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Sample records for ala ser cys

  1. Association of the hOGG1 Ser326Cys polymorphism with sporadic amyotrophic lateral sclerosis.

    PubMed

    Coppedè, Fabio; Mancuso, Michelangelo; Lo Gerfo, Annalisa; Carlesi, Cecilia; Piazza, Selina; Rocchi, Anna; Petrozzi, Lucia; Nesti, Claudia; Micheli, Dario; Bacci, Andrea; Migliore, Lucia; Murri, Luigi; Siciliano, Gabriele

    2007-06-13

    Amyotropic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disease causing the loss of motoneurons of the brain and the spinal cord. The etiology of ALS is still uncertain, but males are at increased risk for the disease than females. Several studies have suggested that motoneurons in ALS might be subjected to the double insult of increased DNA oxidative damage and deficiencies in DNA repair systems. Particularly, increased levels of 8-oxoguanine and impairments of the DNA base excision repair system have been observed in neurons of ALS patients. There is evidence that the Ser326Cys polymorphism of the human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene is associated with a reduced DNA repair activity. To evaluate the role of the hOGG1 Ser326Cys polymorphism in sporadic ALS (sALS), we screened 136 patients and 129 matched controls. In the total population, we observed association between both the Cys326 allele (p=0.02) and the combined Ser326Cys+Cys326Cys genotype (OR=1.65, 95% CI=1.06-2.88) and increased risk of disease. After stratification by gender, the Cys326 allele (p=0.01), both the Ser326Cys genotype (OR=2.14, 95% CI=1.09-4.19) and the combined Ser326Cys+Cys326Cys genotype (OR=2.15, 95% CI=1.16-4.01) were associated with sALS risk only in males. No significant association between the Ser326Cys polymorphism and disease phenotype, including age and site of onset and disease progression, was observed. Present results suggest a possible involvement of the hOGG1 Ser326Cys polymorphism in sALS pathogenesis. PMID:17531381

  2. Catalytic Thr or Ser Residue Modulates Structural Switches in 2-Cys Peroxiredoxin by Distinct Mechanisms

    PubMed Central

    Tairum, Carlos A.; Santos, Melina Cardoso; Breyer, Carlos A.; Geyer, R. Ryan; Nieves, Cecilia J.; Portillo-Ledesma, Stephanie; Ferrer-Sueta, Gerardo; Toledo, José Carlos; Toyama, Marcos H.; Augusto, Ohara; Netto, Luis E. S.; de Oliveira, Marcos A.

    2016-01-01

    Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (CP), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-π bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states. PMID:27629822

  3. Catalytic Thr or Ser Residue Modulates Structural Switches in 2-Cys Peroxiredoxin by Distinct Mechanisms.

    PubMed

    Tairum, Carlos A; Santos, Melina Cardoso; Breyer, Carlos A; Geyer, R Ryan; Nieves, Cecilia J; Portillo-Ledesma, Stephanie; Ferrer-Sueta, Gerardo; Toledo, José Carlos; Toyama, Marcos H; Augusto, Ohara; Netto, Luis E S; de Oliveira, Marcos A

    2016-09-15

    Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (CP), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-π bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states.

  4. Catalytic Thr or Ser Residue Modulates Structural Switches in 2-Cys Peroxiredoxin by Distinct Mechanisms.

    PubMed

    Tairum, Carlos A; Santos, Melina Cardoso; Breyer, Carlos A; Geyer, R Ryan; Nieves, Cecilia J; Portillo-Ledesma, Stephanie; Ferrer-Sueta, Gerardo; Toledo, José Carlos; Toyama, Marcos H; Augusto, Ohara; Netto, Luis E S; de Oliveira, Marcos A

    2016-01-01

    Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (CP), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-π bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states. PMID:27629822

  5. Towards understanding the tandem mass spectra of protonated oligopeptides. 2: The proline effect in collision-induced dissociation of protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp).

    PubMed

    Bleiholder, Christian; Suhai, Sándor; Harrison, Alex G; Paizs, Béla

    2011-06-01

    The product ion spectra of proline-containing peptides are commonly dominated by y(n) ions generated by cleavage at the N-terminal side of proline residues. This proline effect is investigated in the current work by collision-induced dissociation (CID) of protonated Ala-Ala-Xxx-Pro-Ala (Xxx includes Ala, Ser, Leu, Val, Phe, and Trp) in an electrospray/quadrupole/time-of-flight (QqTOF) mass spectrometer and by quantum chemical calculations on protonated Ala-Ala-Ala-Pro-Ala. The CID spectra of all investigated peptides show a dominant y(2) ion (Pro-Ala sequence). Our computational results show that the proline effect mainly arises from the particularly low threshold energy for the amide bond cleavage N-terminal to the proline residue, and from the high proton affinity of the proline-containing C-terminal fragment produced by this cleavage. These theoretical results are qualitatively supported by the experimentally observed y(2)/b(3) abundance ratios for protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). In the post-cleavage phase of fragmentation the N-terminal oxazolone fragment with the Ala-Ala-Xxx sequence and Pro-Ala compete for the ionizing proton for these peptides. As the proton affinity of the oxazolone fragment increases, the y(2)/b(3) abundance ratio decreases.

  6. Association of OGG1 Ser326Cys polymorphism and pancreatic cancer susceptibility: evidence from a meta-analysis.

    PubMed

    Yan, Yulan; Chen, Xu; Li, Taijie; Li, Meng; Liang, Hongjie

    2014-03-01

    The 8-oxoguanine DNA glycosylase (OGG1) gene has been considered to be associated with cancer susceptibility. The OGG1 Ser326Cys polymorphism has been reported to be associated with pancreatic cancer (PC), but the published studies have yielded inconsistent results. For better understanding of the effect of OGG1 Ser326Cys polymorphism on PC susceptibility, a meta-analysis was performed. All eligible studies were identified through a search of PubMed, Excerpta Medica Database (Embase), Elsevier Science Direct, and Chinese Biomedical Literature Database before May 2013. The association between the OGG1 Ser326Cys polymorphism and PC risk was conducted by odds ratios (ORs) and 95% confidence intervals (CIs). A total of five case-control studies with 1,690 cases and 3,650 controls were eventually collected. Overall, we found that OGG1 Ser326Cys polymorphism was not associated with PC susceptibility (Cys/Cys vs. Ser/Ser: OR = 0.95, 95% CI = 0.80-1.14; Cys/Cys vs. Ser/Ser + Ser/Cys: OR = 0.95, 95% CI = 0.78-1.14; Cys/Cys + Ser/Cys vs. Ser/Ser (OR = 1.00, 95% CI = 0.89-1.12)). In the subgroup analysis based on ethnicity, source of control, sample size, and genotyping method, no significant association was found in any genetic models. This meta-analysis suggests that the OGG1 Ser326Cys polymorphism may not associated with PC susceptibility. Considering the limited sample size and ethnicity included in the meta-analysis, further larger scaled and well-designed studies are needed to confirm our results.

  7. Relationship between hOGG1 Ser326Cys gene polymorphism and coronary artery lesions in patients with diabetes mellitus

    PubMed Central

    Wu, Zhi-Yong; Wang, Meng-Hong; Qi, Hong-Mei; Wu, Mei-Hua; Ge, Yu-Zhi; Li, Hua-Tai

    2015-01-01

    To study the relationship between human 8-oxoguanine glycosylase (hOGG1) Ser326Cys gene polymorphism and coronary artery lesions in patients with diabetes mellitus, we analyzed 323 patients with diabetic mellitus, who underwent coronary angiography. Using PCR-RFLP, these patients were grouped into three genotypes: Cys/Cys (n=85), Ser/Ser (n=121), and Ser/Cys (n=117). Several clinical data, including history of diseases and biochemical indices were recorded. hOGG1 mRNA expression and 8-hydroxy deoxyguanosine (8-OHdG) were measured by RT-PCR and ELISA, respectively. The quantities and severity of coronary artery with lesions were analyzed from coronary angiography. The Gensini and SYNTAX scores were detected by the unitary criteria. The 8-OHdG levels showed statistical difference among the three genotypes (F=21.56, P<0.05). Also, 8-OHdG in Cys/Cys genotype was higher than Ser/Ser and Ser/Cys genotype (q=2.32, q=3.12, P<0.05). In terms of the expression of hOGGl mRNA, the measure of hOGGl/β-actin showed significant difference among the three groups (F=12.56, P<0.05). On comparing two groups, hOGGl/β-actin in Cys/Cys genotype was higher thanSer/Ser and Ser/Cys genotypes (q=2.32, q=3.12, P<0.05). Percentage of 3-vessel lesions was high in Cys/Cys genotype and percentage of 1-vessel lesions was low in Ser/Cys genotype. Gensini and SYNTAX scores and ratio of complex lesions were significantly higher in the Cys/Cys genotype than the other two genotypes (FGensini=47.16, FSYNTAX=55.12; P<0.05). hOGG1 Ser326Cys polymorphism showed correlation with coronary artery lesions in patients with diabetes mellitus, and Cys/Cys genotype may have more impact on the severity of lesions. PMID:26770476

  8. Adsorption of amino acids (ALA, CYS, HIS, MET) on zeolites: fourier transform infrared and Raman spectroscopy investigations.

    PubMed

    Carneiro, Cristine E A; de Santana, Henrique; Casado, Clara; Coronas, Joaquin; Zaia, Dimas A M

    2011-06-01

    Minerals adsorb more amino acids with charged R-groups than amino acids with uncharged R-groups. Thus, the peptides that form from the condensation of amino acids on the surface of minerals should be composed of amino acid residues that are more charged than uncharged. However, most of the amino acids (74%) in today's proteins have an uncharged R-group. One mechanism with which to solve this paradox is the use of organophilic minerals such as zeolites. Over the range of pH (pH 2.66-4.50) used in these experiments, the R-group of histidine (His) is positively charged and neutral for alanine (Ala), cysteine (Cys), and methionine (Met). In acidic hydrothermal environments, the pH could be even lower than those used in this study. For the pH range studied, the zeolites were negatively charged, and the overall charge of all amino acids was positive. The conditions used here approximate those of prebiotic Earth. The most important finding of this work is that the relative concentrations of each amino acid (X=His, Met, Cys) to alanine (X/Ala) are close to 1.00. This is an important result with regard to prebiotic chemistry because it could be a solution for the paradox stated above. Pore size did not affect the adsorption of Cys and Met on zeolites, and the Si/Al ratio did not affect the adsorption of Cys, His, and Met. ZSM-5 could be used for the purification of Cys from other amino acids (Student-Newman-Keuls test, p<0.05), and mordenite could be used for separation of amino acids from each other (Student-Newman-Keuls test, p<0.05). As shown by Fourier transform infrared (FT-IR) spectra, Ala interacts with zeolites through the [Formula: see text] group, and methionine-zeolite interactions involve the COO, [Formula: see text], and CH(3) groups. FT-IR spectra show that the interaction between the zeolites and His is weak. Cys showed higher adsorption on all zeolites; however, the hydrophobic Van der Waals interaction between zeolites and Cys is too weak to produce any

  9. Adsorption of Amino Acids (Ala, Cys, His, Met) on Zeolites: Fourier Transform Infrared and Raman Spectroscopy Investigations

    NASA Astrophysics Data System (ADS)

    Carneiro, Cristine E. A.; de Santana, Henrique; Casado, Clara; Coronas, Joaquin; Zaia, Dimas A. M.

    2011-06-01

    Minerals adsorb more amino acids with charged R-groups than amino acids with uncharged R-groups. Thus, the peptides that form from the condensation of amino acids on the surface of minerals should be composed of amino acid residues that are more charged than uncharged. However, most of the amino acids (74%) in today's proteins have an uncharged R-group. One mechanism with which to solve this paradox is the use of organophilic minerals such as zeolites. Over the range of pH (pH 2.66-4.50) used in these experiments, the R-group of histidine (His) is positively charged and neutral for alanine (Ala), cysteine (Cys), and methionine (Met). In acidic hydrothermal environments, the pH could be even lower than those used in this study. For the pH range studied, the zeolites were negatively charged, and the overall charge of all amino acids was positive. The conditions used here approximate those of prebiotic Earth. The most important finding of this work is that the relative concentrations of each amino acid (X=His, Met, Cys) to alanine (X/Ala) are close to 1.00. This is an important result with regard to prebiotic chemistry because it could be a solution for the paradox stated above. Pore size did not affect the adsorption of Cys and Met on zeolites, and the Si/Al ratio did not affect the adsorption of Cys, His, and Met. ZSM-5 could be used for the purification of Cys from other amino acids (Student-Newman-Keuls test, p<0.05), and mordenite could be used for separation of amino acids from each other (Student-Newman-Keuls test, p<0.05). As shown by Fourier transform infrared (FT-IR) spectra, Ala interacts with zeolites through the group, and methionine-zeolite interactions involve the COO, , and CH3 groups. FT-IR spectra show that the interaction between the zeolites and His is weak. Cys showed higher adsorption on all zeolites; however, the hydrophobic Van der Waals interaction between zeolites and Cys is too weak to produce any structural changes in the Cys groups (amine

  10. The Association between OGG1 Ser326Cys Polymorphism and Lung Cancer Susceptibility: A Meta-Analysis of 27 Studies

    PubMed Central

    Shen, Li-Jun; Jin, Zhen-Xiao; Zhao, Yu-Hong; Yi, Ding-Hua; Chen, Wen-Sheng; Yu, Shi-Qiang

    2012-01-01

    Background Numerous studies have investigated association of OGG1 Ser326Cys polymorphism with lung cancer susceptibility; however, the findings are inconsistent. Therefore, we performed a meta-analysis based on 27 publications encompass 9663 cases and 11348 controls to comprehensively evaluate such associations. Methods We searched publications from MEDLINE and EMBASE which were assessing the associations between OGG1 Ser326Cys polymorphism and lung cancer risk. We calculated pooled odds ratio (OR) and 95% confidence interval (CI) by using either fixed-effects or random-effects model. We used genotype based mRNA expression data from HapMap for SNP rs1052133 in normal cell lines among 270 subjects with four different ethnicities. Results The results showed that individuals carrying the Cys/Cys genotype did not have significantly increased risk for lung cancer (OR = 1.15, 95% CI = 0.98–1.36) when compared with the Ser/Ser genotype; similarly, no significant association was found in recessive, dominant or heterozygous co-dominant model (Ser/Cys vs. Cys/Cys). However, markedly increased risks were found in relatively large sample size (Ser/Ser vs. Cys/Cys: OR = 1.29, 95% CI = 1.13–1.48, and recessive model: OR = 1.19, 95% CI = 1.07–1.32). As to histological types, we found the Cys/Cys was associated with adenocarcinoma risk (Ser/Ser vs. Cys/Cys: OR = 1.32, 95% CI = 1.12–1.56; Ser/Cys vs. Cys/Cys: OR = 1.19, 95% CI = 1.04–1.37, and recessive model OR = 1.23, 95% CI = 1.08–1.40). No significant difference of OGG1 mRNA expression was found among genotypes between different ethnicities. Conclusions Despite some limitations, this meta-analysis established solid statistical evidence for an association between the OGG1 Cys/Cys genotype and lung cancer risk, particularly for studies with large sample size and adenocarcinoma, but this association warrants additional validation in larger and well designed studies. PMID

  11. Activation of superoxide formation and lysozyme release in human neutrophils by the synthetic lipopeptide Pam3Cys-Ser-(Lys)4. Involvement of guanine-nucleotide-binding proteins and synergism with chemotactic peptides.

    PubMed Central

    Seifert, R; Schultz, G; Richter-Freund, M; Metzger, J; Wiesmüller, K H; Jung, G; Bessler, W G; Hauschildt, S

    1990-01-01

    Upon exposure to the bacterial chemotactic peptide fMet-Leu-Phe, human neutrophils release lysozyme and generate superoxide anions (O2.-). The synthetic lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3Cys), which is derived from the N-terminus of bacterial lipoprotein, when attached to Ser-(Lys)4 [giving Pam3Cys-Ser-(Lys)4], activated O2.- formation and lysozyme release in human neutrophils with an effectiveness amounting to about 15% of that of fMet-Leu-Phe. Palmitic acid, muramyl dipeptide, lipopolysaccharide and the lipopeptides Pam3Cys-Ala-Gly, Pam3Cys-Ser-Gly, Pam3Cys-Ser, Pam3Cys-OMe and Pam3Cys-OH did not activate O2.- formation. Pertussis toxin, which ADP-ribosylates guanine-nucleotide-binding proteins (G-proteins) and functionally uncouples formyl peptide receptors from G-proteins, prevented activation of O2.- formation by fMet-Leu-Phe and inhibited Pam3Cys-Ser-(Lys)4-induced O2.- formation by 85%. Lipopeptide-induced exocytosis was pertussis-toxin-insensitive. O2.- formation induced by Pam3Cys-Ser-(Lys)4 and fMet-Leu-Phe was enhanced by cytochalasin B, by a phorbol ester and by a diacylglycerol kinase inhibitor. Addition of activators of adenylate cyclase and removal of extracellular Ca2+ inhibited O2.- formation by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 to different extents. Pam3Cys-Ser-(Lys)4 synergistically enhanced fMet-Leu-Phe-induced O2.- formation and primed neutrophils to respond to the chemotactic peptide at non-stimulatory concentrations. Our data suggest the following. (1) Pam3Cys-Ser-(Lys)4 activates neutrophils through G-proteins, involving pertussis-toxin-sensitive and -insensitive processes. (2) The signal transduction pathways activated by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 are similar but not identical. (3) In inflammatory processes, bacterial lipoproteins and chemotactic peptides may interact synergistically to activate O2.- formation, leading to enhanced bactericidal activity. PMID:2160237

  12. Functional characterization of the human organic cation transporter 2 variant p.270Ala>Ser.

    PubMed

    Zolk, Oliver; Solbach, Thomas F; König, Jörg; Fromm, Martin F

    2009-06-01

    The organic cation transporter 2 (OCT2, SLC22A2) plays an important role for renal drug elimination. Recent clinical studies indicate an impact of the frequent nonsynonymous c.808G>T (p.270Ala>Ser) polymorphism on renal clearance of metformin and the extent of the metformin-cimetidine interaction. The role of this polymorphism for renal disposition of endogenous compounds and drugs other than metformin has not been investigated. In addition, it is unclear whether the observed genotype dependence of an OCT2-mediated drug-drug interaction might occur also with other OCT inhibitors. To address these issues, we generated human embryonic kidney cells stably expressing wild-type OCT2 or the p.270Ala>Ser variant. No differences in protein expression levels and membrane incorporation pattern were observed between the two cell lines. The p.270Ala>Ser variant significantly impaired uptake kinetics of 1-methyl-4-phenylpyridinium, dopamine, norepinephrine, and propranolol. V(max) values were significantly reduced for uptake of all four compounds mediated by the p.270Ala>Ser variant compared with wild-type OCT2. In addition, a significant difference in the affinity to wild-type and mutant OCT2 was observed for dopamine (K(m) dopamine: 932 +/- 77 versus 1285 +/- 132 microM). Moreover, out of a set of 27 compounds p.270Ala>Ser OCT2 was significantly less sensitive to inhibition by cimetidine, flurazepam, metformin, mexiletine, propranolol, and verapamil than wild-type OCT2 (e.g., for propranolol: IC(50) wild type versus p.270Ala>Ser 189 versus 895 microM, P < 0.001). Our results indicate that the common OCT2 c.808G>T single nucleotide polymorphism significantly alters uptake of endogenous compounds and drugs. Moreover, for selected compounds the extent of OCT2-mediated drug interactions could depend on OCT2 c.808G>T genotype. PMID:19251820

  13. The 5-HT2C receptor gene Cys23Ser polymorphism influences the intravaginal ejaculation latency time in Dutch Caucasian men with lifelong premature ejaculation.

    PubMed

    Janssen, Paddy Kc; Schaik, Ron van; Olivier, Berend; Waldinger, Marcel D

    2014-01-01

    It has been postulated that the persistent short intravaginal ejaculation latency time (IELT) of men with lifelong premature ejaculation (LPE) is related to 5-hydroxytryptamine (HT)2C receptor functioning. The aim of this study was to investigate the relationship of Cys23Ser 5-HT2C receptor gene polymorphism and the duration of IELT in men with LPE. Therefore, a prospective study was conducted in 64 Dutch Caucasian men with LPE. Baseline IELT during coitus was assessed by stopwatch over a 1-month period. All men were genotyped for Cys23Ser 5-HT2C receptor gene polymorphism. Allele frequencies and genotypes of Cys and Ser variants of 5-HT2C receptor gene polymorphism were determined. Association between Cys/Cys and Ser/Ser genotypes and the natural logarithm of the IELT in men with LPE were investigated. As a result, the geometric mean, median and natural mean IELT were 25.2, 27.0, 33.9 s, respectively. Of all men, 20.0%, 10.8%, 23.1% and 41.5% ejaculated within 10, 10-20, 20-30 and 30-60 s after vaginal penetration. Of the 64 men, the Cys/Cys and Ser/Ser genotype frequency for the Cys23Ser polymorphism of the 5-HT2C receptor gene was 81% and 19%, respectively. The geometric mean IELT of the wildtypes (Cys/Cys) is significantly lower (22.6 s; 95% CI 18.3-27.8 s) than in male homozygous mutants (Ser/Ser) (40.4 s; 95% CI 20.3-80.4 s) (P = 0.03). It is concluded that Cys23Ser 5-HT2C receptor gene polymorphism is associated with the IELT in men with LPE. Men with Cys/Cys genotype have shorter IELTs than men with Ser/Ser genotypes.

  14. The 5-HT2C receptor gene Cys23Ser polymorphism influences the intravaginal ejaculation latency time in Dutch Caucasian men with lifelong premature ejaculation

    PubMed Central

    Janssen, Paddy KC; van Schaik, Ron; Olivier, Berend; Waldinger, Marcel D

    2014-01-01

    It has been postulated that the persistent short intravaginal ejaculation latency time (IELT) of men with lifelong premature ejaculation (LPE) is related to 5-hydroxytryptamine (HT)2C receptor functioning. The aim of this study was to investigate the relationship of Cys23Ser 5-HT2C receptor gene polymorphism and the duration of IELT in men with LPE. Therefore, a prospective study was conducted in 64 Dutch Caucasian men with LPE. Baseline IELT during coitus was assessed by stopwatch over a 1-month period. All men were genotyped for Cys23Ser 5-HT2C receptor gene polymorphism. Allele frequencies and genotypes of Cys and Ser variants of 5-HT2C receptor gene polymorphism were determined. Association between Cys/Cys and Ser/Ser genotypes and the natural logarithm of the IELT in men with LPE were investigated. As a result, the geometric mean, median and natural mean IELT were 25.2, 27.0, 33.9 s, respectively. Of all men, 20.0%, 10.8%, 23.1% and 41.5% ejaculated within 10, 10–20, 20–30 and 30–60 s after vaginal penetration. Of the 64 men, the Cys/Cys and Ser/Ser genotype frequency for the Cys23Ser polymorphism of the 5-HT2C receptor gene was 81% and 19%, respectively. The geometric mean IELT of the wildtypes (Cys/Cys) is significantly lower (22.6 s; 95% CI 18.3–27.8 s) than in male homozygous mutants (Ser/Ser) (40.4 s; 95% CI 20.3–80.4 s) (P = 0.03). It is concluded that Cys23Ser 5-HT2C receptor gene polymorphism is associated with the IELT in men with LPE. Men with Cys/Cys genotype have shorter IELTs than men with Ser/Ser genotypes. PMID:24799636

  15. The 5-HT2C receptor gene Cys23Ser polymorphism influences the intravaginal ejaculation latency time in Dutch Caucasian men with lifelong premature ejaculation.

    PubMed

    Janssen, Paddy Kc; Schaik, Ron van; Olivier, Berend; Waldinger, Marcel D

    2014-01-01

    It has been postulated that the persistent short intravaginal ejaculation latency time (IELT) of men with lifelong premature ejaculation (LPE) is related to 5-hydroxytryptamine (HT)2C receptor functioning. The aim of this study was to investigate the relationship of Cys23Ser 5-HT2C receptor gene polymorphism and the duration of IELT in men with LPE. Therefore, a prospective study was conducted in 64 Dutch Caucasian men with LPE. Baseline IELT during coitus was assessed by stopwatch over a 1-month period. All men were genotyped for Cys23Ser 5-HT2C receptor gene polymorphism. Allele frequencies and genotypes of Cys and Ser variants of 5-HT2C receptor gene polymorphism were determined. Association between Cys/Cys and Ser/Ser genotypes and the natural logarithm of the IELT in men with LPE were investigated. As a result, the geometric mean, median and natural mean IELT were 25.2, 27.0, 33.9 s, respectively. Of all men, 20.0%, 10.8%, 23.1% and 41.5% ejaculated within 10, 10-20, 20-30 and 30-60 s after vaginal penetration. Of the 64 men, the Cys/Cys and Ser/Ser genotype frequency for the Cys23Ser polymorphism of the 5-HT2C receptor gene was 81% and 19%, respectively. The geometric mean IELT of the wildtypes (Cys/Cys) is significantly lower (22.6 s; 95% CI 18.3-27.8 s) than in male homozygous mutants (Ser/Ser) (40.4 s; 95% CI 20.3-80.4 s) (P = 0.03). It is concluded that Cys23Ser 5-HT2C receptor gene polymorphism is associated with the IELT in men with LPE. Men with Cys/Cys genotype have shorter IELTs than men with Ser/Ser genotypes. PMID:24799636

  16. Radical formation of amino acid precursors in interstellar regions? Ser, Cys and Asp.

    PubMed

    Knowles, Daniel J; Wang, Tianfang; Bowie, John H

    2010-11-01

    It is proposed that the glycine precursor NH(2)CH(2)CN may be synthesised in interstellar dust clouds by the radical combination reactions NH(2)˙ + ˙CH(2)CN → NH(2)CH(2)CN (ΔG = -302 kJ mol(-1)) and/or NH(2)CH(2)˙ + ˙CN → NH(2)CH(2)CN (ΔG = -414 kJ mol(-1)). All calculations at the CCSD(T)/aug-cc-pVDZ//B3LYP/6-31+G(d) level of theory. This paper extends that concept to radical/radical coupling reactions to form Ser, Cys and Asp precursor nitriles. The hydrogen abstraction process NH(2)CH(2)CN + HO˙→ NH(2)˙CHCN + H(2)O (ΔG = -130 kJ mol(-1)) is suggested to precede the radical coupling reactions NH(2)˙CHCN + R˙→ NH(2)CHRCN (R˙ = ˙CH(2)OH, ˙CH(2)SH and ˙CH(2)CN) to form nitrile precursors of the amino acids Ser, Cys and Asp. These three reactions are all favourable (ΔG = -240, -227 and -223 kJ mol(-1)). The radical species ˙CH(2)NH(2), ˙CH(2)OH, ˙CH(2)SH and ˙CH(2)CN are shown to be stable for the microsecond timeframe by a combination of theoretical calculations and the experimental mass spectrometric neutralization/reionization procedure.

  17. The hOGG1 Ser326Cys polymorphism contributes to digestive system cancer susceptibility: evidence from 48 case-control studies.

    PubMed

    Wang, Yang; Gao, Xujie; Wei, Feng; Zhang, Xinwei; Yu, Jinpu; Zhao, Hua; Sun, Qian; Yan, Fan; Yan, Cihui; Li, Hui; Ren, Xiubao

    2015-02-01

    The Ser326Cys polymorphism in the human 8-oxogunaine DNA glycosylase (hOGG1) gene had been implicated in cancer susceptibility. Studies investigating the associations between the Ser326Cys polymorphism and digestion cancer susceptibility showed conflicting results. Therefore, a meta-analysis was performed to derive a more precise estimation of the relationship. We conducted a meta-analysis of 48 studies that included 12,073 cancer cases and 19,557 case-free controls. We assessed the strength of the association using odds ratios (ORs) with 95% confidence intervals (CIs). In our analysis, the hOGG1 Ser326Cys polymorphism was significantly associated with the risk of digestive system cancers (Cys/Cys vs. Ser/Ser: OR = 1.17, 95% CI = 1.00-1.35, P < 0.001; Cys/Cys vs. Cys/Ser + Ser/Ser: OR = 1.14, 95% CI = 1.00-1.29, P < 0.001). In subgroup analyses by cancer types, we found that the hOGG1 Ser326Cys polymorphism may increase hepatocellular cancer and colorectal cancer risks, but decrease the risk of oral cancer. These findings supported that hOGG1 Ser326Cys polymorphism may contribute to the susceptibility of digestive cancers.

  18. A Ser311Cys mutation in the human dopamine receptor D2 gene is associated with reduced energy expenditure.

    PubMed

    Tataranni, P A; Baier, L; Jenkinson, C; Harper, I; Del Parigi, A; Bogardus, C

    2001-04-01

    Brain dopaminergic pathways play a major role in the control of movement. Absence of the murine dopamine D2 receptor gene (drd2) produces bradykinesia and hypothermia. A Ser311Cys mutation of the human DRD2 produces a marked functional impairment of the receptor and is associated with higher BMI in some populations. We hypothesized that the Ser311Cys mutation of DRD2 may inhibit energy expenditure. Here we report that total energy expenditure (doubly labeled water) measured in 89 nondiabetic Pima Indians was 244 kcal/ day lower in homozygotes for the Cys311-encoding allele when compared with those heterozygous and homozygous for the Ser311-encoding allele (P = 0.056). The 24-h resting energy expenditure (respiratory chamber) measured in 320 nondiabetic Pimas was also 87 kcal/day lower in homozygotes for the Cys311-encoding allele when compared with those heterozygous and homozygous for the Ser311-encoding allele (P = 0.026). These findings are the first evidence that a genetic mutation is associated with reduced energy expenditure in humans. Because the impact of this mutation on human obesity is small, we suggest that either the energy deficit induced is not large enough to significantly influence body weight in this population and/or that the Cys311-encoding allele is also associated with reduced energy intake.

  19. Improving the Specificity of the Prostate-Specific Antigen Substrate Glutaryl-Hyp-Ala-Ser-Chg-Gln as a Promoiety.

    PubMed

    Aloysius, Herve; Hu, Longqin

    2015-10-01

    To develop PSA peptide substrates with improved specificity and plasma stability from the known substrate sequence glutaryl-Hyp-Ala-Ser-Chg-Gln, systematic replacements of the N-terminal segment with D-retro-inverso-peptides were performed with the incorporation of 7-amino-4-methylcoumarin (7-AMC) after Gln for convenient fluorometric determination and ranking of the PSA substrate activity. The D-retro-inverso-peptide conjugates with P2-P5 D-amino acid substitutions were moderate but poorer PSA substrates as compared to the original peptide, suggesting that inversion of the amide bonds and/or incorporation of the additional atom as in the urea linker adversely affected PSA binding. However, P5 substitution of Hyp with Ser showed significant improvements in PSA cleavage rate; the resulting AMC conjugate, glutaryl-Ser-Ala-Ser-Chg-Gln-AMC (11), exhibited the fastest PSA cleavage rate of 351 pmol/min/100 nmol PSA. In addition, GABA←mGly-Ala-Ser-Chg-Gln-AMC (conjugate 6) was the second best PSA substrate and released 7-AMC at a rate of 225 pmol/min/100 nmol PSA as compared to 171 pmol/min/100 nmol PSA for the control conjugate glutaryl-Hyp-Ala-Ser-Chg-Gln-AMC. Incubations of selected AMC conjugates with mouse and human plasma revealed that GABA←D-Ser-ψ[NH-CO-NH]-Ala-Ser-Chg-Gln-AMC (5) and GABA←mGly-Ala-Ser-Chg-Gln-AMC (6) were most stable to non-PSA-mediated proteolysis. Our results suggest that the PSA specificity of glutaryl-Hyp-Ala-Ser-Chg-Gln is improved with Ser and mGly substitutions of Hyp at the P5.

  20. Structural and kinetic studies on the Ser101Ala variant of choline oxidase: Catalysis by compromise

    SciTech Connect

    Finnegan, S.; Orville, A.; Yuan, H.; Wang, Y.-F.; Weber, I. T.; Gadda, G.

    2010-09-15

    The oxidation of choline catalyzed by choline oxidase includes two reductive half-reactions where FAD is reduced by the alcohol substrate and by an aldehyde intermediate transiently formed in the reaction. Each reductive half-reaction is followed by an oxidative half-reaction where the reduced flavin is oxidized by oxygen. Here, we have used mutagenesis to prepare the Ser101Ala mutant of choline oxidase and have investigated the impact of this mutation on the structural and kinetic properties of the enzyme. The crystallographic structure of the Ser101Ala enzyme indicates that the only differences between the mutant and wild-type enzymes are the lack of a hydroxyl group on residue 101 and a more planar configuration of the flavin in the mutant enzyme. Kinetics established that replacement of Ser101 with alanine yields a mutant enzyme with increased efficiencies in the oxidative half-reactions and decreased efficiencies in the reductive half-reactions. This is accompanied by a significant decrease in the overall rate of turnover with choline. Thus, this mutation has revealed the importance of a specific residue for the optimization of the overall turnover of choline oxidase, which requires fine-tuning of four consecutive half-reactions for the conversion of an alcohol to a carboxylic acid.

  1. Association Between Paraoxonase 2 Ser311Cys Polymorphism and Coronary Heart Disease Risk: A Meta-Analysis.

    PubMed

    Chen, Min-Li; Zhao, Hua; Liao, Ning; Xie, Zheng-Fu

    2016-01-01

    BACKGROUND The relationship between coronary heart disease (CHD) and the paraoxonase 2 (PON2) Ser311Cys polymorphism has received much attention. We conducted a meta-analysis on the results from published case-control studies examining this relation. MATERIAL AND METHODS A literature search was performed using PubMed and ISI Web of Knowledge databases until October 2015. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using Stata version 11.0 software. Data were pooled using the random-effects model. RESULTS Nine studies were eligible for statistical analysis and included a total of 5278 participants. The results did not support an association between the Ser311Cys polymorphism and CHD in the overall populations (Asians, Caucasians, and a Hispanic mixed population) under dominant (OR 1.07; 95% CI 0.91-1.28; Pz=0.413), recessive (OR 1.19; 95% CI 0.72-1.95; Pz=0.500), homozygote (OR 1.20; 95% CI 0.71-2.03; Pz=0.489), and allelic comparison (OR 1.08; 95% CI 0.91-1.28; Pz=0.390) models. However, in subgroup analysis according to ethnicity, we found that the Ser311Cys polymorphism was associated with CHD risk in Caucasians under recessive (OR 2.08; 95% CI 1.30-3.34; Pz=0.002) and homozygote (OR 2.16; 95% CI 1.33-3.50; Pz=0.002) models. Subgroup analysis indicated no significant association of this polymorphism with CHD in either Asian or Hispanic populations. CONCLUSIONS The PON2 Ser311Cys polymorphism is associated with CHD risk in Caucasians, but there is no association between this polymorphism and CHD in Asians or Hispanic populations. PMID:27609416

  2. Association Between Paraoxonase 2 Ser311Cys Polymorphism and Coronary Heart Disease Risk: A Meta-Analysis

    PubMed Central

    Chen, Min-Li; Zhao, Hua; Liao, Ning; Xie, Zheng-Fu

    2016-01-01

    Background The relationship between coronary heart disease (CHD) and the paraoxonase 2 (PON2) Ser311Cys polymorphism has received much attention. We conducted a meta-analysis on the results from published case-control studies examining this relation. Material/Methods A literature search was performed using PubMed and ISI Web of Knowledge databases until October 2015. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using Stata version 11.0 software. Data were pooled using the random-effects model. Results Nine studies were eligible for statistical analysis and included a total of 5278 participants. The results did not support an association between the Ser311Cys polymorphism and CHD in the overall populations (Asians, Caucasians, and a Hispanic mixed population) under dominant (OR 1.07; 95% CI 0.91–1.28; Pz=0.413), recessive (OR 1.19; 95% CI 0.72–1.95; Pz=0.500), homozygote (OR 1.20; 95% CI 0.71–2.03; Pz=0.489), and allelic comparison (OR 1.08; 95% CI 0.91–1.28; Pz=0.390) models. However, in subgroup analysis according to ethnicity, we found that the Ser311Cys polymorphism was associated with CHD risk in Caucasians under recessive (OR 2.08; 95% CI 1.30–3.34; Pz=0.002) and homozygote (OR 2.16; 95% CI 1.33–3.50; Pz=0.002) models. Subgroup analysis indicated no significant association of this polymorphism with CHD in either Asian or Hispanic populations. Conclusions The PON2 Ser311Cys polymorphism is associated with CHD risk in Caucasians, but there is no association between this polymorphism and CHD in Asians or Hispanic populations. PMID:27609416

  3. Association between the OGG1 Ser326Cys Polymorphism and Cancer Risk: Evidence from 152 Case-Control Studies.

    PubMed

    Zou, Hua; Li, Qing; Xia, Wei; Liu, Yong; Wei, Xi; Wang, Dong

    2016-01-01

    Although it has been suggested that the 8-oxoguanine DNA glycosylase (OGG1) gene Ser326Cys polymorphism may be a risk factor for cancer, the conclusions from previous studies are inconsistent. Thus, we conducted an updated meta-analysis to estimate the effect of OGG1 variant genotypes on cancer susceptibility. We searched the PubMed for all eligible studies published in English for the period ending September 2014. We found the association between OGG1 Ser326Cys polymorphism and cancer susceptibility based on 152 case-control studies in different genetic model comparisons (dominant model: OR = 1.053, P = 0.018; recessive model: OR = 1.108, P < 0.001; homozygote: OR = 1.135, P < 0.001; additive model: OR = 1.059, P < 0.001). However, the results from the subgroup analyses based on types of cancer, health population as controls or studies with relatively large sample size did not support the conclusion. Although the overall results of this meta-analysis showed a positive association between OGG1 variant genotypes and cancer susceptibility, the subgroup analyses by cancer type, sample size, and source of controls presented inconsistent results. Therefore, the current evidence from the meta-analysis did not support the hypothesis of OGG1 Ser326Cys polymorphism as a risk factor of cancer. PMID:27390603

  4. Association between the OGG1 Ser326Cys Polymorphism and Cancer Risk: Evidence from 152 Case-Control Studies

    PubMed Central

    Zou, Hua; Li, Qing; Xia, Wei; Liu, Yong; Wei, Xi; Wang, Dong

    2016-01-01

    Although it has been suggested that the 8-oxoguanine DNA glycosylase (OGG1) gene Ser326Cys polymorphism may be a risk factor for cancer, the conclusions from previous studies are inconsistent. Thus, we conducted an updated meta-analysis to estimate the effect of OGG1 variant genotypes on cancer susceptibility. We searched the PubMed for all eligible studies published in English for the period ending September 2014. We found the association between OGG1 Ser326Cys polymorphism and cancer susceptibility based on 152 case-control studies in different genetic model comparisons (dominant model: OR = 1.053, P = 0.018; recessive model: OR = 1.108, P < 0.001; homozygote: OR = 1.135, P < 0.001; additive model: OR = 1.059, P < 0.001). However, the results from the subgroup analyses based on types of cancer, health population as controls or studies with relatively large sample size did not support the conclusion. Although the overall results of this meta-analysis showed a positive association between OGG1 variant genotypes and cancer susceptibility, the subgroup analyses by cancer type, sample size, and source of controls presented inconsistent results. Therefore, the current evidence from the meta-analysis did not support the hypothesis of OGG1 Ser326Cys polymorphism as a risk factor of cancer. PMID:27390603

  5. No evidence for association of dopamine D2 receptor variant (Ser311/Cys311) with major psychosis

    SciTech Connect

    Sasaki, Tsukasa; Macciardi, F.M.; Badri, F.

    1996-07-26

    We investigated a variant of the dopamine D2 receptor gene (Ser311/Cys311 substitution) in Caucasian patients with schizophrenia (n = 273), delusional disorder (n = 62), bipolar I affective disorder (n = 63), and controls (n = 255). No evidence for association between the receptor variant and any of the diseases was found, even when patients with younger age-of-onset (<25 years) were compared with controls. Futhermore, in a subgroup of schizophrenia patients whom we assessed for negative symptoms, those with the Cys allele did not differ from the remainder of the group. Also, the bipolar affective disorder patients with psychotic features did not show evidence for association with the receptor variant. Thus, our results do not provide evidence for an association between this D2 receptor variant and schizophrenia, or delusional disorder, or bipolar affective disorder. 11 refs., 1 tab.

  6. The hOGG1 Ser326Cys gene polymorphism and susceptibility for bladder cancer: a meta-analysis

    PubMed Central

    Wenjuan, Cao; Jianzhong, Lu; Chong, Li; Yanjun, Gao; Keqing, Lu; Hanzhang, Wang; Zhiping, Wang

    2016-01-01

    ABSTRACT Objective: To assess the susceptibility of the hOGG1 genetic polymorphism for bladder cancer and evaluate the impact of smoking exposure. Materials and Methods: Articles included in PubMed, Medline and Springer databases were retrieved using the following key words: “human 8-oxoguanine DNA glycosylase”, “OGG”, “OGG1”, “hOGG1”, “genetic variation”, “polymorphism” , “bladder cancer”, and “bladder carcinoma” to Meta-analysis was performed to detect whether there were differences between the bladder cancer group and the control group about the distribution of genotypes of the hOGG1 gene. Results: The results showed that there are no significant associations between the hOGG1 326Cys polymorphism and bladder cancer: GG vs. CC (OR: 1.09, 95% CI: 0.85–1.40, p=0.480); GC vs. CC (OR: 1.05, 95% CI: 0.85–1.28, p=0.662); GG+GC vs. CC (OR: 1.04, 95% CI: 0.89–1.21, p=0.619); GG vs. GC+CC(OR: 1.02, 95% CI: 0.78–1.33, p=0.888); G vs. C (OR: 1.01, 95% CI: 0.91–1.13, p=0.818). In the smoker population, no significant associations between the hOGG1 326Cys polymorphism and bladder cancer were observed for all the models. However, individuals carrying the hOGG1 Cys326Cys genotype have increased risk for bladder cancer compared to those carrying the hOGG1 Ser326Ser genotype in the non-smoker Asian population. Conclusion: The hOGG1 326Cys polymorphisms aren't a risk factor for bladder cancer, especially in the smoker population. But GG genotype is a risk factor for bladder cancer to the non-smoker Asian population compared with CC genotype. PMID:27583352

  7. Novel Association of WNK4 Gene, Ala589Ser Polymorphism in Essential Hypertension, and Type 2 Diabetes Mellitus in Malaysia.

