute diffusion coefficients are a strong function of modifier concentration. Several sub-1min separations, including a 7.5s separation, are presented. Copyright © 2017 Elsevier B.V. All rights reserved.

Neto-Mediated Intracellular Interactions Shape Postsynaptic Composition at the Drosophila Neuromuscular Junction

PubMed Central

Ramos, Cathy I.; Igiesuorobo, Oghomwen; Wang, Qi; Serpe, Mihaela

2015-01-01

The molecular mechanisms controlling the subunit composition of glutamate receptors are crucial for the formation of neural circuits and for the long-term plasticity underlying learning and memory. Here we use the Drosophila neuromuscular junction (NMJ) to examine how specific receptor subtypes are recruited and stabilized at synaptic locations. In flies, clustering of ionotropic glutamate receptors (iGluRs) requires Neto (Neuropillin and Tolloid-like), a highly conserved auxiliary subunit that is essential for NMJ assembly and development. Drosophila neto encodes two isoforms, Neto-α and Neto-β, with common extracellular parts and distinct cytoplasmic domains. Mutations that specifically eliminate Neto-β or its intracellular domain were generated. When Neto-β is missing or is truncated, the larval NMJs show profound changes in the subtype composition of iGluRs due to reduced synaptic accumulation of the GluRIIA subunit. Furthermore, neto-β mutant NMJs fail to accumulate p21-activated kinase (PAK), a critical postsynaptic component implicated in the synaptic stabilization of GluRIIA. Muscle expression of either Neto-α or Neto-β rescued the synaptic transmission at neto null NMJs, indicating that Neto conserved domains mediate iGluRs clustering. However, only Neto-β restored PAK synaptic accumulation at neto null NMJs. Thus, Neto engages in intracellular interactions that regulate the iGluR subtype composition by preferentially recruiting and/or stabilizing selective receptor subtypes. PMID:25905467

Directional control of WAVE2 membrane targeting by EB1 and phosphatidylinositol 3,4,5-triphosphate.

PubMed

Takahashi, Kazuhide; Tanaka, Tacu; Suzuki, Katsuo

2010-03-01

Membrane targeting of WAVE2 along microtubules is mediated by a motor protein kinesin and requires Pak1, a downstream effector of Rac1. However, the mechanism by which WAVE2 targeting to the leading edge is directionally controlled remains largely unknown. Here we demonstrate that EB1, a microtubule plus-end-binding protein, constitutively associates with stathmin, a microtubule-destabilizing protein, in human breast cancer cells. Stimulation of the cells with insulin-like growth factor I (IGF-I) induced Pak1-dependent binding of the EB1-stathmin complex to microtubules that bear WAVE2 and colocalization of the complex with WAVE2 at the leading edge. Depletion of EB1 by small interfering RNA (siRNA) abrogated the IGF-I-induced WAVE2 targeting and stathmin binding to microtubules. On the other hand, chemotaxis chamber assays indicated that the IGF-I receptor (IGF-IR) was locally activated in the region facing toward IGF-I. In addition, IGF-I caused phosphatidylinositol 3-kinase (PI 3-kinase)-dependent production of phosphatidylinositol 3,4,5-triphosphate (PIP3) near activated IGF-IR and WAVE2 colocalization with it. Collectively, WAVE2-membrane targeting is directionally controlled by binding of the EB1-stathmin complex to WAVE2-bearing microtubules and by the interaction between WAVE2 and PIP3 produced near IGF-IR that is locally activated by IGF-I.

Synthesis of carbon-11 labeled 1-(3,4-dimethoxybenzyl)-2,2-dimethyl-1,2,3,4-tetrahydroisoquinolinium derivatives as new potential PET SKCa channel imaging agents.

PubMed

Gao, Mingzhang; Wang, Min; Zheng, Qi-Huang

2008-02-01

Small conductance Ca2+-activated K+ (SKCa) channels play an important role in many functions such as neuronal communication and behavioral plasticity, secretion, and cell proliferation. SKCa channel modulation is associated with various brain, heart, and cancer diseases. N-methyl-laudanosine and its structurally related derivatives, substituted 1-(3,4-dimethoxybenzyl)-2,2-dimethyl-1,2,3,4-tetrahydroisoquinoliniums, are reversible and selective SKCa channel blockers. Carbon-11 labeled N-methyl-laudanosine and its tetrahydroisoquinolinium derivatives may serve as new probes for positron emission tomography (PET) to image SKCa channels in the brain, heart, and cancer. The key intermediates, substituted isoquinolines (3a-c), were synthesized using a modification of the Pomeranz-Fritsch procedure. The precursors, substituted 1-(3,4-dimethoxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinolines (8a-c), and their corresponding reference standards, substituted 1-(3,4-dimethoxybenzyl)-2,2-dimethyl-1,2,3,4-tetrahydroisoquinoliniums (9a-c), were synthesized from compounds 3a-c with 3,4-dimethoxybenzyl chloride (2) in multiple steps with moderate to excellent chemical yields. The precursor 6,7-dimethoxy-1-(3,4-dimethoxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinoline (10) was commercially available, and the methylation of compound 10 with methyl iodide provided N-methyl-laudanosine (11). The target quaternary ammonium tracers, carbon-11 labeled 1-(3,4-dimethoxybenzyl)-2,2-dimethyl-1,2,3,4-tetrahydroisoquinoliniums ([11C]9a-c and [11C]11), were prepared by N-[11C]methylation of the tertiary amine precursors (8a-c and 10) with [11C]methyl triflate and isolated by a simplified solid-phase extraction (SPE) purification using a SiO2 or cation-exchange CM Sep-Pak cartridge in 40-65% radiochemical yields.

A 32-country comparison of tobacco smoke derived particle levels in indoor public places

PubMed Central

Hyland, A; Travers, M J; Dresler, C; Higbee, C; Cummings, K M

2018-01-01

Objective To compare tobacco smoke-derived particulate levels in transportation and hospitality venues with and without smoking in 32 countries using a standardised measurement protocol. Methods The TSI SidePak AM510 Personal Aerosol Monitor was used to measure the concentration of particulate matter less than 2.5 microns in diameter (PM2.5) in 1822 bars, restaurants, retail outlets, airports and other workplaces in 32 geographically dispersed countries between 2003 and 2007. Results Geometric mean PM2.5 levels were highest in Syria (372 μg/m3), Romania (366 μg/m3) and Lebanon (346 μg/m3), while they were lowest in the three countries that have nationwide laws prohibiting smoking in indoor public places (Ireland at 22 μg/m3, Uruguay at 18 μg/m3 and New Zealand at 8 μg/m3). On average, the PM2.5 levels in places where smoking was observed was 8.9 times greater (95% CI 8.0 to 10) than levels in places where smoking was not observed. Conclusions Levels of indoor fine particle air pollution in places where smoking is observed are typically greater than levels that the World Health Organization and US Environmental Protection Agency have concluded are harmful to human health. PMID:18303089

40 CFR 721.3025 - Fatty acids C12-18, C18 unsaturated, C12-18 alkyl esters (generic).

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Fatty acids C12-18, C18 unsaturated... Significant New Uses for Specific Chemical Substances § 721.3025 Fatty acids C12-18, C18 unsaturated, C12-18... chemical substance identified generically as fatty acids C12-18, C18 unsaturated, C12-18 alkyl esters (PMNs...

Folate content in tomato ( Lycopersicon esculentum ). influence of cultivar, ripeness, year of harvest, and pasteurization and storage temperatures.

PubMed

Iniesta, M Dolores; Pérez-Conesa, Darío; García-Alonso, Javier; Ros, Gaspar; Periago, M Jesús

2009-06-10

The effects of cultivar, on-vine ripening, and year of harvest on the folate content of raw tomatoes were studied. Folate content in hot-break tomato puree (HTP) subjected to pasteurization at different temperatures and its evolution during the shelf life of tomato juice were also investigated. 5-Methyltetrahydrofolate (5-CH(3)-H(4)-folate) was the only folate compound identified in raw tomatoes and HTP, but tetrahydrofolate (H(4)-folate) was 10% of the folate detected in tomato juice. The content of folates in raw tomatoes ranged from 4.1 to 35.3 microg/100 g of fresh weight and was highly influenced by all of the factors studied. No clear trend of folate content with ripening stage was observed. The extractability of 5-CH(3)-H(4)-folate from HTP increased significantly after pasteurization at 98 degrees C for 40 s, but higher temperatures decreased its content. Tomato juice showed folate losses during storage independent of the storage temperature. Folate losses were higher when tomato juice was packed in glass bottles than in Tetra Pak.

Direct anti-HCV agents

PubMed Central

Zhang, Xingquan

2015-01-01

Unlike human immunodeficiency virus (HIV) and hepatitis B virus (HBV), hepatitis C virus (HCV) infection is a curable disease. Current direct antiviral agent (DAA) targets are focused on HCV NS3/4A protein (protease), NS5B protein (polymerase) and NS5A protein. The first generation of DAAs includes boceprevir and telaprevir, which are protease inhibitors and were approved for clinical use in 2011. The cure rate for genotype 1 patients increased from 45% to 70% when boceprevir or telaprevir was added to standard PEG-IFN/ribavirin. More effective and less toxic second generation DAAs supplanted these drugs by 2013. The second generation of DAAs includes sofosbuvir (Sovaldi), simeprevir (Olysio), and fixed combination medicines Harvoni and Viekira Pak. These drugs increase cure rates to over 90% without the need for interferon and effectively treat all HCV genotypes. With these drugs the “cure HCV” goal has become a reality. Concerns remain about drug resistance mutations and the high cost of these drugs. The investigation of new HCV drugs is progressing rapidly; fixed dose combination medicines in phase III clinical trials include Viekirax, asunaprevir+daclatasvir+beclabuvir, grazoprevir+elbasvir and others. PMID:26904396

Acute Necrotizing Ulcerative Gingivitis: Microbial and Immunologic Studies.

DTIC Science & Technology

1984-08-05

Erythromycin (CVE) agar; MM10 agar; and Trypticase Soy Agar (TSA) with hemin and menadione (TSAHK). CVE agar (pH 7.2 contains in g/l the following... menadione in 95% ETOH and 50 ml of defibrinated sheep blood were added. Mlcrobioloaical Culturing of ANLIG Lesion Samples of subgingival plaque were...supplemented with 5 g/ml hemin and 2 g/ml menadione using the BBL anaerobe jar-Gas Pak system. After 24 to 72 h of growth, the organisms were harvested by

Translations on North Korea, Number 563

DTIC Science & Technology

1977-11-29

units are subordinate to the Unit to Which Comrade Kim ;e"J°\\S *elonf £nit leader Kim Yong - ung ) : The Triple Red Banner Unit to Which Comrade...No. 563 CONTENTS PAGE Development and Prosperity of Anju Described (Sonu Song-ok; MINJU CHOSON, 30 Aug 77) 1 Kim and Pak Receive Reply From GDR...CHOSON in Korean 30 Aug 77 p 4 [Article by Sonu Song-ok: "Anju Thriving With New Life"] [Text] The respected and beloved leader Comrade Kim Il

Translations on North Korea, Number 546.

DTIC Science & Technology

1977-08-26

Comrade Ho Tam " Kye Ung -t’ae " Pak Yong -sok Functionary of the sector concerned Yi Ch’ang-son " Chon In-ch’ol " [Pyongyang NODONG SINMUN in Korean 14...functionary of the sector concerned Kim Tu- yong . [Pyongyang NODONG SINMUN in Korean 15 Jun 77 p 2] ARAB DELEGATION WELCOME—The following participated...Hwa-son . Functionary of the sector concerned Kim Yong -sun " [Pyongyang NODONG SINMUN in Korean 15 Jun 77 p 3] ARAB DELEGATION BANQUET—Attending a

The Precarious Void of Extremist Ideology in Bangladesh: A Path Towards Understanding the Phenomenology of Militant Violence

DTIC Science & Technology

2012-06-01

55 viii Figure 1 Map of Bangladesh x 2 The Muslim World 43 3 Terrorist Incidents by Militant...identities shape the population’s view of the outside world .7 Regional expertise beyond Af-Pak Hands has become a national security imperative in a...post-9/11 world , in which increased focus must be placed on all of South Asia. The region contains more than half the world’s Muslim population within

betaPIX controls cell motility and neurite extension by regulating the distribution of GIT1.

PubMed

Za, Lorena; Albertinazzi, Chiara; Paris, Simona; Gagliani, Mariacristina; Tacchetti, Carlo; de Curtis, Ivan

2006-07-01

Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. GIT1/p95-APP1 is a member of a family of GTPase-activating proteins for ARF GTPases that affect endocytosis, adhesion and migration. GIT1 associates with paxillin and a complex including the Rac/Cdc42 exchanging factors PIX/Cool and the kinase PAK. In this study, we show that overexpression of betaPIX induces the accumulation of endogenous and overexpressed GIT1 at large structures similar to those induced by an ArfGAP-defective mutant of GIT1 (p95-C2). Immunohistochemical analysis and immunoelectron microscopy reveal that these structures include the endogenous transferrin receptor. Time-lapse analysis during motogenic stimuli shows that the formation and perinuclear accumulation of the p95-C2-positive structures is paralleled by inhibition of lamellipodium formation and cell retraction. Both dimerization and a functional SH3 domain of betaPIX are required for the accumulation of GIT1 in fibroblasts, which is prevented by the monomeric PIX-PG-DeltaLZ. This mutant also prevents the formation of endocytic aggregates and inhibition of neurite outgrowth in retinal neurons expressing p95-C2. Our results indicate that betaPIX is an important regulator of the subcellular distribution of GIT1, and suggest that alteration in the level of expression of the complex affects the endocytic compartment and cell motility.