    PubMed

    Ghodsian, Nooshin; Ismail, Patimah; Ahmadloo, Salma; Heidari, Farzad; Haghvirdizadeh, Polin; Ataollahi Eshkoor, Sima; Etemad, Ali

    2016-01-01

    With-no-lysine (K) Kinase-4 (WNK4) consisted of unique serine and threonine protein kinases, genetically associated with an autosomal dominant form of hypertension. Argumentative consequences have lately arisen on the association of specific single nucleotide polymorphisms of WNK4 gene and essential hypertension (EHT). The aim of this study was to determine the association of Ala589Ser polymorphism of WNK4 gene with essential hypertensive patients in Malaysia. WNK4 gene polymorphism was specified utilizing mutagenically separated polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) method in 320 subjects including 163 cases and 157 controls. Close relation between Ala589Ser polymorphism and elevated systolic and diastolic blood pressure (SBP and DBP) was recognized. Sociodemographic factors including body mass index (BMI), age, the level of fasting blood sugar (FBS), low density lipoprotein (LDL), and triglyceride (TG) in the cases and healthy subjects exhibited strong differences (p < 0.05). The distribution of allele frequency and genotype of WNK4 gene Ala589Ser polymorphism showed significant differences (p < 0.05) between EHT subjects with or without type 2 diabetes mellitus (T2DM) and normotensive subjects, statistically. The WNK4 gene variation influences significantly blood pressure increase. Ala589Ser probably has effects on the enzymic activity leading to enhanced predisposition to the disorder. PMID:27314050

  8. Novel Association of WNK4 Gene, Ala589Ser Polymorphism in Essential Hypertension, and Type 2 Diabetes Mellitus in Malaysia

    PubMed Central

    Ghodsian, Nooshin; Ismail, Patimah; Ahmadloo, Salma; Heidari, Farzad; Haghvirdizadeh, Polin; Ataollahi Eshkoor, Sima; Etemad, Ali

    2016-01-01

    With-no-lysine (K) Kinase-4 (WNK4) consisted of unique serine and threonine protein kinases, genetically associated with an autosomal dominant form of hypertension. Argumentative consequences have lately arisen on the association of specific single nucleotide polymorphisms of WNK4 gene and essential hypertension (EHT). The aim of this study was to determine the association of Ala589Ser polymorphism of WNK4 gene with essential hypertensive patients in Malaysia. WNK4 gene polymorphism was specified utilizing mutagenically separated polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) method in 320 subjects including 163 cases and 157 controls. Close relation between Ala589Ser polymorphism and elevated systolic and diastolic blood pressure (SBP and DBP) was recognized. Sociodemographic factors including body mass index (BMI), age, the level of fasting blood sugar (FBS), low density lipoprotein (LDL), and triglyceride (TG) in the cases and healthy subjects exhibited strong differences (p < 0.05). The distribution of allele frequency and genotype of WNK4 gene Ala589Ser polymorphism showed significant differences (p < 0.05) between EHT subjects with or without type 2 diabetes mellitus (T2DM) and normotensive subjects, statistically. The WNK4 gene variation influences significantly blood pressure increase. Ala589Ser probably has effects on the enzymic activity leading to enhanced predisposition to the disorder. PMID:27314050

  9. Vasohibins: new transglutaminase-like cysteine proteases possessing a non-canonical Cys-His-Ser catalytic triad

    PubMed Central

    Sanchez-Pulido, Luis; Ponting, Chris P.

    2016-01-01

    Summary: Vasohibin-1 and Vasohibin-2 regulate angiogenesis, tumour growth and metastasis. Their molecular functions, however, were previously unknown, in large part owing to their perceived lack of homology to proteins of known structure and function. To identify their functional amino acids and domains, their molecular activity and their evolutionary history, we undertook an in-depth analysis of Vasohibin sequences. We find that Vasohibin proteins are previously undetected members of the transglutaminase-like cysteine protease superfamily, and all possess a non-canonical Cys-His-Ser catalytic triad. We further propose a calcium-dependent activation mechanism for Vasohibin proteins. These findings can now be used to design constructs for protein structure determination and to develop enzyme inhibitors as angiogenic regulators to treat metastasis and tumour growth. Contact: luis.sanchezpulido@dpag.ox.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26794318

  10. The influence of Ser-154, Cys-113, and the phosphorylated threonine residue on the catalytic reaction mechanism of Pin1.

    PubMed

    Vöhringer-Martinez, Esteban; Verstraelen, Toon; Ayers, Paul W

    2014-08-21

    Pin1 is an enzyme that specifically catalyzes the cis-trans isomerization of proline amide bonds in peptides that contain a phosphorylated threonine or serine residue in the position preceding proline. In the cell, the isomerization reaction is associated with cellular signaling and has been related to diseases such as Alzheimer and cancer. The catalytic mechanism by which Pin1 accelerates the isomerization reaction, however, is still unknown. In this study, we use molecular dynamics simulation in combination with the QM/MM methodology to disclose the influence of the residues Ser-154 and Cys-113 in the enzyme and the phosphorylated threonine residue in the peptide on the reaction mechanism. To account for the correct electrostatic interaction between the three residues and the reactive center, we derive atomic charges that account for the varying electrostatic field in the catalytic cavity. Different methods based on reproducing the molecular electrostatic potential or an atoms in molecules approach were investigated. Finally, the reaction mechanism is analyzed with the mean reaction force and the influence of the three residues is disclosed. Our results show that Pin1 specifically catalyzes the isomerization of the trans conformer in a jump-rope type of motion, as suggested by us and confirmed experimentally by others. This is accomplished by anchoring the threonine phosphate residue on one end of the peptide through electrostatic interactions with the basic triad of the enzyme and at the other end through specific enzyme-peptide hydrogen bonds. Cys-113 reduces the structural contribution to the activation free energy through the stabilization of the cis conformer, and Ser-154 in combination with Gln-131 assist in the isomerization reaction of the trans isomer. PMID:25059768

  11. Anatomy of a Red Copper Center: Spectroscopic Identification and Reactivity of the Copper Centers of Bacillus Subtilis Sco and its Cys to Ala Variants

    PubMed Central

    Siluvai, Gnana S; Mayfield, Mary; Nilges, Mark J; George, Serena DeBeer; Blackburn, Ninian J

    2010-01-01

    Sco is a mononuclear red copper protein involved in the assembly of cytochrome c oxidase. It is spectroscopically similar to red copper nitrosocyanin, but unlike the latter, which has one copper cysteine thiolate, the former has two. In addition to the two cysteine ligands (C45 and C49), the WT protein from Bacillus subtilis (hereafter named BSco) has a histidine (H135) and an unknown endogenous protein oxygen ligand in a distorted tetragonal array. We have compared the properties of the WT protein to variants in which each of the two coordinating Cys residues has been individually mutated to Ala, using UV/vis, Cu and S K edge XAS, EPR, and resonance Raman spectroscopy. Unlike the Cu(II) form of native Sco, the Cu(II) complexes of the Cys variants are unstable. The copper center of C49A undergoes autoreduction to the Cu(I) form which is shown by EXAFS to be composed of a novel 2-coordinate center with one Cys and one His ligand. C45A rearranges to a new stable Cu(II) species coordinated by C49 H135 and a second His ligand recruited from a previously uncoordinated protein side chain. The different chemistry exhibited by the Cys variants can be rationalized by whether a stable Cu(I) species can be formed by autoredox chemistry. For C49A, the remaining Cys and His residues are trans which facilitate the formation of the highly stable 2-coordinate Cu(I) species, while for C45A such a configuration cannot be attained. Resonance Raman spectroscopy of the WT protein indicates a net weak Cu–S bond strength at ~ 2.24 Å corresponding to the two thiolate copper bonds, whereas the single variant C45A shows a moderately strong Cu–S bond at ~ 2.16 Å. S K-edge data gives a total covalency of 28% for both Cu-S bonds in the WT protein. These data suggest an average covalency per Cu-S bond lower than nitrosocyanin and close to that expected for type-2 Cu(II)-thiolate systems. The data are discussed relative to the unique Cu-S characteristics of cupredoxins, whence it is

  12. Variability of 5-HT2C receptor cys23ser polymorphism among European populations and vulnerability to affective disorder.

    PubMed

    Lerer, B; Macciardi, F; Segman, R H; Adolfsson, R; Blackwood, D; Blairy, S; Del Favero, J; Dikeos, D G; Kaneva, R; Lilli, R; Massat, I; Milanova, V; Muir, W; Noethen, M; Oruc, L; Petrova, T; Papadimitriou, G N; Rietschel, M; Serretti, A; Souery, D; Van Gestel, S; Van Broeckhoven, C; Mendlewicz, J

    2001-09-01

    Substantial evidence supports a role for dysfunction of brain serotonergic (5-HT) systems in the pathogenesis of major affective disorder, both unipolar (recurrent major depression) and bipolar.(1) Modification of serotonergic neurotransmission is pivotally implicated in the mechanism of action of antidepressant drugs(2) and also in the action of mood stabilizing agents, particularly lithium carbonate.(3) Accordingly, genes that code for the multiple subtypes of serotonin receptors that have been cloned and are expressed in brain,(4) are strong candidates for a role in the genetic etiology of affective illness. We examined a structural variant of the serotonin 2C (5-HT2C) receptor gene (HTR2C) that gives rise to a cysteine to serine substitution in the N terminal extracellular domain of the receptor protein (cys23ser),(5) in 513 patients with recurrent major depression (MDD-R), 649 patients with bipolar (BP) affective disorder and 901 normal controls. The subjects were drawn from nine European countries participating in the European Collaborative Project on Affective Disorders. There was significant variation in the frequency of the HT2CR ser23 allele among the 10 population groups included in the sample (from 24.6% in Greek control subjects to 9.2% in Scots, chi(2) = 20.9, df 9, P = 0.01). Logistic regression analysis demonstrated that over and above this inter-population variability, there was a significant excess of HT2CR ser23 allele carriers in patients compared to normal controls that was demonstrable for both the MDD (chi(2) = 7.34, df 1, P = 0.006) and BP (chi(2) = 5.45, df 1, P = 0.02) patients. These findings support a possible role for genetically based structural variation in 5-HT2C receptors in the pathogenesis of major affective disorder.

  13. Repair of oxidative DNA damage is delayed in the Ser326Cys polymorphic variant of the base excision repair protein OGG1.

    PubMed

    Kershaw, Rachael M; Hodges, Nikolas J

    2012-07-01

    Gene-environment interactions influence an individual's risk of disease development. A common human 8-oxoguanine DNA glycosylase 1 (OGG1) variant, Cys326-hOGG1, has been associated with increased cancer risk. Evidence suggests that this is due to reduced repair ability, particularly under oxidising conditions but the underlying mechanism is poorly understood. Oxidising conditions may arise due to internal cellular processes, such as inflammation or external chemical or radiation exposure. To investigate wild-type and variant OGG1 regulation and activity under oxidising conditions, we generated mOgg1 (-/-) null mouse embryonic fibroblasts cells stably expressing Ser326- and Cys326-hOGG1 and measured activity, gene expression, protein expression and localisation following treatment with the glutathione-depleting compound L-buthionine-S-sulfoximine (BSO). Assessment of OGG1 activity using a 7,8-dihydro-8-oxodeoxyguanine (8-oxo dG) containing molecular beacon demonstrated that the activity of both Ser326- and Cys326-hOGG1 was increased following oxidative treatment but with different kinetics. Peak activity of Ser326-hOGG1 occurred 12 h prior to that of Cys326-hOGG1. In both variants, the increased activity was not associated with any gene expression or protein increase or change in protein localisation. These findings suggest that up-regulation of OGG1 activity in response to BSO-induced oxidative stress is via post-transcriptional regulation and provide further evidence for impaired Cys326-hOGG1 repair ability under conditions of oxidative stress. This may have important implications for increased mutation frequency resulting from increased oxidative stress in individuals homozygous for the Cys326 hOGG1 allele.

  14. OGG1 Ser326Cys polymorphism interacts with cigarette smoking to increase oxidative DNA damage in human sperm and the risk of male infertility.

    PubMed

    Ji, Guixiang; Yan, Lifeng; Liu, Wei; Qu, Jianhua; Gu, Aihua

    2013-04-12

    8-Oxoguanine DNA glycosylase 1 (OGG1) plays an important role in repairing oxidative DNA damage induced by chemical agents, such as tobacco. This study examined the effects of OGG1 Ser326Cys polymorphism and cigarette smoking, alone or combined, on sperm oxidative DNA damage and the risk of male infertility. A total of 620 idiopathic infertile subjects and 480 fertile controls were recruited in this study. Sperm 8-hydroxydeoxyguanine (8-OHdG) was measured by immunofluorescent assay using flow cytometry and genotypes were determined by OpenArray platform with a chip-based Taq-Man genotyping technology. Our results demonstrated that both cigarette smoking and OGG1 polymorphism can affect the sperm 8-OHdG levels. Individuals with variant Cys/Cys homozygote showed higher levels of sperm 8-OHdG than wide-type homozygote carriers (Ser/Ser). Stratified analysis found that the association between OGG1 polymorphism and sperm 8-OHdG levels was only observed among smokers with pack-years ≥5 but not among those subjects with pack-years<5 (pack-years=packs smoked per day×years as a smoker). Further analysis based on the case-control study revealed that variant allele (Cys) of OGG1 was significantly associated with male infertility risk in a dominant model (OR=1.35, 95% CI: 1.01-1.82; trend P<0.001). Furthermore, we found a significant gene-environment interaction between OGG1 Ser326Cys polymorphism and cigarette smoking in relation to male infertility risk (Pinteration=0.0003). These findings provided the first evidence about potential interactive effects of OGG1 polymorphism and cigarette smoking on male infertility risk.

  15. Association between OGG1 Ser326Cys polymorphism and risk of upper aero-digestive tract and gastrointestinal cancers: a meta-analysis.

    PubMed

    Das, Sambuddha; Nath, Sayantan; Bhowmik, Aditi; Ghosh, Sankar Kumar; Choudhury, Yashmin

    2016-01-01

    Cancers of the upper aero-digestive and gastrointestinal tract are one of the major causes of mortality around the world. DNA repair genes play a vital role in preventing carcinogenesis by maintaining genomic integrity. Polymorphisms in the nucleotide sequence of DNA repair genes are often reported to be associated with an increased risk for different cancers. The OGG1 gene encodes the enzyme 8-oxoguanine DNA glycosylase which removes oxidatively damaged bases of DNA. Several studies report that the OGG1 Ser326Cys polymorphism increases the risk for cancers of the upper aero-digestive and gastrointestinal tract. However, other studies provide evidence that such an association does not exist. A meta-analysis to assess the role of OGG1 Ser326Cys polymorphism in the cancers of the upper aero-digestive and gastrointestinal tract was therefore undertaken in order to resolve this ambiguity. Seventeen studies were recruited for this meta-analysis after screening 58 articles with a total of 5533 cases and 6834 controls for which the odds ratio with 95 % confidence interval was calculated. Begg's funnel test and Egger's test were performed for calculating publication bias. Our study reveals an association between OGG1 Ser326Cys polymorphism and cancer susceptibility of the upper aero-digestive and gastrointestinal tract (CG + GG vs CC; odds ratio, OR 1.22; 95 % CI 1.05-1.41; GG vs CG + CC; OR 1.36; 95 % CI 1.09-1.70; GG vs CC; OR 1.46; 95 % CI 1.12-1.92). Subgroup analysis based on cancer types and ethnicity also revealed the association of OGG1 Ser326Cys polymorphism to the risk for upper aero-digestive and gastrointestinal tract cancers among both the Asian and the Caucasian populations. No risk was however observed for smoking habits and OGG1 Ser326Cys polymorphism. In conclusion, OGG1 Ser326Cys polymorphism may be associated with the increased risk for aero-digestive tract and gastro-intestinal cancers in both Asian and Caucasian populations. PMID:27026921

  16. The effect of disulfide bond introduction and related Cys/Ser mutations on the stability of a cyclohexanone monooxygenase.

    PubMed

    Schmidt, Sandy; Genz, Maika; Balke, Kathleen; Bornscheuer, Uwe T

    2015-11-20

    Baeyer-Villiger monooxygenases (BVMO) belong to the class B of flavin-dependent monooxygenases (type I BVMOs) and catalyze the oxidation of (cyclic) ketones into esters and lactones. The prototype BVMO is the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. This enzyme shows an impressive substrate scope with a high chemo-, regio- and/or enantioselectivity. BVMO reactions are often difficult, if not impossible to achieve by chemical approaches and this makes these enzymes thus highly desired candidates for industrial applications. Unfortunately, the industrial use is hampered by several factors related to the lack of stability of these biocatalysts. Thus, the aim of this study was to improve the CHMO's long-term stability, one of the most relevant parameter for biocatalytic processes, and additionally its stability against oxidation. We used an easy computational method for the prediction of stabilizing disulfide bonds in the CHMO-scaffold. The three most promising predicted disulfide pairs were created and biochemically characterized. The most oxidatively stable variant (Y411C-A463C) retained nearly 60% activity after incubation with 25 mM H2O2 whereas the wild type retained only 16%. In addition, one extra disulfide pair (T415C-A463C) was created and tested for increased stability. The melting temperature (Tm) of this variant was increased by 5°C with simultaneous improved long-term stability. After verification by ABD-F labeling that this mutant does not form a disulfide bond, single and double Cys/Ser mutants were prepared and investigated. Subsequent analysis revealed that the T415C single point variant is the most stable variant with a 30-fold increased long-term stability (33% residual activity after 24h incubation at 25°C) showcasing a great achievement for practical applications.

  17. Advanced research on dopamine signaling to develop drugs for the treatment of mental disorders: Ser311Cys polymorphisms of the dopamine D2-receptor gene and schizophrenia.

    PubMed

    Itokawa, Masanari; Arinami, Tadao; Toru, Michio

    2010-01-01

    Schizophrenia is a debilitating and complex mental disorder with a prevalence of approximately 1% worldwide. The etiology remains unclear, despite massive research efforts. Hyperactive dopaminergic signal transduction in the central nervous system is suggested to be involved in the pathophysiology of schizophrenia (the dopamine hypothesis). The dopamine D(2)-receptor (DRD2) gene is thus a promising candidate for associations with risk of schizophrenia. We investigated DRD2 and found a novel missense nucleotide change causing an amino acid substitution of serine with cysteine at codon 311 (Ser311Cys). We performed an association study using 156 schizophrenia patients and 300 controls. Cys311 in DRD2 was significantly associated with schizophrenia. Patients with the Cys311 allele displayed shorter duration of hospitalization and less severe negative symptoms and were more frequently married compared to patients without this allele, suggesting good response to treatment. We expanded samples to 291 patients with schizophrenia (including 11 postmortem brain samples), 579 controls, and 78 patients with affective disorders in a further case-control study. Cys311 was associated with schizophrenia, particularly in patients without negative symptoms, and bipolar disorder with mood-incongruent psychotic symptoms. Three meta-analyses using over 20 published studies confirmed the association. In vitro studies showed that Cys311-type D(2) receptor impairs dopamine-induced sequestration, which appears to be consistent with the dopamine hypothesis.

  18. Nonconserved Residues Ala287 and Ser290 of the Cryptosporidium hominis Thymidylate Synthase Domain Facilitate Its Rapid Rate of Catalysis

    SciTech Connect

    Doan,L.; Martucci, W.; Vargo, M.; Atreya, C.; Anderson, K.

    2007-01-01

    Cryptosporidium hominis TS-DHFR exhibits an unusually high rate of catalysis at the TS domain, at least 10-fold greater than those of other TS enzymes. Using site-directed mutagenesis, we have mutated residues Ala287 and Ser290 in the folate-binding helix to phenylalanine and glycine, respectively, the corresponding residues in human and most other TS enzymes. Our results show that the mutant A287F, the mutant S290G, and the double mutant all have reduced affinities for methylene tetrahydrofolate and reduced rates of reaction at the TS domain. Interestingly, the S290G mutant enzyme had the lowest TS activity, with a catalytic efficiency {approx}200-fold lower than that of the wild type (WT). The rate of conformational change of the S290G mutant is {approx}80 times slower than that of WT, resulting in a change in the rate-limiting step from hydride transfer to covalent ternary complex formation. We have determined the crystal structure of ligand-bound S290G mutant enzyme, which shows that the primary effect of the mutation is an increase in the distance between the TS ligands. The kinetic and crystal structure data presented here provide the first evidence explaining the unusually fast TS rate in C. hominis.

  19. Further Characterization of Cys-Type and Ser-Type Anaerobic Sulfatase Maturating Enzymes Suggests a Commonality in Mechanism of Catalysis†

    PubMed Central

    Grove, Tyler L.; Ahlum, Jessica H.; Qin, Rosie M.; Lanz, Nicholas D.; Radle, Matthew I.; Krebs, Carsten; Booker, Squire J.

    2013-01-01

    The anaerobic sulfatase maturating enzyme from Clostridium perfringens (anSMEcpe) catalyzes the two-electron oxidation of a cysteinyl residue on a cognate protein to a formyglycyl residue (FGly) using a mechanism that involves organic radicals. The FGly residue plays a unique role as a cofactor in a class of enzymes termed arylsulfatases, which catalyze the hydrolysis of various organosulfate monoesters. anSMEcpe has been shown to be a member of the radical S-adenosylmethionine (SAM) family of enzymes, [4Fe–4S] cluster–requiring proteins that use a 5’-deoxyadenosyl 5’-radical (5’-dA•) generated from a reductive cleavage of SAM to initiate radical-based catalysis. Herein, we show that anSMEcpe contains in addition to the [4Fe–4S] cluster harbored by all radical SAM (RS) enzymes, two additional [4Fe–4S] clusters, similar to the radical SAM protein AtsB, which catalyzes the two-electron oxidation of a seryl residue to a FGly residue. We show by size-exclusion chromatography that both AtsB and anSMEcpe are monomeric proteins, and site-directed mutagenesis studies on AtsB reveal that individual Cys→Ala substitutions at seven conserved positions result in insoluble protein, consistent with those residues acting as ligands to the two additional [4Fe–4S] clusters. Ala substitutions at an additional conserved Cys residue (C291 in AtsB; C276 in anSMEcpe) afford proteins that display intermediate behavior. These proteins exhibit reduced solubility and drastically reduced activity, behavior that is conspicuously similar to that of a critical Cys residue in BtrN, another radical SAM dehydrogenase [Grove, T. L., et al (2010) Biochemistry, 49, 3783–3785]. We also show that wild-type anSMEcpe acts on peptides containing other oxidizeable amino acids at the target position. Moreover, we show that the enzyme will convert threonyl peptides to the corresponding ketone product, and also allo-threonyl peptides, but with a significantly reduced efficiency

  20. Identification of a cys-ser substitution in the 5-HT{sub 2C} (HTR2C) receptor gene and allelic association to violent behavior and alcoholism

    SciTech Connect

    Lappalainen, J.; Ozaki, N.; Goldman, D.

    1994-09-01

    Several lines of evidence suggest that brain serotonergic functions, including behavioral and neurochemical responses to 5-HT{sub 2C} agonist, are abnormal in some individuals with alcoholism and aggressive behaviors. The aim of the present study was to identify coding sequence variants in the human 5-HT{sub 2C} receptor gene which may cause abnormal or variant function of this receptor. Using SSCP analysis, a non-conservative cys-ser substitution was found in the 5-HT{sub 2C} receptor (designated 5-HT{sub 2Ccys} and 5-HT{sub 2Cser}). The polymorphism was typed in CEPH families to genetically map the gene. To test for association of the variant to alcoholism, violent behavior and serotonin function, the 5-HT{sub 2C} genotypes of 151 non-related Finnish male alcoholic violent offenders and impulsive fire setters and 127 Finnish psychiatrically interviewed healthy male volunteers were determined. CSF 5-HIAA concentrations were available for 74 alcoholic violent offenders and 25 healthy volunteers. Linkage analysis placed the 5-HT{sub 2C} gene on Xq21, a region that has been previously shown to contain genes for several mental retardation syndromes. The 5-HT{sub 2Ccys}/5-HT{sub 2Cser} genotype frequencies in alcoholic violent offenders and controls differed significantly (0.90/0.10 and 0.82/0.18, respectively, P=0.048). The association was found to be strongest in the violent offenders who did not fulfill the criteria for antisocial personality disorder (5-HT{sub 2Ccys}/5-HT{sub 2Cser} 0.93/0.07, p=0.021). No association was found between CSF 5-HIAA concentrations and 5-HT{sub 2C} genotype. These results implicate a 5-HT{sub 2C} receptor amino acid substitution in predisposition to alcohol abuse and violent behavior in a subgroup of alcoholics.

  1. Two new beta-chain variants: Hb Tripoli [beta26(B8)Glu-->Ala] and Hb Tizi-Ouzou [beta29(B11)Gly-->Ser].

    PubMed

    Lacan, Philippe; Becchi, Michel; Zanella-Cleon, Isabelle; Aubry, Martine; Ffrench, Martine; Couprie, Nicole; Francina, Alain

    2004-08-01

    Two new beta-globin chain variants: Hb Tripoli: codon 26, GAG-->GCG [beta26(B8)Glu-->Ala] and Hb Tizi-Ouzou: codon 29, GGC-->AGC [beta29(B11)Gly-->Ser] are described on the first exon of the beta-globin gene. The two variants are characterized by DNA sequencing and mass spectrometry (MS). Hematological abnormalities were found in the two carriers. The presence of microcytosis and hypochromia is explained by an additional homozygous 3.7 kb alpha(+) thalassemic deletion for the carrier of Hb Tizi-Ouzou. Hb Tizi-Ouzou showed a slight instability in vitro. The same hematological abnormalities associated with anemia are difficult to explain for Hb Tripoli's carrier in the absence of an alpha-globin genes abnormality and could suggest a possible abnormal splicing.

  2. Functional characterization of the PCLO p.Ser4814Ala variant associated with major depressive disorder reveals cellular but not behavioral differences.

    PubMed

    Giniatullina, A; Maroteaux, G; Geerts, C J; Koopmans, B; Loos, M; Klaassen, R; Chen, N; van der Schors, R C; van Nierop, P; Li, K W; de Jong, J; Altrock, W D; Cornelisse, L N; Toonen, R F; van der Sluis, S; Sullivan, P F; Stiedl, O; Posthuma, D; Smit, A B; Groffen, A J; Verhage, M

    2015-08-01

    Genome-wide association studies have suggested a role for a genetic variation in the presynaptic gene PCLO in major depressive disorder (MDD). As with many complex traits, the PCLO variant has a small contribution to the overall heritability and the association does not always replicate. One variant (rs2522833, p.Ser4814Ala) is of particular interest given that it is a common, nonsynonymous exon variant near a calcium-sensing part of PCLO. It has been suggested that the molecular effects of such variations penetrate to a variable extent in the population due to phenotypic and genotypic heterogeneity at the population level. More robust effects may be exposed by studying such variations in isolation, in a more homogeneous context. We tested this idea by modeling PCLO variation in a mouse knock-in model expressing the Pclo(SA)(/)(SA) variant. In the highly homogeneous background of inbred mice, two functional effects of the SA-variation were observed at the cellular level: increased synaptic Piccolo levels, and 30% increased excitatory synaptic transmission in cultured neurons. Other aspects of Piccolo function were unaltered: calcium-dependent phospholipid binding, synapse formation in vitro, and synaptic accumulation of synaptic vesicles. Moreover, anxiety, cognition and depressive-like behavior were normal in Pclo(SA)(/)(SA) mice. We conclude that the PCLO p.Ser4814Ala missense variant produces mild cellular phenotypes, which do not translate into behavioral phenotypes. We propose a model explaining how (subtle) cellular phenotypes do not penetrate to the mouse behavioral level but, due to genetic and phenotypic heterogeneity and non-linearity, can produce association signals in human population studies. PMID:26045179

  3. Functional characterization of the PCLO p.Ser4814Ala variant associated with major depressive disorder reveals cellular but not behavioral differences.

    PubMed

    Giniatullina, A; Maroteaux, G; Geerts, C J; Koopmans, B; Loos, M; Klaassen, R; Chen, N; van der Schors, R C; van Nierop, P; Li, K W; de Jong, J; Altrock, W D; Cornelisse, L N; Toonen, R F; van der Sluis, S; Sullivan, P F; Stiedl, O; Posthuma, D; Smit, A B; Groffen, A J; Verhage, M

    2015-08-01

    Genome-wide association studies have suggested a role for a genetic variation in the presynaptic gene PCLO in major depressive disorder (MDD). As with many complex traits, the PCLO variant has a small contribution to the overall heritability and the association does not always replicate. One variant (rs2522833, p.Ser4814Ala) is of particular interest given that it is a common, nonsynonymous exon variant near a calcium-sensing part of PCLO. It has been suggested that the molecular effects of such variations penetrate to a variable extent in the population due to phenotypic and genotypic heterogeneity at the population level. More robust effects may be exposed by studying such variations in isolation, in a more homogeneous context. We tested this idea by modeling PCLO variation in a mouse knock-in model expressing the Pclo(SA)(/)(SA) variant. In the highly homogeneous background of inbred mice, two functional effects of the SA-variation were observed at the cellular level: increased synaptic Piccolo levels, and 30% increased excitatory synaptic transmission in cultured neurons. Other aspects of Piccolo function were unaltered: calcium-dependent phospholipid binding, synapse formation in vitro, and synaptic accumulation of synaptic vesicles. Moreover, anxiety, cognition and depressive-like behavior were normal in Pclo(SA)(/)(SA) mice. We conclude that the PCLO p.Ser4814Ala missense variant produces mild cellular phenotypes, which do not translate into behavioral phenotypes. We propose a model explaining how (subtle) cellular phenotypes do not penetrate to the mouse behavioral level but, due to genetic and phenotypic heterogeneity and non-linearity, can produce association signals in human population studies.

  4. Substance P primes lipoteichoic acid- and Pam3CysSerLys4-mediated activation of human mast cells by up-regulating Toll-like receptor 2.

    PubMed

    Tancowny, Brian P; Karpov, Victor; Schleimer, Robert P; Kulka, Marianna

    2010-10-01

    Substance P (SP) is a neuropeptide with neuroimmunoregulatory activity that may play a role in susceptibility to infection. Human mast cells, which are important in innate immune responses, were analysed for their responses to pathogen-associated molecules via Toll-like receptors (TLRs) in the presence of SP. Human cultured mast cells (LAD2) were activated by SP and TLR ligands including lipopolysaccharide (LPS), Pam3CysSerLys4 (Pam3CSK4) and lipoteichoic acid (LTA), and mast cell leukotriene and chemokine production was assessed by enzyme-linked immunosorbent assay (ELISA) and gene expression by quantitative PCR (qPCR). Mast cell degranulation was determined using a β-hexosaminidase (β-hex) assay. SP treatment of LAD2 up-regulated mRNA for TLR2, TLR4, TLR8 and TLR9 while anti-immunoglobulin E (IgE) stimulation up-regulated expression of TLR4 only. Flow cytometry and western blot confirmed up-regulation of TLR2 and TLR8. Pretreatment of LAD2 with SP followed by stimulation with Pam3CSK4 or LTA increased production of leukotriene C4 (LTC(4) ) and interleukin (IL)-8 compared with treatment with Pam3CSK4 or LTA alone (>2-fold; P<0·01). SP alone activated 5-lipoxygenase (5-LO) nuclear translocation but also augmented Pam3CSK4 and LTA-mediated 5-LO translocation. Pam3CSK4, LPS and LTA did not induce LAD2 degranulation. SP primed LTA and Pam3CSK4-mediated activation of JNK, p38 and extracellular-signal-regulated kinase (ERK) and activated the nuclear translocation of c-Jun, nuclear factor (NF)-κB, activating transcription factor 2 (ATF-2) and cyclic-AMP-responsive element binding protein (CREB) transcription factors. Pretreatment with SP followed by LTA stimulation synergistically induced production of chemokine (C-X-C motif) ligand 8 (CXCL8)/IL-8, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP-1), tumour necrosis factor (TNF) and IL-6 protein. SP primes TLR2-mediated activation of human mast cells by up-regulating TLR expression and

  5. A novel hemoglobin variant found on the α1 chain: Hb KSVGH (HBA1: p.Lys57_Gly58insSerHisGlySerAlaGlnValLys).

    PubMed

    Wang, Mei-Chun; Tsai, Kuo-Wang; Chu, Chih-Hsun; Yu, Ming-Sun; Lam, Hing-Chung

    2015-01-01

    Glycosylated hemoglobin (Hb A1C) is a crucial indicator for the long-term control and the diagnosis of diabetes. However, the presence of hemoglobin (Hb) variants may affect the measured value of Hb A1C and result in an abnormal graph trend and inconsistency between the clinical blood sugar test and Hb A1C values. In this study, laboratory data of 41,267 patients with diabetes were collected. The Hb A1C levels and the graph results were examined. We identified 74 cases containing abnormal Hb A1C graph trends. The conducted blood cell counts and capillary Hb electrophoresis were used to analyze Hb variants. We also determined gene variation for the Hb variants by a sequence approach. Fifteen different types of Hb variants were identified in this study. Among these, we found a novel variant in which the α1 subunit of Hb showed an insertion of 24 nucleotides (nts) between the 56th and 57th residues. We named this novel variant Hb Kaohsiung Veterans General Hospital (Hb KSVGH) (HBA1: p.Lys57_Gly58insSerHisGlySerAlaGlnValLys).

  6. Possible interference between tissue-non-specific alkaline phosphatase with an Arg54-->Cys substitution and acounterpart with an Asp277-->Ala substitution found in a compound heterozygote associated with severe hypophosphatasia.

    PubMed Central

    Fukushi-Irié, M; Ito, M; Amaya, Y; Amizuka, N; Ozawa, H; Omura, S; Ikehara, Y; Oda, K

    2000-01-01

    Tissue-non-specific alkaline phosphatase (TNSALP) with an Arg(54)-->Cys (R54C) or an Asp(277)-->Ala (D277A)substitution was found in a patient with hypophosphatasia [Henthorn,Raducha, Fedde, Lafferty and Whyte (1992) Proc. Natl. Acad. Sci. U.S.A.89, 9924-9928]. To examine effects of these missense mutations onproperties of TNSALP, the TNSALP mutants were expressed ectopically inCOS-1 cells. The wild-type TNSALP was synthesized as a 66-kDa endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form, and processed to an 80-kDa mature form, which is anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). Although the mutant proteins were found to be modified by GPI, digestion with phosphatidylinositol-specific phospholipase C, cell-surface biotinylation and immunofluorescence observation demonstrated that the cell-surface appearance of TNSALP (R54C) and TNSALP (D277A) was either almost totally or partially retarded respectively. The 66-kDa Endo H-sensitive band was the only form, and was rapidly degraded in the cells expressing TNSALP (R54C). In contrast with cells expressing TNSALP(R54C), where alkaline phosphatase activity was negligible, significant enzyme activity was detected and, furthermore, the 80-kDa mature form appeared on the surface of the cells expressing TNSALP (D277A). Analysis by sedimentation on sucrose gradients showed that a considerable fraction of newly synthesized TNSALP (R54C) and TNSALP(D277A) formed large aggregates, indicating improper folding and incorrect oligomerization of the mutant enzymes. When co-expressed with TNSALP (R54C), the level of the 80-kDa mature form of TNSALP (D277A)was decreased dramatically, with a concomitant reduction in enzyme activity in the co-transfected cell. These findings suggest that TNSALP(R54C) interferes with folding and assembly of TNSALP (D277A) intrans when expressed in the same cell, thus probably explaining why a compound heterozygote for these mutant alleles developed severe

  7. The atypical N-glycosylation motif, Asn-Cys-Cys, in human GPR109A is required for normal cell surface expression and intracellular signaling

    PubMed Central

    Yasuda, Daisuke; Imura, Yuki; Ishii, Satoshi; Shimizu, Takao; Nakamura, Motonao

    2015-01-01

    Asparagine-linked glycosylation (N-glycosylation) is necessary for the proper folding of secreted and membrane proteins, including GPCRs. Thus, many GPCRs possess the N-glycosylation motif Asn-X-Ser/Thr at their N-termini and/or extracellular loops. We found that human GPR109A (hGPR109A) has an N-glycosylation site at Asn17 in the N-terminal atypical motif, Asn17-Cys18-Cys19. Why does hGPR109A require the atypical motif, rather than the typical sequence? Here we show that Asn17-Cys18-Cys19 sequence of hGPR109A possesses 2 biologic roles. First, Asn17-X-Cys19 contributed to hGPR109A N-glycosylation by acting as an atypical motif. This modification is required for the normal surface expression of hGPR109A, as evidenced by the reduced surface expression of the nonglycosylated mutants, hGPR109A/N17A, and the finding that hGPR109A/C19S and hGPR109A/C19T, which are N-glycosylated at Asn17, exhibited expression similar to the wild-type receptor. Second, the X-Cys18-Cys19 dicysteine is indispensable for hGPR109A function. Substitution of Cys18 or Cys19 residue to Ala impaired Gi-mediated signaling via hGPR109A. We propose the disulfide bond formations of these residues with other Cys existed in the extracellular loops for the proper folding. Together, these results suggest that the atypical motif Asn17-Cys18-Cys19 is crucial for the normal surface trafficking and function of hGPR109A.—Yasuda, D., Imura, Y., Ishii, S., Shimizu, T., and Nakamura, M. The atypical N-glycosylation motif, Asn-Cys-Cys, in human GPR109A is required for normal cell surface expression and intracellular signaling. PMID:25690651

  8. Substitution of the Lys linker with the β-Ala linker dramatically decreased the renal uptake of 99mTc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone peptides.

    PubMed

    Flook, Adam M; Yang, Jianquan; Miao, Yubin

    2014-11-13

    The purpose of this study was to examine whether the substitution of the Lys linker with the β-Ala could reduce the renal uptake of (99m)Tc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-β-Ala-(Arg(11))CCMSH (1) {c[Arg-Ser-Asp-dTyr-Asp]-β-Ala-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RTD-β-Ala-(Arg(11))CCMSH (2), RVD-β-Ala-(Arg(11))CCMSH (3), RAD-β-Ala-(Arg(11))CCMSH (4), NAD-β-Ala-(Arg(11))CCMSH (5), and EAD-β-Ala-(Arg(11))CCMSH (6) peptides were synthesized and evaluated for their melanocortin 1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of their (99m)Tc-conjugates were determined in B16/F1 melanoma-bearing C57 mice. The substitution of the Lys linker with β-Ala linker dramatically reduced the renal uptake of all six (99m)Tc-peptides. (99m)Tc-4 exhibited the highest melanoma uptake (15.66 ± 6.19% ID/g) and the lowest kidney uptake (20.18 ± 3.86% ID/g) among these (99m)Tc-peptides at 2 h postinjection. The B16/F1 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT)/CT using (99m)Tc-4 as an imaging probe.

  9. The role of conserved Cys residues in Brassica rapa auxin amidohydrolase: Cys139 is crucial for the enzyme activity and Cys320 regulates enzyme stability.

    PubMed

    Smolko, Ana; Šupljika, Filip; Martinčić, Jelena; Jajčanin-Jozić, Nina; Grabar-Branilović, Marina; Tomić, Sanja; Ludwig-Müller, Jutta; Piantanida, Ivo; Salopek-Sondi, Branka

    2016-04-01

    Brassica rapa auxin amidohydrolase (BrILL2) participates in the homeostasis of the plant hormones auxins by hydrolyzing the amino acid conjugates of auxins, thereby releasing the free active form of hormones. Herein, the potential role of the two conserved Cys residues of BrILL2 (at sequence positions 139 and 320) has been investigated by using interdisciplinary approaches and methods of molecular biology, biochemistry, biophysics and molecular modelling. The obtained results show that both Cys residues participate in the regulation of enzyme activity. Cys320 located in the satellite domain of the enzyme is mainly responsible for protein stability and regulation of enzyme activity through polymer formation, as has been revealed by enzyme kinetics and differential scanning calorimetry analysis of the BrILL2 wild type and mutants C320S and C139S. Cys139 positioned in the active site of the catalytic domain is involved in the coordination of one Mn(2+) ion of the bimetal center and is crucial for the enzymatic activity. Although the point mutation Cys139 to Ser causes the loss of enzyme activity, it does not affect the metal binding to the BrILL2 enzyme, as has been shown by isothermal titration calorimetry, circular dichroism spectropolarimetry and differential scanning calorimetry data. MD simulations (200 ns) revealed a different active site architecture of the BrILL2C139S mutant in comparison to the wild type enzyme. Additional possible reasons for the inactivity of the BrILL2C139S mutant have been discussed based on MD simulations and MM-PBSA free energy calculations of BrILL2 enzyme complexes (wt and C139S mutant) with IPA-Ala as a substrate.

  10. The role of conserved Cys residues in Brassica rapa auxin amidohydrolase: Cys139 is crucial for the enzyme activity and Cys320 regulates enzyme stability.

    PubMed

    Smolko, Ana; Šupljika, Filip; Martinčić, Jelena; Jajčanin-Jozić, Nina; Grabar-Branilović, Marina; Tomić, Sanja; Ludwig-Müller, Jutta; Piantanida, Ivo; Salopek-Sondi, Branka

    2016-04-01

    Brassica rapa auxin amidohydrolase (BrILL2) participates in the homeostasis of the plant hormones auxins by hydrolyzing the amino acid conjugates of auxins, thereby releasing the free active form of hormones. Herein, the potential role of the two conserved Cys residues of BrILL2 (at sequence positions 139 and 320) has been investigated by using interdisciplinary approaches and methods of molecular biology, biochemistry, biophysics and molecular modelling. The obtained results show that both Cys residues participate in the regulation of enzyme activity. Cys320 located in the satellite domain of the enzyme is mainly responsible for protein stability and regulation of enzyme activity through polymer formation, as has been revealed by enzyme kinetics and differential scanning calorimetry analysis of the BrILL2 wild type and mutants C320S and C139S. Cys139 positioned in the active site of the catalytic domain is involved in the coordination of one Mn(2+) ion of the bimetal center and is crucial for the enzymatic activity. Although the point mutation Cys139 to Ser causes the loss of enzyme activity, it does not affect the metal binding to the BrILL2 enzyme, as has been shown by isothermal titration calorimetry, circular dichroism spectropolarimetry and differential scanning calorimetry data. MD simulations (200 ns) revealed a different active site architecture of the BrILL2C139S mutant in comparison to the wild type enzyme. Additional possible reasons for the inactivity of the BrILL2C139S mutant have been discussed based on MD simulations and MM-PBSA free energy calculations of BrILL2 enzyme complexes (wt and C139S mutant) with IPA-Ala as a substrate. PMID:26959939

  11. The effect of artificial seawater on SERS spectra of amino acids-Ag colloids: An experiment of prebiotic chemistry

    NASA Astrophysics Data System (ADS)

    Nascimento, Fernanda C.; Carneiro, Cristine E. A.; Santana, Henrique de; Zaia, Dimas A. M.

    2014-01-01

    The large enhancement of signal observed in surface enhanced Raman spectroscopy (SERS) could be helpful for identifying amino acids on the surface of other planets, in particular for Mars, as well as in prebiotic chemistry experiments of interaction minerals/amino acids. This paper reports the effect of several substances (NaCl, MgCl2, KBr, CaSO4, K2SO4, MgSO4, KI, NH4Cl, SrCl2, CaCl2, Na2SO4, KOH, NaOH, H3BO3) on the SERS spectra of colloid of sodium citrate-CSC and colloid of sodium borohydride-CSB. The effect of four different artificial seawaters and these artificial seawaters plus amino acids (α-Ala-alanine, Gly-glycine, Cys-cysteine, AIB-2-aminoisobutiric acid) on SERS spectra using both CSC and CSB was also studied. For CSC, the effect of water, after dilution of the colloid, was the appearance of several absorption bands belonging to sodium citrate in the SERS spectrum. In general, artificial seawaters enhanced several bands in SERS spectra using CSC and CSB and CSC was more sensitive to those artificial seawaters than CSB. The identification of Gly, α-Ala and AIB using CSC or CSB was not possible because several bands belonging to artificial seawaters, sodium citrate or sodium borohydride were enhanced. On the other hand, artificial seawaters did not interfere in the SERS spectra of Cys using CSC or CSB, although the interaction of Cys with each colloid was different. For CSC the band at 2568 cm-1 (S-H stretching) of Cys vanished and for CSB the intensity of this band decreased, indicating the -SH of Cys was bonded to Ag to form -S-Ag. Thus SERS spectroscopy could be used for Cys detection on Mars soils using Mars land rovers as well as to study the interaction between Cys and minerals in prebiotic chemistry experiments.

  12. Identification, expression, and pharmacology of a Cys{sub 23}-Ser{sub 23} substitution in the human 5-HT{sub 2C} receptor gene (HTR2C)

    SciTech Connect

    Lappalainen, J.; Ozaki, N.; Goldman, D.

    1995-05-20

    The function of brain serotonin-2C (5-HT{sub 2C}) receptors, including behavioral and neurochemical responses to 5-HT{sub 2C} agonist challenge, has been suggested to be abnormal in individuals with neuropsychiatric disorders. Thus, it is important to identify polymorphisms and functional variants within this gene. Using SSCP analysis, the authors identified a Cys{sub 23}-Ser{sub 23} substitution (designated 5-HT{sub 2Ccys} and 5-HT{sub 2Cser}) in the first hydrophobic region of the human 5-HT{sub 2C} receptor. Allele frequencies in unrelated Caucasians were 0.13 and 0.87 for 5-HT{sub 2Cser} and 5-HT{sub 2Ccys}, respectively. DNAs from informative CEPH families were typed for this polymorphism and analyzed with respect to 20 linked markers on the X chromosome. Linkage analysis placed the 5-HT{sub 2C} receptor gene (HTR2C) on Xq24. To evaluate whether this amino acid substitution causes a variant function of this receptor, recombinant human 5-HT{sub 2Ccys} and 5-HT{sub 2Cser} receptors were expressed in Xenopus oocytes and tested for responses to 5-HT using electrophysiological techniques. Concentration-response curves for 5-HT were not significantly different in oocytes expressing either form of the receptor, suggesting that the 5-HT{sub 2Ccys} and 5-HT{sub 2Cser} receptor proteins may not differ in their responses to serotonin under baseline physiological conditions. 43 refs., 3 figs., 1 tab.

  13. Survey of organophosphate resistance and an Ala216Ser substitution of acetylcholinesterase-1 gene associated with chlorpyrifos resistance in Apolygus lucorum (Meyer-Dür) collected from the transgenic Bt cotton fields in China.

    PubMed

    Zhen, Congai; Miao, Ling; Liang, Pei; Gao, Xiwu

    2016-09-01

    The mirid bug is frequently controlled by the application of organophosphorus insecticides in the transgenic Bt cotton field of China. A topical bioassay method was performed to evaluate the toxicities of chlorpyrifos and malathion towards field-collected Chinese populations of Apolygus lucorum from transgenic Bt cotton fields. For chlorpyrifos, the resistance ratios ranged from 0.8 to 9.4-fold compared to a susceptible strain. For malathion, the resistance levels relative to the susceptible strain ranged from 1.2 to 14.4-fold. Compared to a susceptible strain, the Cangzhou population from Hebei province showed the highest resistance ratios towards these insecticides. A comparison of the detoxifying and target enzyme activities between the Cangzhou population and a susceptible strain revealed that altered acetylcholinesterase possibly account for the chlorpyrifos and malathion resistance in the Cangzhou population. Two acetylcholinesterase (AChE-encoding) genes (designated Alace1 and Alace2) from the green mirid bug (A. lucorum) were identified. The Alace1 and Alace2 genes encoded 597 and 645 amino acids, respectively. Both AChE proteins had conserved motifs including a catalytic triad, a choline-binding site, and an acyl pocket. Quantitative real-time PCR analysis showed that Alace1 had a much higher transcriptional level than Alace2, for the expression profiles of both spatial and time distributions. One amino acid substitution, Ala216Ser in Alace1, was found in the Cangzhou population. These results suggest that the mutation Ala216Ser should be most likely involved in organophosphorus resistance in A. lucorum. PMID:27521910

  14. Structure of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser of Aβ-peptide with phospholipase A2 from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution.

    PubMed

    Mirza, Zeenat; Pillai, Vikram Gopalakrishna; Zhong, Wei-Zhu

    2014-01-01

    Alzheimer's disease (AD) is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ) are the prime player of AD's neuropathology. Studies have implicated the varied role of phospholipase A2 (PLA2) in brain where it contributes to neuronal growth and inflammatory response. Overall contour and chemical nature of the substrate-binding channel in the low molecular weight PLA2s are similar. This study involves the reductionist fragment-based approach to understand the structure adopted by N-terminal fragment of Alzheimer's Aβ peptide in its complex with PLA2. In the current communication, we report the structure determined by X-ray crystallography of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser (DAEFRHDS) of Aβ-peptide with a Group I PLA2 purified from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution (Protein Data Bank (PDB) Code: 3JQ5). This is probably the first attempt to structurally establish interaction between amyloid-β peptide fragment and hydrophobic substrate binding site of PLA2 involving H bond and van der Waals interactions. We speculate that higher affinity between Aβ and PLA2 has the therapeutic potential of decreasing the Aβ-Aβ interaction, thereby reducing the amyloid aggregation and plaque formation in AD. PMID:24619194

  15. Structure of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser of Aβ-peptide with phospholipase A2 from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution.

    PubMed

    Mirza, Zeenat; Pillai, Vikram Gopalakrishna; Zhong, Wei-Zhu

    2014-01-01

    Alzheimer's disease (AD) is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ) are the prime player of AD's neuropathology. Studies have implicated the varied role of phospholipase A2 (PLA2) in brain where it contributes to neuronal growth and inflammatory response. Overall contour and chemical nature of the substrate-binding channel in the low molecular weight PLA2s are similar. This study involves the reductionist fragment-based approach to understand the structure adopted by N-terminal fragment of Alzheimer's Aβ peptide in its complex with PLA2. In the current communication, we report the structure determined by X-ray crystallography of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser (DAEFRHDS) of Aβ-peptide with a Group I PLA2 purified from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution (Protein Data Bank (PDB) Code: 3JQ5). This is probably the first attempt to structurally establish interaction between amyloid-β peptide fragment and hydrophobic substrate binding site of PLA2 involving H bond and van der Waals interactions. We speculate that higher affinity between Aβ and PLA2 has the therapeutic potential of decreasing the Aβ-Aβ interaction, thereby reducing the amyloid aggregation and plaque formation in AD.

  16. ALA Candidates: Presidential Timbre

    ERIC Educational Resources Information Center

    Berry, John N., III

    2010-01-01

    This article presents an interview with two effective spokespeople, notable school librarian Sara Kelly Johns and retired public library administrator Molly Raphael, who compete to be American Library Association (ALA) president. One of them will be elected president of ALA for a year's term beginning in July 2011. Each candidate comes from a…

  17. Hemoglobin βCys93 is essential for cardiovascular function and integrated response to hypoxia.

    PubMed

    Zhang, Rongli; Hess, Douglas T; Qian, Zhaoxia; Hausladen, Alfred; Fonseca, Fabio; Chaube, Ruchi; Reynolds, James D; Stamler, Jonathan S

    2015-05-19

    Oxygen delivery by Hb is essential for vertebrate life. Three amino acids in Hb are strictly conserved in all mammals and birds, but only two of those, a His and a Phe that stabilize the heme moiety, are needed to carry O2. The third conserved residue is a Cys within the β-chain (βCys93) that has been assigned a role in S-nitrosothiol (SNO)-based hypoxic vasodilation by RBCs. Under this model, the delivery of SNO-based NO bioactivity by Hb redefines the respiratory cycle as a triune system (NO/O2/CO2). However, the physiological ramifications of RBC-mediated vasodilation are unknown, and the apparently essential nature of βCys93 remains unclear. Here we report that mice with a βCys93Ala mutation are deficient in hypoxic vasodilation that governs blood flow autoregulation, the classic physiological mechanism that controls tissue oxygenation but whose molecular basis has been a longstanding mystery. Peripheral blood flow and tissue oxygenation are decreased at baseline in mutant animals and decline excessively during hypoxia. In addition, βCys93Ala mutation results in myocardial ischemia under basal normoxic conditions and in acute cardiac decompensation and enhanced mortality during transient hypoxia. Fetal viability is diminished also. Thus, βCys93-derived SNO bioactivity is essential for tissue oxygenation by RBCs within the respiratory cycle that is required for both normal cardiovascular function and circulatory adaptation to hypoxia.

  18. Site-directed mutagenesis substituting cysteine for serine in 2-Cys peroxiredoxin (2-Cys Prx A) of Arabidopsis thaliana effectively improves its peroxidase and chaperone functions

    PubMed Central

    Lee, Eun Mi; Lee, Seung Sik; Tripathi, Bhumi Nath; Jung, Hyun Suk; Cao, Guang Ping; Lee, Yuno; Singh, Sudhir; Hong, Sung Hyun; Lee, Keun Woo; Lee, Sang Yeol; Cho, Jae-Young; Chung, Byung Yeoup

    2015-01-01

    Background and Aims The 2-Cys peroxiredoxin (Prx) A protein of Arabidopsis thaliana performs the dual functions of a peroxidase and a molecular chaperone depending on its conformation and the metabolic conditions. However, the precise mechanism responsible for the functional switching of 2-Cys Prx A is poorly known. This study examines various serine-to-cysteine substitutions on α-helix regions of 2-Cys Prx A in Arabidopsis mutants and the effects they have on the dual function of the protein. Methods Various mutants of 2-Cys Prx A were generated by replacing serine (Ser) with cysteine (Cys) at different locations by site-directed mutagenesis. The mutants were then over-expressed in Escherichia coli. The purified protein was further analysed by size exclusion chromatography, polyacrylamide gel electrophoresis, circular dichroism spectroscopy and transmission electron microscopy (TEM) and image analysis. Peroxidase activity, molecular chaperone activity and hydrophobicity of the proteins were also determined. Molecular modelling analysis was performed in order to demonstrate the relationship between mutation positions and switching of 2-Cys Prx A activity. Key Results Replacement of Ser150 with Cys150 led to a marked increase in holdase chaperone and peroxidase activities of 2-Cys Prx A, which was associated with a change in the structure of an important domain of the protein. Molecular modelling demonstrated the relationship between mutation positions and the switching of 2-Cys Prx A activity. Examination of the α2 helix, dimer–dimer interface and C-term loop indicated that the peroxidase function is associated with a fully folded α2 helix and easy formation of a stable reduced decamer, while a more flexible C-term loop makes the chaperone function less likely. Conclusions Substitution of Cys for Ser at amino acid location 150 of the α-helix of 2-Cys Prx A regulates/enhances the dual enzymatic functions of the 2-Cys Prx A protein. If confirmed in planta, this

  19. cAMP-specific phosphodiesterase HSPDE4D3 mutants which mimic activation and changes in rolipram inhibition triggered by protein kinase A phosphorylation of Ser-54: generation of a molecular model.

    PubMed Central

    Hoffmann, R; Wilkinson, I R; McCallum, J F; Engels, P; Houslay, M D

    1998-01-01

    Ser-13 and Ser-54 were shown to provide the sole sites for the protein kinase A (PKA)-mediated phosphorylation of the human cAMP-specific phosphodiesterase isoform HSPDE4D3. The ability of PKA to phosphorylate and activate HSPDE4D3 was mimicked by replacing Ser-54 with either of the negatively charged amino acids, aspartate or glutamate, within the consensus motif of RRES54. The PDE4 selective inhibitor rolipram ¿4-[3-(cyclopentoxy)-4-methoxyphenyl]-2-pyrrolidone¿ inhibited both PKA-phosphorylated HSPDE4D3 and the Ser-54-->Asp mutant, with an IC50 value that was approximately 8-fold lower than that seen for the non-PKA-phosphorylated enzyme. Lower IC50 values for inhibition by rolipram were seen for a wide range of non-activated residue 54 mutants, except for those which had side-chains able to serve as hydrogen-bond donors, namely the Ser-54-->Thr, Ser-54-->Tyr and Ser-54-->Cys mutants. The Glu-53-->Ala mutant exhibited an activity comparable with that of the PKA phosphorylated native enzyme and the Ser-54-->Asp mutant but, in contrast to the native enzyme, was insensitive to activation by PKA, despite being more rapidly phosphorylated by this protein kinase. The activated Glu-53-->Ala mutant exhibited a sensitivity to inhibition by rolipram which was unchanged from that of the native enzyme. The double mutant, Arg-51-->Ala/Arg-52-->Ala, showed no change in either enzyme activity or rolipram inhibition from the native enzyme and was incapable of providing a substrate for PKA phosphorylation at Ser-54. No difference in inhibition by dipyridamole was seen for the native enzyme and the Ser-54-->Asp and Ser-54-->Ala mutants. A model is proposed which envisages that phosphorylation by PKA triggers at least two distinct conformational changes in HSPDE4D3; one of these gives rise to enzyme activation and another enhances sensitivity to inhibition by rolipram. Activation of HSPDE4D3 by PKA-mediated phosphorylation is suggested to involve disruption of an ion

  20. Dopamine DRD2/Cys311 is not associated with chronic schizophrenia

    SciTech Connect

    Crawford, F.; Hoyne, J.; Cai, Xingang

    1996-09-20

    A mutation in the DRD2 receptor gene has been reported in association with schizophrenia in Japanese and Caucasian populations. The variation, Ser to Cys at codon 311, occurs in the third intracellular loop of the receptor and is therefore putatively functional. We report the results of screening US Caucasian schizophrenic and nonschizophrenic populations. We detected the occurrence of the DRD2 Cys311 variant in both schizophrenics and controls. Our data demonstrates no significant difference between the frequency of Cys311 in Caucasian schizophrenic and non-schizophrenic populations, indicating no association with schizophrenia. 8 refs., 1 fig., 1 tab.

  1. No association of dopamine D2 receptor molecular variant Cys311 and schizophrenia in Chinese patients

    SciTech Connect

    Chia-Hsiang Chen; Shih-Hsiang Chien; Hai-Gwo Hwu

    1996-07-26

    A serine-to-cysteine mutation of dopamine D2 receptor at codon 311 (Cys311) was found to have higher frequency in schizophrenic patients than in normal controls in Japanese by Arinami et al. The Cys311 allele was found to be associated with patients with younger age-of-onset, positive family history, and more positive symptoms. To investigate the possible involvement of Cys311 in schizophrenia in the Chinese population, 114 unrelated Taiwanese Chinese schizophrenic patients with positive family history and 88 normal controls were genotyped for Cys311. Four patients and 5 normal controls were heterozygotes of Ser311/Cys311; no homozygotes of Cys311 were identified in either group. The allele frequencies of Cys311 in Chinese schizophrenic patients and normal controls were 2% and 3%, respectively. No significant difference was detected between the two groups. Our results do not support the argument that the Cys311 allele of DRD2 poses a genetic risk for certain types of schizophrenia in Chinese populations. 18 refs.

  2. Specific affinity-labeling of the nociceptin ORL1 receptor using a thiol-activated Cys(Npys)-containing peptide ligand.

    PubMed

    Matsushima, Ayami; Nishimura, Hirokazu; Matsuyama, Yutaka; Liu, Xiaohui; Costa, Tommaso; Shimohigashi, Yasuyuki

    2016-11-01

    We previously showed that an antagonist-based peptide ligand, H-Cys(Npys)-Arg-Tyr-Tyr-Arg- Ile-Lys-NH2 , captures the free thiol groups in the ligand-binding site of the nociceptin receptor ORL1. However, the exact receptor sites of this thiol-disulfide exchange reaction have not been uncovered, although such identification would help to clarify the ligand recognition site. Since the Cys→Ala substitution prevents the reaction, we performed the so-called Ala scanning for all the Cys residues in the transmembrane (TM) domains of the ORL1 receptor. Seven different mutant receptors were soundly expressed in the COS-7 cells and examined for their specific affinity labeling by a competitive binding assay using nociceptin and [(3) H]nociceptin. The results of in vitro Ala scanning analyses revealed that the labeled residues were Cys59 in TM1, Cys215 and Cys231 in TM5, and Cys310 in TM7. The present study has provided a novel method of Cys(Npys)-affinity labeling for identification of the ligand-binding sites in the ORL1 receptor. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 460-469, 2016.

  3. Delta-ALA urine test

    MedlinePlus

    Delta-aminolevulinic acid ... This test looks for an increased level of delta-ALA. It may be used to help diagnose ... An increased level of urinary delta-ALA may indicate: Lead poisoning ... level may occur with chronic (long-term) liver disease .

  4. Chk1 phosphorylation at Ser286 and Ser301 occurs with both stalled DNA replication and damage checkpoint stimulation

    SciTech Connect

    Ikegami, Yosuke; Goto, Hidemasa Kiyono, Tohru; Enomoto, Masato; Kasahara, Kousuke; Tomono, Yasuko; Tozawa, Keiichi; Morita, Akimichi; Kohri, Kenjiro; Inagaki, Masaki

    2008-12-26

    We previously reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase (Cdk) 1 during mitosis [T. Shiromizu et al., Genes Cells 11 (2006) 477-485]. Here, we demonstrated that Chk1-Ser286 and -Ser301 phosphorylation also occurs in hydroxyurea (HU)-treated or ultraviolet (UV)-irradiated cells. Unlike the mitosis case, however, Chk1 was phosphorylated not only at Ser286 and Ser301 but also at Ser317 and Ser345 in the checkpoint response. Treatment with Cdk inhibitors diminished Chk1 phosphorylation at Ser286 and Ser301 but not at Ser317 and Ser345 with the latter. In vitro analyses revealed Ser286 and Ser301 on Chk1 to serve as two major phosphorylation sites for Cdk2. Immunoprecipitation analyses further demonstrated that Ser286/Ser301 and Ser317/Ser345 phosphorylation occur in the same Chk1 molecule during the checkpoint response. In addition, Ser286/Ser301 phosphorylation by Cdk2 was observed in Chk1 mutated to Ala at Ser317 and Ser345 (S317A/S345A), as well as Ser317/Ser345 phosphorylation by ATR was in S286A/S301A. Therefore, Chk1 phosphorylation in the checkpoint response is regulated not only by ATR but also by Cdk2.

  5. The role of short-range Cys171-Cys178 disulfide bond in maintaining cutinase active site integrity: A molecular dynamics simulation

    SciTech Connect

    Matak, Mehdi Youssefi; Moghaddam, Majid Erfani

    2009-12-11

    Understanding structural determinants in enzyme active site integrity can provide a good knowledge to design efficient novel catalytic machineries. Fusarium solani pisi cutinase with classic triad Ser-His-Asp is a promising enzyme to scrutinize these structural determinants. We performed two MD simulations: one, with the native structure, and the other with the broken Cys171-Cys178 disulfide bond. This disulfide bond stabilizes a turn in active site on which catalytic Asp175 is located. Functionally important H-bonds and atomic fluctuations in catalytic pocket have been changed. We proposed that this disulfide bond within active site can be considered as an important determinant of cutinase active site structural integrity.

  6. Crystallographic analysis of a subcomplex of the transsulfursome with tRNA for Cys-tRNA(Cys) synthesis.

    PubMed

    Chen, Meirong; Nakazawa, Yuto; Kubo, Yume; Asano, Nozomi; Kato, Koji; Tanaka, Isao; Yao, Min

    2016-07-01

    In most organisms, Cys-tRNA(Cys) is directly synthesized by cysteinyl-tRNA synthetase (CysRS). Many methanogenic archaea, however, use a two-step, indirect pathway to synthesize Cys-tRNA(Cys) owing to a lack of CysRS and cysteine-biosynthesis systems. This reaction is catalyzed by O-phosphoseryl-tRNA synthetase (SepRS), Sep-tRNA:Cys-tRNA synthase (SepCysS) and SepRS/SepCysS pathway enhancer (SepCysE) as the transsulfursome, in which SepCysE connects both SepRS and SepCysS. On the transsulfursome, SepRS first ligates an O-phosphoserine to tRNA(Cys), and the mischarged intermediate Sep-tRNA(Cys) is then transferred to SepCysS, where it is further modified to Cys-tRNA(Cys). In this study, a subcomplex of the transsulfursome with tRNA(Cys) (SepCysS-SepCysE-tRNA(Cys)), which is involved in the second reaction step of the indirect pathway, was constructed and then crystallized. The crystals diffracted X-rays to a resolution of 2.6 Å and belonged to space group P6522, with unit-cell parameters a = b = 107.2, c = 551.1 Å. The structure determined by molecular replacement showed that the complex consists of a SepCysS dimer, a SepCysE dimer and one tRNA(Cys) in the asymmetric unit. PMID:27380375

  7. The Disulfide Bond Cys255-Cys279 in the Immunoglobulin-Like Domain of Anthrax Toxin Receptor 2 Is Required for Membrane Insertion of Anthrax Protective Antigen Pore

    PubMed Central

    Boone, Kyle; Altiyev, Agamyrat; Puschhof, Jens; Sauter, Roland; Arigi, Emma; Ruiz, Blanca; Peng, Xiuli; Almeida, Igor; Sherman, Michael; Xiao, Chuan; Sun, Jianjun

    2015-01-01

    Anthrax toxin receptors act as molecular clamps or switches that control anthrax toxin entry, pH-dependent pore formation, and translocation of enzymatic moieties across the endosomal membranes. We previously reported that reduction of the disulfide bonds in the immunoglobulin-like (Ig) domain of the anthrax toxin receptor 2 (ANTXR2) inhibited the function of the protective antigen (PA) pore. In the present study, the disulfide linkage in the Ig domain was identified as Cys255-Cys279 and Cys230-Cys315. Specific disulfide bond deletion mutants were achieved by replacing Cys residues with Ala residues. Deletion of the disulfide bond C255-C279, but not C230-C315, inhibited the PA pore-induced release of the fluorescence dyes from the liposomes, suggesting that C255-C279 is essential for PA pore function. Furthermore, we found that deletion of C255-C279 did not affect PA prepore-to-pore conversion, but inhibited PA pore membrane insertion by trapping the PA membrane-inserting loops in proteinaceous hydrophobic pockets. Fluorescence spectra of Trp59, a residue adjacent to the PA-binding motif in von Willebrand factor A (VWA) domain of ANTXR2, showed that deletion of C255-C279 resulted in a significant conformational change on the receptor ectodomain. The disulfide deletion-induced conformational change on the VWA domain was further confirmed by single-particle 3D reconstruction of the negatively stained PA-receptor heptameric complexes. Together, the biochemical and structural data obtained in this study provides a mechanistic insight into the role of the receptor disulfide bond C255-C279 in anthrax toxin action. Manipulation of the redox states of the receptor, specifically targeting to C255-C279, may become a novel strategy to treat anthrax. PMID:26107617

  8. ALA 2010: The Costly Cornucopia

    ERIC Educational Resources Information Center

    Berry, John N., III

    2010-01-01

    Every librarian wants to go to the American Library Association (ALA) annual conference in Washington, DC, June 24-29. Despite that, more than half of those asked informally said they can't afford it. The good news is a cornucopia of programs aimed at nearly every need of librarians of all types and including every best practice in libraries. Many…

  9. ALA Conference 2009: Chicago Hope

    ERIC Educational Resources Information Center

    Berry, John N., III

    2009-01-01

    There is joy among those who have the funds to go to Chicago for the American Library Association (ALA) annual conference, July 9-15. Every librarian knows there is nothing better than a Chicago gathering, with the city's wonderful haunts, museums, restaurants, and fine memories of past conferences. The conference program covers nearly every…

  10. Stick to the ALA Plan

    ERIC Educational Resources Information Center

    Berry, John N., III

    2007-01-01

    One candidate for president-elect of the American Library Association (ALA) is a woman, the other is a man. One can tell them apart by looking at them. But Nancy Davenport and James Rettig are not that far apart on the issues that confront the old association and the profession it serves. They have selected slightly different emphases for their…

  11. Description of two new alpha variants: Hb Canuts [alpha85(F6)Asp-->His (alpha1)] and Hb Ambroise Pare [alpha117(GH5)Phe-->Ile (alpha2)]; two new beta variants: Hb Beaujolais [beta84(EF8)Thr-->Asn] and Hb Monplaisir [beta147 (Tyr-Lys-Leu-Ala-Phe-Phe-Leu-Leu-Ser-Asn-Phe-Tyr-158-COOH)] and one new delta variant: Hb (A2)North Africa [delta59(E3)Lys-->Met].

    PubMed

    Joly, Philippe; Lacan, Philippe; Bererd, Martine; Garcia, Caroline; Zanella-Cleon, Isabelle; Becchi, Michel; Aubry, Martine; Couprie, Nicole; Francina, Alain

    2009-01-01

    We present here five new hemoglobin (Hb) variants which have been identified during routine Hb analysis before their genotypic characterization. Four of these result from a classical missense mutation: Hb Canuts [alpha85(F6)Asp-->His (alpha1)], Hb Ambroise Pare [alpha117(GH5)Phe-->Ile (alpha2)], Hb Beaujolais [beta84(EF8)Thr-->Asn] and HbA(2)-North Africa [delta59(E3)Lys-->Met]. The last one, Hb Monplaisir [beta147 (Tyr-Lys-Leu-Ala-Phe-Phe-Leu-Leu-Ser-Asn-Phe-Tyr-158-COOH)], results from a frameshift mutation at the stop codon of the beta-globin gene which leads to a modified C-terminal sequence in the beta-globin chain. None of these variants seem to have a particular clinical expression in the heterozygous state. The circumstances of the discovery of these five new Hb variants emphasize the fact that an association of techniques is necessary for a complete screening of Hb variants during routine Hb analysis. Globin chain separation by reversed phase liquid chromatography (RP-LC) appears to be the most relevant method.

  12. Sunset at the ALaMO

    NASA Video Gallery

    A new color all-sky camera has opened its eyes at the ALaMO, or Automated Lunar and Meteor Observatory, at NASA's Marshall Space Flight Center in Huntsville, Ala. Watch its inaugural video below, s...

  13. The ALA Yearbook: 1976 Centennial Edition.

    ERIC Educational Resources Information Center

    Wedgeworth, Robert, Ed.; Dell, Richard, Ed.

    The first American Library Association (ALA) yearbook appears in the ALA's centennial year. The yearbook brings together in alphabetical sequence a series of articles on topics of enduring as well as current interest to the library community. Included are also reports of the activities of the ALA units, affiliate organizations, and other…

  14. The functional role of Cys3-Cys4 loop in hydrophobin HGFI.

    PubMed

    Niu, Baolong; Gong, Yanbo; Gao, Xianghua; Xu, Haijin; Qiao, Mingqiang; Li, Wenfeng

    2014-11-01

    Hydrophobins are a large group of low-molecular weight proteins. These proteins are highly surface-active and can form amphipathic membranes by self-assembling at hydrophobic-hydrophilic interfaces. Based on physical properties and hydropathy profiles, hydrophobins are divided into two classes. Upon the analysis of amino acid sequences and higher structures, some models suggest that the Cys3-Cys4 loop regions in class I and II hydrophobins can exhibit remarkable difference in their alignment and conformation, and have a critical role in the rodlets structure formation. To examine the requirement for the Cys3-Cys4 loop in class I hydrophobins, we used protein fusion technology to obtain a mutant protein HGFI-AR by replacing the amino acids between Cys3 and Cys4 of the class I hydrophobin HGFI from Grifola frondosa with those ones between Cys3 and Cys4 of the class II hydrophobin HFBI from Trichoderma reesei. The gene of the mutant protein HGFI-AR was successfully expressed in Pichia pastoris. Water contact angle (WCA) and X-ray photoelectron spectroscopy (XPS) measurements demonstrated that the purified HGFI-AR could form amphipathic membranes by self-assembling at mica and hydrophobic polystyrene surfaces. This property enabled them to alter the surface wettabilities of polystyrene and mica and change the elemental composition of siliconized glass. In comparison to recombinant class I hydrophobin HGFI (rHGFI), the membranes formed on hydrophobic surfaces by HGFI-AR were not robust enough to resist 1 % hot SDS washing. Atomic force microscopy (AFM) measurements indicated that unlike rHGFI, no rodlet structure was observed on the mutant protein HGFI-AR coated mica surface. In addition, when compared to rHGFI, no secondary structural change was detected by Circular Dichroism (CD) spectroscopy after HGFI-AR self-assembled at the water-air interface. HGFI-AR could not either be deemed responsible for the fluorescence intensity increase of Thioflavin T (THT) and the

  15. Toward Homogeneous Erythropoietin: Chemical Synthesis of the Ala1-Gly28 Glycopeptide Domain by “Alanine” Ligation

    PubMed Central

    Kan, Cindy; Trzupek, John D.; Wu, Bin; Wan, Qian; Chen, Gong; Tan, Zhongping; Yuan, Yu; Danishefsky, Samuel J.

    2009-01-01

    The Ala1—Gly28 glycopeptide fragment (28) of EPO was prepared by chemical synthesis as a single glycoform. Key steps in the synthesis include attachment of a complex dodecasaccharide (7) to a seven amino acid peptide via Lansbury aspartylation, native chemical ligation to join peptide 19 with the glycopeptide domain 18, and a selective desulfurization at the ligation site to reveal the natural Ala19. This glycopeptide fragment (28) contains both the requisite N-linked dodecasaccharide and a C-terminal αthioester handle, the latter feature permitting direct coupling with a glycopeptide fragment bearing N-terminal Cys29 without further functionalization. PMID:19334679

  16. Geometrical analysis of Cys-Cys bridges in proteins and their prediction from incomplete structural information

    NASA Technical Reports Server (NTRS)

    Goldblum, A.; Rein, R.

    1987-01-01

    Analysis of C-alpha atom positions from cysteines involved in disulphide bridges in protein crystals shows that their geometric characteristics are unique with respect to other Cys-Cys, non-bridging pairs. They may be used for predicting disulphide connections in incompletely determined protein structures, such as low resolution crystallography or theoretical folding experiments. The basic unit for analysis and prediction is the 3 x 3 distance matrix for Cx positions of residues (i - 1), Cys(i), (i +1) with (j - 1), Cys(j), (j + 1). In each of its columns, row and diagonal vector--outer distances are larger than the central distance. This analysis is compared with some analytical models.

  17. Vancomycin resistance: modeling backbone variants with D-Ala-D-Ala and D-Ala-D-Lac peptides.

    PubMed

    Leung, Siegfried S F; Tirado-Rives, Julian; Jorgensen, William L

    2009-02-15

    To seek vancomycin analogs with broader antibacterial activity, effects of backbone modifications for the agylcon 2 on binding with D-Ala-D-Ala- and D-Ala-D-Lac-containing peptides were investigated by Monte Carlo/free energy perturbation (MC/FEP) calculations. The experimental trend in binding affinities for 2 with three tripeptides was well reproduced. Possible modifications of the peptide bond between residues 4 and 5 were then considered, specifically for conversion of the OCNH linkage to CH(2)NH(2)(+) (6), FCCH (7), HCCH (8), and HNCO (9). The MC/FEP results did not yield binding improvements for 7, 8, and 9, though the fluorovinyl replacement is relatively benign. The previously reported analog 6 remains as the only variant that exhibits improved affinity for the D-Ala-D-Lac sequence and acceptable affinity for the D-Ala-D-Ala sequence. PMID:19128968

  18. Energy-transfer measurements of the Cys35-Cys84 distance in bovine cardiac troponin C.

    PubMed

    Liou, Y M; Fuchs, F

    1993-09-01

    Bovine cardiac troponin C (cTnC) has cysteine residues located in the non-functional Ca(2+)-binding loop I (Cys-35) and at the N-terminal end of the central helix (Cys-84) near site II, the regulatory Ca(2+)-binding site. Recently, we reported that the excimer fluorescence resulting from the dimerization of adjacent pyrene groups attached to the two Cys residues is reduced by Ca2+ binding to site II (Liou, Y.-M. and Fuchs, F. (1992) Biophys. J. 61, 892-901). This result would suggest that Ca2+ binding causes a separation of the two Cys residues, a conclusion at variance with predictions from molecular modeling studies (Herzberg, O., Moult, J. and James, M.N.G. (1986) J. Biol. Chem. 261, 2638-2644). Alternatively, the reduction in excimer fluorescence could be accounted for by an immobilization of the pyrene attached to Cys-84 by a Ca(2+)-induced hydrophobic pocket. To arrive at a more definitive interpretation of these experiments, we carried out steady-state fluorescence resonance energy-transfer measurements of the Cys35-Cys84 distance. We used three different donor-acceptor pairs: 2-(4'-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS) and 4-dimethylamino-phenylazophenyl-4-maleimide (DABMI), IAANS and N-(4-(dimethyl-amino)-3,5-dinitrophenyl) maleimide (DDPM), and 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) and DDPM. At pCa 8.0, the distances were 23.8, 21.0, and 22.0 A with the donor-acceptor pairs, IAANS-DABMI, IAANS-DDPM and IAEDAN-DDPM, respectively. At pCa 4.0, the distances were 25.8, 24.1 and 21.2 A. The distances at pCa 8 and pCa 4.0 were not significantly altered when labeled cTnC was complexed with cardiac troponin I (cTnI). Thus, Ca2+ has little, if any, effect on the Cys35-Cys84 distance. These results are consistent with a model in which Ca2+ binding induces a separation of helices B and C from helix D, without any relative movement of the two N-terminal Ca(2+)-binding domains.

  19. Asymmetric processing of mutant factor X Arg386Cys reveals differences between intrinsic and extrinsic pathway activation.

    PubMed

    Baroni, M; Pavani, G; Pinotti, M; Branchini, A; Bernardi, F; Camire, R M

    2015-10-01

    Alterations in coagulation factor X (FX) activation, mediated by the extrinsic VIIa/tissue factor (FVIIa/TF) or the intrinsic factor IXa/factor VIIIa (FIXa/FVIIIa) complexes, can result in hemorrhagic/prothrombotic tendencies. However, the molecular determinants involved in substrate recognition by these enzymes are poorly defined. Here, we investigated the role of arginine 386 (chymotrypsin numbering c202), a surface-exposed residue on the FX catalytic domain. The naturally occurring FX386Cys mutant and FX386Ala variant were characterized. Despite the unpaired cysteine, recombinant (r)FX386Cys was efficiently secreted (88.6±21.3% of rFXwt) and possessed normal clearance in mice. rFX386Cys was also normally activated by FVIIa/TF and displayed intact amidolytic activity. In contrast, rFX386Cys activation by the FIXa/FVIIIa complex was 4.5-fold reduced, which was driven by a decrease in the kcat (1.6∗10(-4) s(-1) vs 5.8∗10(-4) s(-1), rFXwt). The virtually unaltered Km (70.6 nM vs 55.6nM, rFXwt) suggested no major alterations in the FX substrate exosite. Functional assays in plasma supplemented with rFX386Cys indicated a remarkable reduction in the thrombin generation rate and thus in coagulation efficiency. Consistently, the rFX386Ala variant displayed similar biochemical features suggesting that global changes at position 386 impact the intrinsic pathway activation. These data indicate that the FXArg386 is involved in FIXa/FVIIIa-mediated FX activation and help in elucidating the bleeding tendency associated with the FX386Cys in a rare FX deficiency case. Taking advantage of the unpaired cysteine, the rFX386Cys mutant may be efficiently targeted by thiol-specific ligands and represent a valuable tool to study FX structure-function relationships both in vitro and in vivo. PMID:26012870

  20. Arg126 and Asp49 Are Essential for the Catalytic Function of Microsomal Prostaglandin E2 Synthase 1 and Ser127 Is Not

    PubMed Central

    Rafique, Nazmi; Goodman, Michael Christopher; Idborg, Helena; Bergqvist, Filip; Jakobsson, Per-Johan

    2016-01-01

    Introduction Prostaglandins are signaling molecules that regulate different physiological processes, involving allergic and inflammatory responses and cardiovascular control. They are involved in several pathophysiological processes, including inflammation and cancer. The inducible terminal enzyme, microsomal prostaglandin E synthase 1 (MPGES1), catalyses prostaglandin E2 production during inflammation. MPGES1 has therefore been intensively studied as a pharmaceutical target and many competitive inhibitors targeting its active site have been developed. However, little is known about its catalytic mechanism. Aim The objective of this study was to investigate which amino acids play a key role in the catalytic mechanism of MPGES1. Materials and Methods Based on results and predictions from previous structural studies, the amino acid residues Asp49, Arg73, Arg126, and Ser127 were chosen and altered by site-directed mutagenesis. The mutated enzyme variants were cloned and expressed in both the E. coli and the Baculovirus expression systems. Their catalytic significance was evaluated by activity measurements with prostanoid profiling. Results and Conclusions Our study shows that Arg126 and Asp49 are absolutely required for the catalytic activity of MPGES1, as when exchanged, the enzyme variants loose activity. Ser127 and Arg73 on the other hand, don't seem to be central to the catalytic mechanism because when exchanged, their variants retain considerable activity. Our finding that the Ser127Ala variant retains activity was surprising since high-resolution structural data supported a role in glutathione activation. The close proximity of Ser127 to the active site is, however, supported since the Ser127Cys variant displays 80% lowered activity. PMID:27684486

  1. Topical application of ALA and ALA hexyl ester on a subcutaneous murine mammary adenocarcinoma: tissue distribution.

    PubMed

    Perotti, C; Casas, A; Fukuda, H; Sacca, P; Batlle, A

    2003-02-10

    Although 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has proven to be clinically beneficial for the treatment of certain cancers, including a variety of skin cancers, optimal tissue localisation still remains a problem. An approach to improve the bioavailability of protoporphyrin IX (PpIX) is the use of ALA derivatives instead of ALA. In this work, we employed a subcutaneous murine mammary adenocarcinoma to study the tissue distribution pattern of the ALA hexyl ester (He-ALA) in comparison with ALA after their topical application in different vehicles. He-ALA induced porphyrin synthesis in the skin overlying the tumour (SOT), but it did not reach the tumour tissue as efficiently. Only 5 h after He-ALA lotion application, tumour porphyrin levels surpassed control values. He-ALA delivered in cream induced a substantially lower porphyrin synthesis in SOT, reinforcing the importance of the vehicle in the use of topical PDT. Porphyrin levels in internal organs remained almost within control values when He-ALA was employed. The addition of DMSO to ALA formulation slightly increased tumour and SOT porphyrin biosynthesis, but it did not when added to He-ALA lotion.