A second generation SNP and SSR integrated linkage map and QTL mapping for the Chinese mitten crab Eriocheir sinensis

PubMed Central

Qiu, Gao-Feng; Xiong, Liang-Wei; Han, Zhi-Ke; Liu, Zhi-Qiang; Feng, Jian-Bin; Wu, Xu-Gan; Yan, Yin-Long; Shen, Hong; Huang, Long; Chen, Li

2017-01-01

The Chinese mitten crab Eriocheir sinensis is the most economically important cultivated crab species in China, and its genome has a high number of chromosomes (2n = 146). To obtain sufficient markers for construction of a dense genetic map for this species, we employed the recently developed specific-locus amplified fragment sequencing (SLAF-seq) method for large-scale SNPs screening and genotyping in a F1 full-sib family of 149 individuals. SLAF-seq generated 127,677 polymorphic SNP markers, of which 20,803 valid markers were assigned into five segregation types and were used together with previous SSR markers for linkage map construction. The final integrated genetic map included 17,680 SNP and 629 SSR markers on the 73 linkage groups (LG), and spanned 14,894.9 cM with an average marker interval of 0.81 cM. QTL mapping localized three significant growth-related QTL to a 1.2 cM region in LG53 as well as 146 sex-linked markers in LG48. Genome-wide QTL-association analysis further identified four growth-related QTL genes named LNX2, PAK2, FMRFamide and octopamine receptors. These genes are involved in a variety of different signaling pathways including cell proliferation and growth. The map and SNP markers described here will be a valuable resource for the E. sinensis genome project and selective breeding programs. PMID:28045132

Evidence and potential risk factors of tuberculosis among captive Asian elephants and wildlife staff in Peninsular Malaysia.

PubMed

Yakubu, Yusuf; Ong, Bee Lee; Zakaria, Zunita; Hassan, Latiffah; Mutalib, Abdul Rahim; Ngeow, Yun Fong; Verasahib, Khebir; Razak, Mohd Firdaus Ariff Abdul

2016-03-01

Elephant tuberculosis (TB) caused by Mycobacterium tuberculosis is an important re-emerging zoonosis with considerable conservation and public health risk. We conducted prospective cohort and cross-sectional studies in elephants and wildlife staff respectively in order to identify potential risk factors associated with TB in captive Asian elephants and their handlers in Peninsular Malaysia. Sixty elephants in six different facilities were screened for TB longitudinally using the ElephantTB STAT-PAK and DPP VetTB assays from February 2012 to May 2014, and 149 wildlife staff were examined for tuberculosis infection using the QuantiFERON-TB Gold In-tube (QFT) assay from January to April, 2012. Information on potential risk factors associated with infection in both elephants and staff were collected using questionnaires and facility records. The overall seroprevalence of TB amongst the elephants was 23.3% (95% CI: 13.8-36.3) and the risk of seroconversion was significantly higher among elephants with assigned mahouts [p=0.022, OR=4.9 (95% CI: 1.3-18.2)]. The percentage of QFT responders among wildlife staff was 24.8% (95% CI: 18.3-32.7) and the risk of infection was observed to be significantly associated with being a zoo employee [p=0.018, OR=2.7 (95% CI: 1.2-6.3)] or elephant handler [p=0.035, OR=4.1 (95% CI: 1.1-15.5)]. These findings revealed a potential risk of TB infection in captive elephants and handlers in Malaysia, and emphasize the need for TB screening of newly acquired elephants, isolating sero-positive elephants and performing further diagnostic tests to determine their infection status, and screening elephant handlers for TB, pre- and post-employment. Copyright © 2016 Elsevier B.V. All rights reserved.

Metal oxide coating of carbon supports for supercapacitor applications.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boyle, Timothy J.; Tribby, Louis, J; Lakeman, Charles D. E.

2008-07-01

The global market for wireless sensor networks in 2010 will be valued close to $10 B, or 200 M units. TPL, Inc. is a small Albuquerque based business that has positioned itself to be a leader in providing uninterruptible power supplies in this growing market with projected revenues expected to exceed $26 M in 5 years. This project focused on improving TPL, Inc.'s patent-pending EnerPak{trademark} device which converts small amounts of energy from the environment (e.g., vibrations, light or temperature differences) into electrical energy that can be used to charge small energy storage devices. A critical component of the EnerPak{trademark}more » is the supercapacitor that handles high power delivery for wireless communications; however, optimization and miniaturization of this critical component is required. This proposal aimed to produce prototype microsupercapacitors through the integration of novel materials and fabrication processes developed at New Mexico Technology Research Collaborative (NMTRC) member institutions. In particular, we focused on developing novel ruthenium oxide nanomaterials and placed them into carbon supports to significantly increase the energy density of the supercapacitor. These improvements were expected to reduce maintenance costs and expand the utility of the TPL, Inc.'s device, enabling New Mexico to become the leader in the growing global wireless power supply market. By dominating this niche, new customers were expected to be attracted to TPL, Inc. yielding new technical opportunities and increased job opportunities for New Mexico.« less

[Optimization of solid-phase extraction for enrichment of toxic organic compounds in water samples].

PubMed

Zhang, Ming-quan; Li, Feng-min; Wu, Qian-yuan; Hu, Hong-ying

2013-05-01

A concentration method for enrichment of toxic organic compounds in water samples has been developed based on combined solid-phase extraction (SPE) to reduce impurities and improve recoveries of target compounds. This SPE method was evaluated in every stage to identify the source of impurities. Based on the analysis of Waters Oasis HLB without water samples, the eluent of SPE sorbent after dichloromethane and acetone contributed 85% of impurities during SPE process. In order to reduce the impurities from SPE sorbent, soxhlet extraction of dichloromethane followed by acetone and lastly methanol was applied to the sorbents for 24 hours and the results had proven that impurities were reduced significantly. In addition to soxhlet extraction, six types of prevalent SPE sorbents were used to absorb 40 target compounds, the lgK(ow) values of which were within the range of 1.46 and 8.1, and recovery rates were compared. It was noticed and confirmed that Waters Oasis HLB had shown the best recovery results for most of the common testing samples among all three styrenedivinylbenzene (SDB) polymer sorbents, which were 77% on average. Furthermore, Waters SepPak AC-2 provided good recovery results for pesticides among three types of activated carbon sorbents and the average recovery rates reached 74%. Therefore, Waters Oasis HLB and Waters SepPak AC-2 were combined to obtain a better recovery and the average recovery rate for the tested 40 compounds of this new SPE method was 87%.

Minor Merger Origin for the Circumnuclear Starburst in NGC 7742

NASA Technical Reports Server (NTRS)

Mazzuca, Lisa M.; Sarzi, M.; Knapen, J. H.; Veilleux, S.; Swaters, R.

2006-01-01

We present an emission-line diagnostic analysis of integral-field spectroscopic observations that cover the central kiloparsec of NGC 7742. This Sa galaxy hosts a spectacular nuclear starburst ring and nuclear regions characterized by low-ionization emission. The gas in the ring rotates in the opposite sense to the stars in the galaxy, suggesting a recent merging or acquisition event. The combination of integral-field measurements for the H alpha+[N II] emission lines from DensePak and the H beta and [O 111] emission from SAURON allow the construction of diagnostic diagrams that highlight the transition from star formation in the nuclear ring to excitation by high-velocity shocks or by a central AGN towards the center. DensePak measurements for the [S II] line ratio reveal very low gas densities in the nuclear ring, N(sub e) less than 100 per cubic centimeters, characteristic of massive H II regions. Comparison with MAPPINGS III models for starbursts with low gas densities show that the ring is of roughly solar metallicity. This suggests that the gas in the nuclear ring originated in a stellar system capable of substantially enriching the gas metallicity through sustained star formation. We propose that NGC 7742 cannibalised a smaller galaxy rich in metal-poor gas, and that star formation episodes in the ring have since increased the metallicity to its present value. The techniques explored here can be widely used to study similar systems, including composite (AGN+starburst) galaxies.

Evaluation of simple rapid HIV assays and development of national rapid HIV test algorithms in Dar es Salaam, Tanzania

PubMed Central

2009-01-01

Background Suitable algorithms based on a combination of two or more simple rapid HIV assays have been shown to have a diagnostic accuracy comparable to double enzyme-linked immunosorbent assay (ELISA) or double ELISA with Western Blot strategies. The aims of this study were to evaluate the performance of five simple rapid HIV assays using whole blood samples from HIV-infected patients, pregnant women, voluntary counseling and testing attendees and blood donors, and to formulate an alternative confirmatory strategy based on rapid HIV testing algorithms suitable for use in Tanzania. Methods Five rapid HIV assays: Determine™ HIV-1/2 (Inverness Medical), SD Bioline HIV 1/2 3.0 (Standard Diagnostics Inc.), First Response HIV Card 1–2.0 (PMC Medical India Pvt Ltd), HIV1/2 Stat-Pak Dipstick (Chembio Diagnostic System, Inc) and Uni-Gold™ HIV-1/2 (Trinity Biotech) were evaluated between June and September 2006 using 1433 whole blood samples from hospital patients, pregnant women, voluntary counseling and testing attendees and blood donors. All samples that were reactive on all or any of the five rapid assays and 10% of non-reactive samples were tested on a confirmatory Inno-Lia HIV I/II immunoblot assay (Immunogenetics). Results Three hundred and ninety samples were confirmed HIV-1 antibody positive, while 1043 were HIV negative. The sensitivity at initial testing of Determine, SD Bioline and Uni-Gold™ was 100% (95% CI; 99.1–100) while First Response and Stat-Pak had sensitivity of 99.5% (95% CI; 98.2–99.9) and 97.7% (95% CI; 95.7–98.9), respectively, which increased to 100% (95% CI; 99.1–100) on repeat testing. The initial specificity of the Uni-Gold™ assay was 100% (95% CI; 99.6–100) while specificities were 99.6% (95% CI; 99–99.9), 99.4% (95% CI; 98.8–99.7), 99.6% (95% CI; 99–99.9) and 99.8% (95% CI; 99.3–99.9) for Determine, SD Bioline, First Response and Stat-Pak assays, respectively. There was no any sample which was concordantly false positive in Uni-Gold™, Determine and SD Bioline assays. Conclusion An alternative confirmatory HIV testing strategy based on initial testing on either SD Bioline or Determine assays followed by testing of reactive samples on the Determine or SD Bioline gave 100% sensitivity (95% CI; 99.1–100) and 100% specificity (95% CI; 96–99.1) with Uni-Gold™ as tiebreaker for discordant results. PMID:19226452

Rapid Immuno-Chromatographic Assay for the Detection of Antibodies to HIV Compare with Elisa among Voluntary and Replacement Blood Donor of Mymensingh Medical College Hospital.

PubMed

Chakrabarty, P; Rudra, S; Hossain, M A; Begum, S A; Mirza, T T; Rudra, M

2015-04-01

Suitable algorithms based on a combination of two or more simple rapid HIV assays have been shown to have a diagnostic accuracy comparable to double enzyme-linked immunosorbent assay (ELISA) or double ELISA with Western Blot strategies. The aims of this study were to evaluate the performance of five simple rapid HIV assays using whole blood samples from voluntary and replacement blood donors & HIV-infected patients (positive samples from BSMMU, Dhaka). Five rapid HIV assays: Determine™ HIV-1/2 (Inverness Medical), SD Bioline HIV 1/2 3.0 (Standard Diagnostics Inc.), First Response HIV Card 1-2.0 (PMC Medical India Pvt Ltd.), HIV1/2 Stat-Pak Dipstick (Chembio Diagnostic System, Inc) and Uni-Gold™ HIV-1/2 (Biotech) were evaluated between 1st February to 30th June, 2013 using 400 whole blood samples from voluntary and replacement blood donors. All samples that were reactive on all or any of the five rapid assays and 10% of non-reactive samples were tested on a confirmatory Inno-Lia HIV I/II immunoblot assay (Immunogenetics). Only 01 sample including ten positive samples from BSMMU were confirmed HIV-1 antibody positive, while 399 were HIV negative. The sensitivity at initial testing of Determine, SD Bioline and Uni-Gold™ was 100% (95% CI; 99.1-100) while First Response and Stat-Pak had sensitivity of 99.5% (95% CI; 98.2-99.9) and 97.7% (95% CI; 95.7-98.9) respectively, which increased to 100% (95% CI; 99.1-100) on repeat testing. The initial specificity of the Uni-Gold™ assay was 100% (95% CI; 99.6-100) while specificities were 99.6% (95% CI; 99-99.9), 99.4% (95% CI; 98.8-99.7), 99.6% (95% CI; 99-99.9) and 99.8% (95% CI; 99.3-99.9) for Determine, SD Bioline, First Response and Stat-Pak assays, respectively. There was no any sample which was concordantly false positive in Uni-Gold™, Determine and SD Bioline assays. An alternative confirmatory HIV testing strategy based on initial testing on either SD Bioline or Determine assays followed by testing of reactive samples on the Determine or SD Bioline gave 100% sensitivity (95% CI; 99.1-100) and 100% specificity (95% CI; 96-99.1) with Uni-Gold™ as tiebreaker for discordant results.

Evaluation of simple rapid HIV assays and development of national rapid HIV test algorithms in Dar es Salaam, Tanzania.