  2. ALA Midwinter 2011 Preview: A Better Place

    ERIC Educational Resources Information Center

    Berry, John N., III

    2010-01-01

    There has been an effort to make the American Library Association (ALA) Midwinter Meeting "member-friendly," so that more ALA members will attend. Held January 7-11 in beautiful San Diego, the conference program is loaded with interesting events that look suspiciously like entertainment, plus learning opportunities, and the usual parties and…

  3. Comparison between mALA- and ALA-PDT in the treatment of basal cell carcinomas

    NASA Astrophysics Data System (ADS)

    Schleier, Peter; Zenk, Witold; Hyckel, Peter; Berndt, Alexander

    2006-02-01

    Introduction: The external application of aminoleavulinic acid (ALA), which is a substrate of physiologic cell metabolism, represents a possible treatment option in superficial basal cell carcinomas (BCC). The development of new ALA-esters (mALA) with potential for higher penetration depths promises higher therapeutic success. This research aimed to prove the following hypothesis: The cytotoxic effect of the mALA- photodynamic therapy (mALA-PDT), when compared to the ALA-PDT, leads to a higher clinical success rate. Material and Methods: 24 patients with multiple facial tumors, after having received several local surgical excisions with known histology, were treated with either ALA- or mALA-PDT, during the past two years. In total, 89 basal cell carcinoma, 45 actinic keratoses, 6 keratoacanthoma, and 2 squamous cell carcinomas were treated. ALA-PDT: A thermo gel with 40 % mALA or ALA was applied from a cooled syringe. Three to five hours after gel application the skin was cleaned from any gel residues. Irradiation was done with a diode laser and was performed in two sessions, each 10 min long. After intervals of 2, 4 and 12 weeks, the patients were recalled to assess therapeutic efficacy. This was followed by photographic documentation. Results: More than 80% of the tumors treated primarily were resolved successfully. A recurrence rate of approximately 15% was observed. Three per cent of the tumors showed no reaction to therapy. There were no statistically significant differences between the two therapeutic groups. Discussion: The advantage of the use of ALA lies foremost in the fast metabolic use of the body's own photosensitizer PpIX. There are no known side effects of this therapy. Moreover, external application is superior to systemic application with regard to patient management. The method can be combined with other therapies. Although the mALA should have a better penetration in tumor tissue, the therapeutic outcome is similar to the use of ALA.

  4. Engineering of 2-Cys Peroxiredoxin for Enhanced Stress-Tolerance

    PubMed Central

    An, Byung Chull; Lee, Seung Sik; Lee, Jae Taek; Hong, Sung Hyun; Wi, Seung Gon; Chung, Byung Yeoup

    2011-01-01

    A typical 2-cysteine peroxiredoxin (2-Cys Prx)-like protein (PpPrx) that alternatively acts as a peroxidase or a molecular chaperone in Pseudomonas putida KT2440 was previously characterized. The dual functions of PpPrx are regulated by the existence of an additional Cys112 between the active Cys51 and Cys171 residues. In the present study, additional Cys residues (Cys31, Cys112, and Cys192) were added to PpPrx variants to improve their enzymatic function. The optimal position of the additional Cys residues for the dual functionality was assessed. The peroxidase activities of the S31C and Y192C mutants were increased 3- to 4-fold compared to the wild-type, while the chaperone activity was maintained at > 66% of PpPrx. To investigate whether optimization of the dual functions could enhance stress-tolerance in vivo, a complementation study was performed. The S31C and Y192C mutants showed a much greater tolerance than other variants under a complex condition of heat and oxidative stresses. The optimized dual functions of PpPrx could be adapted for use in bioengineering systems and industries, such as to develop organisms that are more resistant to extreme environments. PMID:21773675

  5. [Cys(O2NH2)2]enkephalin analogues and dalargin: selectivity for delta-opioid receptors.

    PubMed

    Pencheva, N; Bocheva, A; Dimitrov, E; Ivancheva, C; Radomirov, R

    1996-05-23

    To investigate the structure-activity relationships for potent and selective action of enkephalins at the delta-opioid receptors, two newly synthesized analogues, [Cys(O2NH2)2,Leu5]enkephalin and [Cys(O2NH2)2, Met5] enkephalin and the hexapeptide [D-Ala2,Leu5]enkephalyl-Arg (dalargin) were tested and compared with [Leu5]enkephalin and [Met5]enkephalin, for their effectiveness to inhibit electrically evoked contractions of the mouse vas deferens (predominantly enkephalin-selective delta-opioid receptors) and the guinea pig ileum (mu- and kappa-opioid receptors). The mouse vas deferens assays included evaluation of the effects of opioid agonists on the first, purinergic, and the second, adrenergic, components of electrically evoked biphasic responses (10 Hz and 20 Hz) and on ATP- or noradrenaline-evoked, tetrodotoxin-resistant responses. The opioids tested inhibited in a similar manner: (i) the purinergic and the adrenergic components of the electrically evoked contractions; and (ii) the ATP- and noradrenaline-induced postjunctional responses of the mouse vas deferens. Extremely low IC50 values (of 2-5 orders) were found for [Cys(O2NH2)2,Leu5] enkephalin, whose relative potency was between 239 and 1316 times higher than that of [Leu5]enkephalin. The order of potency for the other peptides in this tissue was: [Cys(O2NH2)2,Met5]enkephalin > [Leu5]enkephalin > dalargin > [Met5]enkephalin. The highest IC50 values in the guinea pig ileum assays, indicating the lowest affinity for mu-/kappa-opioid receptors, were obtained for the cysteine sulfonamide analogues, while dalargin showed a potency four times higher than that of [Met5]enkephalin. The order of potency in this tissue was: dalargin > [Met5]enkephalin > [Leu5]enkephalin > [Cys(O2NH2)2,Met5]enkephalin > [Cys(O2NH2)2,Leu5]enkephalin. The ratio, IC50 in guinea pig ileum: IC50 in mouse vas deferens, indicating selectivity of the respective peptide for delta-opioid receptors, was extremely high for [Cys(O2NH2)2,Leu5

  6. Identification of the amine-polyamine-choline transporter superfamily 'consensus amphipathic region' as the target for inactivation of the Escherichia coli GABA transporter GabP by thiol modification reagents. Role of Cys-300 in restoring thiol sensitivity to Gabp lacking Cys.

    PubMed Central

    Hu, L A; King, S C

    1999-01-01

    The Escherichia coli gamma-aminobutyric acid transporter GabP (gab permease) contains a functionally significant cysteine residue (Cys-300) within its consensus amphipathic region (CAR), a putative channel-forming structure that extends out of transmembrane helix 8 and into the adjoining cytoplasmic loop 8-9 of transporters from the amine-polyamine-choline (APC) superfamily. Here we show that of the five cysteine residues (positions 158, 251, 291, 300 and 443) in the E. coli GabP, Cys-300 is the one that renders the transport activity sensitive to inhibition by thiol modification reagents: whereas substituting Ala for Cys-300 mimics the inhibitory effect of thiol modification, substituting Ala at position 158, 251, 291 or 443 preserves robust transport activity and confers no resistance to thiol inactivation; and whereas the robustly active Cys-300 single-Cys mutant is fully sensitive to thiol modification, other single-Cys mutants (Cys at 158, 251, 291 or 443) exhibit kinetically compromised transport activities that resist further chemical inactivation by thiol reagents. The present study reveals additionally that Cys-300 exhibits (1) sensitivity to hydrophobic thiol reagents, (2) general resistance to bulky (fluorescein 5-maleimide) and/or charged ¿2-sulphonatoethyl methanethiosulphonate or [2-(trimethylammonium)ethyl] methanethiosulphonate¿ thiol reagents and (3) a peculiar sensitivity to p-chloromercuribenzenesulphonate (PCMBS). The accessibility of PCMBS to Cys-300 (located midway through the lipid bilayer) might be related to the structural similarity that it shares with guvacine (1, 2,3,6-tetrahydro-3-pyridinecarboxylic acid), a transported GabP substrate. These structural requirements for thiol sensitivity provide the first chemical evidence consistent with channel-like access to the polar surface of the CAR, a physical configuration that might provide a basis for understanding how this region impacts the function of APC transporters generally [Closs

  7. Dalargin and [Cys-(O2NH2)]2 analogues of enkephalins and their selectivity for mu opioid receptors.

    PubMed

    Pencheva, N; Ivancheva, C; Dimitrov, E; Bocheva, A; Radomirov, R

    1995-07-01

    1. Effects of the enkephalins Met-enk (M) and Leu-enk (L), of two newly synthesized analogues--[Cys-(O2NH2)]2-Met-enk (CM) and [Cys-(O2NH2)]2-Leu-enk (CL)--and of a hexapeptide--D-Ala2-Leu5-Arg6 (Dalargin; DL) on the spontaneous and electrically stimulated activity were examined with respect to their selectivity for the mu opioid receptors in the longitudinal layer of guinea pig ileum. 2. M and CM exerted relaxing and contractile effects on the spontaneous contractile activity while L, CL and DL produced only relaxation. The order of potency towards the relaxatory phase was DL > M > CM > L > CL and towards the contractile phase CM > M. 3. The effects of enkephalins on the spontaneous activity were naloxone and TTX sensitive except for the contractile phase of M and CM which persisted in the presence of TTX. NO was not involved in the neurotransmission of the relaxatory responses, while the blockade of alpha and beta adrenoceptors showed the participation of adrenergic mechanisms. Relaxation and contraction induced by enkephalins could not be directly attributed to cholinergic neurotransmission. 4. The naloxone-sensitive and concentration-dependent inhibitory effects of enkephalins and their analogues on the electrically stimulated cholinergic contractions were established. The order of the relative potency of opioids was: DL-3.8; M-1.0; L-0.4; CM-0.01; CL-0.005. 5. These data indicated that the D-Ala2 substitution and lengthening of the peptide chain by Arg6 in the molecule of L increased the potency at the mu opiate receptors, while the substitution in position 2 with Cys-(O2NH2) in the molecule of M and L yielded a less potent and selective mu agonists.

  8. Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression

    SciTech Connect

    Lee, Seung Heon; Kang, Sukmo; Dong, Mi Sook; Park, Jung-Duck; Park, Jinseo; Rhee, Sangkee; Ryu, Doug-Young

    2015-06-15

    Cytochrome P450 (CYP) 1A1 is a heme-containing enzyme involved in detoxification of hydrophobic pollutants. Its Ala62Pro variant has been identified previously. Ala62 is located in α-helix A of CYP1A1. Residues such as Pro and Gly are α-helix breakers. In this study, the Ala62Pro variant was characterized using heterologous expression. E. coli expressing the Ala62Pro variant, and the purified variant protein, had lower CYP (i.e. holoenzyme) contents than their wild-type (WT) equivalents. The CYP variant from E. coli and mammalian cells exhibited lower 7-ethoxyresorufin O-dealkylation (EROD) and benzo[a]pyrene hydroxylation activities than the WT. Enhanced supplementation of a heme precursor during E. coli culture did not increase CYP content in E. coli expressing the variant, but did for the WT. As for Ala62Pro, E. coli expressing an Ala62Gly variant had a lower CYP content than the WT counterpart, but substitution of Ala62 with α-helix-compatible residues such as Ser and Val partially recovered the level of CYP produced. Microsomes from mammalian cells expressing Ala62Pro and Ala62Gly variants exhibited lower EROD activities than those expressing the WT or Ala62Val variant. A region harboring α-helix A has interactions with another region containing heme-interacting residues. Site-directed mutagenesis analyses suggest the importance of interactions between the two regions on holoenzyme expression. Together, these findings suggest that the Ala62Pro substitution leads to changes in protein characteristics and function of CYP1A1 via structural disturbance of the region where the residue is located. - Highlights: • Ala62 is located in α-helix A of the carcinogen-metabolizing enzyme CYP1A1. • Pro acts as an α-helix breaker. • A variant protein of CYP1A1, Ala62Pro, had lower heme content than the wild-type. • The variant of CYP1A1 had lower enzyme activities than the wild-type.

  9. Toward practical SERS sensing

    NASA Astrophysics Data System (ADS)

    Zhao, Yiping

    2012-06-01

    Since its discovery more than 30 years ago, surface-enhanced Raman scattering (SERS) has been recognized as a highly sensitive detection technique for chemical and biological sensing and medical diagnostics. However, the practical application of this remarkably sensitive technique has not been widely accepted as a viable diagnostic method due to the difficulty in preparing robust and reproducible substrates that provide maximum SERS enhancement. Here, we demonstrate that the aligned silver nanorod (AgNR) array substrates engineered by the oblique angle deposition method are capable of providing extremely high SERS enhancement factors (>108). The substrates are large area, uniform, reproducible, and compatible with general microfabrication process. The enhancement factor depends strongly on the length and shape of the Ag nanorods and the underlying substrate coating. By optimizing AgNR SERS substrates, we show that SERS is able to detect trace amount of toxins, virus, bacteria, or other chemical and biological molecules, and distinguish different viruses/bacteria and virus/bacteria strains. The substrate can be tailored into a multi-well chip for high throughput screening, integrated into fiber tip for portable sensing, incorporated into fluid/microfluidic devices for in situ real-time monitoring, fabricated onto a flexible substrate for tracking and identification, or used as on-chip separation device for ultra-thin layer chromatography and diagnostics. By combining the unique SERS substrates with a handheld Raman system, it can become a practical and portable sensor system for field applications. All these developments have demonstrated that AgNR SERS substrates could play an important role in the future for practical clinical, industrial, defense, and security sensing applications.

  10. ALA 2010: Where to Eat in DC

    ERIC Educational Resources Information Center

    Library Journal, 2010

    2010-01-01

    As host to visitors and transplants from around the world, Washington, DC, benefits from the constant infusion of different cultures. Although most neighborhoods lack a unified culinary flavor, make no mistake: DC is a city of distinctive areas, each with its own style, ensuring that hungry American Library Association (ALA) 2010 conference…

  11. Expression Levels of ALA Dehydratase as a Marker of ALA-PDT Efficacy

    NASA Astrophysics Data System (ADS)

    Avital, Schauder; Tamar, Feuerstein; Zvi, Malik

    2010-05-01

    Accelerated synthesis of protoporphyrinIX (PpIX) following ALA pre-treatment followed by light irradiation is the principle of ALA-PDT. Several limiting enzymes were suggested to control PpIX accumulation and PDT efficacy, among them porphobilinogen deaminase (PBGD) and ferrochelatase. Here we reveal the centrality of ALA dehydratase (ALAD) activity in predicting ALA-PDT efficacy. Silencing of ALAD expression and activity was carried out in leukemic cells using shRNA plasmid transfection or Pb2+ intoxication. ALAD activity, porphyrin synthesis and mitochondrial activity were determined versus PDT efficacy. In K562 ALAD-silenced cells, ALAD activity and expression were reduced and as a result, PpIX synthesis was almost abolished. Following ALA treatment and irradiation, ALAD-silenced cells depicted normal mitochondrial activity, in contrast to control and non-silencing transfected cells where accumulated PpIX and irradiation caused ROS formation and mitochondrial damage. Morphological analysis by scanning electron microscopy (SEM) of ALA-PDT treated cells showed no morphological changes in ALAD-silenced cells, while controls exhibited cell deformations and lysis. Annexin V-FITC/PI staining as well as LDH-L leakage testing showed that membrane integrity was undamaged following ALA-PDT in ALAD silenced cells. Pb2+ treatment in MEL cells impaired ALAD activity and reduced PpIX synthesis but to a lesser extent. In conclusion, we show that a dramatic reduction in PpIX accumulation following down regulation of ALAD expression prevents an efficient PDT. Thus, ALAD has a major role in regulating PpIX synthesis and ALA-PDT therapeutic outcome. Monitoring ALAD expression or activity in various tumors may be useful as prognostic tool to predict PDT efficacy.

  12. Opioid profiles of Cys2-containing enkephalin analogues.

    PubMed

    Pencheva, Nevena; Milanov, Peter; Vezenkov, Lubomir; Pajpanova, Tamara; Naydenova, Emilia

    2004-09-13

    To elucidate the structural features determining delta-opioid receptor properties of enkephalin analogues containing Cys(O2NH2) in position 2, a series of Cys2-containing derivatives were synthesized and tested for their effectiveness in depressing electrically evoked contractions of the mouse vas deferens (predominantly enkephalin-selective delta-opioid receptors) and the guinea-pig ileum (mu- and kappa-opioid receptors). The peptidase resistance of the compounds was also tested. The ratio IC50 in the guinea-pig ileum/IC50 in the mouse vas deferens, indicating selectivity for delta-opioid receptors, was high for Cys(O2NH2)2-containing analogues and especially for [Cys(O2NH2)2, Leu5]enkephalin, which was about seven times more selective than delta-opioid receptor selective ligand cyclic [D-Pen2, D-Pen5]enkephalin (DPDPE). The dissociation constant (KA) and relative efficacy (e(rel)) of the compounds in the mouse-isolated vas deferens were determined using explicit formulae derived by fitting of the data points with two-parametric hyperbolic function. The obtained values for KA and e(rel) suggest that: (i) incorporation of Cys(O2NH2)2 in the molecule of [Leu5]enkephalin highly increases the efficacy and does not change significantly the affinity of the respective analogues to delta-opioid receptors; [Cys(O2NH2)2, Leu5]enkephalin has higher affinity than DPDPE, but is less resistant to enzyme degradation; the effect of this modification on the efficacy is decreased when methionine is in position 5; (ii) D-configuration of Cys(O2NH2)2-containing analogues increases their peptidase resistance, but reduces efficacy and affinity of the peptides towards delta-opioid receptors; (iii) the substitution of Cys(O2NH2) with Hcy(O2NH2) reduces the efficacy, affinity and potency of the respective analogues and maintains their sensitivity to endogenous peptidases; (iv) the substitution of the sulfonamide group with benzyl group in the molecule of Cys in position 2 decreases their

  13. ALA-Butyrate prodrugs for Photo-Dynamic Therapy

    NASA Astrophysics Data System (ADS)

    Berkovitch, G.; Nudelman, A.; Ehenberg, B.; Rephaeli, A.; Malik, Z.

    2010-05-01

    The use of 5-aminolevulinic acid (ALA) administration has led to many applications of photodynamic therapy (PDT) in cancer. However, the hydrophilic nature of ALA limits its ability to penetrate the cells and tissues, and therefore the need for ALA derivatives became an urgent research target. In this study we investigated the activity of novel multifunctional acyloxyalkyl ester prodrugs of ALA that upon metabolic hydrolysis release active components such as, formaldehyde, and the histone deacetylase inhibitory moiety, butyric acid. Evaluation of these prodrugs under photo-irradiation conditions showed that butyryloxyethyl 5-amino-4-oxopentanoate (ALA-BAC) generated the most efficient photodynamic destruction compared to ALA. ALA-BAC stimulated a rapid biosynthesis of protoporphyrin IX (PpIX) in human glioblastoma U-251 cells which resulted in generation of intracellular ROS, reduction of mitochondrial activity, leading to apoptotic and necrotic death of the cells. The apoptotic cell death induced by ALA / ALA-BAC followed by PDT equally activate intrinsic and extrinsic apoptotic signals and both pathways may occur simultaneously. The main advantage of ALA-BAC over ALA stems from its ability to induce photo-damage at a significantly lower dose than ALA.

  14. Distinct expression patterns of two Arabidopsis phytocystatin genes, AtCYS1 and AtCYS2, during development and abiotic stresses

    PubMed Central

    Hwang, Jung Eun; Hong, Joon Ki; Lim, Chan Ju; Chen, Huan; Je, Jihyun; Yang, Kyung Ae; Kim, Dool Yi; Choi, Young Ju; Lee, Sang Yeol

    2010-01-01

    The phytocystatins of plants are members of the cystatin superfamily of proteins, which are potent inhibitors of cysteine proteases. The Arabidopsis genome encodes seven phytocystatin isoforms (AtCYSs) in two distantly related AtCYS gene clusters. We selected AtCYS1 and AtCYS2 as representatives for each cluster and then generated transgenic plants expressing the GUS reporter gene under the control of each gene promoter. These plants were used to examine AtCYS expression at various stages of plant development and in response to abiotic stresses. Histochemical analysis of AtCYS1 promoter- and AtCYS2 promoter-GUS transgenic plants revealed that these genes have similar but distinct spatial and temporal expression patterns during normal development. In particular, AtCYS1 was preferentially expressed in the vascular tissue of all organs, whereas AtCYS2 was expressed in trichomes and guard cells in young leaves, caps of roots, and in connecting regions of the immature anthers and filaments and the style and stigma in flowers. In addition, each AtCYS gene has a unique expression profile during abiotic stresses. High temperature and wounding stress enhanced the expression of both AtCYS1 and AtCYS2, but the temporal and spatial patterns of induction differed. From these data, we propose that these two AtCYS genes play important, but distinct, roles in plant development and stress responses. PMID:20526604

  15. The desensitization gate of inhibitory Cys-loop receptors

    NASA Astrophysics Data System (ADS)

    Gielen, Marc; Thomas, Philip; Smart, Trevor G.

    2015-04-01

    Cys-loop neurotransmitter-gated ion channels are vital for communication throughout the nervous system. Following activation, these receptors enter into a desensitized state in which the ion channel shuts even though the neurotransmitter molecules remain bound. To date, the molecular determinants underlying this most fundamental property of Cys-loop receptors have remained elusive. Here we present a generic mechanism for the desensitization of Cys-loop GABAA (GABAARs) and glycine receptors (GlyRs), which both mediate fast inhibitory synaptic transmission. Desensitization is regulated by interactions between the second and third transmembrane segments, which affect the ion channel lumen near its intracellular end. The GABAAR and GlyR pore blocker picrotoxin prevented desensitization, consistent with its deep channel-binding site overlapping a physical desensitization gate.

  16. The desensitization gate of inhibitory Cys-loop receptors.

    PubMed

    Gielen, Marc; Thomas, Philip; Smart, Trevor G

    2015-01-01

    Cys-loop neurotransmitter-gated ion channels are vital for communication throughout the nervous system. Following activation, these receptors enter into a desensitized state in which the ion channel shuts even though the neurotransmitter molecules remain bound. To date, the molecular determinants underlying this most fundamental property of Cys-loop receptors have remained elusive. Here we present a generic mechanism for the desensitization of Cys-loop GABAA (GABAARs) and glycine receptors (GlyRs), which both mediate fast inhibitory synaptic transmission. Desensitization is regulated by interactions between the second and third transmembrane segments, which affect the ion channel lumen near its intracellular end. The GABAAR and GlyR pore blocker picrotoxin prevented desensitization, consistent with its deep channel-binding site overlapping a physical desensitization gate. PMID:25891813

  17. Security effectiveness review (SER)

    SciTech Connect

    Kouprianova, I.; Ek, D.; Showalter, R.; Bergman, M.

    1998-08-01

    As part of the on-going DOE/Russian MPC and A activities at the Institute of Physics and Power Engineering (IPPE) and in order to provide a basis for planning MPC and A enhancements, an expedient method to review the effectiveness of the MPC and A system has been adopted. These reviews involve the identification of appropriate and cost-effective enhancements of facilities at IPPE. This effort requires a process that is thorough but far less intensive than a traditional vulnerability assessment. The SER results in a quick assessment of current and needed enhancements. The process requires preparation and coordination between US and Russian analysts before, during, and after information gathering at the facilities in order that the analysis is accurate, effective, and mutually agreeable. The goal of this paper is to discuss the SER process, including the objectives, time scale, and lessons learned at IPPE.

  18. ALA Pretreatment Improves Waterlogging Tolerance of Fig Plants

    PubMed Central

    An, Yuyan; Qi, Lin; Wang, Liangju

    2016-01-01

    5-aminolevulinic acid (ALA), a natural and environmentally friendly plant growth regulator, can improve plant tolerance to various environmental stresses. However, whether ALA can improve plant waterlogging tolerance is unknown. Here, we investigated the effects of ALA pretreatment on the waterlogging-induced damage of fig (Ficus carica Linn.) plants, which often suffer from waterlogging stress. ALA pretreatment significantly alleviated stress-induced morphological damage, increased leaf relative water content (RWC), and reduced leaf superoxide anion (O2⋅¯) production rate and malonaldehyde (MDA) content in fig leaves, indicating ALA mitigates waterlogging stress of fig plants. We further demonstrated that ALA pretreatment largely promoted leaf chlorophyll content, photosynthetic electron transfer ability, and photosynthetic performance index, indicating ALA significantly improves plant photosynthetic efficiency under waterlogging stress. Moreover, ALA pretreatment significantly increased activities of leaf superoxide dismutase (SOD) and peroxidase (POD), root vigor, and activities of root alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH), indicating ALA also significantly improves antioxidant ability and root function of fig plants under waterlogging stress. Taken together, ALA pretreatment improves waterlogging tolerance of fig plants significantly, and the promoted root respiration, leaf photosynthesis, and antioxidant ability may contribute greatly to this improvement. Our data firstly shows that ALA can improve plant waterlogging tolerance. PMID:26789407

  19. ALA Pretreatment Improves Waterlogging Tolerance of Fig Plants.

    PubMed

    An, Yuyan; Qi, Lin; Wang, Liangju

    2016-01-01

    5-aminolevulinic acid (ALA), a natural and environmentally friendly plant growth regulator, can improve plant tolerance to various environmental stresses. However, whether ALA can improve plant waterlogging tolerance is unknown. Here, we investigated the effects of ALA pretreatment on the waterlogging-induced damage of fig (Ficus carica Linn.) plants, which often suffer from waterlogging stress. ALA pretreatment significantly alleviated stress-induced morphological damage, increased leaf relative water content (RWC), and reduced leaf superoxide anion ([Formula: see text]) production rate and malonaldehyde (MDA) content in fig leaves, indicating ALA mitigates waterlogging stress of fig plants. We further demonstrated that ALA pretreatment largely promoted leaf chlorophyll content, photosynthetic electron transfer ability, and photosynthetic performance index, indicating ALA significantly improves plant photosynthetic efficiency under waterlogging stress. Moreover, ALA pretreatment significantly increased activities of leaf superoxide dismutase (SOD) and peroxidase (POD), root vigor, and activities of root alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH), indicating ALA also significantly improves antioxidant ability and root function of fig plants under waterlogging stress. Taken together, ALA pretreatment improves waterlogging tolerance of fig plants significantly, and the promoted root respiration, leaf photosynthesis, and antioxidant ability may contribute greatly to this improvement. Our data firstly shows that ALA can improve plant waterlogging tolerance. PMID:26789407

  20. ALA Pretreatment Improves Waterlogging Tolerance of Fig Plants.

    PubMed

    An, Yuyan; Qi, Lin; Wang, Liangju

    2016-01-01

    5-aminolevulinic acid (ALA), a natural and environmentally friendly plant growth regulator, can improve plant tolerance to various environmental stresses. However, whether ALA can improve plant waterlogging tolerance is unknown. Here, we investigated the effects of ALA pretreatment on the waterlogging-induced damage of fig (Ficus carica Linn.) plants, which often suffer from waterlogging stress. ALA pretreatment significantly alleviated stress-induced morphological damage, increased leaf relative water content (RWC), and reduced leaf superoxide anion ([Formula: see text]) production rate and malonaldehyde (MDA) content in fig leaves, indicating ALA mitigates waterlogging stress of fig plants. We further demonstrated that ALA pretreatment largely promoted leaf chlorophyll content, photosynthetic electron transfer ability, and photosynthetic performance index, indicating ALA significantly improves plant photosynthetic efficiency under waterlogging stress. Moreover, ALA pretreatment significantly increased activities of leaf superoxide dismutase (SOD) and peroxidase (POD), root vigor, and activities of root alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH), indicating ALA also significantly improves antioxidant ability and root function of fig plants under waterlogging stress. Taken together, ALA pretreatment improves waterlogging tolerance of fig plants significantly, and the promoted root respiration, leaf photosynthesis, and antioxidant ability may contribute greatly to this improvement. Our data firstly shows that ALA can improve plant waterlogging tolerance.

  1. Mechanisms of mitochondrial holocytochrome c synthase and the key roles played by cysteines and histidine of the heme attachment site, Cys-XX-Cys-His.

    PubMed

    Babbitt, Shalon E; San Francisco, Brian; Mendez, Deanna L; Lukat-Rodgers, Gudrun S; Rodgers, Kenton R; Bretsnyder, Eric C; Kranz, Robert G

    2014-10-17

    Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19.

  2. Microbial 2-Cys Peroxiredoxins: Insights into Their Complex Physiological Roles

    PubMed Central

    Toledano, Michel B.; Huang, Bo

    2016-01-01

    The peroxiredoxins (Prxs) constitute a very large and highly conserved family of thiol-based peroxidases that has been discovered only very recently. We consider here these enzymes through the angle of their discovery, and of some features of their molecular and physiological functions, focusing on complex phenotypes of the gene mutations of the 2-Cys Prxs subtype in yeast. As scavengers of the low levels of H2O2 and as H2O2 receptors and transducers, 2-Cys Prxs have been highly instrumental to understand the biological impact of H2O2, and in particular its signaling function. 2-Cys Prxs can also become potent chaperone holdases, and unveiling the in vivo relevance of this function, which is still not established, should further increase our knowledge of the biological impact and toxicity of H2O2. The diverse molecular functions of 2-Cys Prx explain the often-hard task of relating them to peroxiredoxin genes phenotypes, which underscores the pleiotropic physiological role of these enzymes and complex biologic impact of H2O2. PMID:26813659

  3. PKC{delta}-mediated IRS-1 Ser24 phosphorylation negatively regulates IRS-1 function

    SciTech Connect

    Greene, Michael W. . E-mail: michael.greene@bassett.org; Ruhoff, Mary S.; Roth, Richard A.; Kim, Jeong-a; Quon, Michael J.; Krause, Jean A.

    2006-10-27

    The IRS-1 PH and PTB domains are essential for insulin-stimulated IRS-1 Tyr phosphorylation and insulin signaling, while Ser/Thr phosphorylation of IRS-1 disrupts these signaling events. To investigate consensus PKC phosphorylation sites in the PH-PTB domains of human IRS-1, we changed Ser24, Ser58, and Thr191 to Ala (3A) or Glu (3E), to block or mimic phosphorylation, respectively. The 3A mutant abrogated the inhibitory effect of PKC{delta} on insulin-stimulated IRS-1 Tyr phosphorylation, while reductions in insulin-stimulated IRS-1 Tyr phosphorylation, cellular proliferation, and Akt activation were observed with the 3E mutant. When single Glu mutants were tested, the Ser24 to Glu mutant had the greatest inhibitory effect on insulin-stimulated IRS-1 Tyr phosphorylation. PKC{delta}-mediated IRS-1 Ser24 phosphorylation was confirmed in cells with PKC{delta} catalytic domain mutants and by an RNAi method. Mechanistic studies revealed that IRS-1 with Ala and Glu point mutations at Ser24 impaired phosphatidylinositol-4,5-bisphosphate binding. In summary, our data are consistent with the hypothesis that Ser24 is a negative regulatory phosphorylation site in IRS-1.

  4. Positive selection of digestive Cys proteases in herbivorous Coleoptera.

    PubMed

    Vorster, Juan; Rasoolizadeh, Asieh; Goulet, Marie-Claire; Cloutier, Conrad; Sainsbury, Frank; Michaud, Dominique

    2015-10-01

    Positive selection is thought to contribute to the functional diversification of insect-inducible protease inhibitors in plants in response to selective pressures exerted by the digestive proteases of their herbivorous enemies. Here we assessed whether a reciprocal evolutionary process takes place on the insect side, and whether ingestion of a positively selected plant inhibitor may translate into a measurable rebalancing of midgut proteases in vivo. Midgut Cys proteases of herbivorous Coleoptera, including the major pest Colorado potato beetle (Leptinotarsa decemlineata), were first compared using a codon-based evolutionary model to look for the occurrence of hypervariable, positively selected amino acid sites among the tested sequences. Hypervariable sites were found, distributed within -or close to- amino acid regions interacting with Cys-type inhibitors of the plant cystatin protein family. A close examination of L. decemlineata sequences indicated a link between their assignment to protease functional families and amino acid identity at positively selected sites. A function-diversifying role for positive selection was further suggested empirically by in vitro protease assays and a shotgun proteomic analysis of L. decemlineata Cys proteases showing a differential rebalancing of protease functional family complements in larvae fed single variants of a model cystatin mutated at positively selected amino acid sites. These data confirm overall the occurrence of hypervariable, positively selected amino acid sites in herbivorous Coleoptera digestive Cys proteases. They also support the idea of an adaptive role for positive selection, useful to generate functionally diverse proteases in insect herbivores ingesting functionally diverse, rapidly evolving dietary cystatins. PMID:26264818

  5. Recent trends in Cys- and Ser/Thr-based synthetic strategies for the elaboration of peptide constructs.

    PubMed

    Monbaliu, Jean-Christophe M; Katritzky, Alan R

    2012-12-11

    Recent conceptual advances in the chemical synthesis of peptide constructs are described, encompassing native chemical ligation (i.e. the chemoselective covalent condensation of unprotected peptide segments) and O-, S-acyl isopeptide strategies (i.e. internal O,S-to-N acyl transfer within peptides).

  6. Role of Cys-1327 and Cys-1337 in redox sensitivity and allosteric monitoring in human carbamoyl phosphate synthetase.

    PubMed

    Hart, Emily J; Powers-Lee, Susan G

    2009-02-27

    Human carbamoyl phosphate synthetase (hCPS) has evolved critical features that allow it to remove excess and potentially neurotoxic ammonia via the urea cycle, including use of only free ammonia as a nitrogen donor, a K(m) for ammonia 100-fold lower than for CPSs that also use glutamine as a nitrogen donor, and required allosteric activation by N-acetylglutamate (AGA), a sensor of excess amino acids. The recent availability of a Schizosaccharomyces pombe expression system for hCPS allowed us to utilize protein engineering approaches to elucidate the distinctive hCPS properties. Although the site of AGA interaction is not defined, it is known that the binding of AGA to CPS leads to a conformational change in which a pair of cysteine side chains become proximate and can then be selectively induced to undergo disulfide bonding. We analyzed the response of hCPS cysteine mutants to thiol-specific reagents and identified Cys-1327 and Cys-1337 as the AGA-responsive proximate cysteines. Possibly two of the features unique to urea-specific CPSs, relative to other CPSs (the conserved Cys-1327/Cys-1337 pair and the occurrence at very high concentrations in the liver mitochondrial matrix) co-evolved to provide buffering against reactive oxygen species. Reciprocal mutation analysis of Escherichia coli CPS (eCPS), creating P909C and G919C and establishing the ability of these engineered cysteine residues to share a disulfide bond, indicated an eCPS conformational change at least partly similar to the hCPS conformational change induced by AGA. These findings strongly suggested an alternative eCPS conformation relative to the single crystal conformation thus far identified. PMID:19106093

  7. Low micromolar zinc accelerates the fibrillization of human tau via bridging of Cys-291 and Cys-322.

    PubMed

    Mo, Zhong-Ying; Zhu, Ying-Zhu; Zhu, Hai-Li; Fan, Jun-Bao; Chen, Jie; Liang, Yi

    2009-12-11

    A hallmark of a group of neurodegenerative diseases such as Alzheimer disease is the formation of neurofibrillary tangles, which are principally composed of bundles of filaments formed by microtubule-associated protein Tau. Clarifying how natively unstructured Tau protein forms abnormal aggregates is of central importance for elucidating the etiology of these diseases. There is considerable evidence showing that zinc, as an essential element that is highly concentrated in brain, is linked to the development or progression of these diseases. Herein, by using recombinant human Tau fragment Tau(244-372) and its mutants, we have investigated the effect of zinc on the aggregation of Tau. Low micromolar concentrations of Zn(2+) dramatically accelerate fibril formation of wild-type Tau(244-372) under reducing conditions, compared with no Zn(2+). Higher concentrations of Zn(2+), however, induce wild-type Tau(244-372) to form granular aggregates in reducing conditions. Moreover, these non-fibrillar aggregates assemble into mature Tau filaments when Zn(2+) has been chelated by EDTA. Unlike wild-type Tau(244-372), low micromolar concentrations of Zn(2+) have no obvious effects on fibrillization kinetics of single mutants C291A and C322A and double mutant C291A/C322A under reducing conditions. The results from isothermal titration calorimetry show that one Zn(2+) binds to one Tau molecule via tetrahedral coordination to Cys-291 and Cys-322 as well as two histidines, with moderate, micromolar affinity. Our data demonstrate that low micromolar zinc accelerates the fibrillization of human Tau protein via bridging Cys-291 and Cys-322 in physiological reducing conditions, providing clues to understanding the relationship between zinc dyshomeostasis and the etiology of neurodegenerative diseases.

  8. "Ser" or Not "Ser:" That Is the Question.

    ERIC Educational Resources Information Center

    Higgs, Theodore V.

    1985-01-01

    Discusses a general strategy for helping students acquire vocabulary, specifically how to master the vocabulary problem of "ser" and "estar." This is done by showing students, in interesting and recognizable ways, that English is sensitive to many of the same semantic differences of "ser" and "estar." (SED)

  9. Branched Intermediate Formation Is the Slowest Step in the Protein Splicing Reaction of the Ala1 KlbA Intein from Methanococcus jannaschii

    PubMed Central

    2011-01-01

    We report the first detailed investigation of the kinetics of protein splicing by the Methanococcus jannaschii KlbA (Mja KlbA) intein. This intein has an N-terminal Ala in place of the nucleophilic Cys or Ser residue that normally initiates splicing but nevertheless splices efficiently in vivo [Southworth, M. W., Benner, J., and Perler, F. B. (2000) EMBO J.19, 5019–5026]. To date, the spontaneous nature of the cis splicing reaction has hindered its examination in vitro. For this reason, we constructed an Mja KlbA intein–mini-extein precursor using intein-mediated protein ligation and engineered a disulfide redox switch that permits initiation of the splicing reaction by the addition of a reducing agent such as dithiothreitol (DTT). A fluorescent tag at the C-terminus of the C-extein permits monitoring of the progress of the reaction. Kinetic analysis of the splicing reaction of the wild-type precursor (with no substitutions in known nucleophiles or assisting groups) at various DTT concentrations shows that formation of the branched intermediate from the precursor is reversible (forward rate constant of 1.5 × 10–3 s–1 and reverse rate constant of 1.7 × 10–5 s–1 at 42 °C), whereas the productive decay of this intermediate to form the ligated exteins is faster and occurs with a rate constant of 2.2 × 10–3 s–1. This finding conflicts with reports about standard inteins, for which Asn cyclization has been assigned as the rate-determining step of the splicing reaction. Despite being the slowest step of the reaction, branched intermediate formation in the Mja KlbA intein is efficient in comparison with those of other intein systems. Interestingly, it also appears that this intermediate is protected against thiolysis by DTT, in contrast to other inteins. Evidence is presented in support of a tight coupling between the N-terminal and C-terminal cleavage steps, despite the fact that the C-terminal single-cleavage reaction occurs in variant Mja Klb

  10. Disposable plasmonic plastic SERS sensor.

    PubMed

    Oo, S Z; Chen, R Y; Siitonen, S; Kontturi, V; Eustace, D A; Tuominen, J; Aikio, S; Charlton, M D B

    2013-07-29

    The 'Klarite™' SERS sensor platform consisting of an array of gold coated inverted square pyramids patterned onto a silicon substrate has become the industry standard over the last decade, providing highly reproducible SERS signals. In this paper, we report successful transfer from silicon to plastic base platform of an optimized SERS substrate design which provides 8 times improvement in sensitivity for a Benzenethiol test molecule compared to standard production Klarite. Transfer is achieved using roll-to-roll and sheet-level nanoimprint fabrication techniques. The new generation plastic SERS sensors provide the added benefit of cheap low cost mass-manufacture, and easy disposal. The plastic replicated SERS sensors are shown to provide ~10(7) enhancement factor with good reproducibility (5%).

  11. ATP-dependent modulation and autophosphorylation of rapeseed 2-Cys peroxiredoxin.

    PubMed

    Aran, Martin; Caporaletti, Daniel; Senn, Alejandro M; Tellez de Iñon, María T; Girotti, María R; Llera, Andrea S; Wolosiuk, Ricardo A

    2008-04-01

    2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous thiol-containing peroxidases that have been implicated in antioxidant defense and signal transduction. Although their biochemical features have been extensively studied, little is known about the mechanisms that link the redox activity and non-redox processes. Here we report that the concerted action of a nucleoside triphosphate and Mg(2+) on rapeseed 2-Cys Prx reversibly impairs the peroxidase activity and promotes the formation of high molecular mass species. Using protein intrinsic fluorescence in the analysis of site-directed mutants, we demonstrate that ATP quenches the emission intensity of Trp179, a residue close to the conserved Cys175. More importantly, we found that ATP facilitates the autophosphorylation of 2-Cys Prx when the protein is successively reduced with thiol-bearing compounds and oxidized with hydroperoxides or quinones. MS analyses reveal that 2-Cys Prx incorporates the phosphoryl group into the Cys175 residue yielding the sulfinic-phosphoryl [Prx-(Cys175)-SO(2)PO(3)(2-)] and the sulfonic-phosphoryl [Prx-(Cys175)-SO(3)PO(3)(2-)] anhydrides. Hence, the functional coupling between ATP and 2-Cys Prx gives novel insights into not only the removal of reactive oxygen species, but also mechanisms that link the energy status of the cell and the oxidation of cysteine residues.

  12. Phosphorylation of septin 3 on Ser-91 by cGMP-dependent protein kinase-I in nerve terminals

    PubMed Central

    2004-01-01

    The septins are a family of GTPase enzymes required for cytokinesis and play a role in exocytosis. Among the ten vertebrate septins, Sept5 (CDCrel-1) and Sept3 (G-septin) are primarily concentrated in the brain, wherein Sept3 is a substrate for PKG-I (cGMP-dependent protein kinase-I) in nerve terminals. There are two motifs for potential PKG-I phosphorylation in Sept3, Thr-55 and Ser-91, but phosphoamino acid analysis revealed that the primary site is a serine. Derivatization of phosphoserine to S-propylcysteine followed by N-terminal sequence analysis revealed Ser-91 as a major phosphorylation site. Tandem MS revealed a single phosphorylation site at Ser-91. Substitution of Ser-91 with Ala in a synthetic peptide abolished phosphorylation. Mutation of Ser-91 to Ala in recombinant Sept3 also abolished PKG phosphorylation, confirming that Ser-91 is the major site in vitro. Antibodies raised against a peptide containing phospho-Ser-91 detected phospho-Sept3 only in the cytosol of nerve terminals, whereas Sept3 was located in a peripheral membrane extract. Therefore Sept3 is phosphorylated on Ser-91 in nerve terminals and its phosphorylation may contribute to the regulation of its subcellular localization in neurons. PMID:15107017

  13. John Ash, ALA., Photographer August 1997. VIEW OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, ALA., Photographer August 1997. VIEW OF LOS ANGELES CITY HALL NINTH FLOOR NORTH OFFICE WING SHOWING PARTITIONS, WINDOWS AND RADIATOR, FACING SOUTHWEST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  14. Synthesis and characterization of Poly[VBTMA]Ala

    NASA Astrophysics Data System (ADS)

    Shahrom, M. S. Raja; Wilfred, C. D.; Chong, F. K.

    2014-10-01

    Polymerized ionic liquids (PILs) were successfully prepared by using 4-(vinylbenzyltrimethyl)ammonium, [VBTMA] as the cation and alanine as the anion. The monomer [VBTMA]Ala was reacted with AIBN as radical initiator to produce poly[VBTMA]Ala. The polymer was characterized by using NMR and FTIR. Thermal degradation behavior for Poly[VBTMA]Ala was 168.70 °C and glass transition temperature (Tg) was not detected even at second cycle. The surface area, pore size and pore volume were determined by using BET surface area and pore size analyzer which showed that Poly[VBTMA]Ala has mesoporous structure. The morphology was determined by FESEM. The CO2 adsorption was measured by gas adsorption cell which showed that the mole of CO2 adsorb increased as pressure increased.

  15. PARP1 Val762Ala polymorphism reduces enzymatic activity

    SciTech Connect

    Wang Xiaogan; Wang Zhaoqi; Tong Weimin . E-mail: tong@iarc.fr; Shen Yan

    2007-03-02

    Poly(ADP-ribose) polymerase 1 (PARP1) modifies a variety of nuclear proteins by poly(ADP-ribosyl)ation, and plays diverse roles in molecular and cellular processes. A common PARP1 single nucleotide polymorphism (SNP) at codon 762, resulting in the substitution of alanine (Ala) for valine (Val) in the catalytic domain has been implicated in susceptibility to cancer. To characterize the functional effect of this polymorphism on PARP1, we performed in vitro enzymatic analysis on PARP1-Ala762 and PARP1-Val762. We found that PARP1-Ala762 displayed 57.2% of the activity of PARP1-Val762 for auto-poly(ADP-ribosyl)ation and 61.9% of the activity of PARP1-Val762 for trans-poly(ADP-ribosyl)ation of histone H1. The kinetic characterization revealed that the K {sub m} of PARP1-Ala762 was increased to a 1.2-fold of the K {sub m} of PARP1-Val762 for trans-poly(ADP-ribosyl)ation. Thus, the PARP1 Val762Ala polymorphism reduces the enzymatic activity of PARP1 by increasing K {sub m}. This finding suggests that different levels of poly(ADP-ribosyl)ation by PARP1 might aid in understanding Cancer risk of carriers of the PARP1 Val762Ala polymorphism.

  16. Carbohydrate recognition factors of a Talpha (Galbeta1-->3GalNAcalpha1-->Ser/Thr) and Tn (GalNAcalpha1-->Ser/Thr) specific lectin isolated from the seeds of Artocarpus lakoocha.

    PubMed

    Singh, Tanuja; Chatterjee, Urmimala; Wu, June H; Chatterjee, Bishnu P; Wu, Albert M

    2005-01-01

    Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha fruit, is a galactose-binding lectin and a potent mitogen of T and B cells. Knowledge obtained from previous studies on the affinity of ALA was limited to molecular and submolecular levels of Galbeta1-->3GalNAc (T) and its derivatives. In the present study, the carbohydrate specificity of ALA was characterized at the macromolecular level according to the mammalian Gal/GalNAc structural units and corresponding glycoconjugates by an enzyme-linked lectinosorbent (ELLSA) and inhibition assays. The results indicate that ALA binds specifically to tumor-associated carbohydrate antigens GalNAcalpha1-->Ser/Thr (Tn) and Galbeta1-->3 GalNAcalpha1-->Ser/Thr (Talpha). It barely cross-reacts with other common glycotopes on glycoproteins, including ABH blood group antigens, Galbeta1-->3/4GlcNAc (I/II) determinants, T/Tn covered by sialic acids, and N-linked plasma glycoproteins. Dense clustering structure of Tn/Talpha-containing glycoproteins tested resulted in 2.4 x 10(5)-6.7 x 10(5)-fold higher affinities to ALA than the respective GalNAc and Gal monomer. According to our results, the overall affinity of ALA for glycans can be ranked respectively: polyvalent Tn/Talpha glycotopes > monomeric Talpha and simple clustered Tn > monomeric Tn > GalNAc > Gal; while other glycotopes: Galalpha1-->3/4Gal (B/E), Galbeta1-->3/4GlcNAc (I/II), GalNAcalpha1-->3Gal/GalNAc (A/F), and GalNAcbeta1-->3/4Gal (P/S) were inactive. The strong specificity of ALA for Tn/Talpha cluster suggests the importance of glycotope polyvalency during carbohydrate-receptor interactions and emphasizes its value as an anti-Tn/T lectin for analysis of glycoconjugate mixtures or transformed carbohydrates.

  17. Genome-wide analysis of the Zn(II)2Cys6 zinc cluster-encoding gene family in Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins with a Zn(II)2Cys6 domain, Cys-X2-Cys-X6-Cys-X5-12-Cys-X2-Cys-X6-9-Cys (hereafter, referred to as the C6 domain), form a subclass of zinc finger proteins found exclusively in fungi and yeast. Genome sequence databases of Saccharomyces cerevisiae and Candida albicans have provided an overvie...

  18. Synthesis, DNA recognition and cleavage studies of novel tetrapeptide complexes, Cu(II)/Zn(II)-Ala-Pro-Ala-Pro

    NASA Astrophysics Data System (ADS)

    Arjmand, Farukh; Jamsheera, A.; Mohapatra, D. K.

    2013-05-01

    New tetrapeptide complexes Cu(II)·Ala-Pro-Ala-Pro (1) and Zn(II)·Ala-Pro-Ala-Pro (2) were synthesized from the reaction of tetrapeptide, Ala-Pro-Ala-Pro and CuCl2/ZnCl2 and were thoroughly characterized by elemental analysis, IR,1H and 13C NMR (in case of 2), ESI-MS, UV and molar conductance measurements. The solution stability study was carried out employing UV-vis absorption titrations over a broad range of pH which suggested the stability of the complexes in solution. In vitro interaction of complexes 1 and 2 with CT-DNA was studied employing UV-vis, fluorescence, circular dichroic and viscometry studies. To throw insight into molecular binding event at the target site, UV-vis titrations of 1 and 2 with mononucleotides of interest viz.; 5'-GMP and 5'-TMP were carried out. Cleavage activity of the complexes with pBR322 plasmid DNA was evaluated by agarose gel electrophoresis and, the electrophoresis pattern demonstrated that both the complexes 1 and 2 are efficient cleavage agents. Further, the Cu(II) complex displayed efficient oxidative cleavage of supercoiled DNA while various reactive oxygen species are responsible for the cleavage in Zn(II) complex.

  19. CASE REPORT: Phenotypic presentation of the Ser63Del MPZ mutation

    PubMed Central

    Miller, Lindsey J.; Patzko, Agnes; Lewis, Richard A.; Shy, Michael E.

    2013-01-01

    Mutations in MPZ cause CMT1B, the second most frequent cause of CMT1. Elegant studies with Ser63del mice suggest that Ser63del MPZ is retained in the ER where it activates the unfolded protein response (UPR) that contributes to the neuropathy. Clinical information about patients with this mutation is limited. We present clinical and electrophysiological data on a large multigenerational family with CMT1B caused by Ser63del MPZ. The patients have a classical CMT1 phenotype that is much less severe than that of patients with Arg98Cys MPZ that also activates the UPR. These results suggest that clinical presentation along cannot predict which MPZ mutations will be retained in the ER and activate the UPR. PMID:22734905

  20. Stamping SERS for creatinine sensing

    NASA Astrophysics Data System (ADS)

    Li, Ming; Du, Yong; Zhao, Fusheng; Zeng, Jianbo; Santos, Greggy M.; Mohan, Chandra; Shih, Wei-Chuan

    2015-03-01

    Urine can be obtained easily, readily and non-invasively. The analysis of urine can provide metabolic information of the body and the condition of renal function. Creatinine is one of the major components of human urine associated with muscle metabolism. Since the content of creatinine excreted into urine is relatively constant, it is used as an internal standard to normalize water variations. Moreover, the detection of creatinine concentration in urine is important for the renal clearance test, which can monitor the filtration function of kidney and health status. In more details, kidney failure can be imminent when the creatinine concentration in urine is high. A simple device and protocol for creatinine sensing in urine samples can be valuable for point-of-care applications. We reported quantitative analysis of creatinine in urine samples by using stamping surface enhanced Raman scattering (S-SERS) technique with nanoporous gold disk (NPGD) based SERS substrate. S-SERS technique enables label-free and multiplexed molecular sensing under dry condition, while NPGD provides a robust, controllable, and high-sensitivity SERS substrate. The performance of S-SERS with NGPDs is evaluated by the detection and quantification of pure creatinine and creatinine in artificial urine within physiologically relevant concentration ranges.

  1. Wide mutation spectrum and frequent variant Ala27Thr of FBN1 identified in a large cohort of Chinese patients with sporadic TAAD

    PubMed Central

    Guo, Jun; Cai, Lun; Jia, Lixin; Li, Xiaoyan; Xi, Xin; Zheng, Shuai; Liu, Xuxia; Piao, Chunmei; Liu, Tingting; Sun, Zhongsheng; Cai, Tao; Du, Jie

    2015-01-01

    Genetic etiology in majority of patients with sporadic thoracic aortic aneurysm and dissections (STAAD) remains unknown. Recent GWAS study suggested common variant(s) in FBN1 is associated with STAAD. The present study aims to test this hypothesis and to identify mutation spectrum by targeted exome sequencing of the FBN1 gene in 146 unrelated patients with STAAD. Totally, 15.75% of FBN1 variants in STAAD were identified, including 5 disruptive and 18 missense mutations. Most of the variants were novel. Genotype-phenotype correlation analysis suggested that the maximum aortic diameter in the disruptive mutation group was significantly larger than that in the non-Cys missense mutation group. Interestingly, the variant Ala27Thr at −1 position, which is predicted to change the cleavage site of the signal peptidase of fibrillin-1, was detected in two unrelated patients. Furthermore, genotyping analysis of this variant detected 10 heterozygous Ala27Thr from additional 666 unrelated patients (1.50%), versus 7 from 1500 controls (0.47%), indicating a significant association of this variant with STAAD. Collectively, the identification of the variant Ala27Thr may represent a relatively common genetic predisposition and a novel pathogenetic mechanism for STAAD. Also, expansion of the mutation spectrum in FBN1 will be helpful in genetic counselling for Chinese patients with STAAD. PMID:26272055

  2. Loss of p27 phosphorylation at Ser10 accelerates early atherogenesis by promoting leukocyte recruitment via RhoA/ROCK.

    PubMed

    Molina-Sánchez, P; Chèvre, R; Rius, C; Fuster, J J; Andrés, V

    2015-07-01

    Reduced phosphorylation of the tumor suppressor p27(Kip1) (p27) at serine 10 (Ser10) is a hallmark of advanced human and mouse atherosclerosis. Apolipoprotein E-null mice defective for this posttranslational modification (apoE(-/-)p27Ser10Ala) exhibited increased atherosclerosis burden at late disease states. Here, we investigated the regulation of p27 phosphorylation in Ser10 at the very initial stages of atherosclerosis and its impact on endothelial-leukocyte interaction and early plaque formation. Hypercholesterolemia in fat-fed apoE(-/-) mice is associated with a rapid downregulation of p27-phospho-Ser10 in primary endothelial cells (ECs) and in aorta prior to the development of macroscopically-visible lesions. We find that lack of p27 phosphorylation at Ser10 enhances the expression of adhesion molecules in aorta of apoE(-/-) mice and ECs, and augments endothelial-leukocyte interactions and leukocyte recruitment in vivo. These effects correlated with increased RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) signaling in ECs, and inhibition of this pathway with fasudil reduced leukocyte-EC interactions to control levels in the microvasculature of p27Ser10Ala mice. Moreover, apoE(-/-)p27Ser10Ala mice displayed increased leukocyte recruitment and homing to atherosusceptible arteries and augmented early plaque development, which could be blunted with fasudil. In conclusion, our studies demonstrate a very rapid reduction in p27-phospho-Ser10 levels at the onset of atherogenesis, which contributes to early plaque build-up through RhoA/ROCK-induced integrin expression in ECs and enhanced leukocyte recruitment.

  3. Actin Cys374 as a nucleophilic target of alpha,beta-unsaturated aldehydes.

    PubMed

    Dalle-Donne, Isabella; Carini, Marina; Vistoli, Giulio; Gamberoni, Luca; Giustarini, Daniela; Colombo, Roberto; Maffei Facino, Roberto; Rossi, Ranieri; Milzani, Aldo; Aldini, Giancarlo

    2007-03-01

    We have recently shown that actin can be modified by the Michael addition of 4-hydroxynonenal to Cys374. Here, we have exposed purified actin at increasing acrolein concentrations and have identified the sites of acrolein addition using LC-ESI-MS/MS. Acrolein reacted with Cys374, His87, His173, and, minimally, His40. Cys374 adduction by both 4-hydroxynonenal and acrolein negligibly affected the polymerization of aldehyde-modified (carbonylated) actin, as shown by fluorescence measurements. Differently, acrolein binding at histidine residues, when Cys374 was completely saturated, inhibited polymerization in a dose-dependent manner. Molecular modeling analyses indicated that structural distortions of the ATP-binding site, induced by four acrolein-Michael adducts, could explain the changes in the polymerization process. Aldehyde binding to Cys374 does not alter significantly actin polymerization because this residue is located in a very flexible region, whose covalent modifications do not alter the protein folding. These data demonstrate that Cys374 represents the primary target site of alpha,beta-unsaturated aldehyde addition to actin in vitro. As Cys374 is a preferential target for various oxidative/nitrosative modifications, and actin is one of the main carbonylated proteins in vivo, these findings also suggest that the highly reactive Cys374 could serve as a carbonyl scavenger of reactive alpha,beta-unsaturated aldehydes and other electrophilic lipids.

  4. A Redox 2-Cys Mechanism Regulates the Catalytic Activity of Divergent Cyclophilins1[W

    PubMed Central

    Campos, Bruna Medéia; Sforça, Mauricio Luis; Ambrosio, Andre Luis Berteli; Domingues, Mariane Noronha; Brasil de Souza, Tatiana de Arruda Campos; Barbosa, João Alexandre Ribeiro Gonçalvez; Leme, Adriana Franco Paes; Perez, Carlos Alberto; Whittaker, Sara Britt-Marie; Murakami, Mario Tyago; Zeri, Ana Carolina de Matos; Benedetti, Celso Eduardo

    2013-01-01

    The citrus (Citrus sinensis) cyclophilin CsCyp is a target of the Xanthomonas citri transcription activator-like effector PthA, required to elicit cankers on citrus. CsCyp binds the citrus thioredoxin CsTdx and the carboxyl-terminal domain of RNA polymerase II and is a divergent cyclophilin that carries the additional loop KSGKPLH, invariable cysteine (Cys) residues Cys-40 and Cys-168, and the conserved glutamate (Glu) Glu-83. Despite the suggested roles in ATP and metal binding, the functions of these unique structural elements remain unknown. Here, we show that the conserved Cys residues form a disulfide bond that inactivates the enzyme, whereas Glu-83, which belongs to the catalytic loop and is also critical for enzyme activity, is anchored to the divergent loop to maintain the active site open. In addition, we demonstrate that Cys-40 and Cys-168 are required for the interaction with CsTdx and that CsCyp binds the citrus carboxyl-terminal domain of RNA polymerase II YSPSAP repeat. Our data support a model where formation of the Cys-40-Cys-168 disulfide bond induces a conformational change that disrupts the interaction of the divergent and catalytic loops, via Glu-83, causing the active site to close. This suggests a new type of allosteric regulation in divergent cyclophilins, involving disulfide bond formation and a loop-displacement mechanism. PMID:23709667

  5. Single Bacterium Detection Using Sers

    NASA Astrophysics Data System (ADS)

    Gonchukov, S. A.; Baikova, T. V.; Alushin, M. V.; Svistunova, T. S.; Minaeva, S. A.; Ionin, A. A.; Kudryashov, S. I.; Saraeva, I. N.; Zayarny, D. A.

    2016-02-01

    This work is devoted to the study of a single Staphylococcus aureus bacterium detection using surface-enhanced Raman spectroscopy (SERS) and resonant Raman spectroscopy (RS). It was shown that SERS allows increasing sensitivity of predominantly low frequency lines connected with the vibrations of Amide, Proteins and DNA. At the same time the lines of carotenoids inherent to this kind of bacterium are well-detected due to the resonance Raman scattering mechanism. The reproducibility and stability of Raman spectra strongly depend on the characteristics of nanostructured substrate, and molecular structure and size of the tested biological object.

  6. Fluorescence-Guided Resection of Malignant Glioma with 5-ALA.

    PubMed

    Kaneko, Sadahiro; Kaneko, Sadao

    2016-01-01

    Malignant gliomas are extremely difficult to treat with no specific curative treatment. On the other hand, photodynamic medicine represents a promising technique for neurosurgeons in the treatment of malignant glioma. The resection rate of malignant glioma has increased from 40% to 80% owing to 5-aminolevulinic acid-photodynamic diagnosis (ALA-PDD). Furthermore, ALA is very useful because it has no serious complications. Based on previous research, it is apparent that protoporphyrin IX (PpIX) accumulates abundantly in malignant glioma tissues after ALA administration. Moreover, it is evident that the mechanism underlying PpIX accumulation in malignant glioma tissues involves an abnormality in porphyrin-heme metabolism, specifically decreased ferrochelatase enzyme activity. During resection surgery, the macroscopic fluorescence of PpIX to the naked eye is more sensitive than magnetic resonance imaging, and the alert real time spectrum of PpIX is the most sensitive method. In the future, chemotherapy with new anticancer agents, immunotherapy, and new methods of radiotherapy and gene therapy will be developed; however, ALA will play a key role in malignant glioma treatment before the development of these new treatments. In this paper, we provide an overview and present the results of our clinical research on ALA-PDD. PMID:27429612

  7. Fluorescence-Guided Resection of Malignant Glioma with 5-ALA

    PubMed Central

    Kaneko, Sadahiro

    2016-01-01

    Malignant gliomas are extremely difficult to treat with no specific curative treatment. On the other hand, photodynamic medicine represents a promising technique for neurosurgeons in the treatment of malignant glioma. The resection rate of malignant glioma has increased from 40% to 80% owing to 5-aminolevulinic acid-photodynamic diagnosis (ALA-PDD). Furthermore, ALA is very useful because it has no serious complications. Based on previous research, it is apparent that protoporphyrin IX (PpIX) accumulates abundantly in malignant glioma tissues after ALA administration. Moreover, it is evident that the mechanism underlying PpIX accumulation in malignant glioma tissues involves an abnormality in porphyrin-heme metabolism, specifically decreased ferrochelatase enzyme activity. During resection surgery, the macroscopic fluorescence of PpIX to the naked eye is more sensitive than magnetic resonance imaging, and the alert real time spectrum of PpIX is the most sensitive method. In the future, chemotherapy with new anticancer agents, immunotherapy, and new methods of radiotherapy and gene therapy will be developed; however, ALA will play a key role in malignant glioma treatment before the development of these new treatments. In this paper, we provide an overview and present the results of our clinical research on ALA-PDD. PMID:27429612

  8. The 482Ser of PPARGC1A and 12Pro of PPARG2 Alleles Are Associated with Reduction of Metabolic Risk Factors Even Obesity in a Mexican-Mestizo Population.

    PubMed

    Vázquez-Del Mercado, Mónica; Guzmán-Ornelas, Milton-Omar; Corona Meraz, Fernanda-Isadora; Ríos-Ibarra, Clara-Patricia; Reyes-Serratos, Eduardo-Alejandro; Castro-Albarran, Jorge; Ruíz-Quezada, Sandra-Luz; Navarro-Hernández, Rosa-Elena

    2015-01-01

    The aim of this study was to investigate the relationship between functional polymorphisms Gly482Ser in PPARGC1A and Pro12Ala in PPARG2 with the presence of obesity and metabolic risk factors. We included 375 individuals characterized as Mexican-Mestizos and classified by the body mass index (BMI). Body dimensions and distribution of body fat were measured. The HOMA-IR and adiposity indexes were calculated. Adipokines and metabolic profile quantification were performed by ELISA and routine methods. Genetic polymorphisms were determined by polymerase chain reaction restriction fragment length polymorphism analysis. A difference between obese and nonobese subjects in polymorphism PPARGC1A distribution was observed. Among obese individuals, carriers of genotype 482Gly/Gly were observed to have decreased body fat, BMI, and body fat ratio versus 482Ser/Ser carriers and increased resistin and leptin levels in carriers Gly+ phenotype versus Gly- phenotype. Subjects with PPARG2 Ala- phenotype (genotype 12Pro/Pro) showed a decreased HOMA-IR index versus individuals with Ala+ phenotype (genotypes 12Pro/Ala plus 12Ala/Ala). We propose that, in obese Mexican-Mestizos, the combination of alleles 482Ser in PPARGC1A and 12Pro in PPARG2 represents a reduced metabolic risk profile, even when the adiposity indexes are increased.

  9. Site-Selective Glycosylation of Hemoglobin on Cys β93

    PubMed Central

    Zhang, Yalong; Bhatt, Veer S.; Sun, Guoyong; Wang, Peng G.; Palmer, Andre F.

    2009-01-01

    In this work, we describe the synthesis and characterization of a novel glycosylated hemoglobin (Hb) with high oxygen affinity as a potential Hb-based oxygen carrier. Site-selective glycosylation of bovine Hb was achieved by conjugating a lactose derivative to Cys 93 on the β subunit of Hb. LC-MS analysis indicates that the reaction was quantitative, with no unmodified Hb present in the reaction product. The glycosylation site was identified by chymotrypsin digestion of the glycosylated bovine Hb followed with LC-MS/MS and from the X-ray crystal structure of the glycosylated Hb. The chemical conjugation of the lactose derivative at Cys β93 yields an oxygen carrier with a high oxygen affinity (P50 of 4.94 mmHg) and low cooperativity coefficient (n) of 1.20. Asymmetric flow field-flow fractionation (AFFFF) coupled with multi-angle static light scattering (MASLS) was used to measure the absolute molecular weight of the glycosylated Hb. AFFFF-MASLS analysis indicates that glycosylation of Hb significantly altered the α2β2-αβ equilibrium compared to native Hb. Subsequent X-ray analysis of the glycosylated Hb crystal showed that the covalently linked lactose derivative is sandwiched between the β1 and α2 (and hence by symmetry the β2 and α1) subunits of the tetramer, and the interaction between the saccharide and amino acid residues located at the interface is apparently stabilized by hydrogen bonding interactions. The resultant structural analysis of the glycosylated Hb helps to explain the shift in the α2β2-αβ equilibrium in terms of the hydrogen bonding interactions at the β1α2/β2α1 interface. Taken together, all of these results indicate that it is feasible to site-specifically glycosylate Hb. This work has great potential in developing an oxygen carrier with defined chemistry that can target oxygen delivery to low pO2 tissues and organs. PMID:18925771

  10. SERS of dithiocarbamates and xanthates

    NASA Astrophysics Data System (ADS)

    Koh, Tse Yuen

    1995-11-01

    The surface-enhanced Raman spectra (SERS) of several simple dithiocarbamates and xanthates on silver colloids have been obtained. The dithiocarbamates studied are thought to adsorb with the -NCS 2 moiety edge-on though the -OCS 2 groups of adsorbed xanthates are parallel to the surface.

  11. Application of 5-ALA for differential diagnostics of stomach diseases

    NASA Astrophysics Data System (ADS)

    Okhotnikova, Natalja L.; Dadvany, Sergey A.; Kuszin, Michail I.; Kharnas, Sergey S.; Zavodnov, Victor Y.; Sklyanskaya, Olga A.; Loschenov, Victor B.; Volkova, Anna I.; Agafonov, Valery V.

    2001-01-01

    59 patients with stomach diseases including gastric cancer or polyp, gastritis, esofagus disease were investigated. Before gastroscopy all patients were given 5-ALA in doses 5mg, 10mg and 20mg per 1kg of body weight orally. Fluorescence diagnostics which estimates concentration of ALA-induced PPIX in regular and alternated tissues of gastric mucosa were carried out in 2-4 hours. Using of 5-ALA has shown high diagnostic effectiveness for differential diagnostics of stomach diseases. This technique has proved 10 diagnosis of cancer and revealed 15 malignant stomach diseases including 4 cancer in situ for patients with preliminary diagnosis of gastric ulcer. It also revealed 5 patients with enhanced fluorescence for which aimed biopsy has shown high degree of inflammation process. The latter were assigned as a risk group.

  12. Mechanisms of control of alae nasi muscle activity.

    PubMed

    Mezzanotte, W S; Tangel, D J; White, D P

    1992-03-01

    Human upper airway dilator muscles are clearly influenced by chemical stimuli such as hypoxia and hypercapnia. Whether in humans there are upper airway receptors capable of modifying the activity of such muscles is unclear. We studied alae nasi electromyography (EMG) in normal men in an attempt to determine 1) whether increasing negative intraluminal pressure influences the activity of the alae nasi muscle, 2) whether nasal airway feedback mechanisms modify the activity of this muscle, and 3) if so, whether these receptor mechanisms are responding to mucosal temperature/pressure changes or to airway deformation. Alae nasi EMG was recorded in 10 normal men under the following conditions: 1) nasal breathing (all potential nasal receptors exposed), 2) oral breathing (nasal receptors not exposed), 3) nasal breathing with splints (airway deformation prevented), and 4) nasal breathing after nasal anesthesia (mucosal receptors anesthetized). In addition, in a separate group, the combined effects of anesthesia and nasal splints were assessed. Under each condition, EMG activity was monitored during basal breathing, progressive hypercapnia, and inspiratory resistive loading. Under all four conditions, both load and hypercapnia produced a significant increase in alae nasi EMG, with hypercapnia producing a similar increment in EMG regardless of nasal receptor exposure. On the other hand, loading produced greater increments in EMG during nasal than during oral breathing, with combined anesthesia plus splinting producing a load response similar to that observed during oral respiration. These observations suggest that nasal airway receptors have little effect on the alae nasi response to hypercapnia but appear to mediate the alae nasi response to loading or negative airway pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. ALA and ALA hexyl ester-induced porphyrin synthesis in chemically induced skin tumours: the role of different vehicles on improving photosensitization.

    PubMed

    Casas, A; Perotti, C; Fukuda, H; Rogers, L; Butler, A R; Batlle, A

    2001-11-30

    Exogenous administration of 5-aminolevulinic acid (ALA) is becoming widely used to enhance the endogenous synthesis of Protoporphyrin IX (PpIX) in photodynamic therapy. We analysed porphyrin formation in chemically induced squamous papillomas, after topical application of ALA and ALA hexyl ester (He-ALA) administered in different formulations, as well as the pattern of distribution in the internal organs, and the synthesis of porphyrins in distant tumoural and normal skins. A lotion formulation containing DMSO and ethanol was the best vehicle for topical ALA delivery to papillomas, whereas cream was the most efficient formulation for He-ALA application. Similar porphyrin concentration can be accumulated in the skin tumours employing either ALA or He-ALA delivered in their optimal formulations. The use of cream as a vehicle of both ALA and He-ALA, induces highest porphyrin tumour/normal skin ratios. The main advantage of using He-ALA is that porphyrins synthesized from the ester are more confined to the site of application, thus inducing low porphyrin levels in normal skin, liver, blood and spleen, as well as in papillomas distant from the point of application, independently on the vehicle employed, so reducing potential side effects of photodynamic therapy.

  14. ALA and ALA hexyl ester-induced porphyrin synthesis in chemically induced skin tumours: the role of different vehicles on improving photosensitization

    PubMed Central

    Casas, A; Perotti, C; Fukuda, H; Rogers, L; Butler, A R; Batlle, A

    2001-01-01

    Exogenous administration of 5-aminolevulinic acid (ALA) is becoming widely used to enhance the endogenous synthesis of Protoporphyrin IX (PpIX) in photodynamic therapy. We analysed porphyrin formation in chemically induced squamous papillomas, after topical application of ALA and ALA hexyl ester (He-ALA) administered in different formulations, as well as the pattern of distribution in the internal organs, and the synthesis of porphyrins in distant tumoural and normal skins. A lotion formulation containing DMSO and ethanol was the best vehicle for topical ALA delivery to papillomas, whereas cream was the most efficient formulation for He-ALA application. Similar porphyrin concentration can be accumulated in the skin tumours employing either ALA or He-ALA delivered in their optimal formulations. The use of cream as a vehicle of both ALA and He-ALA, induces highest porphyrin tumour/normal skin ratios. The main advantage of using He-ALA is that porphyrins synthesized from the ester are more confined to the site of application, thus inducing low porphyrin levels in normal skin, liver, blood and spleen, as well as in papillomas distant from the point of application, independently on the vehicle employed, so reducing potential side effects of photodynamic therapy. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11742504

  15. Deciphering a Molecular Mechanism of Neonatal Diabetes Mellitus by the Chemical Synthesis of a Protein Diastereomer, [d-AlaB8]Human Proinsulin*

    PubMed Central

    Avital-Shmilovici, Michal; Whittaker, Jonathan; Weiss, Michael A.; Kent, Stephen B. H.

    2014-01-01

    Misfolding of proinsulin variants in the pancreatic β-cell, a monogenic cause of permanent neonatal-onset diabetes mellitus, provides a model for a disease of protein toxicity. A hot spot for such clinical mutations is found at position B8, conserved as glycine within the vertebrate insulin superfamily. We set out to investigate the molecular basis of the aberrant properties of a proinsulin clinical mutant in which residue GlyB8 is replaced by SerB8. Modular total chemical synthesis was used to prepare the wild-type [GlyB8]proinsulin molecule and three analogs: [d-AlaB8]proinsulin, [l-AlaB8]proinsulin, and the clinical mutant [l-SerB8]proinsulin. The protein diastereomer [d-AlaB8]proinsulin produced higher folding yields at all pH values compared with the wild-type proinsulin and the other two analogs, but showed only very weak binding to the insulin receptor. The clinical mutant [l-SerB8]proinsulin impaired folding at pH 7.5 even in the presence of protein-disulfide isomerase. Surprisingly, although [l-SerB8]proinsulin did not fold well under the physiological conditions investigated, once folded the [l-SerB8]proinsulin protein molecule bound to the insulin receptor more effectively than wild-type proinsulin. Such paradoxical gain of function (not pertinent in vivo due to impaired secretion of the mutant insulin) presumably reflects induced fit in the native mechanism of hormone-receptor engagement. This work provides insight into the molecular mechanism of a clinical mutation in the insulin gene associated with diabetes mellitus. These results dramatically illustrate the power of total protein synthesis, as enabled by modern chemical ligation methods, for the investigation of protein folding and misfolding. PMID:25002580

  16. SERS Quantification of Entacapone Isomers

    NASA Astrophysics Data System (ADS)

    Marković, Marina; Biljan, Tomislav

    2010-08-01

    Raman spectroscopy, due to its non-destructive character and speed, has found widespread use in pharmaceutical applications [1]. It is also being used for quantifying various isomer mixtures, best known being the quantification of xylene isomers [2-3]. Solid-state isomer quantification of entacapone was earlier reported [4]. Here, we report quantification of isomer mixture of an active pharmaceutical substance, in solution, by SERS.

  17. Fluorescence energy transfer between Cys-10 residues in F-actin filaments.

    PubMed

    Miki, M; Barden, J A; Hambly, B D; dos Remedios, C G

    1986-05-01

    Fluorescence energy transfer was measured between Cys-10 residues in an F-actin filament using 5-[2-((iodoacetyl)amino)-ethyl]aminonaphthalene-1-sulphonic acid (1,5-IAEDANS) as a fluorescence energy donor and 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI) as the acceptor. Both labels were covalently attached to Cys-10 residues in an F-actin filament. Taking the helical structure of the F-actin filament into consideration, the radial coordinate of Cys-10 was calculated to be 23 A. This corresponds to a distance between adjacent sites along the long pitch helix of 56.1 A and along the genetic helix of 53.3 A.

  18. Leading by Example? ALA Division Publications, Open Access, and Sustainability

    ERIC Educational Resources Information Center

    Hall, Nathan; Arnold-Garza, Sara; Gong, Regina; Shorish, Yasmeen

    2016-01-01

    This investigation explores scholarly communication business models in American Library Association (ALA) division peer-reviewed academic journals. Previous studies reveal the numerous issues organizations and publishers face in the academic publishing environment. Through an analysis of documented procedures, policies, and finances of five ALA…

  19. LJ Q&A "ALA Candidates": Library Advocacy x 2

    ERIC Educational Resources Information Center

    Berry, John N., III

    2008-01-01

    Library advocacy in one of two directions is the top priority of both Camila Alire and J. Linda Williams, the candidates campaigning to capture the 2009-10 term as president of the American Library Association (ALA). Alire, dean emeritus of the libraries of both the University of New Mexico and Colorado State University, will push for enhancements…

  20. ALA 2010 Midwinter Meeting: The Price to Participate

    ERIC Educational Resources Information Center

    Berry, John N., III

    2009-01-01

    While the library economy continues its downward slide, the cost of attending the American Library Association (ALA) Midwinter Meeting seems as high as ever. That is the price of professional participation. These days it seems a bit too high and tends to limit involvement in the old association to librarians in the higher echelons of the field.…

  1. Discussion and Dissent: ALA 1991 Midwinter Conference Report.

    ERIC Educational Resources Information Center

    Wilson Library Bulletin, 1991

    1991-01-01

    Provides an overview of the American Library Association (ALA) 1991 midwinter conference. Highlights include federal legislation, the organization of library support staff, fees for library services, library collection preservation policies, electronic access, access policy guidelines for academic libraries, reference staffing for electronic…

  2. ALA-induced PpIX fluorescence in epileptogenic tissue

    NASA Astrophysics Data System (ADS)

    Kleen, Jonathan K.; Valdes, Pablo A.; Harris, Brent T.; Holmes, Gregory L.; Paulsen, Keith D.; Roberts, David W.

    2011-03-01

    Astrogliotic tissue displays markedly increased levels of ALA-induced PpIX fluorescence, making it useful for fluorescence-guided resection in glioma surgery. In patients with temporal lobe epilepsy (TLE) and corresponding animal models, there are areas of astrogliosis that often co-localize with the epileptic focus, which can be resected to eliminate seizures in the majority of treated patients. If this epileptogenic tissue can exhibit PpIX fluorescence that is sufficiently localized, it could potentially help identify margins in epilepsy surgery. We tested the hypothesis that ALA-induced PpIX fluorescence could visually accentuate epileptogenic tissue, using an established animal model of chronic TLE. An acute dose of pilocarpine was used to induce chronic seizure activity in a rat. This rat and a normal control were given ALA, euthanized, and brains examined post-mortem for PpIX fluorescence and neuropathology. Preliminary evidence indicates increased PpIX fluorescence in areas associated with chronic epileptic changes and seizure generation in TLE, including the hippocampus and parahippocampal areas. In addition, strong PpIX fluorescence was clearly observed in layer II of the piriform cortex, a region known for epileptic reorganization and involvement in the generation of seizures in animal studies. We are further investigating whether ALA-induced PpIX fluorescence can consistently identify epileptogenic zones, which could warrant the extension of this technique to clinical studies for use as an adjuvant guidance technology in the resection of epileptic tissue.

  3. Ising Quantum Hall Ferromagnetism in AlAs Quantum Wells.

    NASA Astrophysics Data System (ADS)

    de Poortere, Etienne

    2002-03-01

    Though quantum Hall ferromagnetic transitions in two-dimensional (2D) systems are observed in several materials, such transitions in AlAs 2D electrons offer a unique combination of two remarkable properties: (1) the resistance of the carrier system increases sharply at the transition, and (2) these resistance spikes are hysteretic at low temperatures [1]. We have been able to uncover these properties thanks to recent improvements in the quality of our AlAs samples [2], which now attain a mobility as high as 31 m^2/Vs at a density 5 × 10^11 cm-2. These transport phenomena at Ising transitions result in part from the electronic properties of AlAs, which favor a strong competition between exchange, cyclotron and Zeeman energies. Indeed, 2D electrons in AlAs have a high and anisotropic effective band mass comparable to that of Si, and a band g-factor close to 2. In addition, high-density AlAs 2D electrons occupy two X-point valleys of the Brillouin zone, allowing for inter-valley Ising transitions. In this talk we present results from our study of Ising transitions in AlAs 2D electrons. We observe that the hysteretic resistance of a given transition depends sensitively on the occupation of the two levels involved in the transition, increasing in amplitude as these levels are depleted. We also analyze the spike temperature dependence, which shows that unlike the nearby quantum Hall resistance minima, the resistance spikes themselves are not activated. Other parameters are also varied, such as total carrier density and transverse electric field in the AlAs quantum well. A Hartree-Fock picture of these Ising transitions has been drawn, involving magnetic domains and increased scattering at the domain boundaries [3]. Nevertheless, many of the measured dependencies of the Ising transition resistance spikes are not yet qualitatively understood, forming thus a jigsaw puzzle of many parts. [1] E. P. De Poortere et al., Science 290, 1546 (2000). [2] E. P. De Poortere et al

  4. The role of the Cys-X-X-X-Cys motif on the kinetics of cupric ion loading to the Streptomyces lividans Sco protein.

    PubMed

    Blundell, Katie L I M; Wilson, Michael T; Vijgenboom, Erik; Worrall, Jonathan A R

    2013-08-01

    The mechanisms and spectroscopic properties generated by intermediate states upon cupric ion binding to flexible peptide motifs in proteins are of considerable interest. One such motif is the Cys-X-X-X-Cys motif characteristic to members of the Sco family of proteins. In the antibiotic producing bacterium, Streptomyces lividans, a role for its Sco protein (Sco(Sl)) as a cupric metallochaperone to the extracytoplasmic CuA domain of cytochrome c oxidase has been revealed. Stopped-flow kinetic studies have revealed a mechanism of cupric ion capture by Sco(Sl), which passes through a monothiolate intermediate, with distinct spectral features. In the present study we have used two site directed mutants of Sco(Sl), C86A and C90A, to determine which Cys in the CXXXC motif acts as the capture ligand. Comparison of kinetic and thermodynamic parameters obtained from cupric ion binding to the C86A and C90A mutants clearly indicate that Cys86 is the capture ligand and this finding can be reconciled with structural data. At subsaturating levels of cupric ions both mutants bind copper rapidly, but the absorbance properties are distinctly different from wild type Sco(Sl). This is most extreme for the C86A mutant where the Cys90 thiolate bond is considered to be weaker than the Cys86 thiolate bond in the C90A mutant. We put forward an explanation for this behaviour whereby we propose that the cupric ion is moving to a second site with no thiolate coordination.

  5. The role of the Cys2-Cys7 disulfide bridge in the early steps of Islet amyloid polypeptide aggregation: A molecular dynamics study

    NASA Astrophysics Data System (ADS)

    Milardi, Danilo; Pappalardo, Matteo; Pannuzzo, Martina; Grasso, Domenico M.; Rosa, Carmelo La

    2008-10-01

    Aggregation of Islet amyloid polypeptide (IAPP) is believed to play a critical role in the pathogenesis of Type II Diabetes Mellitus. In an attempt to gain details on the early events of this process, here we performed MD simulations of the spontaneous assembly of three replicas of human IAPP. Systems containing the Cys2-Cys7 disulfide bridge exhibited a greater stability and a decreased tendency to evolve into β-sheet rich structures if compared to the disulfide-depleted variants. Conversely, the stability of assemblies constituted by the rat isoforms was shown to be independent from the presence of the disulfide bridge.

  6. New Insight of Tetraphenylethylene-based Raman Signatures for Targeted SERS Nanoprobe Construction Toward Prostate Cancer Cell Detection.

    PubMed

    Ramya, Adukkadan N; Joseph, Manu M; Nair, Jyothi B; Karunakaran, Varsha; Narayanan, Nisha; Maiti, Kaustabh Kumar

    2016-04-27

    We have designed and synthesized novel tetraphenylethylene (TPE) appended organic fluorogens and unfold their unique Raman fingerprinting reflected by surface-enhanced Raman scattering (SERS) upon adsorption on nanoroughened gold surface as a new insight in addition to their prevalent aggregation-induced emission (AIE) and aggregation-caused quenching (ACQ) phenomena. A series of five TPE analogues has been synthesized consisting of different electron donors such as (1) indoline with propyl (TPE-In), (2) indoline with lipoic acid (TPE-In-L), (3) indoline with Boc-protected propyl amine (TPE-In-Boc), (4) benzothaizole (TPE-B), and (5) quinaldine (TPE-Q). Interestingly, all five TPE analogues produced multiplexing Raman signal pattern, out of which TPE-In-Boc showed a significant increase in signal intensity in the fingerprint region. An efficient SERS nanoprobe has been constructed using gold nanoparticles as SERS substrate, and the TPE-In as the Raman reporter, which conjugated with a specific peptide substrate, Cys-Ser-Lys-Leu-Gln-OH, well-known for the recognition of prostate-specific antigen (PSA). The designated nanoprobe TPE-In-PSA@Au acted as SERS "ON/OFF" probe in peace with the vicinity of PSA protease, which distinctly recognizes PSA expression with a limit of detection of 0.5 ng in SERS platform. Furthermore, TPE-In-PSA@Au nanoprobe was efficiently recognized the overexpressed PSA in human LNCaP cells, which can be visualized through SERS spectral analysis and SERS mapping. PMID:27049934

  7. SLC6A3 coding variant Ala559Val found in two autism probands alters dopamine transporter function and trafficking.

    PubMed

    Bowton, E; Saunders, C; Reddy, I A; Campbell, N G; Hamilton, P J; Henry, L K; Coon, H; Sakrikar, D; Veenstra-VanderWeele, J M; Blakely, R D; Sutcliffe, J; Matthies, H J G; Erreger, K; Galli, A

    2014-10-14

    Emerging evidence associates dysfunction in the dopamine (DA) transporter (DAT) with the pathophysiology of autism spectrum disorder (ASD). The human DAT (hDAT; SLC6A3) rare variant with an Ala to Val substitution at amino acid 559 (hDAT A559V) was previously reported in individuals with bipolar disorder or attention-deficit hyperactivity disorder (ADHD). We have demonstrated that this variant is hyper-phosphorylated at the amino (N)-terminal serine (Ser) residues and promotes an anomalous DA efflux phenotype. Here, we report the novel identification of hDAT A559V in two unrelated ASD subjects and provide the first mechanistic description of its impaired trafficking phenotype. DAT surface expression is dynamically regulated by DAT substrates including the psychostimulant amphetamine (AMPH), which causes hDAT trafficking away from the plasma membrane. The integrity of DAT trafficking directly impacts DA transport capacity and therefore dopaminergic neurotransmission. Here, we show that hDAT A559V is resistant to AMPH-induced cell surface redistribution. This unique trafficking phenotype is conferred by altered protein kinase C β (PKCβ) activity. Cells expressing hDAT A559V exhibit constitutively elevated PKCβ activity, inhibition of which restores the AMPH-induced hDAT A559V membrane redistribution. Mechanistically, we link the inability of hDAT A559V to traffic in response to AMPH to the phosphorylation of the five most distal DAT N-terminal Ser. Mutation of these N-terminal Ser to Ala restores AMPH-induced trafficking. Furthermore, hDAT A559V has a diminished ability to transport AMPH, and therefore lacks AMPH-induced DA efflux. Pharmacological inhibition of PKCβ or Ser to Ala substitution in the hDAT A559V background restores AMPH-induced DA efflux while promoting intracellular AMPH accumulation. Although hDAT A559V is a rare variant, it has been found in multiple probands with neuropsychiatric disorders associated with imbalances in DA neurotransmission

  8. Expression of 6-Cys Gene Superfamily Defines Babesia bovis Sexual Stage Development within Rhipicephalus microplus

    PubMed Central

    Alzan, Heba F.; Herndon, David R.; Ueti, Massaro W.; Scoles, Glen A.; Kappmeyer, Lowell S.; Suarez, Carlos E.