PubMed

Lyamuya, Eligius F; Aboud, Said; Urassa, Willy K; Sufi, Jaffer; Mbwana, Judica; Ndugulile, Faustin; Massambu, Charles

2009-02-18

Suitable algorithms based on a combination of two or more simple rapid HIV assays have been shown to have a diagnostic accuracy comparable to double enzyme-linked immunosorbent assay (ELISA) or double ELISA with Western Blot strategies. The aims of this study were to evaluate the performance of five simple rapid HIV assays using whole blood samples from HIV-infected patients, pregnant women, voluntary counseling and testing attendees and blood donors, and to formulate an alternative confirmatory strategy based on rapid HIV testing algorithms suitable for use in Tanzania. Five rapid HIV assays: Determine HIV-1/2 (Inverness Medical), SD Bioline HIV 1/2 3.0 (Standard Diagnostics Inc.), First Response HIV Card 1-2.0 (PMC Medical India Pvt Ltd), HIV1/2 Stat-Pak Dipstick (Chembio Diagnostic System, Inc) and Uni-Gold HIV-1/2 (Trinity Biotech) were evaluated between June and September 2006 using 1433 whole blood samples from hospital patients, pregnant women, voluntary counseling and testing attendees and blood donors. All samples that were reactive on all or any of the five rapid assays and 10% of non-reactive samples were tested on a confirmatory Inno-Lia HIV I/II immunoblot assay (Immunogenetics). Three hundred and ninety samples were confirmed HIV-1 antibody positive, while 1043 were HIV negative. The sensitivity at initial testing of Determine, SD Bioline and Uni-Gold was 100% (95% CI; 99.1-100) while First Response and Stat-Pak had sensitivity of 99.5% (95% CI; 98.2-99.9) and 97.7% (95% CI; 95.7-98.9), respectively, which increased to 100% (95% CI; 99.1-100) on repeat testing. The initial specificity of the Uni-Gold assay was 100% (95% CI; 99.6-100) while specificities were 99.6% (95% CI; 99-99.9), 99.4% (95% CI; 98.8-99.7), 99.6% (95% CI; 99-99.9) and 99.8% (95% CI; 99.3-99.9) for Determine, SD Bioline, First Response and Stat-Pak assays, respectively. There was no any sample which was concordantly false positive in Uni-Gold, Determine and SD Bioline assays. An alternative confirmatory HIV testing strategy based on initial testing on either SD Bioline or Determine assays followed by testing of reactive samples on the Determine or SD Bioline gave 100% sensitivity (95% CI; 99.1-100) and 100% specificity (95% CI; 96-99.1) with Uni-Gold as tiebreaker for discordant results.

Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries.

PubMed

Huang, Renhua; Fang, Pete; Kay, Brian K

2012-09-01

Site-directed mutagenesis is routinely performed in protein engineering experiments. One method, termed Kunkel mutagenesis, is frequently used for constructing libraries of peptide or protein variants in M13 bacteriophage, followed by affinity selection of phage particles. To make this method more efficient, the following two modifications were introduced: culture was incubated at 25°C for phage replication, which yielded two- to sevenfold more single-stranded DNA template compared to growth at 37°C, and restriction endonuclease recognition sites were used to remove non-recombinants. With both of the improvements, we could construct primary libraries of high complexity and that were 99-100% recombinant. Finally, with a third modification to the standard protocol of Kunkel mutagenesis, two secondary (mutagenic) libraries of a fibronectin type III (FN3) monobody were constructed with DNA segments that were amplified by error-prone and asymmetric PCR. Two advantages of this modification are that it bypasses the lengthy steps of restriction enzyme digestion and ligation, and that the pool of phage clones, recovered after affinity selection, can be used directly to generate a secondary library. Screening one of the two mutagenic libraries yielded variants that bound two- to fourfold tighter to human Pak1 kinase than the starting clone. The protocols described in this study should accelerate the discovery of phage-displayed recombinant affinity reagents. Copyright © 2012 Elsevier Inc. All rights reserved.

40 CFR 721.10297 - Tin, C16-18 and C18-unsatd. fatty acids castor-oil fatty acids complexes.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Tin, C16-18 and C18-unsatd. fatty... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10297 Tin, C16-18 and C18-unsatd... to reporting. (1) The chemical substance identified as tin, C16-18 and C18-unsatd. fatty acids castor...

40 CFR 721.10297 - Tin, C16-18 and C18-unsatd. fatty acids castor-oil fatty acids complexes.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Tin, C16-18 and C18-unsatd. fatty... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10297 Tin, C16-18 and C18-unsatd... to reporting. (1) The chemical substance identified as tin, C16-18 and C18-unsatd. fatty acids castor...

40 CFR 721.10297 - Tin, C16-18 and C18-unsatd. fatty acids castor-oil fatty acids complexes.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Tin, C16-18 and C18-unsatd. fatty... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10297 Tin, C16-18 and C18-unsatd... to reporting. (1) The chemical substance identified as tin, C16-18 and C18-unsatd. fatty acids castor...

Bulk Scale Formulation of Therapeutic Doses of Clinical Grade Ready-to-Use 177Lu-DOTA-TATE: The Intricate Radiochemistry Aspects.

PubMed

Mathur, Anupam; Prashant, Vrinda; Sakhare, Navin; Chakraborty, Sudipta; Vimalnath, K V; Mohan, Repaka Krishna; Arjun, Chanda; Karkhanis, Barkha; Seshan, Ravi; Basu, Sandip; Korde, Aruna; Banerjee, Sharmila; Dash, Ashutosh; Sachdev, Satbir Singh

2017-09-01

177 Lu-DOTA-TATE is a clinically useful and promising therapeutic radiopharmaceutical for peptide receptor radionuclide therapy of neuroendocrine tumors (NETs) overexpressing somatostatin receptors. Currently, the radiopharmaceutical is prepared in-house at nuclear medicine centers, thereby restricting its use to limited centers only. In this article, the authors describe systematic studies toward bulk scale formulation of "ready-to-use" 177 Lu-DOTA-TATE using medium specific activity 177 Lu (740-1110 GBq/mg) at a centralized radiopharmacy facility. In an optimized protocol, 177 Lu-DOTA-TATE synthesis was carried out by direct heating of 177 LuCl 3 (Sp. act. 740-1110 GBq/mg) with DOTA-TATE peptide (1.5-3.0 equivalents) in ammonium acetate buffer (0.2 M) containing 2,5-dihydroxy benzoic acid (gentisic acid). Thereafter, the crude labeled product was purified using a Sep-Pak ® C18 column and diluted with acetate buffer-gentisic acid (1.5% w/v) solution to final radioactive concentration of 740 MBq/mL. This was further sterilized and dispensed as 7.4 GBq patient dose/vial with 2 days postformulation calibration. A peptide/metal ratio of 1.5-3.0 is essential for complexation wherein radiolabeling yields >90% are obtained minimizing free 177 Lu waste. For formulation of 7.4 GBq patient dose (2 days postproduction), even specific activity of about 555 GBq/mg was found to be adequate for the radiometal. The ready-to-use 740 MBq/mL 177 Lu-DOTA-TATE formulation with gentisic acid (1.5% w/v) is observed to be safe for human use for more than 1 week (radiochemical purity >98%) from the day of production when stored at -70°C. However, the target specificity may get affected beyond 2 days as the total peptide content for 7.4 GBq dose may exceed the critical peptide limit of 300 μg. Patient treatment carried with several batches of present formulation in diseased NET patients exhibited desired distribution at the tumor and its metastatic site. A ready-to-use formulation of 177 Lu-DOTA-TATE was successfully prepared and optimized for regular bulk scale production and supply to distant nuclear medicine centers.

The Petroglyphs at Lake Pend Oreille, Bonner County, Northern Idaho.

DTIC Science & Technology

1985-12-01

LAKE PEND OREILLE BONNER COUNTY, NORTHERN IDAHO 00 00 DTIC rCbELECTE DEC 2 8 1987 US Army Corps 87 12 140 of Engineers 1 Seattle District .t bdaaP O... IDAHO 6. PERFORMING ORG. REPORT NUMBERrNs * 1 7. AUTHOR(e) 8. CONTRACT OR GRANT NUMBER(s) K. Boreson and W.R. Peterson DACW67-85-M-0025 9. PERFORMING...CLASSIFICATION OF THIS PAkGE(Won Data Fntered) % .4 %. %~~ % I lr- rv-tTVK tm V THE PETROGLYPHS AT LAKE PEND OREILLE, BONNER COUNTY, NORTHERN IDAHO By

Regulation of Adhesion and Migration by the Rsu1- and PINCH1-mediated Inhibition of Focal Adhesion Formation and Actin Polymerization

DTIC Science & Technology

2012-06-08

RT-PCR and sequencing of the Rsu1 open reading frame revealed that exon 8 is missing in the alternatively spliced Rsu1 RNA in human gliomas and...concentration of 75 nM. Cells were collected 72-96 hours post-transfection. 16 Western blotting Cell lysates were collected in RIPA buffer...cell lysates were collected and bound to the glutathione beads loaded with the GST-fusion of the Rac1/cdc42 binding domain of Pak. The bound Rac1

Bepaling Referentiewaarden voor Ergonomie en Warmtebelasting van Lichtgewicht Bommenpakken (Determination of Ergonomic and Thermal Load Tests and Assessment of Reference Values With Light Weight Bomb Disposal Suits)

DTIC Science & Technology

2008-05-01

bommenpakken kunnen geven bij bewegingen worden gemeten met de volgende bewegingbeperkingtests: sit-and-reach, stand-and-reach, abductie van de armen ...anteflexie van de armen en beperking van zicht. Bij de sit-and-reach test komt een eventuele belernmering bij het voorover buigen naar voren. De...proefpersoon zit op de grond met de benen en armen gestrekt naar voren. Daarbij wordt de afstand tussen de tenen en de vingers gemeten (figuur 4). Als het pak

Testing the Role of p21 Activated Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely Phenocopies Human NF2 Disease

DTIC Science & Technology

2017-06-01

Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely Phenocopies Human NF2 Disease The views, opinions and...Role of p21 Activated Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely Phenocopies Human NF2 Disease Form...NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. The major goal of this research project was to genetically and pharmacologically test the requirement of PAK

Second-hand smoke in four English prisons: an air quality monitoring study.

PubMed

Jayes, Leah R; Ratschen, Elena; Murray, Rachael L; Dymond-White, Suzy; Britton, John

2016-02-04

To measure levels of indoor pollution in relation to smoking in four English prisons. TSI SidePak AM510 Personal Aerosol Monitors were used to measure concentrations of particulate matter less than 2.5 μm in diameter (PM2.5) for periods of up to 9 h in selected smoking and non-smoking areas, and personal exposure monitoring of prison staff during a work shift, in four prisons. PM2.5 data were collected for average periods of 6.5 h from 48 locations on 25 wing landings where smoking was permitted in cells, on 5 non-smoking wings, 13 prisoner cells, and personal monitoring of 22 staff members. Arithmetic mean PM2.5 concentrations were significantly higher on smoking than non-smoking wing landings (43.9 μg/m(3) and 5.9 μg/m(3) respectively, p < 0.001) and in smoking than non-smoking cells (226.2 μg/m(3) and 17.0 μg/m(3) respectively, p < 0.001). Staff members wore monitors for an average of 4.18 h, during which they were exposed to arithmetic mean PM2.5 concentration of 23.5 μg/m(3). The concentration of PM2.5 pollution in smoking areas of prisons are extremely high. Smoking in prisons therefore represents a significant health hazard to prisoners and staff members.

[Isolation and determination of the seeds of Pachyrrhizus errosus protein by high performance gel filtration chromatography (GFC)].

PubMed

Wu, H; Hao, B; Tang, G; Lin, Y

1997-03-01

From the seeds of Pachyrrhizus errosus, three protein constituents, namel PE1, PE2 and PE3, have been isolated and purified by extraction with 5mmol/L phosphate saline (0.9% NaCl) buffer (PB) at pH 7.2, and S-Sepharose Fast Flow Column (2.6cm x 15cm) chromatography which eluted with 5mmol/L phosphate buffer (pH 7.0) containing 1mmol/L NaCl. Three proteins were burther separated on two connected Protein-Pak 60+Protein-Pak 125 [7.5mm x 39cm, 10microm] columns with mobile phase of 0.2mol/L phosphate buffer (pH 6.5). The flow rate was kept constant at 0.8mL/min by YSB-2 type high press pump. The effluent was monitored at a wavelength of 280nm on photodiode array detector. These three proteins are proved to be homogeneous by SDS-PAGE, IEF and HPGFC experiments, and all present the typical absorption spectra in ultraviolet region. The moleculer weights of the three proteins are approxiamtely 33000D, 14500D and 14000D respectively by SDS-PAGE. But as using HPGFC analysis, the MW value of PE2 is 28000D. This indicates PE2 may be composed of two chains joined by disulfide bond, which is further proved from the latter amino acid composition analysis. The isoelectric points of three proteins are 4.5, 6.5 and 7.5 respectively by using IEF. The amion acids compositions of the three proteins were determined with OPA post-column derivatization/fluorescence detection.

Signaling Cascades Governing Cdc42-Mediated Chondrogenic Differentiation and Mensenchymal Condensation.

PubMed

Wang, Jirong R; Wang, Chaojun J; Xu, Chengyun Y; Wu, Xiaokai K; Hong, Dun; Shi, Wei; Gong, Ying; Chen, Haixiao X; Long, Fanxin; Wu, Ximei M

2016-03-01

Endochondral ossification consists of successive steps of chondrocyte differentiation, including mesenchymal condensation, differentiation of chondrocytes, and hypertrophy followed by mineralization and ossification. Loss-of-function studies have revealed that abnormal growth plate cartilage of the Cdc42 mutant contributes to the defects in endochondral bone formation. Here, we have investigated the roles of Cdc42 in osteogenesis and signaling cascades governing Cdc42-mediated chondrogenic differentiation. Though deletion of Cdc42 in limb mesenchymal progenitors led to severe defects in endochondral ossification, either ablation of Cdc42 in limb preosteoblasts or knockdown of Cdc42 in vitro had no obvious effects on bone formation and osteoblast differentiation. However, in Cdc42 mutant limb buds, loss of Cdc42 in mesenchymal progenitors led to marked inactivation of p38 and Smad1/5, and in micromass cultures, Cdc42 lay on the upstream of p38 to activate Smad1/5 in bone morphogenetic protein-2-induced mesenchymal condensation. Finally, Cdc42 also lay on the upstream of protein kinase B to transactivate Sox9 and subsequently induced the expression of chondrocyte differential marker in transforming growth factor-β1-induced chondrogenesis. Taken together, by using biochemical and genetic approaches, we have demonstrated that Cdc42 is involved not in osteogenesis but in chondrogenesis in which the BMP2/Cdc42/Pak/p38/Smad signaling module promotes mesenchymal condensation and the TGF-β/Cdc42/Pak/Akt/Sox9 signaling module facilitates chondrogenic differentiation. Copyright © 2016 by the Genetics Society of America.

Infectivity of wild bird-origin avian paramyxovirus serotype 1 and vaccine effectiveness in chickens.

PubMed

Shabbir, Muhammad Zubair; Akhtar, Sameera; Tang, Yi; Yaqub, Tahir; Ahmad, Arfan; Mustafa, Ghulam; Alam, Muhammad Azhar; Santhakumar, Diwakar; Nair, Venugopal; Munir, Muhammad

2016-12-01

Newcastle disease virus, a prototype avian paramyxovirus serotype 1 (APMV-1), causes economically devastating disease in avian species around the world. Newcastle disease is enzootic in Pakistan and recurrent outbreaks are frequent in multiple avian species even after continuous and extensive use of vaccines. A number of APMV-1 and pigeon paramyxovirus serotype 1 (PPMV-1) strains have been isolated and genetically characterized in recent years. However, the impact of recently characterized wild bird-origin APMVs in domestic poultry, and the potency of routinely used vaccines against these novel and genetically diverse viruses remain unknown. Here, we applied next-generation sequencing for unbiased complete genome characterization of APMV-1 and PPMV-1 strains isolated from clinically diseased peacocks (Pavocristatus) and pigeons (Columbalivia), respectively. Global phylodynamics and evolutionary analysis demonstrates Pigeon/MZS-UVAS-Pak/2014 is clustered into lineage 4 (or genotype VI) and Peacock/MZS-UVAS-Pak/2014 into lineage 5 (or genotype VII). The genomes of both isolates encoded for polybasic residues (112RRQKR↓F117) at the fusion protein cleavage motif along with a number of important substitutions in the surface glycoproteins compared with the vaccine strains. Clinicopathological and immunological investigations in domesticated chickens indicate that these isolates can potentially transmit between tested avian species, can cause systemic infections, and can induce antibodies that are unable to prevent virus shedding. Collectively, the data from these genomic and biological assessments highlight the potential of wild birds in transmitting APMVs to domesticated chickens. The study also demonstrates that the current vaccine regimens are incapable of providing complete protection against wild bird-origin APMVs and PPMVs.

Estrogen-induced cell signaling in the sexually dimorphic nucleus of the rat preoptic area: potential involvement of cofilin in actin dynamics for cell migration.

PubMed

Wada-Kiyama, Yuko; Suzuki, Chiaki; Hamada, Tomohiro; Rai, Dilip; Kiyama, Ryoiti; Kaneda, Makoto; Sakuma, Yasuo

2013-05-03

Estrogen is a key factor to induce the sexually dimorphic nucleus (SDN) in the preoptic area (POA) of the rat brain. Identification of estrogen-dependent signaling pathways at SDN in POA during the critical period is a prerequisite for elucidating the mechanism. In the present study, we treated female rats with/without 17β-estradiol (E2) at birth, designated as postnatal day 1 (P1), and prepared total RNA from brain slices containing SDN for DNA microarray analysis. Among the estrogen-responsive genes identified, protein kinase C-delta (PKC-δ) was significantly up-regulated by E2 at P5. We examined the downstream effectors of PKC-δ protein by Western blotting and found an E2-induced PKC-δ/Rac1/PAK1/LIMK1/cofilin pathway. In the pathway, E2 suppressed the phosphorylation (inactive form) of cofilin. This result was supported by immunohistochemistry, where the phosphorylation/dephosphorylation of cofilin occurred at SDN, which suggests that cell migration is a cue to create sexual dimorphism in POA. Copyright © 2013 Elsevier Inc. All rights reserved.

LC determination of moclobemide and three metabolites in plasma.

PubMed

Misztal, Genowefa; Skibiński, Robert; Olajossy, Marcin; Paw, Beata

2002-11-07

Moclobemide and three metabolites were quantified using 1 ml of plasma and solid-phase extraction with Bakerbond CN column after the addition of nadolol as the internal standard (I.S.). Separation and detection the analysed substances were achieved isocratically with acetonitrile-methanol-0.067 M phosphate buffer pH 2.65-0.4% triethylamine (12.7:1.9:85:0.4, v/v/v/v), a Nova-pak C(8) column and UV detection at 230 nm. The lower limits of quantitation for moclobemide was 10 ng ml(-1), for M1 (Ro 16-3177)-8 ng ml(-1), for M2 (Ro 12-5637)-10 ng ml(-1) and for M3 (Ro 12-8095)-15 ng ml(-1) (as the metabolites). Accuracies calculated of three concentrations in each of three separate runs were between 84.55 and 93.68 for moclobemide, 83.28 and 92.30 for M1, 86.31 and 92.66 for M2 and 88.42 and 93.40 for M3. Precision data within day were between 5.71 and 7.50 for moclobemide, 2.91 and 6.58 for M1, 4.98 and 6.40 for M2 and 0.94 and 4.73 for M3.

M. tuberculosis-Initiated Human Mannose Receptor Signaling Regulates Macrophage Recognition and Vesicle Trafficking by FcRγ-Chain, Grb2, and SHP-1.

PubMed

Rajaram, Murugesan V S; Arnett, Eusondia; Azad, Abul K; Guirado, Evelyn; Ni, Bin; Gerberick, Abigail D; He, Li-Zhen; Keler, Tibor; Thomas, Lawrence J; Lafuse, William P; Schlesinger, Larry S

2017-10-03

Despite its prominent role as a C-type lectin (CTL) pattern recognition receptor, mannose receptor (MR, CD206)-specific signaling molecules and pathways are unknown. The MR is highly expressed on human macrophages, regulating endocytosis, phagocytosis, and immune responses and mediating Mycobacterium tuberculosis (M.tb) phagocytosis by human macrophages, thereby limiting phagosome-lysosome (P-L) fusion. We identified human MR-associated proteins using phosphorylated and non-phosphorylated MR cytoplasmic tail peptides. We found that MR binds FcRγ-chain, which is required for MR plasma membrane localization and M.tb cell association. Additionally, we discovered that MR-mediated M.tb association triggers immediate MR tyrosine residue phosphorylation and Grb2 recruitment, activating the Rac/Pak/Cdc-42 signaling cascade important for M.tb uptake. MR activation subsequently recruits SHP-1 to the M.tb-containing phagosome, where its activity limits PI(3)P generation at the phagosome and M.tb P-L fusion and promotes M.tb growth. In sum, we identify human MR signaling pathways that temporally regulate phagocytosis and P-L fusion during M.tb infection. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Analysis and theoretical modeling of 18O enriched carbon dioxide spectrum by CRDS near 1.35 μm: (II) 16O13C18O, 16O13C17O, 12C18O2, 17O12C18O, 12C17O2, 13C18O2 and 17O13C18O

NASA Astrophysics Data System (ADS)

Karlovets, E. V.; Campargue, A.; Kassi, S.; Tashkun, S. A.; Perevalov, V. I.

2017-04-01

This contribution is the second part of the analysis of the room temperature absorption spectrum of 18O enriched carbon dioxide by very high sensitivity Cavity Ring Down spectroscopy between 6977 and 7918 cm-1 (1.43-1.26 μm). Overall, more than 8600 lines belonging to 166 bands of eleven carbon dioxide isotopologues were rovibrationnally assigned. In a first part (Kassi et al. J Quant Spectrosc Radiat Transfer 187 (2017) 414-425, http://dx.doi.org/10.1016/j.jqsrt.2016.09.002), the results relative to mono-substituted isotopologues, 16O12C18O, 16O12C17O, 12C16O2 and 13C16O2, were presented. This second contribution is devoted to the multiply-substituted isotopologues or clumped isotopologues of particular importance in geochemistry: 16O13C18O, 16O13C17O, 12C18O2, 17O12C18O, 12C17O2, 13C18O2 and 17O13C18O. On the basis of the predictions of effective Hamiltonian models, a total of 3195 transitions belonging to 73 bands were rovibrationnally assigned for these seven species. Among the 73 observed bands, 55 are newly reported. All the identified bands correspond to ΔP=10 and 11 series of transitions, where P= 2V1+V2+3V3 is the polyad number (Vi are vibrational quantum numbers). The accurate spectroscopic parameters of 70 bands have been determined from the standard band-by-band analysis. Global fits of the measured line intensities of the ΔP=10 series of transitions of 17O12C18O and 16O13C18O and of the ΔP=11 series of transitions of 12C18O2, 17O12C18O, 16O13C18O and 13C18O2 were performed to determine the corresponding sets of the effective dipole moment parameters.

Vegetable Oil Derived Solvent, and Catalyst Free “Click Chemistry” Thermoplastic Polytriazoles

PubMed Central

Floros, Michael C.; Leão, Alcides Lopes; Narine, Suresh S.

2014-01-01

Azide-alkyne Huisgen “click” chemistry provides new synthetic routes for making thermoplastic polytriazole polymers—without solvent or catalyst. This method was used to polymerize three diester dialkyne monomers with a lipid derived 18 carbon diazide to produce a series of polymers (labelled C18C18, C18C9, and C18C4 based on monomer chain lengths) free of residual solvent and catalyst. Three diester dialkyne monomers were synthesized with ester chain lengths of 4, 9, and 18 carbons from renewable sources. Significant differences in thermal and mechanical properties were observed between C18C9 and the two other polymers. C18C9 presented a lower melting temperature, higher elongation at break, and reduced Young's modulus compared to C18C4 and C18C18. This was due to the “odd-even” effect induced by the number of carbon atoms in the monomers which resulted in orientation of the ester linkages of C18C9 in the same direction, thereby reducing hydrogen bonding. The thermoplastic polytriazoles presented are novel polymers derived from vegetable oil with favourable mechanical and thermal properties suitable for a large range of applications where no residual solvent or catalyst can be tolerated. Their added potential biocompatibility and biodegradability make them ideal for applications in the medical and pharmaceutical industries. PMID:25032224

Intracellular delivery and antitumor effects of pH-sensitive liposomes based on zwitterionic oligopeptide lipids.

PubMed

Mo, Ran; Sun, Qiong; Li, Nan; Zhang, Can

2013-04-01

pH-sensitive liposomes (HHG2C(18)-L and PEGHG2C(18)-L) based on zwitterionic oligopeptide lipids as anticancer drug carriers were developed and evaluated for effective intracellular delivery and enhanced antitumor activity. The amino acid-based lipids, 1,5-dioctadecyl-l-glutamyl 2-histidyl-hexahydrobenzoic acid (HHG2C(18)) and 1,5-distearyl N-(N-α-(4-mPEG2000) butanedione)-histidyl-l-glutamate (PEGHG2C(18)), were synthesized, which have the multistage pH-response to tumor microenvironmental pH (pH(e), pH 6.0-7.0) and endosomal/lysosomal pH (pH(i), pH 4.0-6.0) successively. HHG2C(18)-L contains HHG2C(18), while PEGHG2C(18)-L includes HHG2C(18) and PEGHG2C(18). Both of them displayed the capability of charge conversion to the surrounding pH. The zeta potentials of HHG2C(18)-L and PEGHG2C(18)-L were negative at pH 7.4, whereas positive at pH 6.5 and more positive at lower pH. Coumarin 6-loaded HHG2C(18)-L (C6/HHG2C(18)-L) and PEGHG2C(18)-L (C6/PEGHG2C(18)-L) showed higher tumor cellular uptake due to electrostatic absorptive endocytosis at pH(e) (pH 6.5), produced proton sponge effect for endo-lysosomal escape, and accumulated to the mitochondria based on stronger positive charge by the hydrolysis of a pH-sensitive linker at pH(i) (pH 5.5 and pH 4.5). Furthermore, temsirolimus (CCI-779)-loaded HHG2C(18)-L (CCI-779/HHG2C(18)-L) and PEGHG2C(18)-L (CCI-779/PEGHG2C(18)-L) had significantly higher antiproliferative and apoptosis inducing effects toward the human renal carcinoma (A498) cells at pH 6.5 relative to that at pH 7.4. The half maximal inhibitory concentration (IC50) of CCI-779/HHG2C(18)-L and CCI-779/PEGHG2C(18)-L were about 3 μg/mL and 5 μg/mL at pH 6.5, 1.67-fold and 1.60-fold improved relative to that at pH 7.4, respectively. The total apoptotic ratio of CCI-779/HHG2C(18)-L and CCI-779/PEGHG2C(18)-L increased from 9.90% and 7.78% at pH 7.4 to 19.53% and 12.10% at pH 6.5, respectively. In vivo, CCI-779/PEGHG2C(18)-L after intravenous administration presented remarkably higher bioavailability and blood persistence compared with unPEGylated CCI-779/HHG2C(18)-L, and had the strongest antitumor efficacy against xenograft renal cancer (Renca) tumor models. Accordingly, the results provide the feasibility of using pH-sensitive zwitterionic oligopeptide lipids to extend the applications of liposomes to efficient anticancer drug delivery in cancer therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

40 CFR 721.642 - Amines, N-(C14-18 and C16-16 unsaturated alkyl)] dipropylene-tri-, tripropylenetetra-, and...

Code of Federal Regulations, 2010 CFR

2010-07-01

... substances amines, N-(C14-18 and C16-18 unsaturated alkyl)] dipropylenetri-, (PMN P-94-1244... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Amines, N-(C14-18 and C16-16... Amines, N-(C14-18 and C16-16 unsaturated alkyl)] dipropylene-tri-, tripropylenetetra-, and...