    2016-01-01

    Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the causative agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) where their orthologues in Plasmodium parasite have been shown to encode for proteins required for the development of sexual stages. The current study identified four additional 6-Cys genes (G, H, I, J) in the B. bovis genome. These four genes are described in the context of the complete ten 6-Cys gene superfamily. The proteins expressed by this gene family are predicted to be secreted or surface membrane directed. Genetic analysis comparing the 6-Cys superfamily among five distinct B. bovis strains shows limited sequence variation. Additionally, A, B, E, H, I and J genes were transcribed in B. bovis infected tick midgut while genes A, B and E were also transcribed in the subsequent B. bovis kinete stage. Transcription of gene C was found exclusively in the kinete. In contrast, transcription of genes D, F and G in either B. bovis infected midguts or kinetes was not detected. None of the 6-Cys transcripts were detected in B. bovis blood stages. Subsequent protein analysis of 6-Cys A and B is concordant with their transcript profile. The collective data indicate as in Plasmodium parasite, certain B. bovis 6-Cys family members are uniquely expressed during sexual stages and therefore, they are likely required for parasite reproduction. Within B. bovis specifically, proteins encoded by 6-Cys genes A and B are markers for sexual stages and candidate antigens for developing novel vaccines able to interfere with the development of B. bovis within the tick vector. PMID:27668751

  9. Functional analysis of recombinant 2-Cys peroxiredoxin from the hard tick Haemaphysalis longicornis.

    PubMed

    Kusakisako, K; Masatani, T; Miyata, T; Galay, R L; Maeda, H; Talactac, M R; Tsuji, N; Mochizuki, M; Fujisaki, K; Tanaka, T

    2016-02-01

    Ticks are obligate haematophagous arthropods that feed on vertebrate blood containing high levels of iron. The host-derived iron reacts to oxygen in the tick's body, and then high levels of reactive oxygen species, including hydrogen peroxide (H(2)O(2)), may be generated. High levels of H(2)O(2) cause oxidative stress to aerobic organisms. Therefore, antioxidant responses are necessary to control H(2)O(2). We focused on peroxiredoxins (Prxs), H(2)O(2) -scavenging enzymes. The sequence of Haemaphysalis longicornis 2-Cys Prx (HlPrx2) was identified from fat body cDNA libraries of this tick and recombinant HlPrx2 was then prepared using Escherichia coli. By comparison with the 2-Cys Prxs of other organisms, we found two conserved cysteines in HlPrx2, Cys51 and Cys172. We examined the antioxidant activity of HlPrx2 and mutant proteins produced by a single base substitution, converting one or both of these cysteines into serines. The assays revealed that proteins containing Cys51 showed antioxidant activity when H(2)O(2) was removed. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and size-exclusion chromatography demonstrated that only the wild-type HlPrx2 formed homodimers and that all of the proteins that we made had a high molecular weight peak. These results indicate that both Cys51 and Cys172 are essential for the dimerization of HlPrx2, whereas only the Cys51 residue is necessary for antioxidant activity. PMID:26471013

  10. The fragmentations of [M-H]- anions derived from underivatised peptides. The side-chain loss of H2S from Cys. A joint experimental and theoretical study.

    PubMed

    Bilusich, Daniel; Brinkworth, Craig S; McAnoy, Andrew M; Bowie, John H

    2003-01-01

    Loss of H2S is the characteristic Cys side-chain fragmentation of the [M-H]- anions of Cys-containing peptides. A combination of experiment and theory suggests that this reaction is initiated from the Cys enolate anion as follows: RNH-(-)C(CH2SH)CONHR' Ø [RNHC(=CH2)CONHR' (HS-)] Ø [RNHC(=CH2)CO-HNR'-H]-+H2S. This process is facile. Calculations at the HF/6-31G(d)//AM1 level of theory indicate that the initial anion needs only > or =20.1 kcal mol(-1) of excess energy to effect loss of H2S. Loss of CH2S is a minor process, RNHCH(CH2SH)CON(-)-R' Ø RNHCH(CH2S-)CONHR' Ø RNH -CHCONHR+CH2S, requiring an excess energy of > or =50.2 kcal mol(-1). When Cys occupies the C-terminal end of a peptide, the major fragmentation from the [M-H]- species involves loss of (H2S+CO2). A deuterium-labelling study suggests that this could either be a charge-remote reaction (a process which occurs remote from and uninfluenced by the charged centre in the molecule), or an anionic reaction initiated from the C-terminal CO2- group. These processes have barriers requiring the starting material to have an excess energy of > or =79.6 (charge-remote) or > or =67.1 (anion-directed) kcal mol(-1), respectively, at the HF/6-31G(d)//AM1 level of theory. The corresponding losses of CH2O and H2O from the [M-H]- anions of Ser-containing peptides require > or =35.6 and > or =44.4 kcal mol(-1) of excess energy (calculated at the AM1 level of theory), explaining why loss of CH2O is the characteristic side-chain loss of Ser in the negative ion mode. PMID:14608618

  11. Overoxidation of chloroplast 2-Cys peroxiredoxins: balancing toxic and signaling activities of hydrogen peroxide

    PubMed Central

    Puerto-Galán, Leonor; Pérez-Ruiz, Juan M.; Ferrández, Julia; Cano, Beatriz; Naranjo, Belén; Nájera, Victoria A.; González, Maricruz; Lindahl, Anna M.; Cejudo, Francisco J.

    2013-01-01

    Photosynthesis, the primary source of biomass and oxygen into the biosphere, involves the transport of electrons in the presence of oxygen and, therefore, chloroplasts constitute an important source of reactive oxygen species, including hydrogen peroxide. If accumulated at high level, hydrogen peroxide may exert a toxic effect; however, it is as well an important second messenger. In order to balance the toxic and signaling activities of hydrogen peroxide its level has to be tightly controlled. To this end, chloroplasts are equipped with different antioxidant systems such as 2-Cys peroxiredoxins (2-Cys Prxs), thiol-based peroxidases able to reduce hydrogen and organic peroxides. At high peroxide concentrations the peroxidase function of 2-Cys Prxs may become inactivated through a process of overoxidation. This inactivation has been proposed to explain the signaling function of hydrogen peroxide in eukaryotes, whereas in prokaryotes, the 2-Cys Prxs of which were considered to be insensitive to overoxidation, the signaling activity of hydrogen peroxide is less relevant. Here we discuss the current knowledge about the mechanisms controlling 2-Cys Prx overoxidation in chloroplasts, organelles with an important signaling function in plants. Given the prokaryotic origin of chloroplasts, we discuss the occurrence of 2-Cys Prx overoxidation in cyanobacteria with the aim of identifying similarities between chloroplasts and their ancestors regarding their response to hydrogen peroxide. PMID:23967002

  12. Overoxidation of chloroplast 2-Cys peroxiredoxins: balancing toxic and signaling activities of hydrogen peroxide.

    PubMed

    Puerto-Galán, Leonor; Pérez-Ruiz, Juan M; Ferrández, Julia; Cano, Beatriz; Naranjo, Belén; Nájera, Victoria A; González, Maricruz; Lindahl, Anna M; Cejudo, Francisco J

    2013-01-01

    Photosynthesis, the primary source of biomass and oxygen into the biosphere, involves the transport of electrons in the presence of oxygen and, therefore, chloroplasts constitute an important source of reactive oxygen species, including hydrogen peroxide. If accumulated at high level, hydrogen peroxide may exert a toxic effect; however, it is as well an important second messenger. In order to balance the toxic and signaling activities of hydrogen peroxide its level has to be tightly controlled. To this end, chloroplasts are equipped with different antioxidant systems such as 2-Cys peroxiredoxins (2-Cys Prxs), thiol-based peroxidases able to reduce hydrogen and organic peroxides. At high peroxide concentrations the peroxidase function of 2-Cys Prxs may become inactivated through a process of overoxidation. This inactivation has been proposed to explain the signaling function of hydrogen peroxide in eukaryotes, whereas in prokaryotes, the 2-Cys Prxs of which were considered to be insensitive to overoxidation, the signaling activity of hydrogen peroxide is less relevant. Here we discuss the current knowledge about the mechanisms controlling 2-Cys Prx overoxidation in chloroplasts, organelles with an important signaling function in plants. Given the prokaryotic origin of chloroplasts, we discuss the occurrence of 2-Cys Prx overoxidation in cyanobacteria with the aim of identifying similarities between chloroplasts and their ancestors regarding their response to hydrogen peroxide.

  13. Redox activity and multiple copper(I) coordination of 2His-2Cys oligopeptide.

    PubMed

    Choi, DongWon; Alshahrani, Aisha A; Vytla, Yashodharani; Deeconda, Manogna; Serna, Victor J; Saenz, Robert F; Angel, Laurence A

    2015-02-01

    Copper binding motifs with their molecular mechanisms of selective copper(I) recognition are essential molecules for acquiring copper ions, trafficking copper to specific locations and controlling the potentially damaging redox activities of copper in biochemical processes. The redox activity and multiple Cu(I) binding of an analog methanobactin peptide-2 (amb2) with the sequence acetyl-His1-Cys2-Tyr3-Pro4-His5-Cys6 was investigated using ion mobility-mass spectrometry (IM-MS) and UV-Vis spectrophotometry analyses. The Cu(II) titration of amb2 showed oxidation of amb2 via the formation of intra- and intermolecular Cys-Cys disulfide bridges and the multiple Cu(I) coordination by unoxidized amb2 or the partially oxidized dimer and trimer of amb2. The principal product of these reactions was [amb2 + 3Cu(I)](+) which probably coordinates the three Cu(I) ions via two bridging thiolate groups of Cys2 and Cys6 and the δN6 of the imidazole groups of His6, as determined by geometry optimized structures at the B3LYP/LanL2DZ level of theory. The products observed by IM-MS showed direct correlation to spectral changes associated with disulfide bond formation in the UV-Vis spectrophotometric study. The results show that IM-MS analysis is a powerful technique for unambiguously determining the major ion species produced during the redox and metal binding chemistry of oligopeptides.

  14. Enrichment by organomercurial agarose and identification of cys-containing peptides from yeast cell lysates.

    PubMed

    Raftery, Mark J

    2008-05-01

    Dynamic range and the presence of highly abundant proteins limit the number of proteins that may be identified within a complex mixture. Cysteine (Cys) has unique chemical reactivity that may be exploited for chemical tagging/capture with biotin/avidin reagents or affinity chromatography allowing specific isolation and subsequent identification of peptide sequences by mass spectrometry. Organomercurial agarose (Hg-beads) specifically captures Cys-containing peptides and proteins from cell lysates. Tryptic peptides from yeast lysates containing Cys were captured and eluted from Hg-beads after incubation with TCEP and trypsin. From two 1 h nano 1-D LC DDA/MS of the eluate >700 proteins were identified with an estimated false positive rate of approximately 1%. Few peptides were identified with high confidence without Cys within their sequence after capture, and extensive washing, indicating little nonspecific binding. The number of fragmentation spectra was increased using automated 2-D nano-LC/MS and allowed identification of 1496 proteins with an estimated false positive rate of 1.1%. Approximately 4% of the proteins identified were from peptides that did not contain Cys, and these were biased toward higher abundance proteins. Comparison of the 1496 proteins to those reported previously showed that >25% were from yeast proteins not previously observed. Most proteins were identified from a single peptide, and sequence coverage was sacrificed by focusing only on identifying Cys-containing peptides, but large numbers of proteins were rapidly identified by eliminating many of the peptides from the higher abundance proteins.

  15. Protective role of Cys-178 against the inactivation and oligomerization of human insulin-degrading enzyme by oxidation and nitrosylation.

    PubMed

    Ralat, Luis A; Ren, Min; Schilling, Alexander B; Tang, Wei-Jen

    2009-12-01

    Insulin-degrading enzyme (IDE), a 110-kDa metalloendopeptidase, hydrolyzes several physiologically relevant peptides, including insulin and amyloid-beta (Abeta). Human IDE has 13 cysteines and is inhibited by hydrogen peroxide and S-nitrosoglutathione (GSNO), donors of reactive oxygen and nitrogen species, respectively. Here, we report that the oxidative burst of BV-2 microglial cells leads to oxidation or nitrosylation of secreted IDE, leading to the reduced activity. Hydrogen peroxide and GSNO treatment of IDE reduces the V(max) for Abeta degradation, increases IDE oligomerization, and decreases IDE thermostability. Additionally, this inhibitory response of IDE is substrate-dependent, biphasic for Abeta degradation but monophasic for a shorter bradykinin-mimetic substrate. Our mutational analysis of IDE and peptide mass fingerprinting of GSNO-treated IDE using Fourier transform-ion cyclotron resonance mass spectrometer reveal a surprising interplay of Cys-178 with Cys-110 and Cys-819 for catalytic activity and with Cys-789 and Cys-966 for oligomerization. Cys-110 is near the zinc-binding catalytic center and is normally buried. The oxidation and nitrosylation of Cys-819 allow Cys-110 to be oxidized or nitrosylated, leading to complete inactivation of IDE. Cys-789 is spatially adjacent to Cys-966, and their nitrosylation and oxidation together trigger the oligomerization and inhibition of IDE. Interestingly, the Cys-178 modification buffers the inhibition caused by Cys-819 modification and prevents the oxidation or nitrosylation of Cys-110. The Cys-178 modification can also prevent the oligomerization-mediated inhibition. Thus, IDE can be intricately regulated by reactive oxygen or nitrogen species. The structure of IDE reveals the molecular basis for the long distance interactions of these cysteines and how they regulate IDE function.

  16. Loss of the Arabidopsis thaliana P4-ATPases ALA6 and ALA7 impairs pollen fitness and alters the pollen tube plasma membrane.

    PubMed

    McDowell, Stephen C; López-Marqués, Rosa L; Cohen, Taylor; Brown, Elizabeth; Rosenberg, Alexa; Palmgren, Michael G; Harper, Jeffrey F

    2015-01-01

    Members of the P4 subfamily of P-type ATPases are thought to create and maintain lipid asymmetry in biological membranes by flipping specific lipids between membrane leaflets. In Arabidopsis, 7 of the 12 Aminophospholipid ATPase (ALA) family members are expressed in pollen. Here we show that double knockout of ALA6 and ALA7 (ala6/7) results in siliques with a ~2-fold reduction in seed set with a high frequency of empty seed positions near the bottom. Seed set was reduced to near zero when plants were grown under a hot/cold temperature stress. Reciprocal crosses indicate that the ala6/7 reproductive deficiencies are due to a defect related to pollen transmission. In-vitro growth assays provide evidence that ala6/7 pollen tubes are short and slow, with ~2-fold reductions in both maximal growth rate and overall length relative to wild-type. Outcrosses show that when ala6/7 pollen are in competition with wild-type pollen, they have a near 0% success rate in fertilizing ovules near the bottom of the pistil, consistent with ala6/7 pollen having short and slow growth defects. The ala6/7 phenotypes were rescued by the expression of either an ALA6-YFP or GFP-ALA6 fusion protein, which showed localization to both the plasma membrane and highly-mobile endomembrane structures. A mass spectrometry analysis of mature pollen grains revealed significant differences between ala6/7 and wild-type, both in the relative abundance of lipid classes and in the average number of double bonds present in acyl side chains. A change in the properties of the ala6/7 plasma membrane was also indicated by a ~10-fold reduction of labeling by lipophilic FM-dyes relative to wild-type. Together, these results indicate that ALA6 and ALA7 provide redundant activities that function to directly or indirectly change the distribution and abundance of lipids in pollen, and support a model in which ALA6 and ALA7 are critical for pollen fitness under normal and temperature-stress conditions. PMID:25954280

  17. The 482Ser of PPARGC1A and 12Pro of PPARG2 Alleles Are Associated with Reduction of Metabolic Risk Factors Even Obesity in a Mexican-Mestizo Population

    PubMed Central

    Vázquez-Del Mercado, Mónica; Guzmán-Ornelas, Milton-Omar; Corona Meraz, Fernanda-Isadora; Ríos-Ibarra, Clara-Patricia; Reyes-Serratos, Eduardo-Alejandro; Castro-Albarran, Jorge; Ruíz-Quezada, Sandra-Luz; Navarro-Hernández, Rosa-Elena

    2015-01-01

    The aim of this study was to investigate the relationship between functional polymorphisms Gly482Ser in PPARGC1A and Pro12Ala in PPARG2 with the presence of obesity and metabolic risk factors. We included 375 individuals characterized as Mexican-Mestizos and classified by the body mass index (BMI). Body dimensions and distribution of body fat were measured. The HOMA-IR and adiposity indexes were calculated. Adipokines and metabolic profile quantification were performed by ELISA and routine methods. Genetic polymorphisms were determined by polymerase chain reaction restriction fragment length polymorphism analysis. A difference between obese and nonobese subjects in polymorphism PPARGC1A distribution was observed. Among obese individuals, carriers of genotype 482Gly/Gly were observed to have decreased body fat, BMI, and body fat ratio versus 482Ser/Ser carriers and increased resistin and leptin levels in carriers Gly+ phenotype versus Gly− phenotype. Subjects with PPARG2 Ala− phenotype (genotype 12Pro/Pro) showed a decreased HOMA-IR index versus individuals with Ala+ phenotype (genotypes 12Pro/Ala plus 12Ala/Ala). We propose that, in obese Mexican-Mestizos, the combination of alleles 482Ser in PPARGC1A and 12Pro in PPARG2 represents a reduced metabolic risk profile, even when the adiposity indexes are increased. PMID:26185753

  18. Novel O-GlcNAcylation on Ser40 of canonical H2A isoforms specific to viviparity

    PubMed Central

    Hirosawa, Mitsuko; Hayakawa, Koji; Yoneda, Chikako; Arai, Daisuke; Shiota, Hitoshi; Suzuki, Takehiro; Tanaka, Satoshi; Dohmae, Naoshi; Shiota, Kunio

    2016-01-01

    We report here newly discovered O-linked-N-acetylglucosamine (O-GlcNAc) modification of histone H2A at Ser40 (H2AS40Gc). The mouse genome contains 18 H2A isoforms, of which 13 have Ser40 and the other five have Ala40. The combination of production of monoclonal antibody and mass spectrometric analyses with reverse-phase (RP)-high performance liquid chromatography (HPLC) fractionation indicated that the O-GlcNAcylation is specific to the Ser40 isoforms. The H2AS40Gc site is in the L1 loop structure where two H2A molecules interact in the nucleosome. Targets of H2AS40Gc are distributed genome-wide and are dramatically changed during the process of differentiation in mouse trophoblast stem cells. In addition to the mouse, H2AS40Gc was also detected in humans, macaques and cows, whereas non-mammalian species possessing only the Ala40 isoforms, such as silkworms, zebrafish and Xenopus showed no signal. Genome database surveys revealed that Ser40 isoforms of H2A emerged in Marsupialia and persisted thereafter in mammals. We propose that the emergence of H2A Ser40 and its O-GlcNAcylation linked a genetic event to genome-wide epigenetic events that correlate with the evolution of placental animals. PMID:27615797

  19. Novel O-GlcNAcylation on Ser(40) of canonical H2A isoforms specific to viviparity.

    PubMed

    Hirosawa, Mitsuko; Hayakawa, Koji; Yoneda, Chikako; Arai, Daisuke; Shiota, Hitoshi; Suzuki, Takehiro; Tanaka, Satoshi; Dohmae, Naoshi; Shiota, Kunio

    2016-01-01

    We report here newly discovered O-linked-N-acetylglucosamine (O-GlcNAc) modification of histone H2A at Ser(40) (H2AS40Gc). The mouse genome contains 18 H2A isoforms, of which 13 have Ser(40) and the other five have Ala(40). The combination of production of monoclonal antibody and mass spectrometric analyses with reverse-phase (RP)-high performance liquid chromatography (HPLC) fractionation indicated that the O-GlcNAcylation is specific to the Ser(40) isoforms. The H2AS40Gc site is in the L1 loop structure where two H2A molecules interact in the nucleosome. Targets of H2AS40Gc are distributed genome-wide and are dramatically changed during the process of differentiation in mouse trophoblast stem cells. In addition to the mouse, H2AS40Gc was also detected in humans, macaques and cows, whereas non-mammalian species possessing only the Ala(40) isoforms, such as silkworms, zebrafish and Xenopus showed no signal. Genome database surveys revealed that Ser(40) isoforms of H2A emerged in Marsupialia and persisted thereafter in mammals. We propose that the emergence of H2A Ser(40) and its O-GlcNAcylation linked a genetic event to genome-wide epigenetic events that correlate with the evolution of placental animals. PMID:27615797

  20. 75 FR 12254 - Official Trail Marker for the Ala Kahakai National Historic Trail

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-15

    ... National Park Service Official Trail Marker for the Ala Kahakai National Historic Trail AGENCY: National.... SUMMARY: This notice issues the official trail marker insignia of the Ala Kahakai National Historic Trail... prescribed as the official trail marker logo for the Ala Kahakai National Historic Trail, administered by...

  1. Basic principles of fluorescence detection with use of 5-ALA

    NASA Astrophysics Data System (ADS)

    Baumgartner, Reinhold; Stepp, Herbert G.

    2000-06-01

    5-Aminolevulinic acid (5-ALA) has been proven to induce selective accumulation of flourescent Protoporphyrin IX (PPIX) in many types of malignant tissue. According to the target to treatment different routes of topical and systemical application of 5-ALA can be chosen. They include techniques like inhalation, installation and rinsing. For fluorescence detection a lamp based system have been developed in the laser-Forschungslabor in Munich together with Storz company. By skillful balancing of excitation filter centered around 400 nm and the observation filter with transmission above 450 nm images with high color contrast can be obtained. The universal application of the D-LIGHT could be demonstrated in different clinical disciplines like urology, neurosurgery, ENT clinic, gynecology and others.

  2. Dual Role of a Biosynthetic Enzyme, CysK, in Contact Dependent Growth Inhibition in Bacteria

    PubMed Central

    Kaundal, Soni; Uttam, Manju; Thakur, Krishan Gopal

    2016-01-01

    Contact dependent growth inhibition (CDI) is the phenomenon where CDI+ bacterial strain (inhibitor) inhibits the growth of CDI−strain (target) by direct cell to cell contact. CDI is mediated by cdiBAI gene cluster where CdiB facilitates the export of CdiA, an exotoxin, on the cell surface and CdiI acts as an immunity protein to protect CDI+ cells from autoinhibition. CdiA-CT, the C-terminal region of the toxin CdiA, from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires binding of a biosynthetic enzyme CysK (O-acetylserine sulfyhydrylase) for activation in the target cells. CdiA-CT can also interact simultaneously with CysK and immunity protein, CdiI, to form a ternary complex in UPEC536. But the role of CysK in the ternary complex is not clear. We studied the hydrodynamic, thermodynamic and kinetic parameters of binary and ternary complexes using AUC, ITC and SPR respectively, to investigate the role of CysK in UPEC536. We report that CdiA-CT binds CdiI and CysK with nanomolar range affinity. We further report that binding of CysK to CdiA-CT improves its affinity towards CdiI by ~40 fold resulting in the formation of a more stable complex with over ~130 fold decrease in dissociation rate. Thermal melting experiments also suggest the role of CysK in stabilizing CdiA-CT/CdiI complex as Tm of the binary complex shifts ~10°C upon binding CysK. Hence, CysK acts a modulator of CdiA-CT/CdiI interactions by stabilizing CdiA-CT/CdiI complex and may play a crucial role in preventing autoinhibition in UPEC536. This study reports a new moonlighting function of a biosynthetic enzyme, CysK, as a modulator of toxin/immunity interactions in UPEC536 inhibitor cells. PMID:27458806

  3. Ala-His Mediated Peptide Bond Formation Revisited

    NASA Astrophysics Data System (ADS)

    Larkin, Deana C.; Martinis, Susan A.; Roberts, Deborah J.; Fox, George E.

    2001-12-01

    The historical origin of the translation machinery remains unresolved. Although the large 23S ribosomal RNA (rRNA) is almost certainly the catalytic component of the peptidyl transferase center in the modern ribosome, it is likely that greatly simplified systems were initially employed in the late stages of the prebiotic world. In particular, it has been suggested that small RNAs carrying amino acids were important for the genesis of protein synthesis. Consistent with this, a dipeptide, Ala-His, was previously claimed to be a prebiotically feasible catalyst mediating peptide bond formation in the presence of aminoacylated tRNA and cognate mRNA template, in the absence of other ribosomal components (Shimizu, 1996). We herein report a detailed study of putative dipeptide formation by Ala-His and RNAs carrying leucine. Based on the results presented here, it is unlikely that the dipeptide, Ala-His, catalyzes significant levels of Leu-Leu dipeptide formation in solution. A product is produced which can be readily mistaken for a dipeptide in the TLC separation systems employed in earlier work. We offer explanations for the formation of this product as well as another unexpected product. The results presented here are consistent with the notion that the translation machinery was likely based on catalytic RNA from its very inception.

  4. The complex filling of alae crater, Kilauea Volcano, Hawaii

    USGS Publications Warehouse

    Swanson, D.A.; Duffield, W.A.; Jackson, D.B.; Peterson, D.W.

    1972-01-01

    Since February 1969 Alae Crater, a 165-m-deep pit crater on the east rift of Kilauea Volcano, has been completely filled with about 18 million m3 of lava. The filling was episodic and complex. It involved 13 major periods of addition of lava to the crater, including spectacular lava falls as high as 100 m, and three major periods of draining of lava from the crater. Alae was nearly filled by August 3, 1969, largely drained during a violent ground-cracking event on August 4, 1969, and then filled to the low point on its rim on October 10, 1969. From August 1970 to May 1971, the crater acted as a reservoir for lava that entered through subsurface tubes leading from the vent fissure 150 m away. Another tube system drained the crater and carried lava as far as the sea, 11 km to the south. Much of the lava entered Alae by invading the lava lake beneath its crust and buoying the crust upward. This process, together with the overall complexity of the filling, results in a highly complicated lava lake that would doubtless be misinterpreted if found in the fossil record. ?? 1972 Stabilimento Tipografico Francesco Giannini & Figli.

  5. Cyclotetrapeptides with alternating ?-Ala residues: synthesis and spectroscopic studies

    NASA Astrophysics Data System (ADS)

    Ngu-Schwemlein, Maria; Zhou, Zhe; Bowie, Toni; Eden, Rebecca

    2003-07-01

    Three cyclotetrapeptides, c[Leu- D-Ala-Xaa- D-Ala], where Xaa is Leu ( P1), Lys ( P2) and Glu ( P3) were synthesized and studied by 1H and 13C NMR and CD spectroscopy. These cyclotetrapeptides exhibit similar coupling constants, 3JHNHα, in the range of 8.56-9.93 Hz, commonly observed for β-turn structures. All amide proton chemical shifts for P1, P2 and P3 exhibited linear dependence on temperature with moderate temperature coefficients ranging from -3.1 to -9.8 ppb/K. Amide proton signal broadening was observed for all residues in P1, P2 and P3, indicating that they are solvent accessible. The number of resonance observed for P1 was half of the total counts, indicating a C2 symmetric conformation. P2 and P3 exhibit similar CD in solvents of varying dielectric constants and dilutions, with characteristic positive CD bands at ca. 210 and 222 nm, which correspond to a β-turn type structure. Small CD/temperature effect was also observed with isodichroic points, consistent with conformational stability and a well-populated cyclotetrapeptide energy state. These heterochiral cyclotetrapeptides consisting of alternating D-Ala residues adopt stabilized open β-turn conformations and may be useful as a ligand template for further functionalization.

  6. A peroxiredoxin cDNA from Taiwanofungus camphorata: role of Cys31 in dimerization.

    PubMed

    Huang, Chih-Yu; Chen, Yu-Ting; Wen, Lisa; Sheu, Dey-Chyi; Lin, Chi-Tsai

    2014-01-01

    Peroxiredoxins (Prxs) play important roles in antioxidant defense and redox signaling pathways. A Prx isozyme cDNA (TcPrx2, 745 bp, EF552425) was cloned from Taiwanofungus camphorata and its recombinant protein was overexpressed. The purified protein was shown to exist predominantly as a dimer by sodium dodecyl sulfate-polyacrylamide gel electrolysis in the absence of a reducing agent. The protein in its dimeric form showed no detectable Prx activity. However, the protein showed increased Prx activity with increasing dithiothreitol concentration which correlates with dissociation of the dimer into monomer. The TcPrx2 contains two Cys residues. The Cys(60) located in the conserved active site is the putative active peroxidatic Cys. The role of Cys(31) was investigated by site-directed mutagenesis. The C31S mutant (C(31) → S(31)) exists predominantly as a monomer with noticeable Prx activity. The Prx activity of the mutant was higher than that of the corresponding wild-type protein by nearly twofold at 12 μg/mL. The substrate preference of the mutant was H2O2 > cumene peroxide > t-butyl peroxide. The Michaelis constant (K M) value for H2O2 of the mutant was 0.11 mM. The mutant enzyme was active under a broad pH range from 6 to 10. The results suggest a role of Cys(31) in dimerization of the TcPrx2, a role which, at least in part, may be involved in determining the activity of Prx. The C(31) residue does not function as a resolving Cys and therefore the TcPrx2 must follow the reaction mechanism of 1-Cys Prx. This TcPrx2 represents a new isoform of Prx family. PMID:24194195

  7. Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives.

    PubMed

    Kim, Seryoung; Ihara, Kohei; Katsube, Satoshi; Hori, Hatsuhiro; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi

    2015-08-01

    We previously reported that the alaE gene of Escherichia coli encodes the l-alanine exporter AlaE. The objective of this study was to elucidate the mechanism of the AlaE exporter. The minimum inhibitory concentration of l-alanine and l-alanyl-l-alanine in alaE-deficient l-alanine-nonmetabolizing cells MLA301ΔalaE was 4- and >4000-fold lower, respectively, than in the alaE-positive parent cells MLA301, suggesting that AlaE functions as an efflux pump to avoid a toxic-level accumulation of intracellular l-alanine and its derivatives. Furthermore, the growth of the alaE-deficient mutant derived from the l-alanine-metabolizing strain was strongly inhibited in the presence of a physiological level of l-alanyl-l-alanine. Intact MLA301ΔalaE and MLA301ΔalaE/pAlaE cells producing plasmid-borne AlaE, accumulated approximately 200% and 50%, respectively, of the [(3) H]l-alanine detected in MLA301 cells, suggesting that AlaE exports l-alanine. When 200 mmol/L l-alanine-loaded inverted membrane vesicles prepared from MLA301ΔalaE/pAlaE were placed in a solution containing 200 mmol/L or 0.34 μmol/L l-alanine, energy-dependent [(3) H]l-alanine accumulation occurred under either condition. This energy-dependent uphill accumulation of [(3) H]l-alanine was strongly inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone but not by dicyclohexylcarbodiimide, suggesting that the AlaE-mediated l-alanine extrusion was driven by proton motive force. Based on these results, physiological roles of the l-alanine exporter are discussed.

  8. CysLT(1)R antagonists inhibit tumor growth in a xenograft model of colon cancer.

    PubMed

    Savari, Sayeh; Liu, Minghui; Zhang, Yuan; Sime, Wondossen; Sjölander, Anita

    2013-01-01

    The expression of the inflammatory G-protein coupled receptor CysLT1R has been shown to be upregulated in colon cancer patients and associated with poor prognosis. The present study investigated the correlation between CysLT1R and colon cancer development in vivo using CysLT1R antagonists (ZM198,615 or Montelukast) and the nude mouse xenograft model. Two drug administration regimens were established. The first regimen was established to investigate the importance of CysLT1R in tumor initiation. Nude mice were inoculated with 50 µM CysLT1R antagonist-pretreated HCT-116 colon cancer cells and received continued treatment (5 mg/kg/day, intraperitoneally). The second regimen aimed to address the role of CysLT1R in tumor progression. Nude mice were inoculated with non-pretreated HCT-116 cells and did not receive CysLT1R antagonist treatment until recordable tumor appearance. Both regimens resulted in significantly reduced tumor size, attributed to changes in proliferation and apoptosis as determined by reduced Ki-67 levels and increased levels of p21(WAF/Cip1) (P<0.01), cleaved caspase 3, and the caspase-cleaved product of cytokeratin 18. Decreased levels of VEGF (P<0.01) and reduced vessel size (P<0.05) were also observed, the latter only in the ZM198,615-pretreatment group. Furthermore, we performed a series of in vitro studies using the colon cancer cell line HCT-116 and CysLT1R antagonists. In addition to significant reductions in cell proliferation, adhesion and colony formation, we observed induction of cell cycle arrest and apoptosis in a dose-dependent manner. The ability of Montelukast to inhibit growth of human colon cancer xenograft was further validated by using two additional colon cancer cell lines, SW-480 and HT-29. Our results demonstrate that CysLT1R antagonists inhibit growth of colon cancer xenografts primarily by reducing proliferation and inducing apoptosis of the tumor cells.

  9. Mechanism of action of peptidoglycan O-acetyltransferase B involves a Ser-His-Asp catalytic triad.

    PubMed

    Moynihan, Patrick J; Clarke, Anthony J

    2014-10-01

    The O-acetylation of the essential cell wall polymer peptidoglycan is essential in many bacteria for their integrity and survival, and it is catalyzed by peptidoglycan O-acetlytransferase B (PatB). Using PatB from Neisseria gonorrhoeae as the model, we have shown previously that the enzyme has specificity for polymeric muropeptides that possess tri- and tetrapeptide stems and that rates of reaction increase with increasing degrees of polymerization. Here, we present the catalytic mechanism of action of PatB, the first to be described for an O-acetyltransferase of any bacterial exopolysaccharide. The influence of pH on PatB activity was investigated, and pKa values of 6.4-6.45 and 6.25-6.35 for the enzyme-substrate complex (kcat vs pH) and the free enzyme (kcat·KM(-1) vs pH), respectively, were determined for the respective cosubstrates. The enzyme is partially inactivated by sulfonyl fluorides but not by EDTA, suggesting the participation of a serine residue in its catalytic mechanism. Alignment of the known and hypothetical PatB amino acid sequences identified Ser133, Asp302, and His305 as three invariant amino acid residues that could potentially serve as a catalytic triad. Replacement of Asp302 with Ala resulted in an enzyme with less than 20% residual activity, whereas activity was barely detectable with (His305 → Ala)PatB and (Ser133 → Ala)PatB was totally inactive. The reaction intermediate of the transferase reaction involving acetyl- and propionyl-acyl donors was trapped on both the wild-type and (Asp302 → Ala) enzymes and LC-MS/MS analysis of tryptic peptides identified Ser133 as the catalytic nucleophile. A transacetylase mechanism is proposed based on the mechanism of action of serine esterases. PMID:25215566

  10. Non-Conserved Residues in Clostridium acetobutylicum tRNAAla Contribute to tRNA Tuning for Efficient Antitermination of the alaS T Box Riboswitch

    PubMed Central

    Liu, Liang-Chun; Grundy, Frank J.; Henkin, Tina M.

    2015-01-01

    The T box riboswitch regulates expression of amino acid-related genes in Gram-positive bacteria by monitoring the aminoacylation status of a specific tRNA, the binding of which affects the folding of the riboswitch into mutually exclusive terminator or antiterminator structures. Two main pairing interactions between the tRNA and the leader RNA have been demonstrated to be necessary, but not sufficient, for efficient antitermination. In this study, we used the Clostridium acetobutylicum alaS gene, which encodes alanyl-tRNA synthetase, to investigate the specificity of the tRNA response. We show that the homologous C. acetobutylicum tRNAAla directs antitermination of the C. acetobutylicum alaS gene in vitro, but the heterologous Bacillus subtilis tRNAAla (with the same anticodon and acceptor end) does not. Base substitutions at positions that vary between these two tRNAs revealed synergistic and antagonistic effects. Variation occurs primarily at positions that are not conserved in tRNAAla species, which indicates that these non-conserved residues contribute to optimal antitermination of the homologous alaS gene. This study suggests that elements in tRNAAla may have coevolved with the homologous alaS T box leader RNA for efficient antitermination. PMID:26426057

  11. Lack of association between dopamine D2 receptor gene Cys311 variant and schizophrenia

    SciTech Connect

    Tanaka, Toshihisa; Fukushima, Noboru; Takahashi, Makoto; Kameda, Kensuke; Ihda, Shin

    1996-04-09

    Itokawa et al. reported identifying one missense nucleotide mutation from C to G resulting in a substitution of serine with cysteine at codon 311 in the third intracellular loop of the dopamine D2 receptor in schizophrenics. Arinami et al. reported finding a positive association between the Cys311 variant and schizophrenia. In response to the report by Arinami et al. we examined 106 unrelated Japanese schizophrenics and 106 normal controls to determine if there is any association of the Cys311 variant with schizophrenia. However, we found no statistically significant differences in allelic frequencies of Cys311 between schizophrenia and normal controls. The present results as well as those of all previous studies except for that of Arinami et al. indicated that an association between the dopamine D2 receptor gene and schizophrenia is unlikely to exist. 24 refs., 1 fig., 1 tab.

  12. Characterization of the cysK2-ctl1-cysE2 gene cluster involved in sulfur metabolism in Lactobacillus casei.

    PubMed

    Bogicevic, Biljana; Irmler, Stefan; Portmann, Reto; Meile, Leo; Berthoud, Hélène

    2012-01-16

    The up- and downstream regions of ctl1 and ctl2 that encode a cystathionine lyase were analyzed in various Lactobacillus casei strains. ctl1 and ctl2 were found to be part of a gene cluster encoding two other open reading frames. One of the two open reading frames precedes ctl1 and encodes a putative cysteine synthase. The other open reading frame lies downstream of ctl1 and encodes a putative serine acetyltransferase. The gene cluster is not present in the publicly available genome sequences of L. casei ATCC 334, BL23 and Zhang. Apparently, the gene cluster was acquired by a horizontal gene transfer event and can also be found in other lactic acid bacteria such as Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. RT-PCR was used to analyze the expression of the gene cluster. Additionally, an mass spectrometry-based selected reaction monitoring method was developed for quantifying Ctl1 in a cell-free extract of lactic acid bacteria. The gene cluster cysK2-ctl1-cysE2 was expressed as single transcript, and expression was down-regulated by cysteine. In addition, cystathionine lyase activity present in cell-free extracts disappeared when L. casei was grown in the presence of cysteine. Whereas the transcript and the gene product of ctl1 protein were found in all studied ctl1(+)L. casei strains, only the transcript but not the protein or cystathionine lyase activity was detected in L. helveticus FAM2888, L. delbrueckii subsp. bulgaricus ATCC 11842 and S. thermophilus FAM17014, which actually possess a homolog of the cysK2-ctl1-cysE2 gene cluster.

  13. Characterization of the cysK2-ctl1-cysE2 gene cluster involved in sulfur metabolism in Lactobacillus casei.

    PubMed

    Bogicevic, Biljana; Irmler, Stefan; Portmann, Reto; Meile, Leo; Berthoud, Hélène

    2012-01-16

    The up- and downstream regions of ctl1 and ctl2 that encode a cystathionine lyase were analyzed in various Lactobacillus casei strains. ctl1 and ctl2 were found to be part of a gene cluster encoding two other open reading frames. One of the two open reading frames precedes ctl1 and encodes a putative cysteine synthase. The other open reading frame lies downstream of ctl1 and encodes a putative serine acetyltransferase. The gene cluster is not present in the publicly available genome sequences of L. casei ATCC 334, BL23 and Zhang. Apparently, the gene cluster was acquired by a horizontal gene transfer event and can also be found in other lactic acid bacteria such as Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. RT-PCR was used to analyze the expression of the gene cluster. Additionally, an mass spectrometry-based selected reaction monitoring method was developed for quantifying Ctl1 in a cell-free extract of lactic acid bacteria. The gene cluster cysK2-ctl1-cysE2 was expressed as single transcript, and expression was down-regulated by cysteine. In addition, cystathionine lyase activity present in cell-free extracts disappeared when L. casei was grown in the presence of cysteine. Whereas the transcript and the gene product of ctl1 protein were found in all studied ctl1(+)L. casei strains, only the transcript but not the protein or cystathionine lyase activity was detected in L. helveticus FAM2888, L. delbrueckii subsp. bulgaricus ATCC 11842 and S. thermophilus FAM17014, which actually possess a homolog of the cysK2-ctl1-cysE2 gene cluster. PMID:21745695

  14. Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

    PubMed

    Papayannopoulos, I A; Biemann, K

    1992-02-01

    The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.