Intestinal digestibility of long-chain fatty acids in lactating dairy cows: A meta-analysis and meta-regression.

PubMed

Boerman, J P; Firkins, J L; St-Pierre, N R; Lock, A L

2015-12-01

The objective of this analysis was to examine the intestinal digestibility of individual long-chain fatty acids (FA) in lactating dairy cows. Available data were collated from 15 publications containing 61 treatments, which reported total and individual FA duodenal flows and calculations of intestinal digestibility. All studies involved lactating dairy cows, and estimates of digestibility were based on measurements either between the duodenum and ileum (18 treatments) or between the duodenum and feces (43 treatments). Fatty acid digestibility was calculated for C16:0, C18:0, C18:1 (cis and trans isomers), C18:2, and C18:3. Digestibility of C18:0 was lower than for C18:1 and C18:3, with no difference in digestibility between saturated FA (C16:0 and C18:0). We weighted the studies by the reciprocal of the variance to generate best-fit equations to predict individual FA digestibility based on duodenal flow of FA and dietary independent variables. The flow of C18:0 negatively affected the digestibility of C18:0 and was also included in the best-fit equations for all other 18-carbon FA using duodenal flow characteristics. The type of fat supplemented had an effect on digestibility of individual FA, with whole seeds having reduced digestibility. Our meta-analysis results showed minimal differences in the digestibility of individual FA. However, C18:0 flow through the duodenum had a negative effect on the digestibility of several individual FA, with the largest negative effect on C18:0 digestibility. The mechanisms that reduce C18:0 absorption at high concentrations are unknown and warrant further investigation. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Group I p21-activated kinases: emerging roles in immune function and viral pathogenesis.

PubMed

Pacheco, Almudena; Chernoff, Jonathan

2010-01-01

Group I p21-activated kinases are a highly conserved three-member family of serine/threonine kinases that act as key effectors for the small GTPases Cdc42 and Rac. In man, these enzymes have been implicated in a wide range of biological processes and are beginning to draw the attention of the pharmaceutical industry as potential therapeutic targets in cancer and in inflammatory processes. In this review, we summarize basic properties of group I Paks and discuss recently uncovered roles for these kinases in immune function and in viral infection.

Proceedings, U.S. Army Corps of Engineers Seminar on Water Project Financing Held at Fort Belvoir, Virginia on 16-17 May 1984.

DTIC Science & Technology

1984-10-01

DAMmwrams, 2.0 2.7 Minicipal Waer b~pply 3.7 8.0 1ftt t rTreatmnt 5.9 7.3 */ art vatenmya, and flood control. Doss not Includeipadutl $3.7 billion in...Department. Please see PA.KS, Page 3 Cryder was an early advocate of the innovative entrepreneurial strategy By KEVIN RODERICK, and has made the county...Joint Agency Financing." I . .. Finace iviionat mit Baneyinvovin waer n ydreletr* prjet. "-". Robert B. Nolan, Jr. Senior Vice President Blyth Eastman

Glufosinate ammonium clean-up procedure from water samples using SPE

NASA Astrophysics Data System (ADS)

Tayeb M., A.; Ismail B., S.; Mardiana-Jansar, K.; Ta, Goh Choo; Agustar, Hani Kartini

2015-09-01

For the determination of glufosinate ammonium residue in soil and water samples, different solid phase extraction (SPE) sorbent efficiency was studied. Four different SPE sorbents i.e.: CROMABOND PS-H+, CROMABOND PS-OH-, ISOLUTE ENV+, Water Sep-Pak and OASIS HLB were used. Sample clean-up performance was evaluated using high performance liquid chromatography (Agilent 1220 infinity LC) with fluorescence detector. Detection of FMO-derivatives was done at λ ex = 260 nm and λ em= 310 nm. OASIS HLB column was the most suitable for the clean-up in view of the overall feasibility of the analysis.

Response of Selected Microorganisms to Experimental Planetary Environments

NASA Technical Reports Server (NTRS)

Foster, T. L.; Winans, L., Jr.

1977-01-01

Results of studies in anaerobic phosphorus metabolism are presented. Specific topics discussed include: (1) anaerobic utilization of PH3; (2) reduction of phosphate or phosphite; (3) isolation of organisms which utilize phosphite or phosphate anaerobically as a final hydrogen acceptor; and (4) the toxicity of PH3 to the organisms. Techniques of anaerobic microbiology associated with space hardware were also studied. These include: (1) the Brewer anaerobe jar/GasPak system; (2) a new procedure to grow aerobes and anaerobes simultaneously; (3) a culture medium to differentiate oblagate from facultative anaerobes; and (4) a procedure to quantitate O2 sensitivity of anaerobes.

Project W-320 acceptance test report for AY-farm electrical distribution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bevins, R.R.

1998-04-02

This Acceptance Test Procedure (ATP) has been prepared to demonstrate that the AY-Farm Electrical Distribution System functions as required by the design criteria. This test is divided into three parts to support the planned construction schedule; Section 8 tests Mini-Power Pane AY102-PPI and the EES; Section 9 tests the SSS support systems; Section 10 tests the SSS and the Multi-Pak Group Control Panel. This test does not include the operation of end-use components (loads) supplied from the distribution system. Tests of the end-use components (loads) will be performed by other W-320 ATPs.

Characterisation of Liquefaction Effects for Beyond-Design Basis Safety Assessment of Nuclear Power Plants

NASA Astrophysics Data System (ADS)

Bán, Zoltán; Győri, Erzsébet; János Katona, Tamás; Tóth, László

2015-04-01

Preparedness of nuclear power plants to beyond design base external effects became high importance after 11th of March 2011 Great Tohoku Earthquakes. In case of some nuclear power plants constructed at the soft soil sites, liquefaction should be considered as a beyond design basis hazard. The consequences of liquefaction have to be analysed with the aim of definition of post-event plant condition, identification of plant vulnerabilities and planning the necessary measures for accident management. In the paper, the methodology of the analysis of liquefaction effects for nuclear power plants is outlined. The case of Nuclear Power Plant at Paks, Hungary is used as an example for demonstration of practical importance of the presented results and considerations. Contrary to the design, conservatism of the methodology for the evaluation of beyond design basis liquefaction effects for an operating plant has to be limited to a reasonable level. Consequently, applicability of all existing methods has to be considered for the best estimation. The adequacy and conclusiveness of the results is mainly limited by the epistemic uncertainty of the methods used for liquefaction hazard definition and definition of engineering parameters characterizing the consequences of liquefaction. The methods have to comply with controversial requirements. They have to be consistent and widely accepted and used in the practice. They have to be based on the comprehensive database. They have to provide basis for the evaluation of dominating engineering parameters that control the post-liquefaction response of the plant structures. Experience of Kashiwazaki-Kariwa plant hit by Niigata-ken Chuetsu-oki earthquake of 16 July 2007 and analysis of site conditions and plant layout at Paks plant have shown that the differential settlement is found to be the dominating effect in case considered. They have to be based on the probabilistic seismic hazard assessment and allow the integration into logic-tree procedure. Earlier studies have shown that the potentially liquefiable layer at Paks Nuclear Power Plant is situated in relatively large depth. Therefore the applicability and adequacy of the methods at high overburden pressure is important. In case of existing facilities, the geotechnical data gained before construction aren't sufficient for the comprehensive liquefaction analysis. Performance of new geotechnical survey is limited. Consequently, the availability of the data has to be accounted while selection the analysis methods. Considerations have to be made for dealing with aleatory uncertainty related to the knowledge of the soil conditions. It is shown in the paper, a careful comparison and analysis of the results obtained by different methodologies provides the basis of the selection of practicable methods for the safety analysis of nuclear power plant for beyond design basis liquefaction hazard.

Characterization of cis- and trans-octadecenoic acid positional isomers in edible fat and oil using gas chromatography-flame ionisation detector equipped with highly polar ionic liquid capillary column.

PubMed

Yoshinaga, Kazuaki; Asanuma, Masaharu; Mizobe, Hoyo; Kojima, Koichi; Nagai, Toshiharu; Beppu, Fumiaki; Gotoh, Naohiro

2014-10-01

In this study, the characterisation of all cis- and trans-octadecenoic acid (C18:1) positional isomers in partially hydrogenated vegetable oil (PHVO) and milk fat, which contain several cis- and trans-C18:1 positional isomers, was achieved by gas chromatography-flame ionisation detector equipped with a highly polar ionic liquid capillary column (SLB-IL111). Prior to analysis, the cis- and trans-C18:1 fractions in PHVO and milk fat were separated using a silver-ion cartridge. The resolution of all cis-C18:1 positional isomers was successfully accomplished at the optimal isothermal column temperature of 120 °C. Similarly, the positional isomers of trans-C18:1, except for trans-6-C18:1 and trans-7-C18:1, were separated at 120 °C. The resolution of trans-6-C18:1 and trans-7-C18:1 isomers was made possible by increasing the column temperature to 160 °C. This analytical method is suitable for determining the cis- and trans-C18:1 positional isomers in edible fats and oils. Copyright © 2014 Elsevier Ltd. All rights reserved.

Akt mediated ROS-dependent selective targeting of mutant KRAS tumors.

PubMed

Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

2014-10-01

Reactive oxygen species (ROS) play a critical role in a variety of cellular processes, ranging from cell survival and proliferation to cell death. Previously, we reported the ability of a small molecule compound, C1, to induce ROS dependent autophagy associated apoptosis in human cancer cell lines and primary tumor cells (Wong C. et al. 2010). Our ongoing investigations have unraveled a hitherto undefined novel signaling network involving hyper-phosphorylation of Akt and Akt-mediated ROS production in cancer cell lines. Interestingly, drug-induced Akt activation is selectively seen in cell lines that carry mutant KRAS; HCT116 cells that carry the V13D KRAS mutation respond favorably to C1 while HT29 cells expressing wild type KRAS are relatively resistant. Of note, not only does the compound target mutant KRAS expressing cells but also induces RAS activation as evidenced by the PAK pull down assay. Corroborating this, pharmacological inhibition as well as siRNA mediated silencing of KRAS or Akt, blocked C1-induced ROS production and rescued tumor colony forming ability in HCT116 cells. To further confirm the involvement of KRAS, we made use of mutant KRAS transformed RWPE-1 prostate epithelial cells. Notably, drug-induced ROS generation and death sensitivity was significantly higher in RWPE-1-KRAS cells than the RWPE-1-vector cells, thus confirming the results obtained with mutant KRAS colorectal carcinoma cell line. Lastly, we made use of HCT116 mutant KRAS knockout cells (KO) where the mutant KRAS allele had been deleted, thus expressing a single wild-type KRAS allele. Exposure of the KO cells to C1 failed to induce Akt activation and mitochondrial ROS production. Taken together, results show the involvement of activated Akt in ROS-mediated selective targeting of mutant KRAS expressing tumors, which could have therapeutic implications given the paucity of chemotherapeutic strategies specifically targeting KRAS mutant cancers. Copyright © 2014. Published by Elsevier Inc.

Dissolved Gases in Seawater and Sediments (Paper 7R0315)

NASA Astrophysics Data System (ADS)

Key, R. M.

1987-07-01

Certainly the most controversial results derived from the study of any dissolved gas concerned oxygen utilization rates in the North Atlantic. Jenkins (1982) estimated a net oxy-gen utilization rate (OUR) for the Beta triangle region of the North Atlantic (apices 26.5°N x 38.5°W, 32.5°N x 30.0°W, and 22.5°N x 28.5°W) of 5.7 moles of oxygen consumed m-2 yr-1 for the zone below 100m. He assumed that the oxygen distribution below the euphotic zone was stationary and steady state and therfore that the in situ oxygen consumption must be balanced by physical transport of oxygen into the area. His estimates were based on measured distributions of dissolved oxygen and the tracers 3He and 3H and a simple model which assumed lateral advection was small. The derived value or OUR was several times higher that previous estimates based on 14C and 15N incubation techniques. The OUR requires a downward flux of carbon from the photic zone of approximately 50 gCm-2yr-1 , which is again much higher that previous results. Jenkins and Goldman (1985) amplified the arguments in a study of seasonal oxygen cycling and primary production based on a ten year time series of measurements from the Panulirus station near Bermuda. Considering insolation, heat budgets and 3He/3H data they estimated a vertically integrated oxygen production rate of 5 Mm-2yr-1 and a subsequent new production of 50 gCm-2yr-1. The results were supported by calculations based on a second order turbulence closure model (Klein and Coste, 1984). These results have been challenged primarily on the basis of the spatial variability of the phenomenon!. Whatever the final outcome Jenkins has clearly demonstrated that the time has come to take a fresh look at oxygen utilization rates and primary productivity given the tools and modelling capabilities now at hand. The TTO data set will go a long way toward providing the necessary data set for the North Atlantic when the analyses are complete. Other studies have dealt with the photooxidative daylight loss of oxygen from near-surface tropical waters (Gieskes and Kraay, 1982), isotopic fractionation between fresh and seawater and the atmosphere (Benson and Krause, 1984) , edge effects on chemistry in the 02 minimum zone (Mullins et al., 1985), and the relationship between oxygen and other biogeochemical properties (Pak, 1984; Blizard and Pak, 1984; Lewitus and Broenkow, 1985).

Early Blood Lead Levels and Sleep Disturbance in Preadolescence

PubMed Central

Liu, Jianghong; Liu, Xianchen; Pak, Victoria; Wang, Yingjie; Yan, Chonghuai; Pinto-Martin, Jennifer; Dinges, David

2015-01-01

Study Objectives: Little is known about the effect of lead exposure on children's sleep. This study examined the association between blood lead levels (BLL) and sleep problems in a longitudinal study of children. Setting: Four community-based elementary schools in Jintan City, China. Participants: 1,419 Chinese children. Measurement and Results: BLL were measured when children were aged 3–5 y, and sleep was assessed at ages 9–13 y. Sleep was assessed by both parents' report, using the Children's Sleep Habits Questionnaire (CSHQ), and children's report, using an adolescent sleep questionnaire. A total of 665 children with complete data on BLL and sleep at both ages were included in the current study. Mean age of the sample at BLL assessment was 4.74 y (standard deviation [SD] = 0.89) and at sleep assessment was 11.05 y (SD = 0.88). Mean BLL was 6.26 μg/dL (SD = 2.54). There were significant positive correlations between BLL and 3 CSHQ subscales: Sleep onset delay (r = 0.113, P < 0.01), sleep duration (r = 0.139, P < 0.001), and night waking (r = 0.089, P < 0.05). Excessive daytime sleepiness (EDS) (26.1% versus 9.0%, P < 0.001) and use of sleeping pills (6.5% versus 1.8%, P = 0.03) were more prevalent in children BLL ≥ 10.0 μg/dL than in those children BLL < 10.0 μg/dL. After adjusting for demographics, BLL ≥ 10.0 μg/dL was significantly associated with increased risk for insomnia symptoms (odds ratio [OR] = 2.01, 95% confidence interval [CI] = 1.03–3.95) and EDS (OR = 2.90, 95% CI = 1.27–6.61). Conclusion: The findings indicate that elevated blood lead levels in early childhood are associated with increased risk for sleep problems and excessive daytime sleepiness in later childhood. Citation: Liu J, Liu X, Pak V, Wang Y, Yan C, Pinto-Martin J, Dinges D. Early blood lead levels and sleep disturbance in preadolescence. SLEEP 2015;38(12):1869–1874. PMID:26194570

Casticin induced apoptotic cell death and altered associated gene expression in human colon cancer colo 205 cells.

PubMed

Shang, Hung-Sheng; Liu, Jia-You; Lu, Hsu-Feng; Chiang, Han-Sun; Lin, Chia-Hain; Chen, Ann; Lin, Yuh-Feng; Chung, Jing-Gung

2017-08-01

Casticin, a polymethoxyflavone, derived from natural plant Fructus Viticis exhibits biological activities including anti-cancer characteristics. The anti-cancer and alter gene expression of casticin on human colon cancer cells and the underlying mechanisms were investigated. Flow cytometric assay was used to measure viable cell, cell cycle and sub-G1 phase, reactive oxygen species (ROS) and Ca 2+ productions, level of mitochondria membrane potential (ΔΨ m ) and caspase activity. Western blotting assay was used to detect expression of protein level associated with cell death. Casticin induced cell morphological changes, decreased cell viability and induced G2/M phase arrest in colo 205 cells. Casticin increased ROS production but decreased the levels of ΔΨ m , and Ca 2+ , increased caspase-3, -8, and -9 activities. The cDNA microarray indicated that some of the cell cycle associated genes were down-regulated such as cyclin-dependent kinase inhibitor 1A (CDKN1A) (p21, Cip1) and p21 protein (Cdc42/Rac)-activated kinase 3 (PAK3). TNF receptor-associated protein 1 (TRAP1), CREB1 (cAMP responsive element binding protein 1) and cyclin-dependent kinase inhibitor 1B (CDKN1B) (p27, Kip1) genes were increased but matrix metallopeptidase 2 (MMP-2), toll-like receptor 4 (TLR4), PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, bet), and CaMK4 (calcium/calmodulin-dependent protein kinase IV) genes were inhibited. Results suggest that casticin induced cell apoptosis via the activation of the caspase- and/or mitochondria-dependent signaling cascade, the accumulation of ROS and altered associated gene expressions in colo 205 human colon cancer cells. © 2016 Wiley Periodicals, Inc.

Temperature-dependent IR spectroscopic and structural study of 18-crown-6 chelating ligand in the complexation with sodium surfactant salts and potassium picrate.

PubMed

Mihelj, Tea; Tomašić, Vlasta; Biliškov, Nikola; Liu, Feng

2014-04-24

18-crown-6 ether (18C6) complexes with the following anionic surfactants: sodium n-dodecylsulfate (18C6-NaDS), sodium 4-(1-pentylheptyl)benzenesulfonate (18C6-NaDBS); and potassium picrate (18C6-KP) were synthesized and studied in terms of their thermal and structural properties. Physico-chemical properties of new solid 1:1 coordination complexes were characterized by infrared (IR) spectroscopy, thermogravimetry and differential thermal analysis, differential scanning calorimetry, X-ray diffraction and microscopic observations. The strength of coordination between Na(+) and oxygen atoms of 18C6 ligand does not depend on anionic part of the surfactant, as established by thermodynamical parameters obtained by temperature-dependent IR spectroscopy. Each of these complexes exhibit different kinds of endothermic transitions in heating scan. Diffraction maxima obtained by SAXS and WAXS, refer the behavior of the compounds 18C6-NaDS and 18C6-NaDBS as smectic liquid crystalline. Distortion of 18C6-NaDS and 18C6-KP complexes occurs in two steps. Temperature of the decomplexation of solid crystal complex 18C6-KP is considerably higher than of mesophase complexes, 18C6-NaDS, and 18C6-NaDBS. The structural and liquid crystalline properties of novel 18-crown-ether complexes are function of anionic molecule geometry, type of chosen cation (Na(+), K(+)), as well as architecture of self-organized aggregates. A good combination of crown ether unit and amphiphile may provide a possibility for preparing new functionalized materials, opening the research field of ion complexation and of host-guest type behavior. Copyright © 2013 Elsevier B.V. All rights reserved.

Dynamic and thermodynamic response of the Ras protein Cdc42Hs upon association with the effector domain of PAK3

PubMed Central

Moorman, Veronica R.; Valentine, Kathleen G.; Bédard, Sabrina; Kasinath, Vignesh; Dogan, Jakob; Love, Fiona M.; Wand, A. Joshua

2014-01-01

Human cell division cycle protein 42 (Cdc42Hs) is a small, Rho-type GTPase involved in multiple cellular processes through its interactions with downstream effectors. The binding domain of one such effector, the actin cytoskeleton-regulating p21 activated kinase 3 (PAK3) is known as PBD46. Nitrogen-15 backbone and carbon-13 methyl NMR relaxation were measured to investigate the dynamical changes in activated GMPPCP•Cdc42Hs upon PBD46 binding. Changes in internal motion of the Cdc42Hs, as revealed by methyl axis order parameters, were observed not only near the Cdc42Hs–PBD46 interface but also in remote sites on the Cdc42Hs molecule. The binding-induced changes in side chain dynamics propagate along the long axis of Cdc42Hs away from the site of PBD46 binding with a sharp distance dependence. Overall, the binding of the PBD46 effector domain on the dynamics of methyl bearing side chains of Cdc42Hs results in a modest rigidification, which is estimated to correspond to an unfavorable change in conformational entropy of approximately −10 kcal mol−1 at 298 K. A cluster of methyl probes closest to the nucleotide-binding pocket of Cdc42Hs become more rigid upon binding of PBD46 and is proposed to slow the catalytic hydrolysis of the γ phosphate moiety. An additional cluster of methyl probes surrounding the guanine ring become more flexible on binding of PBD46, presumably facilitating nucleotide exchange mediated by a guanosine exchange factor. In addition, the Rho insert helix, which is located at a site remote from the PBD46 binding interface, shows a significant dynamic response to PBD46 binding. PMID:25109462

The sediment and hydrographic characteristics of three horseshoe crab nursery beaches in hong kong

NASA Astrophysics Data System (ADS)

Chiu, Helen M. C.; Morton, Brian

2003-04-01

Horseshoe crab juveniles have been recorded from sand and sandy-mud nursery beaches at Pak Nai (western New Territories), San Tau and Shui Hau (Lantau Island), Hong Kong. In order to provide a better understanding of these beaches and to identify those plausible factors which have made them preferred by spawning horseshoe crabs, environmental parameters, including temperature, salinity, pH and dissolved oxygen content of the water, and particle size distribution and organic matter content of the sediments at the three sites, were determined and compared. The hydrographic, and sediment data obtained for the three study sites have revealed some common environmental features. The three nursery beaches are relatively remote, and far (in Hong Kong terms) from urbanized and densely populated areas. The beaches are generally well sheltered from strong wave action and inundated regularly by estuarine waters. Horseshoe crab adults tend to select these beaches for spawning as their protected features ensures the laid eggs are less likely to be washed out of the sand, and hatched juveniles can feed on the meiofauna and grow. Sediments of the three beaches largely comprise medium-sized sand particles and are moderately sorted, suggesting medium porosity and good water permeability. Such a sand type, with the generally high oxygen levels in incursing waters, may help create a well-oxygenated micro-environment for the normal development of horseshoe crab eggs, larvae and juveniles. Lantau Island beaches at San Tau and Shui Hau are relatively free from organic pollution, as reflected in generally high dissolved oxygen level, and low BOD5 and ammonia nitrogen values. Pak Nai is, however, more polluted.

Effects of aging on the fraction distribution and bioavailability of selenium in three different soils.

PubMed

Li, Jun; Peng, Qin; Liang, Dongli; Liang, Sijie; Chen, Juan; Sun, Huan; Li, Shuqi; Lei, Penghui

2016-02-01

Aging refers to the processes by which the mobility and bioavailability of metals in soil decline with time. Although long-term aging is a key process that needs to be considered in risk assessment of metals, few investigations has been attempted to determine whether and how residence time influences the selenium (Se) fractions and bioavailability in soil. In this study, the fractions of Se in soils was evaluated, and bioavailability were assessed by measuring Se concentration in pak choi (Brassica chinensis L.). Results showed that the change of soil available Se in all tested soils divided into two phases: rapid decrease at the initial time (42 d) and slow decline thereafter. The second-order equation could describe the decrease processes of available Se in tested soils during the entire incubation time (R(2) > 0.99), while parabolic diffusion equation had less goodness of fit. Those results indicated that Se aging was controlled not only by diffusion process but also by other processes such as nucleation/precipitation, adsorption/desorption with soil component, occlusion by organic matter and reduction reaction. Soil available Se fractions tended to transform to more stable fractions during aging. The changes of Se concentration in pak choi were consistent with the variation in soil available Se content. In addition, 21 d could be reference for the time of Se aging reaching stabilization in krasnozems and fluvo-aquic soil, and 30 d for black soil. Results could provide theoretical basis to formulate environmental quality criterion and choose the equilibrium time before implementing a pot experiment in Se-spiked soils. Copyright © 2015 Elsevier Ltd. All rights reserved.

Organic soil production from urban soil, spent mushroom substrate, and other additives

NASA Astrophysics Data System (ADS)

Pham, Nhung Thi Ha

2017-09-01

In recent years, spent mushroom substrate (SMS) is becoming the huge problem in environmental pollution issues from mushroom production. However, SMS is also a nutrient-rich ogranic material with available nutrients and high porosity. Therefore, the value of products made from SMS should be exploited to take full advantage of agricultural by-product, support organic agriculture development without environmental pollution. The research has built 5 experimental formulas (4 mixed formulas and 1 control formulas with only urban soil). The analysis results of soil samples from mixed formulas and the control formula witness a significant increase in moisture and OM of mixed formulas (moisture from 36-42%, OM from 5.5-6.9%) after 20 treatment days, and N-P-K contents are also improved remarkably. 60 days later, soil nutrients in mixed formulas continue to rise, with highest OM (8.679%) at CT1; N (0.154%) at CT4; K2O (0,698%) and P2O5 (0,172%) at CT3, in addition, heavy metal contents in all formulas are under standard limit. Synthetic assessment of all norms indicates that the best organic soil product comes from CT3. The pak choi planting experiments are performed show that the growth of plants cultivated on organic soil products made from mixed formulas are much better than plants are grown on initially soil, and they also have no pestilent insect. Specially, pak choi planted on organic soil from CT3 have sharp developing with excellent tolerance ability, quantity and area of leaves are high. Thus, CT3 is the most suitable formula to increase soil nutrients, to solve spent mushroom subtrate streament problems after harvest, and for sustainable agricultural development.

p53 mediates bcl-2 phosphorylation and apoptosis via activation of the Cdc42/JNK1 pathway.