  15. Interaction of haptoglobin with hemoglobin octamers based on the mutation αAsn78Cys or βGly83Cys

    PubMed Central

    Brillet, Thomas; Marden, Michael C.; Yeh, Joanne I.; Shen, Tong-Jian; Ho, Nancy T.; Kettering, Regina; Du, Shoucheng; Vasseur, Corinne; Domingues-Hamdi, Elisa; Ho, Chien; Baudin-Creuza, Véronique

    2013-01-01

    Octameric hemoglobins have been developed by the introduction of surface cysteines in either the alpha or beta chain. Originally designed as a blood substitute, we report here the structure and ligand binding function; in addition the interaction with haptoglobin was studied. The recombinant Hbs (rHbs) with mutations alpha Asn78Cys or beta Gly83Cys spontaneously form octamers under conditions where the cysteines are oxidized. Oxygen binding curves and CO kinetic studies indicate a correct allosteric transition of the tetramers within the octamer. Crystallographic studies of the two rHbs show two disulfide bonds per octamer. Reducing agents may provoke dissociation to tetramers, but the octamers are stable when mixed with fresh human plasma, indicating that the reduction by plasma is slower than the oxidation by the dissolved oxygen, consistent with an enhanced stability. The octameric rHbs were also mixed with a solution of haptoglobin (Hp), which binds the dimers of Hb: there was little interaction for incubation times of 15 min; however, on longer timescales a complex was formed. Dynamic light scattering was used to follow the interaction of Hp with the alpha Asn78Cys octamer during 24 hours; a transition from a simple complex of 15 nm to a final size of 60 nm was observed. The results indicate a specific orientation of the αβ dimers may be of importance for the binding to haptoglobin. PMID:23847747

  16. Sample multiplexing with cysteine-selective approaches: cysDML and cPILOT.

    PubMed

    Gu, Liqing; Evans, Adam R; Robinson, Renã A S

    2015-04-01

    Cysteine-selective proteomics approaches simplify complex protein mixtures and improve the chance of detecting low abundant proteins. It is possible that cysteinyl-peptide/protein enrichment methods could be coupled to isotopic labeling and isobaric tagging methods for quantitative proteomics analyses in as few as two or up to 10 samples, respectively. Here we present two novel cysteine-selective proteomics approaches: cysteine-selective dimethyl labeling (cysDML) and cysteine-selective combined precursor isotopic labeling and isobaric tagging (cPILOT). CysDML is a duplex precursor quantification technique that couples cysteinyl-peptide enrichment with on-resin stable-isotope dimethyl labeling. Cysteine-selective cPILOT is a novel 12-plex workflow based on cysteinyl-peptide enrichment, on-resin stable-isotope dimethyl labeling, and iodoTMT tagging on cysteine residues. To demonstrate the broad applicability of the approaches, we applied cysDML and cPILOT methods to liver tissues from an Alzheimer's disease (AD) mouse model and wild-type (WT) controls. From the cysDML experiments, an average of 850 proteins were identified and 594 were quantified, whereas from the cPILOT experiment, 330 and 151 proteins were identified and quantified, respectively. Overall, 2259 unique total proteins were detected from both cysDML and cPILOT experiments. There is tremendous overlap in the proteins identified and quantified between both experiments, and many proteins have AD/WT fold-change values that are within ~20% error. A total of 65 statistically significant proteins are differentially expressed in the liver proteome of AD mice relative to WT. The performance of cysDML and cPILOT are demonstrated and advantages and limitations of using multiple duplex experiments versus a single 12-plex experiment are highlighted.

  17. Redox activity and multiple copper(I) coordination of 2His-2Cys oligopeptide.

    PubMed

    Choi, DongWon; Alshahrani, Aisha A; Vytla, Yashodharani; Deeconda, Manogna; Serna, Victor J; Saenz, Robert F; Angel, Laurence A

    2015-02-01

    Copper binding motifs with their molecular mechanisms of selective copper(I) recognition are essential molecules for acquiring copper ions, trafficking copper to specific locations and controlling the potentially damaging redox activities of copper in biochemical processes. The redox activity and multiple Cu(I) binding of an analog methanobactin peptide-2 (amb2) with the sequence acetyl-His1-Cys2-Tyr3-Pro4-His5-Cys6 was investigated using ion mobility-mass spectrometry (IM-MS) and UV-Vis spectrophotometry analyses. The Cu(II) titration of amb2 showed oxidation of amb2 via the formation of intra- and intermolecular Cys-Cys disulfide bridges and the multiple Cu(I) coordination by unoxidized amb2 or the partially oxidized dimer and trimer of amb2. The principal product of these reactions was [amb2 + 3Cu(I)](+) which probably coordinates the three Cu(I) ions via two bridging thiolate groups of Cys2 and Cys6 and the δN6 of the imidazole groups of His6, as determined by geometry optimized structures at the B3LYP/LanL2DZ level of theory. The products observed by IM-MS showed direct correlation to spectral changes associated with disulfide bond formation in the UV-Vis spectrophotometric study. The results show that IM-MS analysis is a powerful technique for unambiguously determining the major ion species produced during the redox and metal binding chemistry of oligopeptides. PMID:25800013

  18. l-Cys/CSE/H2S pathway modulates mouse uterus motility and sildenafil effect.

    PubMed

    Mitidieri, Emma; Tramontano, Teresa; Donnarumma, Erminia; Brancaleone, Vincenzo; Cirino, Giuseppe; d'Emmanuele di Villa Bianca, Roberta; Sorrentino, Raffaella

    2016-09-01

    Sildenafil, a selective phosphodiesterase type 5 (PDE5) inhibitor, commonly used in the oral treatment for erectile dysfunction, relaxes smooth muscle of human bladder through the activation of hydrogen sulfide (H2S) signaling. H2S is an endogenous gaseous transmitter with myorelaxant properties predominantly formed from l-cysteine (l-Cys) by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). Sildenafil also relaxes rat and human myometrium during preterm labor but the underlying mechanism is still unclear. In the present study we investigated the possible involvement of H2S as a mediator of sildenafil-induced effect in uterine mouse contractility. We firstly demonstrated that both enzymes, CBS and CSE were expressed, and able to convert l-Cys into H2S in mouse uterus. Thereafter, sildenafil significantly increased H2S production in mouse uterus and this effect was abrogated by CBS or CSE inhibition. In parallel, l-Cys, sodium hydrogen sulfide or sildenafil but not d-Cys reduced spontaneous uterus contractility in a functional study. The blockage of CBS and CSE reduced this latter effect even if a major role for CSE than CBS was observed. This data was strongly confirmed by using CSE(-/-) mice. Indeed, the increase in H2S production mediated by l-Cys or by sildenafil was not found in CSE(-/-) mice. Besides, the effect of H2S or sildenafil on spontaneous contractility was reduced in CSE(-/-) mice. A decisive proof for the involvement of H2S signaling in sildenafil effect in mice uterus was given by the measurement of cGMP. Sildenafil increased cGMP level that was significantly reduced by CSE inhibition. In conclusion, l-Cys/CSE/H2S signaling modulates the mouse uterus motility and the sildenafil effect. Therefore the study may open different therapeutical approaches for the management of the uterus abnormal contractility disorders.

  19. Sample Multiplexing with Cysteine-Selective Approaches: cysDML and cPILOT

    NASA Astrophysics Data System (ADS)

    Gu, Liqing; Evans, Adam R.; Robinson, Renã A. S.

    2015-04-01

    Cysteine-selective proteomics approaches simplify complex protein mixtures and improve the chance of detecting low abundant proteins. It is possible that cysteinyl-peptide/protein enrichment methods could be coupled to isotopic labeling and isobaric tagging methods for quantitative proteomics analyses in as few as two or up to 10 samples, respectively. Here we present two novel cysteine-selective proteomics approaches: cysteine-selective dimethyl labeling (cysDML) and cysteine-selective combined precursor isotopic labeling and isobaric tagging (cPILOT). CysDML is a duplex precursor quantification technique that couples cysteinyl-peptide enrichment with on-resin stable-isotope dimethyl labeling. Cysteine-selective cPILOT is a novel 12-plex workflow based on cysteinyl-peptide enrichment, on-resin stable-isotope dimethyl labeling, and iodoTMT tagging on cysteine residues. To demonstrate the broad applicability of the approaches, we applied cysDML and cPILOT methods to liver tissues from an Alzheimer's disease (AD) mouse model and wild-type (WT) controls. From the cysDML experiments, an average of 850 proteins were identified and 594 were quantified, whereas from the cPILOT experiment, 330 and 151 proteins were identified and quantified, respectively. Overall, 2259 unique total proteins were detected from both cysDML and cPILOT experiments. There is tremendous overlap in the proteins identified and quantified between both experiments, and many proteins have AD/WT fold-change values that are within ~20% error. A total of 65 statistically significant proteins are differentially expressed in the liver proteome of AD mice relative to WT. The performance of cysDML and cPILOT are demonstrated and advantages and limitations of using multiple duplex experiments versus a single 12-plex experiment are highlighted.

  20. l-Cys/CSE/H2S pathway modulates mouse uterus motility and sildenafil effect.

    PubMed

    Mitidieri, Emma; Tramontano, Teresa; Donnarumma, Erminia; Brancaleone, Vincenzo; Cirino, Giuseppe; d'Emmanuele di Villa Bianca, Roberta; Sorrentino, Raffaella

    2016-09-01

    Sildenafil, a selective phosphodiesterase type 5 (PDE5) inhibitor, commonly used in the oral treatment for erectile dysfunction, relaxes smooth muscle of human bladder through the activation of hydrogen sulfide (H2S) signaling. H2S is an endogenous gaseous transmitter with myorelaxant properties predominantly formed from l-cysteine (l-Cys) by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). Sildenafil also relaxes rat and human myometrium during preterm labor but the underlying mechanism is still unclear. In the present study we investigated the possible involvement of H2S as a mediator of sildenafil-induced effect in uterine mouse contractility. We firstly demonstrated that both enzymes, CBS and CSE were expressed, and able to convert l-Cys into H2S in mouse uterus. Thereafter, sildenafil significantly increased H2S production in mouse uterus and this effect was abrogated by CBS or CSE inhibition. In parallel, l-Cys, sodium hydrogen sulfide or sildenafil but not d-Cys reduced spontaneous uterus contractility in a functional study. The blockage of CBS and CSE reduced this latter effect even if a major role for CSE than CBS was observed. This data was strongly confirmed by using CSE(-/-) mice. Indeed, the increase in H2S production mediated by l-Cys or by sildenafil was not found in CSE(-/-) mice. Besides, the effect of H2S or sildenafil on spontaneous contractility was reduced in CSE(-/-) mice. A decisive proof for the involvement of H2S signaling in sildenafil effect in mice uterus was given by the measurement of cGMP. Sildenafil increased cGMP level that was significantly reduced by CSE inhibition. In conclusion, l-Cys/CSE/H2S signaling modulates the mouse uterus motility and the sildenafil effect. Therefore the study may open different therapeutical approaches for the management of the uterus abnormal contractility disorders. PMID:27326921

  1. Achieving optimal SERS through enhanced experimental design

    PubMed Central

    Fisk, Heidi; Westley, Chloe; Turner, Nicholas J.

    2016-01-01

    One of the current limitations surrounding surface‐enhanced Raman scattering (SERS) is the perceived lack of reproducibility. SERS is indeed challenging, and for analyte detection, it is vital that the analyte interacts with the metal surface. However, as this is analyte dependent, there is not a single set of SERS conditions that are universal. This means that experimental optimisation for optimum SERS response is vital. Most researchers optimise one factor at a time, where a single parameter is altered first before going onto optimise the next. This is a very inefficient way of searching the experimental landscape. In this review, we explore the use of more powerful multivariate approaches to SERS experimental optimisation based on design of experiments and evolutionary computational methods. We particularly focus on colloidal‐based SERS rather than thin film preparations as a result of their popularity. © 2015 The Authors. Journal of Raman Spectroscopy published by John Wiley & Sons, Ltd. PMID:27587905

  2. Evidence for a Contribution of ALA Synthesis to Plastid-To-Nucleus Signaling

    SciTech Connect

    Czarnecki, Olaf; Gläßer, Christine; Chen, Jin-Gui; Mayer, Klaus F. X.; Grimm, Bernhard

    2012-01-01

    The formation of 5-aminolevulinic acid (ALA) in tetrapyrrole biosynthesis is widely controlled by environmental and metabolic feedback cues that determine the influx into the entire metabolic path. Because of its central role as the rate-limiting step, we hypothesized a potential role of ALA biosynthesis in tetrapyrrole-mediated retrograde signaling and exploited the direct impact of ALA biosynthesis on nuclear gene expression (NGE) by using two different approaches. Firstly, the Arabidopsis gun1, hy1 (gun2), hy2 (gun3), gun4 mutants showing uncoupled NGE from the physiological state of chloroplasts were thoroughly examined for regulatory modifications of ALA synthesis and transcriptional control in the nucleus. We found that reduced ALA-synthesizing capacity is common to analyzed gun mutants. Inhibition of ALA synthesis by gabaculine (GAB) that inactivates glutamate-1-semialdehyde aminotransferase and ALA feeding of wild-type and mutant seedlings corroborate the expression data of gun mutants. Transcript level of photosynthetic marker genes were enhanced in norflurazon (NF)-treated seedlings upon additional GAB treatment, while enhanced ALA amounts diminish these RNA levels in NF-treated wild-type in comparison to the solely NF-treated seedlings. Secondly, the impact of posttranslationally down-regulated ALA synthesis on NGE was investigated by global transcriptome analysis of GAB-treated Arabidopsis seedlings and the gun4-1 mutant, which is also characterized by reduced ALA formation. A common set of significantly modulated genes was identified indicating ALA synthesis as a potential signal emitter. The over-represented gene ontology categories of genes with decreased or increased transcript abundance highlight a few biological processes and cellular functions, which are remarkably affected in response to plastid-localized ALA biosynthesis. These results support the hypothesis that ALA biosynthesis correlates with retrograde signaling-mediated control of NGE.

  3. Photosensitization and mechanism of cytotoxicity induced by the use of ALA derivatives in photodynamic therapy

    PubMed Central

    Casas, A; Fukuda, H; Di Venosa, G; Batlle, A

    2001-01-01

    The use of more lipophilic derivatives of 5-aminolevulinic acid (ALA) is expected to have better diffusing properties, and after conversion into the parent ALA, to reach a higher protoporphyrin IX (PPIX) formation rate, thus improving the efficacy of topical photodynamic therapy (PDT). Here we have analysed the behaviour of 3 ALA derivatives (ALA methyl-ester, hexyl ester and a 2-sided derivative) regarding PPIX formation, efficiency in photosensitizing cells and mechanism of cellular death. The maximum amount of porphyrins synthesized from 0.6 mM ALA was 47 ± 8 ng/105 cells. The same amount was formed by a concentration 60-fold lower of hexyl-ALA and 2-fold higher of methyl-ALA. The 2-sided derivative failed to produce PPIX accumulation. Applying a 0.6 J cm−2 light dose, cell viability decreased to 50%. With the 1.5 J cm−2 light dose, less than 20% of the cells survive, and higher light doses produced nearly total cell killing. Comparing the PPIX production and the induced phototoxicity, the more the amount of porphyrins, the greater the cellular killing, and PPIX formed from either ALA or ALA-esters equally sensitize the cells to photoinactivation. ALA-PDT treated cells exhibited features of apoptosis, independently on the pro-photosensitizer employed. ALA-PDT can be improved with the use of ALA derivatives, reducing the amount of ALA necessary to induce efficient photosensitization. ©2001 Cancer Research Campaign http://www.bjcancer.com PMID:11461090

  4. Development of high-throughput phosphorylation profiling method for identification of Ser/Thr kinase specificity.

    PubMed

    Kim, Eun-Mi; Kim, Jaehi; Kim, Yun-Gon; Lee, Peter; Shin, Dong-Sik; Kim, Mira; Hahn, Ji-Sook; Lee, Yoon-Sik; Kim, Byung-Gee

    2011-05-01

    Identification of substrate specificity of kinases is crucial to understand the roles of the kinases in cellular signal transduction pathways. Here, we present an approach applicable for the discovery of substrate specificity of Ser/Thr kinases. The method, which is named as the 'high-throughput phosphorylation profiling (HTPP)' method was developed on the basis of a fully randomized one-bead one-compound (OBOC) combinatorial ladder type peptide library and MALDI-TOF MS. The OBOC ladder peptide library was constructed by the 'split and pool' method on a HiCore resin. The peptide library sequence was Ac-Ala-X-X-X-Ser-X-X-Ala-BEBE-PLL resin. The substrate specificity of murine PKA (cAMP-dependent protein kinase A) and yeast Yak1 kinase was identified using this method. On the basis of the result, we identified Ifh1, which is a co-activator for the transcription of ribosomal protein genes, as a novel substrate of Yak1 kinase. The putative Yak1-dependent phosphorylation site of Ifh1 was verified by in vitro kinase assay.

  5. The Silver Nanorod Array SERS Substrates

    NASA Astrophysics Data System (ADS)

    Zhao, Yiping; Liu, Yongjun

    2010-08-01

    The fabrication of large area, uniform and high enhancement substrates for surface enhanced Raman scattering (SERS) based sensing is a bottle-neck for practical applications of SERS. Recently using oblique angle deposition (OAD) method, we have fabricated silver nanorod arrays with SERS enhancement factor >108, and SERS intensity variation <14%. The SERS spectra from those substrates have been demonstrated to be able to distinguish different viruses and virus strains, bacteria, microRNAs, or other chemical and biological molecules. We have performed a detailed characterization on those Ag nanorod substrates. The SERS enhancement factor depends strongly on the nanorod length and the fabrication conditions. For different deposition angle, there is an optimal nanorod length that gives the maximum enhancement. The SERS enhancement seems to directly depend on the reflectivity of the Ag nanorod substrates at the excitation wavelength regardless of the deposition angles and rod length. The SERS performance also depends strongly on the configurations of the excitation laser beam: the incident angle, the polarization, and the reflectance of the underlayer substrates. A simple modified Greenler's model is proposed to qualitatively explain those effects. The possible origin for the high enhancement of the Ag nanorod substrates has been studied by placing the Raman probe molecules on different locations of the substrates, and we have found that the side surfaces of the nanorod arrays contributes more to the SERS enhancement compared to the ends. We propose that this is due to the anisotropic absorbance nature of the Ag nanorod substrates.

  6. Plasmonic nanostructures for bioanalytical applications of SERS

    NASA Astrophysics Data System (ADS)

    Kahraman, Mehmet; Wachsmann-Hogiu, Sebastian

    2016-03-01

    Surface-enhanced Raman scattering (SERS) is a potential analytical technique for the detection and identification of chemicals and biological molecules and structures in the close vicinity of metallic nanostructures. We present a novel method to fabricate tunable plasmonic nanostructures and perform a comprehensive structural and optical characterization of the structures. Spherical latex particles are uniformly deposited on glass slides and used as templates to obtain nanovoid structures on polydimethylsiloxane surfaces. The diameter and depth of the nanovoids are controlled by the size of the latex particles. The nanovoids are coated with a thin Ag layer for fabrication of uniform plasmonic nanostructures. Structural characterization of the surfaces is performed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Optical properties of these plasmonic nanostructures are evaluated via UV/Vis spectroscopy, and SERS. The sample preparation step is the key point to obtain strong and reproducible SERS spectra from the biological structures. When the colloidal suspension is used as a SERS substrate for the protein detection, the electrostatic interaction of the proteins with the nanoparticles is described by the nature of their charge status, which influences the aggregation properties such as the size and shape of the aggregates, which is critical for the SERS experiment. However, when the solid SERS substrates are fabricated, SERS signal of the proteins that are background free and independent of the protein charge. Pros and cons of using plasmonic nano colloids and nanostructures as SERS substrate will be discussed for label-free detection of proteins using SERS.

  7. Skin laser treatments enhancing transdermal delivery of ALA.

    PubMed

    Gómez, Clara; Costela, Ángel; García-Moreno, Inmaculada; Llanes, Felipe; Teijón, José M; Blanco, M Dolores

    2011-01-01

    Drug delivery across skin has been limited due to barrier properties of the skin, especially those of the stratum corneum (SC). Use of the laser radiation has been suggested for the controlled removal of the SC. The purpose of this study was to study in vitro the influence of infrared radiation from the erbium:yttrium-aluminum-garnet (Er:YAG) laser (λ = 2940  nm), and visible from the 2nd harmonic of a neodymium:yttrium-aluminum-garnet (Nd:YAG) laser (λ = 532  nm) on transdermal delivery of 5-aminolevulinic acid (ALA). Pinna skin of the inner side of rabbit ear was used for skin permeation. The light sources were an Er:YAG laser (Key III Plus KaVo) and a Q-switched Nd:YAG laser (Lotis TII SL-2132). Permeation study, morphological and structural skin examination by histology and differential scanning calorimetry (DSC) were carried out. Permeation profiles and histological observations obtained after irradiation with infrared and visible laser radiation differed due to different biophysical effects on irradiated skin. Wavelength of 2940  nm required lower energy contribution to produce the same level of permeation than visible radiation at 532  nm. Structural analysis by DSC shows a selective impact on the lipidic structure. Laser pretreatment enhanced the delivery of ALA trough the skin by SC ablation.

  8. Monitoring blood flow and photobleaching during topical ALA PDT treatment

    NASA Astrophysics Data System (ADS)

    Sands, Theresa L.; Sunar, Ulas; Foster, Thomas H.; Oseroff, Allan R.

    2009-02-01

    Photodynamic therapy (PDT) using topical aminolevulinic acid (ALA) is currently used as a clinical treatment for nonmelanoma skin cancers. In order to optimize PDT treatment, vascular shutdown early in treatment must be identified and prevented. This is especially important for topical ALA PDT where vascular shutdown is only temporary and is not a primary method of cell death. Shutdown in vasculature would limit the delivery of oxygen which is necessary for effective PDT treatment. Diffuse correlation spectroscopy (DCS) was used to monitor relative blood flow changes in Balb/C mice undergoing PDT at fluence rates of 10mW/cm2 and 75mW/cm2 for colon-26 tumors implanted intradermally. DCS is a preferable method to monitor the blood flow during PDT of lesions due to its ability to be used noninvasively throughout treatment, returning data from differing depths of tissue. Photobleaching of the photosensitizer was also monitored during treatment as an indirect manner of monitoring singlet oxygen production. In this paper, we show the conditions that cause vascular shutdown in our tumor model and its effects on the photobleaching rate.

  9. Fluorescence microscopy studies on ALA-sensitized tissues

    NASA Astrophysics Data System (ADS)

    Huettmann, Gereon; Achtelik, Wolfgang; Loening, Martin; Sommer, Konrad; Diddens, Heyke C.

    1996-12-01

    Fluorescence microscopy has the potential to study the spatial distribution of photosensitizers in tissue samples with cellular or subcellular resolution. A fluorescence microscope was developed to study the distribution of photosensitizer in tissue samples by acquiring fluorescence images in various spectral ranges and spatially resolved fluorescence spectra both from identical samples. Both methods provide complementary information, since the fluorescence images show the distribution of the sensitizers with a high spatial resolution whereas spatially resolved fluorescence spectra can identify the sensitizers and separate their fluorescence from background light emission by the spectral shape of the fluorescence. Protoporphyrin IX (PPIX) distribution induced by 5-aminolevulinic acid (ALA) was studied by fluorescence microscopy in basal cell carcinoma (BCC) and in cervical intraepithelial neoplasia (CIN). In an attempt to understand the varying success in treating BCC with topically applied ALA the PPIX distribution was studied in BCC samples of 10 patients. A strong fluorescence was observed in tumor cells as well as in epidermis, sebaceous glands, and hair follicles. The depth of PPIX sensitization of the BCCs ranged from 0.4 to 3 mm and the ratio of tumor versus epidermal fluorescence of uninvolved skin was near one. In the BCCs an uneven sensitization with a lower fluorescence in the center of the tumor was often observed. Samples of the cervical mucosa also showed PPIX fluorescence in the endothelial layer, the malignant tissues and the glands. No increased fluorescence of the dysplastic lesions compared to the epithelium was observed.

  10. Alae nasi activation and nasal resistance in healthy subjects.

    PubMed

    Strohl, K P; O'Cain, C F; Slutsky, A S

    1982-06-01

    To investigate the effect of alae nasi (AN) activation on nasal resistance, we monitored AN electromyographic (EMG) activity in 17 healthy subjects using surface electrodes placed on either side of the external nares and measured inspiratory nasal resistance utilizing the method of posterior rhinometry. With CO2 inhalation (6 subj), AN EMG activity increased as nasal resistance fell 23 +/- 5% (P less than 0.01). In the same subjects, voluntary flaring of the external nares also increased AN EMG and decreased nasal resistance by 29 +/- 5% (P less than 0.01). Nasal resistance was altered by nasal flaring and CO2 inhalation even after administration of a topical nasal vasoconstrictive spray (8 subj). In six subjects, voluntary nasal flaring or inhibition with the mouth closed produced a 21 +/- 12% change (P less than 0.01) in total airway resistance as measured by body plethysmography. We conclude that activation of the alae nasi will decrease nasal and total airway resistance during voluntary nasal flaring and during CO2 inhalation and thus should be considered in any studies of upper airway resistance.

  11. The effect of Ser 128 substitution on the structure and stability of cAMP receptor protein from Escherichia coli.

    PubMed

    Małecki, J; Wasylewski, Z

    1998-04-01

    Kinetic measurements of denaturation and renaturation of two mutants of cAMP receptor protein (CRP) at position 128, namely Ser --> Ala and Ser --> Pro, were performed in order to assess changes introduced by the mutation in the quaternary structure and protein stability. No significant changes were found in the unfolding/refolding reactions. However, small perturbations in the dissociation of CRP dimer can be seen, which indicate that subunit interactions are influenced by the mutation. Studies of intrinsic fluorescence quenching of these two mutants are also reported, showing changes compared with wild-type protein. Near-UV circular dichroism measurements indicate, however, that Trp residues remain in the same environment as in the wild-type CRP. It is proposed that Ser at position 128 is involved in maintaining the proper domain alignment within CRP subunits. PMID:9588945

  12. Non-reductive modulation of chloroplast fructose-1,6-bisphosphatase by 2-Cys peroxiredoxin.

    PubMed

    Caporaletti, Daniel; D'Alessio, Ana C; Rodriguez-Suarez, Roberto J; Senn, Alejandro M; Duek, Paula D; Wolosiuk, Ricardo A

    2007-04-13

    2-Cys peroxiredoxin (2-Cys Prx) is a large group of proteins that participate in cell proliferation, differentiation, apoptosis, and photosynthesis. In the prevailing view, this ubiquitous peroxidase poises the concentration of H2O2 and, in so doing, regulates signal transduction pathways or protects macromolecules against oxidative damage. Here, we describe the first purification of 2-Cys Prx from higher plants and subsequently we show that the native and the recombinant forms of rapeseed leaves stimulate the activity of chloroplast fructose-1,6-bisphosphatase (CFBPase), a key enzyme of the photosynthetic CO2 assimilation. The absence of reductants, the strict requirement of both fructose 1,6-bisphosphate and Ca2+, and the response of single mutants C174S and C179S CFBPase bring forward clear differences with the well-known stimulation mediated by reduced thioredoxin via the regulatory 170's loop of CFBPase. Taken together, these findings provide an unprecedented insight into chloroplast enzyme regulation wherein both 2-Cys Prx and the 170's loop of CFBPase exhibit novel functions.

  13. Expression of 6-Cys gene superfamily defines babesia bovis sexual stage development within rhipicephalus microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) and t...

  14. Nematode cys-loop GABA receptors: biological function, pharmacology and sites of action for anthelmintics.

    PubMed

    Accardi, Michael V; Beech, Robin N; Forrester, Sean G

    2012-06-01

    Parasitic nematode infection of humans and livestock is a major problem globally. Attempts to control nematode populations have led to the development of several classes of anthelmintic, which target cys-loop ligand-gated ion channels. Unlike the vertebrate nervous system, the nematode nervous system possesses a large and diversified array of ligand-gated chloride channels that comprise key components of the inhibitory neurotransmission system. In particular, cys-loop GABA receptors have evolved to play many fundamental roles in nematode behaviour such as locomotion. Analysis of the genomes of several free-living and parasitic nematodes suggests that there are several groups of cys-loop GABA receptor subunits that, for the most part, are conserved among nematodes. Despite many similarities with vertebrate cys-loop GABA receptors, those in nematodes are quite distinct in sequence similarity, subunit composition and biological function. With rising anthelmintic resistance in many nematode populations worldwide, GABA receptors should become an area of increased scientific investigation in the development of the next generation of anthelmintics. PMID:22430311

  15. Non-reductive modulation of chloroplast fructose-1,6-bisphosphatase by 2-Cys peroxiredoxin

    SciTech Connect

    Caporaletti, Daniel; D'Alessio, Ana C.; Rodriguez-Suarez, Roberto J.; Senn, Alejandro M.; Duek, Paula D.; Wolosiuk, Ricardo A. . E-mail: rwolosiuk@leloir.org.ar

    2007-04-13

    2-Cys peroxiredoxin (2-Cys Prx) is a large group of proteins that participate in cell proliferation, differentiation, apoptosis, and photosynthesis. In the prevailing view, this ubiquitous peroxidase poises the concentration of H{sub 2}O{sub 2} and, in so doing, regulates signal transduction pathways or protects macromolecules against oxidative damage. Here, we describe First purification of 2-Cys Prx from higher plants and subsequently we show that the native and the recombinant forms of rapeseed leaves stimulate the activity of chloroplast fructose-1,6-bisphosphatase (CFBPase), a key enzyme of the photosynthetic CO{sub 2} assimilation. The absence of reductants, the strict requirement of both fructose 1,6-bisphosphate and Ca{sup 2+}, and the response of single mutants C174S and C179S CFBPase bring forward clear differences with the well-known stimulation mediated by reduced thioredoxin via the regulatory 170's loop of CFBPase. Taken together, these findings provide an unprecedented insight into chloroplast enzyme regulation wherein both 2-Cys Prx and the 170's loop of CFBPase exhibit novel functions.

  16. Nematode cys-loop GABA receptors: biological function, pharmacology and sites of action for anthelmintics.

    PubMed

    Accardi, Michael V; Beech, Robin N; Forrester, Sean G

    2012-06-01

    Parasitic nematode infection of humans and livestock is a major problem globally. Attempts to control nematode populations have led to the development of several classes of anthelmintic, which target cys-loop ligand-gated ion channels. Unlike the vertebrate nervous system, the nematode nervous system possesses a large and diversified array of ligand-gated chloride channels that comprise key components of the inhibitory neurotransmission system. In particular, cys-loop GABA receptors have evolved to play many fundamental roles in nematode behaviour such as locomotion. Analysis of the genomes of several free-living and parasitic nematodes suggests that there are several groups of cys-loop GABA receptor subunits that, for the most part, are conserved among nematodes. Despite many similarities with vertebrate cys-loop GABA receptors, those in nematodes are quite distinct in sequence similarity, subunit composition and biological function. With rising anthelmintic resistance in many nematode populations worldwide, GABA receptors should become an area of increased scientific investigation in the development of the next generation of anthelmintics.

  17. SerVermont--The First Year. 1986.

    ERIC Educational Resources Information Center

    Parsons, Cynthia

    SerVermont is a volunteer program for high school students. The program stresses public service in the community and is intended to teach students the value of personal volunteer service to their local communities. During SerVermont's first year of operation, 11 high schools were awarded minigrants to be used in developing programs in which…

  18. Application of SERS in photographic sciences

    NASA Astrophysics Data System (ADS)

    Kneipp, K.

    1990-03-01

    Various possibilities for application of SERS in photographic science will be shortly discussed and illustrated. SERS can give information about the formation of print-out silver in a silver halide emulsion, about the sulfur digestion of photographic emulsions and about the relative efficiencies of sensitization within a series of similar cyanine dyes.

  19. Tissue distribution and kinetics of endogenous porphyrins synthesized after topical application of ALA in different vehicles

    PubMed Central

    Casas, A; Fukuda, H; Batlle, A M del C

    1999-01-01

    The use of 5-aminolaevulinic acid (ALA) is gaining increasing attention for photosensitization in photodynamic therapy of superficially localized tumours. The aim of this work was to determine the kinetics of porphyrin generation in tissues after topical application of ALA delivered in different vehicles on the skin overlying the tumour and normal skin of mice. Maximal accumulation was found in tumour 3 h after ALA application in both cream and lotion preparations. Normal and overlying tumour skin tissues showed different kinetic patterns, reflecting histological changes when the latter is invaded by tumour cells. Liver, kidney, spleen and blood porphyrins also raised from basal levels, showing that ALA and/or ALA-induced porphyrins reach all tissues after topical application. During the first 24 h of ALA topical application, precursors and porphyrins are excreted by both urine and faeces. ALA lotion applied on the skin overlying the tumour induced higher accumulation of tumoural porphyrins than cream, and lotion applied on normal skin appeared to be the most efficient upon inducing total body porphyrins. This work has demonstrated the great influence of the formulation of ALA vehicle on penetration through the skin. Knowledge of the kinetics of porphyrin generation after different conditions of ALA application is needed for the optimization of diagnosis and phototherapy in human tumours. © 1999 Cancer Research Campaign PMID:10487606

  20. Tissue distribution and kinetics of endogenous porphyrins synthesized after topical application of ALA in different vehicles.

    PubMed

    Casas, A; Fukuda, H; Batlle, A M

    1999-09-01

    The use of 5-aminolaevulinic acid (ALA) is gaining increasing attention for photosensitization in photodynamic therapy of superficially localized tumours. The aim of this work was to determine the kinetics of porphyrin generation in tissues after topical application of ALA delivered in different vehicles on the skin overlying the tumour and normal skin of mice. Maximal accumulation was found in tumour 3 h after ALA application in both cream and lotion preparations. Normal and overlying tumour skin tissues showed different kinetic patterns, reflecting histological changes when the latter is invaded by tumour cells. Liver, kidney, spleen and blood porphyrins also raised from basal levels, showing that ALA and/or ALA-induced porphyrins reach all tissues after topical application. During the first 24 h of ALA topical application, precursors and porphyrins are excreted by both urine and faeces. ALA lotion applied on the skin overlying the tumour induced higher accumulation of tumoural porphyrins than cream, and lotion applied on normal skin appeared to be the most efficient upon inducing total body porphyrins. This work has demonstrated the great influence of the formulation of ALA vehicle on penetration through the skin. Knowledge of the kinetics of porphyrin generation after different conditions of ALA application is needed for the optimization of diagnosis and phototherapy in human tumours.

  1. In vivo study of ALA PLGA nanoparticles-mediated PDT for treating cutaneous squamous cell carcinoma

    NASA Astrophysics Data System (ADS)

    Wang, Xiaojie; Shi, Lei; Huang, Zheng; Wang, Xiuli

    2014-09-01

    Background: Squamous cell carcinoma (SCC) is a common skin cancer and its treatment is still a challenge. Although topical photodynamic therapy (PDT) is effective for treating in situ and superficial SCC, the effectiveness of topical ALA delivery to thick SCC can be limited by its bioavailability. Polylactic-co-glycolic acid nanopartieles (PLGA NPs) might provide a promising ALA delivery strategy. The aim of this study was to evaluate the efficacy of ALA PLGA NPs PDT for the treatment of cutaneous SCC in a mouse model. Methods: ALA loaded PLGA NPs were prepared and characterized. The therapeutic efficacy of ALA PLGA NP mediated PDT in treating UV-induced cutaneous SCC in the mice model were examined. Results: In vivo study showed that ALA PLGA NPs PDT were more effective than free ALA of the same concentration in treating mouse cutaneous SCC. Conclusion: ALA PLGA NPs provides a promising strategy for delivering ALA and treating cutaneous SCC.

  2. Standard method for characterizing SERS substrates

    NASA Astrophysics Data System (ADS)

    Guicheteau, Jason A.; Hankus, Mikella E.; Christesen, Steven D.; Fountain, Augustus W., III; Pellegrino, Paul M.; Emmons, Erik D.; Tripathi, Ashish; Wilcox, Phillip; Emge, Darren

    2012-06-01

    We present the methodology and results of a standard assessment protocol to evaluate disparate SERS substrates that were developed for the Defense Advanced Research Programs Agency (DARPA) SERS Science and Technology Fundamentals Program. The results presented are a snapshot of a collaborative effort between the US Army Edgewood Chemical Biological Center, and the US Army Research Laboratory-Aldelphi Laboratory Center to develop a quantitative analytical method with spectroscopic figures of merit to unambiguously compare the sensitivity and reproducibility of various SERS substrates submitted by the program participants. We present the design of a common assessment protocol and the definition of a SERS enhancement value (SEV) in order to effectively compare SERS active surfaces.

  3. Spectroscopy and photometry of X Ser (Nova Ser 1903) during its current DN stunted outburst

    NASA Astrophysics Data System (ADS)

    Munari, U.; Dallaporta, S.

    2016-09-01

    The old nova X Ser (Nova Ser 1903) is known to display stunted dwarf-nova outbursts (Honeycutt et al. 1998, AJ 115, 2527), for which multiband photometry - and spectroscopy in particular - seem acutely rare.

  4. 5-ALA-assisted photodynamic therapy in canine prostates

    NASA Astrophysics Data System (ADS)

    Sroka, Ronald; Muschter, Rolf; Knuechel, Ruth; Steinbach, Pia; Perlmutter, Aaron P.; Martin, Thomas; Baumgartner, Reinhold

    1996-05-01

    Photodynamic therapy (PDT) and interstitial thermotherapy are well known treatment modalities in urology. The approach of this study is to combine both to achieve a selective treatment procedure for benign prostatic hyperplasia (BPH) and prostate carcinoma. Measurements of thy in-vivo pharmacokinetics of 5-ALA induced porphyrins by means of fiber assisted ratiofluorometry showed a maximum fluorescence intensity at time intervals of 3 - 4 h post administration. Fluorescence microscopy at that time showed bright fluorescence in epithelial cells while in the stroma fluorescence could not be observed. Interstitial PDT using a 635-nm dye laser with an irradiation of 50 J/cm2 resulted in a nonthermic hemorrhagic lesion. The lesion size did not change significantly when an irradiation of 100 J/cm2 was used. The usefulness of PDT for treating BPH as well as prostate carcinoma has to be proven in further studies.

  5. The contribution of NADPH thioredoxin reductase C (NTRC) and sulfiredoxin to 2-Cys peroxiredoxin overoxidation in Arabidopsis thaliana chloroplasts.

    PubMed

    Puerto-Galán, Leonor; Pérez-Ruiz, Juan M; Guinea, Manuel; Cejudo, Francisco Javier

    2015-05-01

    Hydrogen peroxide is a harmful by-product of photosynthesis, which also has important signalling activity. Therefore, the level of hydrogen peroxide needs to be tightly controlled. Chloroplasts harbour different antioxidant systems including enzymes such as the 2-Cys peroxiredoxins (2-Cys Prxs). Under oxidizing conditions, 2-Cys Prxs are susceptible to inactivation by overoxidation of their peroxidatic cysteine, which is enzymatically reverted by sulfiredoxin (Srx). In chloroplasts, the redox status of 2-Cys Prxs is highly dependent on NADPH-thioredoxin reductase C (NTRC) and Srx; however, the relationship of these activities in determining the level of 2-Cys Prx overoxidation is unknown. Here we have addressed this question by a combination of genetic and biochemical approaches. An Arabidopsis thaliana double knockout mutant lacking NTRC and Srx shows a phenotype similar to the ntrc mutant, while the srx mutant resembles wild-type plants. The deficiency of NTRC causes reduced overoxidation of 2-Cys Prxs, whereas the deficiency of Srx has the opposite effect. Moreover, in vitro analyses show that the disulfide bond linking the resolving and peroxidatic cysteines protects the latter from overoxidation, thus explaining the dominant role of NTRC on the level of 2-Cys Prx overoxidation in vivo. The overoxidation of chloroplast 2-Cys Prxs shows no circadian oscillation, in agreement with the fact that neither the NTRC nor the SRX genes show circadian regulation of expression. Additionally, the low level of 2-Cys Prx overoxidation in the ntrc mutant is light dependent, suggesting that the redox status of 2-Cys Prxs in chloroplasts depends on light rather than the circadian clock.

  6. The contribution of NADPH thioredoxin reductase C (NTRC) and sulfiredoxin to 2-Cys peroxiredoxin overoxidation in Arabidopsis thaliana chloroplasts

    PubMed Central

    Puerto-Galán, Leonor; Pérez-Ruiz, Juan M.; Guinea, Manuel; Cejudo, Francisco Javier

    2015-01-01

    Hydrogen peroxide is a harmful by-product of photosynthesis, which also has important signalling activity. Therefore, the level of hydrogen peroxide needs to be tightly controlled. Chloroplasts harbour different antioxidant systems including enzymes such as the 2-Cys peroxiredoxins (2-Cys Prxs). Under oxidizing conditions, 2-Cys Prxs are susceptible to inactivation by overoxidation of their peroxidatic cysteine, which is enzymatically reverted by sulfiredoxin (Srx). In chloroplasts, the redox status of 2-Cys Prxs is highly dependent on NADPH-thioredoxin reductase C (NTRC) and Srx; however, the relationship of these activities in determining the level of 2-Cys Prx overoxidation is unknown. Here we have addressed this question by a combination of genetic and biochemical approaches. An Arabidopsis thaliana double knockout mutant lacking NTRC and Srx shows a phenotype similar to the ntrc mutant, while the srx mutant resembles wild-type plants. The deficiency of NTRC causes reduced overoxidation of 2-Cys Prxs, whereas the deficiency of Srx has the opposite effect. Moreover, in vitro analyses show that the disulfide bond linking the resolving and peroxidatic cysteines protects the latter from overoxidation, thus explaining the dominant role of NTRC on the level of 2-Cys Prx overoxidation in vivo. The overoxidation of chloroplast 2-Cys Prxs shows no circadian oscillation, in agreement with the fact that neither the NTRC nor the SRX genes show circadian regulation of expression. Additionally, the low level of 2-Cys Prx overoxidation in the ntrc mutant is light dependent, suggesting that the redox status of 2-Cys Prxs in chloroplasts depends on light rather than the circadian clock. PMID:25560178

  7. The contribution of NADPH thioredoxin reductase C (NTRC) and sulfiredoxin to 2-Cys peroxiredoxin overoxidation in Arabidopsis thaliana chloroplasts.