PubMed

Thomas, A; Giesler, T; White, E

2000-11-02

A member of the small G protein family, cdc42, was isolated from a screen undertaken to identify p53-inducible genes during apoptosis in primary baby rat kidney (BRK) cells transformed with E1A and a temperature-sensitive mutant p53 using a PCR-based subtractive hybridization method. Cdc42 is a GTPase that belongs to the Rho/Rac subfamily of Ras-like GTPases. In response to external stimuli, Cdc42 is known to transduce signals to regulate the organization of the actin cytoskeleton, induce DNA synthesis in quiescent fibroblasts, and promote apoptosis in neuronal and immune cells. In this study, we have demonstrated that cdc42 mRNA and protein were up-regulated in the presence of wild-type p53 in BRK cells, followed by cytoplasmic to plasma membrane translocation of Cdc42. Overexpression of Cdc42 in the presence of a dominant-negative mutant p53 induced apoptosis rapidly, indicating that Cdc42 functions downstream of p53. Furthermore, stable expression of a dominant-negative mutant of Cdc42 partially inhibited p53-mediated apoptosis. The Bcl-2 family members Bcl-xL, and the adenovirus protein E1B 19K, inhibited Cdc42-mediated apoptosis, whereas Bcl-2 did not. We provide evidence that PAK1 and JNK1 may play a role downstream of Cdc42 to transduce its apoptotic signal. Cdc42/PAK1 activates JNK1-induced phosphorylation of Bcl-2, thereby inactivating its function, and that a phosphorylation resistant mutant (Bcl-2S70,87A,T56,74A) gains the ability to inhibit Cdc42- and p53-mediated apoptosis. Thus, one mechanism by which p53 promotes apoptosis is through activation of Cdc42 and inactivation of Bcl-2.

Solid Waste Composition Study at Taman Universiti, Parit Raja, Batu Pahat

NASA Astrophysics Data System (ADS)

Kadir, A. A.; Sani, M. S. A. M.

2016-07-01

Solid waste management is recognised as one of the most challenging issues confronted by both the developed and developing countries. The problems rise due to growing population in current years which results in increased generation of waste with various compositions. The aim of this study was to determine the waste compositions at Taman Universiti. Taman Universiti is a mix residential and commercial area which a preferred residential location amongst students and lecturers due to its proximate location to UTHM main campus. The waste collection was carried out for 50 houses on a daily basis. The collection and sorting out method was conducted according to Malaysian Standard MS 2505:2012 and the data was collected and recorded The result showed that the average generation rate of household waste at Taman Universiti was 0.16kg/person/day and the moisture content was approximately ranging from 61%-68%. Household wastes collected were categorized and it consisted of food and organic, paper, rigid plastics, plastics film, baby diapers, glass, tetra pak, household hazardous waste, metal, rubber, textiles, garden waste and leather. The proportion of each wastes were approximately 64.67%, 9.36%, 9.22%, 5.33%, 3.51%, 2.53%, 1.37%, 1.05%, 0.84%, 0.85%, 0.80%, 0.27%, and 0.23%, respectively. Results from the analyses indicated that the food and organic waste are the major composition of household waste at Taman Universiti followed by the paper, rigid plastics, and plastic film. Meanwhile, the proportion of baby diapers, glass, tetra pak, household hazardous waste, metal, rubber, textiles, and garden decreasing accordingly. In addition, leather was recognized as the least category that contributed to the household waste.

Causes of graft failure in simultaneous pancreas-kidney transplantation by various time periods.

PubMed

Wakil, Kayo; Sugawara, Yasuhiko; Kokudo, Norihiro; Kadowaki, Takashi

2013-01-01

Data collected by the United Network for Organ Sharing from all approved United States transplant programs was analyzed. The data included 26,572 adult diabetic patients who received a primary pancreas transplant between January 1987 and December 2012. Simultaneous pancreas-kidney (SPK) transplantation was the major therapeutic option for diabetes patients. SPK had better graft survival than pancreas transplant alone (PTA) or pancreas-after-kidney (PAK) or pancreas-with-kidney (from a living donor, PWK). The 5-year pancreas graft survival rates for SPK, PWK, PAK, and PTA were 70.0%, 57.2%, 54.0%, and 48.2%, respectively. When long-term SPK pancreas graft survival was examined by various transplant time periods, it was found that survival has remained almost stable since 1996. Graft survival rates were high among the pancreas recipients transplanted in the periods 1996-2000, 2001-2005, and 2006-2012, and the rates were similar: the 5-year rates were 68.9%, 72.4%, and 73.8%, respectively. Technical failure was the leading cause of graft loss during the first year post-transplant, regardless of period: 61.3%, 68.6%, 64.2%, and 71.9% for 1987-1995, 1996-2000, 2001-2005, and 2006-2012, respectively. After one year, chronic rejection was the leading cause of graft loss in all periods: 51.8%, 53.2%, 44.3%, and 40.7% for 1987-1995, 1996-2000, 2001-2005, and 2006-2012, respectively. Chronic rejection accounted for around 50% (or more) of the grafts that survived over five years. Survival of long-term pancreas grafts as well as long-term causes of graft loss remained almost unchanged across the different transplant periods. Clearly, there is a need for a means to identify early markers of chronic rejection, and to control it to improve long-term survival.

The role of drinking water sources, consumption of vegetables and seafood in relation to blood arsenic concentrations of Jamaican children with and without Autism Spectrum Disorders

PubMed Central

Rahbar, Mohammad H.; Samms-Vaughan, Maureen; Ardjomand-Hessabi, Manouchehr; Loveland, Katherine A.; Dickerson, Aisha S.; Chen, Zhongxue; Bressler, Jan; Shakespeare-Pellington, Sydonnie; Grove, Megan L.; Bloom, Kari; Wirth, Julie; Pearson, Deborah A.; Boerwinkle, Eric

2012-01-01

Arsenic is a toxic metal with harmful effects on human health, particularly on cognitive function. Autism Spectrum Disorders (ASDs) are lifelong neurodevelopmental and behavioral disorders manifesting in infancy or early childhood. We used data from 130 children between 2-8 years (65 pairs of ASD cases with age- and sex-matched control), to compare the mean total blood arsenic concentrations in children with and without ASDs in Kingston, Jamaica. Based on univariable analysis, we observed a significant difference between ASD cases and controls (4.03μg/L for cases vs. 4.48μg/L for controls, P < 0.01). In the final multivariable General Linear Model (GLM), after controlling for car ownership, maternal age, parental education levels, source of drinking water, consumption of “yam, sweet potato, or dasheen”, “carrot or pumpkin”, “callaloo, broccoli, or pak choi”, cabbage, avocado, and the frequency of seafood consumption per week, we did not find a significant association between blood arsenic concentrations and ASD status (4.36μg/L for cases vs. 4.65μg/L for controls, P = 0.23). Likewise, in a separate final multivariable GLM, we found that source of drinking water, eating avocado, and eating “callaloo, broccoli, or pak choi” were significantly associated with higher blood arsenic concentrations (all three P < 0.05). Based on our findings, we recommend assessment of arsenic levels in water, fruits, and vegetables, as well as increased awareness among the Jamaican population regarding potential risks for various exposures to arsenic. PMID:22819887

Rapid diagnostic tests duo as alternative to conventional serological assays for conclusive Chagas disease diagnosis.

PubMed

Egüez, Karina E; Alonso-Padilla, Julio; Terán, Carolina; Chipana, Zenobia; García, Wilson; Torrico, Faustino; Gascon, Joaquim; Lozano-Beltran, Daniel-Franz; Pinazo, María-Jesús

2017-04-01

Chagas disease is caused by the parasite Trypanosoma cruzi. It affects several million people, mainly in Latin America, and severe cardiac and/or digestive complications occur in ~30% of the chronically infected patients. Disease acute stage is mostly asymptomatic and infection goes undiagnosed. In the chronic phase direct parasite detection is hampered due to its concealed presence and diagnosis is achieved by serological methods, like ELISA or indirect hemagglutination assays. Agreement in at least two tests must be obtained due to parasite wide antigenic variability. These techniques require equipped labs and trained personnel and are not available in distant regions. As a result, many infected people often remain undiagnosed until it is too late, as the two available chemotherapies show diminished efficacy in the advanced chronic stage. Easy-to-use rapid diagnostic tests have been developed to be implemented in remote areas as an alternative to conventional tests. They do not need electricity, nor cold chain, they can return results within an hour and some even work with whole blood as sample, like Chagas Stat-Pak (ChemBio Inc.) and Chagas Detect Plus (InBIOS Inc.). Nonetheless, in order to qualify a rapidly diagnosed positive patient for treatment, conventional serological confirmation is obligatory, which might risk its start. In this study two rapid tests based on distinct antigen sets were used in parallel as a way to obtain a fast and conclusive Chagas disease diagnosis using whole blood samples. Chagas Stat-Pak and Chagas Detect Plus were validated by comparison with three conventional tests yielding 100% sensitivity and 99.3% specificity over 342 patients seeking Chagas disease diagnosis in a reference centre in Sucre (Bolivia). Combined used of RDTs in distant regions could substitute laborious conventional serology, allowing immediate treatment and favouring better adhesion to it.

Genomic inferences of domestication events are corroborated by written records in Brassica rapa.

PubMed

Qi, Xinshuai; An, Hong; Ragsdale, Aaron P; Hall, Tara E; Gutenkunst, Ryan N; Chris Pires, J; Barker, Michael S

2017-07-01

Demographic modelling is often used with population genomic data to infer the relationships and ages among populations. However, relatively few analyses are able to validate these inferences with independent data. Here, we leverage written records that describe distinct Brassica rapa crops to corroborate demographic models of domestication. Brassica rapa crops are renowned for their outstanding morphological diversity, but the relationships and order of domestication remain unclear. We generated genomewide SNPs from 126 accessions collected globally using high-throughput transcriptome data. Analyses of more than 31,000 SNPs across the B. rapa genome revealed evidence for five distinct genetic groups and supported a European-Central Asian origin of B. rapa crops. Our results supported the traditionally recognized South Asian and East Asian B. rapa groups with evidence that pak choi, Chinese cabbage and yellow sarson are likely monophyletic groups. In contrast, the oil-type B. rapa subsp. oleifera and brown sarson were polyphyletic. We also found no evidence to support the contention that rapini is the wild type or the earliest domesticated subspecies of B. rapa. Demographic analyses suggested that B. rapa was introduced to Asia 2,400-4,100 years ago, and that Chinese cabbage originated 1,200-2,100 years ago via admixture of pak choi and European-Central Asian B. rapa. We also inferred significantly different levels of founder effect among the B. rapa subspecies. Written records from antiquity that document these crops are consistent with these inferences. The concordance between our age estimates of domestication events with historical records provides unique support for our demographic inferences. © 2017 John Wiley & Sons Ltd.

Reliable Diagnosis of Carnitine Palmitoyltransferase Type IA Deficiency by Analysis of Plasma Acylcarnitine Profiles.

PubMed

Heiner-Fokkema, M Rebecca; Vaz, Frédéric M; Maatman, Ronald; Kluijtmans, Leo A J; van Spronsen, Francjan J; Reijngoud, Dirk-Jan

2017-01-01

Carnitine palmitoyltransferase IA (CPT-IA) deficiency is an inherited disorder of the carnitine cycle (MIM #255120). Patients affected by this deficiency might be missed easily because of lack of specific and sensitive biochemical markers. In this study, sensitivity and specificity of plasma free carnitine (C0) and long-chain acylcarnitines (lc-ac: C16:0-, C16:1-, C18:0-, C18:1- and C18:2-ac) was evaluated, including the sum of lc-ac (∑lc-ac) and the molar ratios C0/(C16:0-ac+C18:0-ac) and C0/∑lc-ac. Nine plasma acylcarnitine profiles of 4 CPT-IA deficient patients were compared with profiles of 2,190 subjects suspected of or diagnosed with an inherited disorder of metabolism. Age-dependent reference values were calculated based on the patient population without a definite diagnosis of an inborn error of metabolism (n = 1,600). Sensitivity, specificity, and Receiver Operating Characteristic (ROC) curves were calculated based on samples of the whole patient population. Concentrations of C0 in plasma were normal in all CPT-IA deficient patient samples. ROC analyses showed highest diagnostic values for C18:0-ac, C18:1-ac, and ∑lc-ac (AUC 1.000) and lowest for C0 (AUC 0.738). Combining two markers, i.e., a plasma C18:1-ac concentration <0.05 μmol/L and a molar ratio of C0/(C16:0-ac+C18:0-ac) >587, specificity to diagnose CPT-IA deficiency increased to 99.3% compared with either C18:1-ac (97.4%) or C0/(C16:0-ac+C18:0-ac) (96.9%) alone, all at a sensitivity of 100%. Combination of a low concentration of C18:1-ac with a high molar ratio of C0/(C16:0-ac+C18:0-ac) ratio in plasma has high diagnostic value for CPT-IA deficiency. Patients with a clinical suspicion of CPT-IA deficiency can be diagnosed with this test combination.

Mechanism of Electromagnetic Energy Effects of the Nervous System. Experimental System and Preliminary Results.

DTIC Science & Technology

1982-07-01

17 * 7 Expulsion of pipette by cell... 17 8 Effects of curare on cell studied 27 June 1980 ... 18 9 Pipette experiment, 18 July 1980 ... 27 10...Response of cell studied 18 August 1980 to sudden dc shift... 29 3 3 FIGURES (cont’d) C-Ia-c Baseline period of cell studied 18 June 1980 ... page 49 C-2a-c...Exposure period of cell studied 18 June 1980 ... 50 C-3a-c Post-exposure period of cell studied 18 June 1980 ... 51 C-4a-d Baseline, first exposure

Infrared spectroscopy of 17O- and 18O-enriched carbon dioxide: Line positions and intensities in the 4681-5337 cm-1 region

NASA Astrophysics Data System (ADS)

Borkov, Yu. G.; Jacquemart, D.; Lyulin, O. M.; Tashkun, S. A.; Perevalov, V. I.

2015-07-01

The line positions and intensities of carbon dioxide isotopologues have been retrieved in the 4681-5337 cm-1 spectral range from Fourier transform spectra of carbon dioxide recorded in LADIR (Paris, France) with the Bruker IFS 125-HR [Jacquemart D, et al., J Quant Spectrosc Radiat Transf 2012;113:961-975]. In total 6386 line positions and intensities of 89 bands of 12 isotopologues 16O12C16O, 16O13C16O, 16O12C18O, 16O12C17O, 16O13C18O, 16O13C17O, 18O12C18O, 17O12C18O, 17O12C17O, 18O13C18O, 17O13C18O, and 17O13C17O have been retrieved. 23 bands were newly assigned. All studied bands belong to the ΔP=7 series of transitions, where P = 2V1 +V2 + 3V3 is the polyad number (Vi are vibrational quantum numbers). The accuracy of the line position measurement is about 0.3×10-3 cm-1 for the unblended and not very weak lines. The accuracy of the line intensities varies from 4% to 15% depending on the isotopologue, on the intensity of the line and on the extent of the line overlapping. The observed intensities were used to fit the effective dipole moment parameters for the ΔP=7 series of transitions in 16O12C18O, 16O12C17O, 12C17O2, 17O12C18O, 16O13C17O, 13C17O2 and 17O13C18O isotopologues of carbon dioxide.

40 CFR 180.1068 - C12-C18 fatty acid potassium salts; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false C12-C18 fatty acid potassium salts... RESIDUES IN FOOD Exemptions From Tolerances § 180.1068 C12-C18 fatty acid potassium salts; exemption from the requirement of a tolerance. C12-C18 fatty acids (saturated and unsaturated) potassium salts are...

40 CFR 180.1068 - C12-C18 fatty acid potassium salts; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false C12-C18 fatty acid potassium salts... RESIDUES IN FOOD Exemptions From Tolerances § 180.1068 C12-C18 fatty acid potassium salts; exemption from the requirement of a tolerance. C12-C18 fatty acids (saturated and unsaturated) potassium salts are...

40 CFR 180.1068 - C12-C18 fatty acid potassium salts; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 25 2012-07-01 2012-07-01 false C12-C18 fatty acid potassium salts... RESIDUES IN FOOD Exemptions From Tolerances § 180.1068 C12-C18 fatty acid potassium salts; exemption from the requirement of a tolerance. C12-C18 fatty acids (saturated and unsaturated) potassium salts are...

40 CFR 180.1068 - C12-C18 fatty acid potassium salts; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 24 2011-07-01 2011-07-01 false C12-C18 fatty acid potassium salts... RESIDUES IN FOOD Exemptions From Tolerances § 180.1068 C12-C18 fatty acid potassium salts; exemption from the requirement of a tolerance. C12-C18 fatty acids (saturated and unsaturated) potassium salts are...

40 CFR 180.1068 - C12-C18 fatty acid potassium salts; exemption from the requirement of a tolerance.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 24 2014-07-01 2014-07-01 false C12-C18 fatty acid potassium salts... RESIDUES IN FOOD Exemptions From Tolerances § 180.1068 C12-C18 fatty acid potassium salts; exemption from the requirement of a tolerance. C12-C18 fatty acids (saturated and unsaturated) potassium salts are...

Specific coupling between the 13-keto carbonyl and chlorin skeletal vibrational modes of synthetic 13 1- 18O-(un)labelled metallochlorophyll derivatives

NASA Astrophysics Data System (ADS)

Morishita, Hidetada; Tamiaki, Hitoshi

2009-03-01

Metal complexes of methyl 13 1- 18O-labelled pyropheophorbide- a1-M- 18O (M = Zn, Cu and Ni) were prepared by metallation of the 18O-labelled free base ( 1- 18O) and 18O-labelling of unlabelled nickel complex ( 1-Ni). The FT-IR spectra of 1-Zn and 1-Zn- 18O in CH 2Cl 2 showed that the 13-keto carbonyl stretching vibration mode moved to about a 30-cm -1 lower wavenumber by 18O-labelling of the 13 1-oxo moiety. In 1-Cu- 18O and 1-Ni- 18O, the 13-C dbnd 18O stretching modes were close to the highest-energy wavenumber mode of chlorin skeletal C-C/C-N vibrations at around 1650 cm -1 and they were coupled in CH 2Cl 2 to give two split IR bands (Fermi resonance). A similar coupling was observed in the resonance Raman scattering of 1-Ni- 18O in the solid state. The hydrogen-bonded 13-C dbnd 16O vibration mode of 1-Ni similarly coupled with the skeletal C-C/C-N mode in CCl 4 containing 1% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol, while such a coupling was not observed in a neat CCl 4 solution of 1-Ni possessing the 13-C dbnd 16O free from any interaction. The skeletal C-C/C-N band selectively coupled with the 13-C dbnd O, not with the 3-C dbnd O, when the difference in their peak maxima was less than 20 cm -1.

A bioassay-guided fractionation system to identify endogenous small molecules that activate plasma membrane H+-ATPase activity in Arabidopsis.

PubMed

Han, Xiuli; Yang, Yongqing; Wu, Yujiao; Liu, Xiaohui; Lei, Xiaoguang; Guo, Yan

2017-05-17

Plasma membrane (PM) H+-ATPase is essential for plant growth and development. Various environmental stimuli regulate its activity, a process that involves many protein cofactors. However, whether endogenous small molecules play a role in this regulation remains unknown. Here, we describe a bio-guided isolation method to identify endogenous small molecules that regulate PM H+-ATPase activity. We obtained crude extracts from Arabidopsis seedlings with or without salt treatment and then purified them into fractions based on polarity and molecular mass by repeated column chromatography. By evaluating the effect of each fraction on PM H+-ATPase activity, we found that fractions containing the endogenous, free unsaturated fatty acids oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) extracted from salt-treated seedlings stimulate PM H+-ATPase activity. These results were further confirmed by the addition of exogenous C18:1, C18:2, or C18:3 in the activity assay. The ssi2 mutant, with reduced levels of C18:1, C18:2, and C18:3, displayed reduced PM H+-ATPase activity. Furthermore, C18:1, C18:2, and C18:3 directly bound to the C-terminus of the PM H+-ATPase AHA2. Collectively, our results demonstrate that the binding of free unsaturated fatty acids to the C-terminus of PM H+-ATPase is required for its activation under salt stress. The bio-guided isolation model described in this study could enable the identification of new endogenous small molecules that modulate essential protein functions, as well as signal transduction, in plants. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The interaction between ketamine and some crown ethers in common organic solvents studied by NMR: The effect of donating atoms and ligand structure

NASA Astrophysics Data System (ADS)

Chekin, Fereshteh; Bordbar, Maryam; Fathollahi, Yaghoub; Alizadeh, Naader

2006-02-01

1H NMR spectroscopy was used to investigate the stoichiometry and stability of the drug ketamine cation complexes with some crown ethers, such as 15-crown-5 (15C5), aza-15-crown-5 (A15C5), 18-crown-6 (18C6), aza-18-crown-6 (A18C6), diaza-18-crown-6 (DA18C6), dibenzyl-diaza-18-crown-6 (DBzDA18C6) and cryptant [2,2,2] (C222) in acetonitrile (AN), dimethylsulfoxide (DMSO) and methanol (MeOH) at 27 °C. In order to evaluate the formation constants of the ketamine cation complexes, the CH 3 protons chemical shift (on the nitrogen atom of ketamine) was measured as function of ligand/ketamine mole ratio. The formation constant of resulting complexes were calculated by the computer fitting of chemical shift versus mole ratio data to appropriate equations. A significant chemical shift variation was not observed for 15C5 and 18C6. The stoichiometry of the mono aza and diaza ligands are 1:1 and 1:2 (ligand/ketamine), respectively. In all of the solvents studied, DA18C6 formed more stable complexes than other ligands. The solvent effect on the stability of these complexes is discussed.

Overexpression of ceramide synthase 1 increases C18-ceramide and leads to lethal autophagy in human glioma

PubMed Central

Wang, Zheng; Wen, Lijun; Zhu, Fei; Wang, Yanping; Xie, Qing; Chen, Zijun; Li, Yunsen

2017-01-01

Ceramide synthase 1 (CERS1) is the most highly expressed CERS in the central nervous system, and ceramide with an 18-carbon–containing fatty acid chain (C18-ceramide) in the brain plays important roles in signaling and sphingolipid development. However, the roles of CERS1 and C18-ceramide in glioma are largely unknown. In the present study, measured by electrospray ionization linear ion trap mass spectrometry, C18-ceramide was significantly lower in glioma tumor tissues compared with controls (P < 0.001), indicating that C18-ceramide might have a role in glioma. These roles were examined by reconstitution of C18-ceramide in U251 and A172 glioma cells via addition of exogenous C18-ceramide or overexpression of CERS1, which has been shown to specifically induce the generation of C18-ceramide. Overexpression of CERS1 or adding exogenous C18-ceramide inhibited cell viability and induced cell death by activating endoplasmic reticulum stress, which induced lethal autophagy and inhibited PI3K/AKT signal pathway in U251 and A172 glioma cells. Moreover, overexpression of CERS1 or adding exogenous C18-ceramide increased the sensitivity of U251 and A172 glioma cells to teniposide (VM-26). Thus, the combined therapy of CERS1/C18-ceramide and VM-26 may be a novel therapeutic strategy for the treatment of human glioma. PMID:29262618

Intentional and unintentional poisoning in Pakistan: a pilot study using the Emergency Departments surveillance project.

PubMed

Khan, Nadeem; Pérez-Núñez, Ricardo; Shamim, Nudrat; Khan, Uzma; Naseer, Naureen; Feroze, Asher; Razzak, Junaid; Hyder, Adnan A

2015-01-01

Acute poisoning is one of the most common reasons for emergency department visits around the world. In Pakistan, the epidemiological data on poisoning is limited due to an under developed poison information surveillance system. We aim to describe the characteristics associated with intentional and unintentional poisoning in Pakistan presenting to emergency departments. The data was extracted from the Pakistan National Emergency Department Surveillance (Pak-NEDS) which was an active surveillance conducted between November 2010 and March 2011. All patients, regardless of age, who presented with poisoning to any of Pakistan's seven major tertiary care centers' emergency departments, were included. Information about patient demographics, type of poisoning agent, reason for poisoning and outcomes were collected using a standard questionnaire. Acute poisoning contributed to 1.2% (n = 233) of patients with intentional and unintentional injuries presenting to EDs of participating centers. Of these, 68% were male, 54% were aged 19 to 44 and 19% were children and adolescents (<18 years). Types of poisoning included chemical/gas (43.8%), drug/medicine (27%), alcohol (16.7%) and food/plant (6%). In half of all patients the poisoning was intentional. A total of 11.6% of the patients were admitted and 6.6% died. Poisoning causes more morbidity and mortality in young adults in Pakistan compared to other age groups, half of which is intentional. Improving mental health, regulatory control for hazardous chemicals and better access to care through poison information centers and emergency departments will potentially help control the problem.

Steric/π-electronic insulation of the carbo-benzene ring: dramatic effects of tert-butyl vs phenyl crowns on geometric, chromophoric, redox and magnetic properties.

PubMed

Listunov, Dymytrii; Duhayon, Carine; Poater, Albert; Mazères, Serge; Saquet, Alix; Maraval, Valérie; Chauvin, Remi

2018-04-13

Hexa-tert-butyl-carbo-benzene C18tBu6 and three phenylated counterparts C18tBumPh6-m (m = 4, 2) have been synthesized. The peralkylated version (m = 6) provides experimental access to intrinsic features of the insulated C18 core independently from the influence of π-conjugated substituent. Over the series, structural, spectroscopical and electrochemical properties are compared with those of the hexaphenylated reference (m = 0). Anchoring tBu substituents at the C18 macrocycle is shown to enhance stability and solubility, and to dramatically modify UV-vis absorption and redox properties. Whereas all the carbo-benzenes reported hitherto were obtained as dark-reddish/greenish solids, crystals and solutions of C18tBu6 happen to be yellow (max = 379 nm vs 472 nm for C18Ph6). By comparison to C18Ph6, reduction of C18tBu6 remains reversible but occurs at a twice higher absolute potential (E1/2 = -1.36 V vs -0.72 V). Systematic X-ray diffraction analyses and DFT calculations show that the C18 ring symmetry is the nearest to D6h for m = 6, indicating a maximum geometrical aromaticity. According to calculated nucleus independent chemical shifts, the macrocyclic magnetic aromaticity is also found to be maximum for C18tBu6: NICS(0)=-17.2 ppm, vs -18.0±0.1 ppm for the theoretical references C18H6 and C18F6, and -13.5 ppm for C18Ph6. Accurate correlations of NICS(0) with experimental or calculated maximum UV-vis absorption wavelength max and chemical hardness =ELUMO-EHOMO are evidenced. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.