    PubMed

    Puerto-Galán, Leonor; Pérez-Ruiz, Juan M; Guinea, Manuel; Cejudo, Francisco Javier

    2015-05-01

    Hydrogen peroxide is a harmful by-product of photosynthesis, which also has important signalling activity. Therefore, the level of hydrogen peroxide needs to be tightly controlled. Chloroplasts harbour different antioxidant systems including enzymes such as the 2-Cys peroxiredoxins (2-Cys Prxs). Under oxidizing conditions, 2-Cys Prxs are susceptible to inactivation by overoxidation of their peroxidatic cysteine, which is enzymatically reverted by sulfiredoxin (Srx). In chloroplasts, the redox status of 2-Cys Prxs is highly dependent on NADPH-thioredoxin reductase C (NTRC) and Srx; however, the relationship of these activities in determining the level of 2-Cys Prx overoxidation is unknown. Here we have addressed this question by a combination of genetic and biochemical approaches. An Arabidopsis thaliana double knockout mutant lacking NTRC and Srx shows a phenotype similar to the ntrc mutant, while the srx mutant resembles wild-type plants. The deficiency of NTRC causes reduced overoxidation of 2-Cys Prxs, whereas the deficiency of Srx has the opposite effect. Moreover, in vitro analyses show that the disulfide bond linking the resolving and peroxidatic cysteines protects the latter from overoxidation, thus explaining the dominant role of NTRC on the level of 2-Cys Prx overoxidation in vivo. The overoxidation of chloroplast 2-Cys Prxs shows no circadian oscillation, in agreement with the fact that neither the NTRC nor the SRX genes show circadian regulation of expression. Additionally, the low level of 2-Cys Prx overoxidation in the ntrc mutant is light dependent, suggesting that the redox status of 2-Cys Prxs in chloroplasts depends on light rather than the circadian clock. PMID:25560178

  8. Role of ALA sensitivity in HepG2 cell in the presence of diode laser

    NASA Astrophysics Data System (ADS)

    Fakhar-E-Alam, M.; Atif, M.; Alsalhi, M. S.; Siddique, M.; Kishwar, S.; Qadir, M. I.; Willander, M.

    2011-05-01

    5-aminolevulinic acid (ALA) being an amazing second generation photosensitizer was studied as photodamaging drug on hepatocellular carcinoma (HepG2) cells. The mentioned photosensitizer is converted to PpIX in HepG2 cells in vitro, inducing haem in the cell causing generation of singlet oxygen leading to cell apoptosis. Cell uptake of 5-ALA was evaluated with different concentrations (ranging from 0-800 μg/ml) for 0-49 h incubation period. ALA administered in HepG2 cells is converted into Protoporphyrin IX (PpIX) which has a short half life and constitute a good hematoporphyrin derivative (HPD). Cytotoxicity of ALA in dark and cellular viability without ALA in the presence of light was studied, showing minimal toxic effects in dark with no photodamaging effect on mentioned cells in absence of ALA were observed. The optimal uptake of photosensitizer (5-ALA) in HepG2 cells was investigated by means of spectrophotometeric measurements, cellular viability was determined by means of neutral red assay (NRA). It was observed that with different concentrations (0-800 μg/ml) of ALA or light doses (0-160 J/cm2), there were no significant effect on cellular viability when studied independently. The novel of photocytotoxic study indicates that light dose of 120 J/cm2 produces convincing Photodynamic therapy (PDT) results for HepG2 cells incubated with 262 μg/ml of 5-ALA deducting that HepG2 cell line is sensitive to ALA mediated PDT. Finally morphological changes in HePG2 cells were determined before and after ALA-mediated PDT by confocal microscopy.

  9. Development of SERS substrates for immunoassay applications

    NASA Astrophysics Data System (ADS)

    Celik, Okkes; Kahraman, Mehmet

    2016-03-01

    Surface-enhanced Raman scattering (SERS) is an emerging technique for the detection and identification of biological structures. SERS is based on immunoassay methods are mostly used for the specific detection and identification of bacteria. In this study, SERS substrates are developed with deposition of synthesized spherical 13 nm gold nanoparticles (AuNPs) and 50 nm silver nanoparticles (AgNPs) on regular glass slides with convective assembly method for SERS based immunoassay for the detection and identification of bacteria. The synthesized NPs are characterized by UV-vis absorption spectroscopy, dynamic light scattering (DLS) and atomic force microscopy (AFM). Colloidal suspensions are concentrated by centrifugation to obtain thin films by the deposition of NPs on a regular glass slide with the convective assembly. The experimental parameters for the convective assembly are optimized by changing of NP concentration, stage velocity and NPs volume dropped between two glass slides. Structural characterization of thin films is performed by AFM and SEM. SERS is also used for the optical characterization of the prepared thin films of NPs. In this study, 4- aminothiophenol (4-ATP) is used as probe molecules to evaluate SERS activity of the thin films depending on the type and concentration of NPs. The results demonstrate that, SERS performances of the thin films are dependent on not only the type of NPs but also it depends on the concentration of NPs which forms thin films. The thin film having highest SERS activity could be used for the SERS-based immunoassays for the detection and identification of bacteria.

  10. Cultural Diversity: A Conversation with the Presidents of ALA's Ethnic Caucuses, Part 2.

    ERIC Educational Resources Information Center

    Liu, Carol F. L.

    1994-01-01

    This second in a series highlights the presidents of the Black Caucus of ALA (American Library Association) and the American Indian Library Association. Issues discussed include leaders' roles in supporting diversity; member networking benefits; helping minorities move into leadership positions; integrating diversity into the ALA; and professional…

  11. ALA-PDT inhibits proliferation and promotes apoptosis of SCC cells through STAT3 signal pathway.

    PubMed

    Qiao, Li; Mei, Zhusong; Yang, Zhiyong; Li, Xinji; Cai, Hong; Liu, Wei

    2016-06-01

    Previous studies suggest that apoptosis of carcinoma cells led by photodynamics is mainly intrinsic apoptosis, but whether the extrinsic pathway is involved in the treatment of carcinoma by photodynamic therapy is not confirmed. This research investigated the effect of ALA-PDT on the proliferation and apoptosis of SCC cell A431 and COLO-16, and discussed the role played by JAK/STAT3 signal pathway in this process. Our data showed that the expression levels STAT3 and p-STAT3 protein in the cancer tissue are higher than the corresponding adjacent tissue to carcinoma. The expression level of p-STAT3 in cancerous tissue has a correlation with the tumor size and tissue histopathological differentiation. ALA-PDT could inhibit proliferation of A431 and COLO-16 cells, STAT3 knock down could enhance ALA-PDT's inhibition of cell proliferation, and promote apoptosis induced by ALA-PDT. On the other hand, overexpression of STAT3 has the opposite effect. In addition, ALA-PDT can weaken the protein expression of STAT3 and its target gene Bcl-2 mRNA, and ALA-PDT can strengthen the protein expression of STAT3's target gene Bax mRNA. Overexpression of STAT3 can offset the effect on Bcl-2 and Bax by ALA-PDT; on the other hand, STAT3 knocking down can strengthen ALA-PDT's effect on Bcl-2 and Bax. PMID:26805005

  12. Association of severe micropenis with Gly146Ala polymorphism in the gene for steroidogenic factor-1.

    PubMed

    Wada, Yuka; Okada, Michiyo; Hasegawa, Tomonobu; Ogata, Tsutomu

    2005-08-01

    Steroidogenic factor-1 (SF-1) regulates the transcription of multiple genes involved in the androgen biosynthesis, and SF-1 Gly146Ala polymorphism is known to reduce the transactivation function by approximately 20%. To examine whether the Gly146Ala polymorphism constitutes a susceptibility factor for the development of micropenis (MP), we analyzed this polymorphism in a total of 52 patients with micropenis (T-MP) consisting of 30 patients with severe MP below -2.5 SD (S-MP) and 22 patients with mild MP from -2.1 SD to -2.5 SD (M-MP), together with 115 control males. The Ala allele, the Ala/Gly genotype, and the Ala/Ala plus Ala/Gly genotype frequencies were significantly higher in the S-MP patients than in the control males, whereas the allele and the genotype frequencies were comparable between the M-MP patients and the control males. The results suggest that the SF-1 Gly146Ala polymorphism may constitute a susceptibility factor for the development of S-MP, and that M-MP can be regarded as a normal variation in terms of the polymorphism effect.

  13. A Fifty-Five Year Partnership: ALA and the AFL-CIO.

    ERIC Educational Resources Information Center

    Meyers, Arthur S.

    2002-01-01

    Provides a history of the establishment of the AFL-CIO/ALA (American Federation of Labor (AFL)-Congress of Industrial Organizations (CIO)/American Library Association (ALA)) Joint Committee on Library Service to Labor Groups. Topics include public learning, labor, and libraries; services to labor; problems and issues, including conflicts between…

  14. Alpha linolenic acid (ALA) from Rosa canina, sacha inchi and chia oils may increase ALA accretion and its conversion into n-3 LCPUFA in diverse tissues of the rat.

    PubMed

    Valenzuela B, Rodrigo; Barrera R, Cynthia; González-Astorga, Marcela; Sanhueza C, Julio; Valenzuela B, Alfonso

    2014-07-25

    Alpha-linolenic acid (ALA) is an essential n-3 PUFA; its n-3 LCPUFA derivatives EPA and DHA, which have diverse beneficial effects, are scarce in the human diet. In recent years nontraditional vegetable oils rich in ALA (up to 45%) have been developed as new alternatives to increase ALA consumption. This work evaluated the accretion of ALA, EPA and DHA into the phospholipids extracted from erythrocytes, liver, kidney, small intestine, heart, quadriceps and the brain in rats fed sunflower (SFO), canola (CO), Rosa canina (RCO), sacha inchi (Plukenetia volubilis, SIO) and chia (Salvia hispánica, ChO) oils. Five experimental groups (n = 12 per group) were fed for 21 days with SFO (1% ALA), CO (10% ALA), RCO (33% ALA), SIO (49% ALA), and ChO (64% ALA). SIO and ChO allowed higher ALA accretion in all tissues, except the brain, and a reduction in the content of arachidonic acid in all tissues except the brain. EPA was increased in erythrocytes, liver, kidney, small intestine, heart and quadriceps, but not in the brain. DHA was increased in the liver, small intestine and brain tissues. Our results demonstrate that ALA, when provided in significant amounts, can be converted into n-3 LCPUFA, mostly DHA in the liver and brain. It is suggested that oils rich in ALA, such as SIO and ChO, are good sources for obtaining higher tissue levels of ALA, also allowing its selective conversion into n-3 LCPUFA in some tissues of the rat.

  15. Activatable fluorescent cys-diabody conjugated with indocyanine green derivative: consideration of fluorescent catabolite kinetics on molecular imaging.

    PubMed

    Sano, Kohei; Nakajima, Takahito; Ali, Towhid; Bartlett, Derek W; Wu, Anna M; Kim, Insook; Paik, Chang H; Choyke, Peter L; Kobayashi, Hisataka

    2013-10-01

    Antibody fragments including diabodies have more desirable pharmacokinetic characteristics than whole antibodies. An activatable optical imaging probe based on a cys-diabody targeting prostate-specific membrane antigen conjugated with the near-infrared fluorophore, indocyanine green (ICG), was designed such that it can only be activated when bound to the tumor, leading to high signal-to-background ratios. We employed short polyethylene glycol (PEG) linkers between the ICG and the reactive functional group (Sulfo-OSu group), resulting in covalent conjugation of ICG to the cys-diabody, which led to lower dissociation of ICG from cys-diabody early after injection, reducing hepatic uptake. However, unexpectedly, high and long-term fluorescence was observed in the kidneys, liver, and blood pool more than 1 h after injection of the cys-diabody PEG-ICG conjugate. A biodistribution study using I125-labeled cys-diabody-ICG showed immediate uptake in the kidneys followed by a rapid decrease, while gastric activity increased due to released radioiodine during rapid cys-diabody-ICG catabolism in the kidneys. To avoid this catabolic pathway, it would be preferable to use antibody fragments large enough not to be filtered through glomerulus or to conjugate the fragments with fluorescent dyes that are readily excreted into urine when cleaved from the cys-diabody to achieve high tumor-specific detection.

  16. Activatable fluorescent cys-diabody conjugated with indocyanine green derivative: consideration of fluorescent catabolite kinetics on molecular imaging

    PubMed Central

    Sano, Kohei; Nakajima, Takahito; Ali, Towhid; Bartlett, Derek W.; Wu, Anna M.; Kim, Insook; Paik, Chang H.; Choyke, Peter L.

    2013-01-01

    Abstract. Antibody fragments including diabodies have more desirable pharmacokinetic characteristics than whole antibodies. An activatable optical imaging probe based on a cys-diabody targeting prostate-specific membrane antigen conjugated with the near-infrared fluorophore, indocyanine green (ICG), was designed such that it can only be activated when bound to the tumor, leading to high signal-to-background ratios. We employed short polyethylene glycol (PEG) linkers between the ICG and the reactive functional group (Sulfo-OSu group), resulting in covalent conjugation of ICG to the cys-diabody, which led to lower dissociation of ICG from cys-diabody early after injection, reducing hepatic uptake. However, unexpectedly, high and long-term fluorescence was observed in the kidneys, liver, and blood pool more than 1 h after injection of the cys-diabody PEG-ICG conjugate. A biodistribution study using I125-labeled cys-diabody-ICG showed immediate uptake in the kidneys followed by a rapid decrease, while gastric activity increased due to released radioiodine during rapid cys-diabody-ICG catabolism in the kidneys. To avoid this catabolic pathway, it would be preferable to use antibody fragments large enough not to be filtered through glomerulus or to conjugate the fragments with fluorescent dyes that are readily excreted into urine when cleaved from the cys-diabody to achieve high tumor-specific detection. PMID:23752742

  17. Multiple redox and non-redox interactions define 2-Cys peroxiredoxin as a regulatory hub in the chloroplast.

    PubMed

    Muthuramalingam, Meenakumari; Seidel, Thorsten; Laxa, Miriam; Nunes de Miranda, Susana M; Gärtner, Florian; Ströher, Elke; Kandlbinder, Andrea; Dietz, Karl-Josef

    2009-11-01

    In plants, the highly abundant 2-cysteine peroxiredoxin (2-CysPrx) is associated with the chloroplast and involved in protecting photosynthesis. This work addresses the multiple interactions of the 2-CysPrx in the chloroplast, which depend on its redox state. Transcript co-regulation analysis showed a strong linkage to the peptidyl-prolyl-cis/trans isomerase Cyclophilin 20-3 (Cyp20-3) and other components of the photosynthetic apparatus. Co-expression in protoplasts and quantification of fluorescence resonance energy transfer (FRET) efficiency in vivo confirmed protein interactions of 2-CysPrx with Cyp20-3 as well as NADPH-dependent thioredoxin reductase C (NTRC), while thioredoxin x (Trx-x) did not form complexes that could enable FRET. Likewise, changes in FRET of fluorescently labeled 2-CysPrx in vitro and in vivo proved redox dependent dynamics of 2-CysPrx. Addition of Cyp20-3 to an in vitro peroxidase assay with 2-CysPrx had no significant effect on peroxide reduction. Also, in the presence of NTRC, addition of Cyp20-3 did not further enhance peroxide reduction. In addition, 2-CysPrx functioned as chaperone and inhibited aggregation of citrate synthase during heat treatment. This activity was partly inhibited by Cyp20-3. As a new interaction partner of decameric 2-CysPrx, photosystem II could be identified after chloroplast fractionation and in pull-down assays after reconstitution. In summary, the data indicate a dynamic function of plant 2-CysPrx as redox sensor, chaperone, and regulator in the chloroplast with diverse functions beyond its role as thiol peroxidase.

  18. Fibrinogen residue γAla341 is necessary for calcium binding and 'A-a' interactions.

    PubMed

    Park, Rojin; Ping, Lifang; Song, Jaewoo; Hong, Sung-Yu; Choi, Tae-Youn; Choi, Jong-Rak; Gorkun, Oleg V; Lord, Susan T

    2012-05-01

    The fibrinogen γ-module has several important sites relating to fibrinogen function, which include the high affinity calcium binding site, hole 'a' that binds with knob 'A', and the D:D interface. Residue γAla341, which is located in the vicinity of these sites, is altered in three variant fibrinogens: fibrinogen Seoul (γAla341Asp), Tolaga Bay (γAla341Val), and Lyon III (γAla341Thr). In order to investigate the impaired polymerisation of fibrinogens γAla341Asp and γAla341Val to understand the role of γAla341 in fibrin polymerisation and fibrinogen synthesis, we have expressed γAla341Asp and γAla341Val in Chinese hamster ovary (CHO) cells, purified these fibrinogens from the culture media and performed biochemical tests to elucidate their function. Expression in CHO cells was similar for these variants. For both variants the kinetics of thrombin-catalysed FpA release was not different from normal fibrinogen, while FpB release was slower than that of normal. Thrombin-catalysed polymerisation of both variants was dependent on the calcium concentration. At physiologic calcium (1 mM) the variants showed impaired polymerisation with a longer lag period and a slower Vmax than normal fibrinogen. Scanning electron micrographs showed the clots were less organised than normal, having thicker and more twisted fibers, and larger pores. Analysis by SDS-PAGE showed that factor XIIIa-catalysed γ and α chain cross-linking was delayed, and plasmin-catalysed lysis was not reduced by the presence of 5 mM calcium or 5 mM GPRP (Gly-Pro-Arg-Pro). Our data indicate that fibrinogen residue γAla341 is important for the proper conformation of the γ-module, maintaining calcium-binding site and 'A-a' interactions.

  19. Cognitive deficits and ALA-D-inhibition in children exposed to multiple metals.

    PubMed

    do Nascimento, Sabrina N; Barth, Anelise; Göethel, Gabriela; Baierle, Marília; Charão, Mariele F; Brucker, Natália; Moro, Angela M; Bubols, Guilherme B; Sobreira, Johanna S; Sauer, Elisa; Rocha, Rafael; Gioda, Adriana; Dias, Ana Cristina; Salles, Jerusa F; Garcia, Solange C

    2015-01-01

    Children are especially vulnerable to adverse effects of multiple metals exposure. The aim of this study was to assess some metals concentrations such as lead (Pb), arsenic (As), chromium (Cr), manganese (Mn) and iron (Fe) in whole blood, serum, hair and drinking water samples using inductively coupled plasma-mass spectrometry (ICP-MS) in rural and urban children. In addition, evaluate the adverse effects of multiple metals exposure on cognitive function and δ-aminolevulinate dehydratase (ALA-D) activity. The cognitive ability assessment was performed by the Raven's Colored Progressive Matrices (RCPM) test. The ALA-D activity and ALA-D reactivation index (ALA-RE) activity with DTT and ZnCl2 also were determined. Forty-six rural children and 23 urban children were enrolled in this study. Rural children showed percentile IQ scores in the RCPM test significantly decreased in relation to urban children. According to multiple linear regression analysis, the Mn and Fe in hair may account for the cognitive deficits of children. Manganese and Fe in hair also were positively correlated with Mn and Fe in drinking water, respectively. These results suggest that drinking water is possibly a source of metals exposure in children. ALA-D activity was decreased and ALA-RE with DTT and ZnCl2 was increased in rural children in comparison to urban children. Moreover, ALA-D inhibition was correlated with Cr blood levels and ALA-RE/DDT and ALA-RE/ZnCl2 were correlated with levels of Cr and Hg in blood. Thus, our results indicated some adverse effects of children's exposure to multiple metals, such as cognitive deficits and ALA-D inhibition, mainly associated to Mn, Fe, Cr and Hg.

  20. Evaluation of ALA-induced PpIX as a photosensitizer for PDT in cats

    NASA Astrophysics Data System (ADS)

    Lucroy, Michael D.; Edwards, Benjamin F.; Peavy, George M.; Krasieva, Tatiana B.; Griffey, Stephen M.; Madewell, Bruce R.

    1998-07-01

    Given exogenously, ALA defeats intrinsic regulatory feedback mechanisms allowing intracellular accumulation of protoporphyrin IX (PpIX), a highly efficient photosensitizer. In vivo, PpIX synthesis in neoplastic mammary tissues averages 20-fold higher than in normal mammary tissues. PpIX is retained intracellularly, unlike perivascular localization of other photosensitizers, and it is then cleared quickly from the body. In vitro, ALA induced PpIX production in our laboratory in 6 cell lines tested, including an established feline kidney cell line and dermal fibroblasts from primary skin biopsy explant, resulting in photosensitization. Fluorescent microscopy confirmed PpIX production in skin adnexae following ALA administration in a normal cat. To evaluate toxicity, three cats were treated with a single i.v. dose of ALA (either 100, 200, of 400 mg/kg) and followed for 7 days. Cats receiving 100 or 200 mg/kg ALA i.v. had elevated liver enzymes and bilirubin within 24 hours. Histopathology revealed hydropic changes in the liver and renal fibrosis. The cat receiving 400 mg/kg ALA intravenously had cutaneous flush, bradycardia and apnea associated with ALA administration; within 24 hours the cat was lethargic, anorectic and icteric. ALT, AST and bilirubin concentrations had increased significantly. At necropsy the liver had a prominent lobular pattern; histopathology revealed severe periportal hepatitis and splenic necrosis. Systemically administered ALA induces PpIX production, but toxicity may preclude its clinical application in the cat. PpIX levels seem to be more time dependent than those dependent at these three ALA doses and they are well beyond the saturation point for adequate PpIX conversion. The literature is scant regarding toxicity associated with parenteral administration of ALA.

  1. Cloning and functional characterization of a typical 2-Cys peroxiredoxin from southern bluefin tuna (Thunnus maccoyii).

    PubMed

    Sutton, Drew L; Loo, Grace H; Menz, R Ian; Schuller, Kathryn A

    2010-06-01

    Peroxiredoxins (Prxs, EC: 1.11.1.15) are cysteine-dependent peroxidases proposed to function as antioxidant enzymes and also in H2O2-mediated cell signaling. They have been well characterized in yeast, mammals, protists and bacteria but not yet in fish. Here we describe the cloning and functional characterization of a Prx 2 cDNA from southern bluefin tuna (SBT, Thunnus maccoyii), an important aquaculture species in South Australia. The SBT Prx sequence was closely related (76-92% identical) to Prx 1 and 2 sequences from other fish and mammals and phylogenetic analyses showed that it was most likely a Prx 2. The deduced amino acid sequence contained the peroxidatic and resolving Cys residues characteristic of typical 2-Cys Prx proteins from all kingdoms of life. It also contained the GGLG motif associated with the sensitivity of eukaryotic typical 2-Cys Prx proteins to overoxidation and consequent inactivation by H2O2. When the SBT Prx 2 was expressed in E. coli, it showed thioredoxin (Trx)-dependent peroxidase activity with H2O2, cumene hydroperoxide (CuOOH) and t-butyl hydroperoxide (t-bOOH). The SBT Prx displayed Michaelis-Menten kinetics with Trx but sigmoidal kinetics with H2O2 and CuOOH. The K(m)(Trx) was 12 microM and the S(0.5) values for H2O2 and CuOOH were 29 and 25 microM, respectively. At mM concentrations of H2O2, SBT Prx progressively lost its peroxidase activity as has been observed for other eukaryotic typical 2-Cys Prx proteins. The native SBT Prx enzyme existed as a mixture of dimers, tetramers, decamers and a higher order aggregate.

  2. Insulin-regulated aminopeptidase in adipocyte is Cys-specific and affected by obesity.

    PubMed

    Alponti, Rafaela Fadoni; Viana, Luciana Godoy; Yamanouye, Norma; Silveira, Paulo Flavio

    2015-08-01

    Insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3) in adipocytes is well known to traffic between high (HDM) and low (LDM) density microsomal fractions toward the plasma membrane (MF) under stimulation by insulin. However, its catalytic preference for aminoacyl substrates with N-terminal Leu or Cys is controversial. Furthermore, possible changes in its traffic under metabolic challenges are unknown. The present study investigated the catalytic activity attributable to EC 3.4.11.3 in HDM, LDM and MF from isolated adipocytes of healthy (C), food deprived (FD) and monosodium glutamate (MSG) obese rats on aminoacyl substrates with N-terminal Cys or Leu, in absence or presence of insulin. Efficacy and reproducibility of subcellular adipocyte fractionation procedure were demonstrated. Comparison among HDM vs LDM vs MF intragroup revealed that hydrolytic activity trafficking from LDM to MF under influence of insulin in C, MSG and FD is only on N-terminal Cys. In MSG the same pattern of anterograde traffic and aminoacyl preference occurred independently of insulin stimulation. The pathophysiological significance of IRAP in adipocytes seems to be linked to comprehensive energy metabolism related roles of endogenous substrates with N-terminal cysteine pair such as vasopressin and oxytocin.

  3. In vivo parameters influencing 2-Cys Prx oligomerization: The role of enzyme sulfinylation.

    PubMed

    Noichri, Y; Palais, G; Ruby, V; D'Autreaux, B; Delaunay-Moisan, A; Nyström, T; Molin, M; Toledano, M B

    2015-12-01

    2-Cys Prxs are H2O2-specific antioxidants that become inactivated by enzyme hyperoxidation at elevated H2O2 levels. Although hyperoxidation restricts the antioxidant physiological role of these enzymes, it also allows the enzyme to become an efficient chaperone holdase. The critical molecular event allowing the peroxidase to chaperone switch is thought to be the enzyme assembly into high molecular weight (HMW) structures brought about by enzyme hyperoxidation. How hyperoxidation promotes HMW assembly is not well understood and Prx mutants allowing disentangling its peroxidase and chaperone functions are lacking. To begin addressing the link between enzyme hyperoxidation and HMW structures formation, we have evaluated the in vivo 2-Cys Prxs quaternary structure changes induced by H2O2 by size exclusion chromatography (SEC) on crude lysates, using wild type (Wt) untagged and Myc-tagged S. cerevisiae 2-Cys Prx Tsa1 and derivative Tsa1 mutants or genetic conditions known to inactivate peroxidase or chaperone activity or altering the enzyme sensitivity to hyperoxidation. Our data confirm the strict causative link between H2O2-induced hyperoxidation and HMW formation/stabilization, also raising the question of whether CP hyperoxidation triggers the assembly of HMW structures by the stacking of decamers, which is the prevalent view of the literature, or rather, the stabilization of preassembled stacked decamers.

  4. Molecular structures of two crystalline forms of the cyclic heptapeptide antibiotic ternatin, cyclo[-beta-OH-D-Leu-D-Ile-(NMe)Ala-(NMe)Leu-Leu-(NMe)Ala-D-(NMe)Ala-].

    PubMed

    Miller, R; Galitsky, N M; Duax, W L; Langs, D A; Pletnev, V Z; Ivanov, V T

    1993-12-01

    The crystal structures of two solvated forms of ternatin, cyclo[-beta-OH-D-Leu-D-Ile-(NMe)Ala-(NMe)Leu-Leu-(NMe)Ala-D-(NMe)Ala-] are reported. The first crystallizes with two molecules of peptide and one of dioxane in the asymmetric unit: P2(1)2(1)2(1), a = 11.563(1), b = 21.863(2), c = 36.330(4) A. The second crystallizes with two molecules of peptide and one of water in the asymmetric unit: P2(1)2(1)2(1), a = 14.067(2), b = 16.695(1), c = 36.824(6) A. N-Methylation of four of the seven residues of ternatin appears to reduce the number of low-energy conformations the molecule can assume. The same H-bonded macrocyclic ring conformation is adopted by the backbone of each of the four molecules observed here. All the amino-acid side chains, with the exception of D-Ile2, have similar orientations in each of the four conformers. The heptapeptide macrocycle is characterized by: (i) a cis peptide between (NMe)Ala3 and (NMe)Leu4, (ii) a type II beta-bend, involving residues Leu5-(NMe)Ala6-D-(NMe)Ala7-beta-OH-D-Leu1, stabilized by two H-bonds, N1-->O5 and N5-->O1, between Leu5 and beta-OH-D-Leu1 residues, (iii) a third intramolecular H-bond, observed in each of the four molecules, between the hydroxyl group of beta-OH-D-Leu1 and the carbonyl oxygen of D-Ile2.

  5. Structure of C42D Azotobacter vinelandii FdI. A Cys-X-X-Asp-X-X-Cys motif ligates an air-stable [4Fe-4S]2+/+ cluster.

    PubMed

    Jung, Y S; Bonagura, C A; Tilley, G J; Gao-Sheridan, H S; Armstrong, F A; Stout, C D; Burgess, B K

    2000-11-24

    All naturally occurring ferredoxins that have Cys-X-X-Asp-X-X-Cys motifs contain [4Fe-4S](2+/+) clusters that can be easily and reversibly converted to [3Fe-4S](+/0) clusters. In contrast, ferredoxins with unmodified Cys-X-X-Cys-X-X-Cys motifs assemble [4Fe-4S](2+/+) clusters that cannot be easily interconverted with [3Fe-4S](+/0) clusters. In this study we changed the central cysteine of the Cys(39)-X-X-Cys(42)-X-X-Cys(45) of Azotobacter vinelandii FdI, which coordinates its [4Fe-4S](2+/+) cluster, into an aspartate. UV-visible, EPR, and CD spectroscopies, metal analysis, and x-ray crystallography show that, like native FdI, aerobically purified C42D FdI is a seven-iron protein retaining its [4Fe-4S](2+/+) cluster with monodentate aspartate ligation to one iron. Unlike known clusters of this type the reduced [4Fe-4S](+) cluster of C42D FdI exhibits only an S = 1/2 EPR with no higher spin signals detected. The cluster shows only a minor change in reduction potential relative to the native protein. All attempts to convert the cluster to a 3Fe cluster using conventional methods of oxygen or ferricyanide oxidation or thiol exchange were not successful. The cluster conversion was ultimately accomplished using a new electrochemical method. Hydrophobic and electrostatic interaction and the lack of Gly residues adjacent to the Asp ligand explain the remarkable stability of this cluster.

  6. Phosphorylation of Hepatitis C Virus RNA Polymerases Ser29 and Ser42 by Protein Kinase C-Related Kinase 2 Regulates Viral RNA Replication

    PubMed Central

    Han, Song-Hee; Kim, Seong-Jun; Kim, Eun-Jung; Kim, Tae-Eun; Moon, Jae-Su; Kim, Geon-Woo; Lee, Seung-Hoon; Cho, Kun; Yoo, Jong Shin; Son, Woo Sung; Rhee, Jin-Kyu; Han, Seung Hyun

    2014-01-01

    ABSTRACT Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the Δ1 finger loop region that interacts with the thumb subdomain of NS5B. Colony-forming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its Δ1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication. IMPORTANCE While the role of RNA-dependent RNA polymerases (RdRps) in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated hepatitis C virus (HCV) nonstructural protein 5B (NS5B). Reverse-genetics studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activities revealed

  7. Identification of a novel phosphorylation site, Ser-170, as a regulator of bad pro-apoptotic activity.

    PubMed

    Dramsi, Shaynoor; Scheid, Michael P; Maiti, Arpita; Hojabrpour, Payman; Chen, Xianming; Schubert, Kathryn; Goodlett, David R; Aebersold, Ruedi; Duronio, Vincent

    2002-02-22

    Bad is a pro-apoptotic member of the Bcl-2 family of proteins that is thought to exert a death-promoting effect by heterodimerization with Bcl-X(L), nullifying its anti-apoptotic activity. Growth factors may promote cell survival at least partially through phosphorylation of Bad at one or more of Ser-112, -136, or -155. Our previous work showed that Bad is also phosphorylated in response to cytokines at another site, which we now identify as Ser-170. The functional role of this novel phosphorylation site was assessed by site-directed mutagenesis and analysis of the pro-apoptotic function of Bad in transiently transfected HEK293 and COS-7 cells or by stable expression in the cytokine-dependent cell line, MC/9. In general, mutation of Ser-170 to Ala results in a protein with increased ability to induce apoptosis, similar to the S112A mutant. Mutation of Ser-170 to Asp, mimicking a constitutively phosphorylated site, results in a protein that is virtually unable to induce apoptosis. Similarly, the S112A/S170D double mutant does not cause apoptosis in HEK293 and MC/9 cell lines. These data strongly suggest that phosphorylation of Bad at Ser-170 is a critical event in blocking the pro-apoptotic activity of Bad. PMID:11717309

  8. IRS1Ser³⁰⁷ phosphorylation does not mediate mTORC1-induced insulin resistance.

    PubMed

    Herrema, Hilde; Lee, Jaemin; Zhou, Yingjiang; Copps, Kyle D; White, Morris F; Ozcan, Umut

    2014-01-10

    Increased mammalian target of rapamycin complex 1 (mTORC1) activity has been suggested to play important roles in development of insulin resistance in obesity. mTORC1 hyperactivity also increases endoplasmic reticulum (ER) stress, which in turn contributes to development of insulin resistance and glucose intolerance. Increased IRS1 phosphorylation at Ser307 in vitro is correlated with mTORC1- and ER stress-induced insulin resistance. This phosphorylation site correlates strongly with impaired insulin receptor signaling in diabetic mice and humans. In contrast, evidence from knock-in mice suggests that phosphorylation of IRS1 at Ser307 is actually required to maintain insulin sensitivity. To study the involvement of IRS1(Ser307) phosphorylation in mTORC1-mediated glucose intolerance and insulin sensitivity in vivo, we investigated the effects of liver specific TSC1 depletion in IRS1(Ser307Ala) mice and controls. Our results demonstrate that blockade of IRS1(Ser307) phosphorylation in vivo does not prevent mTORC1-mediated glucose intolerance and insulin resistance. PMID:24333417

  9. PPARg2 Ala¹² variant protects against Graves' orbitopathy and modulates the course of the disease.

    PubMed

    Pawlak-Adamska, Edyta; Daroszewski, Jacek; Bolanowski, Marek; Oficjalska, Jolanta; Janusz, Przemyslaw; Szalinski, Marek; Frydecka, Irena

    2013-07-01

    Orbital fibroblast differentiation to adipocytes is a peroxisome proliferator-activated receptor g (PPARg)-dependent process essential for pathogenic tissue remodeling in Graves' orbitopathy (GO). PPARg2 Pro¹²Ala polymorphism modulates expression and/or function of the molecule encoded by this gene and is a promising locus of GO. Here, we analyzed associations of PPARg2 Pro¹²Ala with clinical manifestation of GO in 742 Polish Caucasians including 276 Graves' disease (GD) patients. In our study, the Ala¹² allele and Ala¹² variant (Ala¹²Ala and/or Pro¹²Ala genotype) decreased the risk of GO (p = 0.000012 and p = 0.00013). Moreover, Ala¹²Ala genotype was observed only in patients without GO (p = 0.002). GD patients with Ala¹² variant had less active and less severe eye symptoms. Female carriers of the Ala¹² allele rarely developed GO, but the marker was not related to symptoms of GO. The opposite finding was recorded in males, in whom the studied polymorphism was related to activity, but not to the development, of GO. In Ala¹² variant carriers without familial history of thyroid disease, risk of GO was lower than in persons with a familial background. The Ala¹² allele seemed to protect smokers from GO, but in nonsmokers, such a relation was not obvious. A multivariate analysis indicated the Pro¹²Ala marker as an independent risk factor of eye symptoms (p = 0.0001) and lack of Ala increases the risk of GO 3.24-fold. In conclusion, the gain-of-function Ala¹² variant protects against GO and modulates the course of the disease.

  10. The role of DAMPS in ALA-PDT for skin squamous cell carcinoma (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Wang, Xiuli; Wang, Xiaojie; Ji, Jie; Zhang, Haiyan; Shi, Lei

    2016-03-01

    5-Aminolevulinic acid mediated photodynamic therapy (ALA-PDT) is an established local approach for skin squamous cell carcinoma. It is believed that dangerous signals damage-associated molecular patterns (DAMPs) play an important role in ALA-PDT. In this study, we evaluated in vitro and in vivo expressions of major DAMPs, calreticulin (CRT), heat shock proteins 70 (HSP70), and high mobility group box 1 (HMGB1), induced by ALA-PDT using immunohistochemistry, western blot, and ELISA in a squamous cell carcinoma (SCC) mouse model. The role of DAMPs in the maturation of DCs potentiated by ALA-PDT-treated tumor cells was detected by FACS and ELISA. Our results showed that ALA-PDT enhanced the expression of CRT, HSP70, and HMGB1. These induced DAMPs played an important role in activating DCs by PDT-treated tumor cells, including phenotypic maturation (upregulation of surface expression of MHC-II, CD80, and CD86) and functional maturation (enhanced capability to secrete IFN-γ and IL-12). Furthermore, injecting ALA-PDT-treated tumor cells into naïve mice resulted in complete protection against cancer cells of the same origin. Our findings indicate that ALA-PDT can upregulate DAMPs and enhance tumor immunogenicity, providing a promising strategy for inducing a systemic anticancer immune response.

  11. The novel dipeptide Tyr-Ala (TA) significantly enhances the lifespan and healthspan of Caenorhabditis elegans.

    PubMed

    Zhang, Z; Zhao, Y; Wang, X; Lin, R; Zhang, Y; Ma, H; Guo, Y; Xu, L; Zhao, B

    2016-04-01

    Food-derived bioactive peptides may have various physiological modulatory and regulatory functions and are now being studied extensively. Recently, the novel dipeptide Tyr-Ala was isolated from hydrolyzed maize protein. Tyr-Ala significantly prolonged the lifespan of wild-type Caenorhabditis elegans and extended the nematode healthspan and lifespan during heat/oxidative stress. Compared with its constituent amino acids, Tyr-Ala was more efficient in enhancing stress resistance. Further studies demonstrated that the significant longevity-extending effects of Tyr-Ala on Caenorhabditis elegans were attributed to its in vitro and in vivo free radical-scavenging effects, in addition to its ability to up-regulate stress resistance-related proteins, such as SOD (Superoxide Dismutase)-3 and HSP (Heat Shock Protein)-16.2. Real-time PCR results showed that the up-regulation of aging-associated genes, such as daf-16, sod-3, hsp-16.2 and skn-1, also contributed to the stress-resistance effect of Tyr-Ala. These results indicate that the novel dipeptide Tyr-Ala can protect against external stress and thus extend the lifespan and healthspan of Caenorhabditis elegans. Thereby, Tyr-Ala could be used as a potential medicine in anti-aging research. PMID:26987062

  12. The Cys4 zinc finger of bacteriophage T7 primase in sequence-specific single-stranded DNA recognition

    PubMed Central

    Kusakabe, Takahiro; Hine, Anna V.; Hyberts, Sven G.; Richardson, Charles C.

    1999-01-01

    Bacteriophage T7 DNA primase recognizes 5′-GTC-3′ in single-stranded DNA. The primase contains a single Cys4 zinc-binding motif that is essential for recognition. Biochemical and mutagenic analyses suggest that the Cys4 motif contacts cytosine of 5′-GTC-3′ and may also contribute to thymine recognition. Residues His33 and Asp31 are critical for these interactions. Biochemical analysis also reveals that T7 primase selectively binds CTP in the absence of DNA. We propose that bound CTP selects the remaining base G, of 5′-GTC-3′, by base pairing. Our deduced mechanism for recognition of ssDNA by Cys4 motifs bears little resemblance to the recognition of trinucleotides of double-stranded DNA by Cys2His2 zinc fingers. PMID:10200256

  13. Molecular recognition in the interaction of chloroplast 2-Cys peroxiredoxin with NADPH-thioredoxin reductase C (NTRC) and thioredoxin x.

    PubMed

    Bernal-Bayard, Pilar; Ojeda, Valle; Hervás, Manuel; Cejudo, Francisco J; Navarro, José A; Velázquez-Campoy, Adrián; Pérez-Ruiz, Juan M

    2014-11-28

    In addition to the standard NADPH thioredoxin reductases (NTRs), plants hold a plastidic NTR (NTRC), with a thioredoxin module fused at the C-terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs). The interaction of NTRC and chloroplastic thioredoxin x with 2-Cys Prxs has been confirmed in vivo, by bimolecular fluorescence complementation (BiFC) assays, and in vitro, by isothermal titration calorimetry (ITC) experiments. In comparison with thioredoxin x, NTRC interacts with 2-Cys Prx with higher affinity, both the thioredoxin and NTR domains of NTRC contributing significantly to this interaction, as demonstrated by using the NTR and thioredoxin modules of the enzyme expressed separately. The presence of the thioredoxin domain seems to prevent the interaction of NTRC with thioredoxin x.

  14. Molecular recognition in the interaction of chloroplast 2-Cys peroxiredoxin with NADPH-thioredoxin reductase C (NTRC) and thioredoxin x.

    PubMed

    Bernal-Bayard, Pilar; Ojeda, Valle; Hervás, Manuel; Cejudo, Francisco J; Navarro, José A; Velázquez-Campoy, Adrián; Pérez-Ruiz, Juan M

    2014-11-28

    In addition to the standard NADPH thioredoxin reductases (NTRs), plants hold a plastidic NTR (NTRC), with a thioredoxin module fused at the C-terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs). The interaction of NTRC and chloroplastic thioredoxin x with 2-Cys Prxs has been confirmed in vivo, by bimolecular fluorescence complementation (BiFC) assays, and in vitro, by isothermal titration calorimetry (ITC) experiments. In comparison with thioredoxin x, NTRC interacts with 2-Cys Prx with higher affinity, both the thioredoxin and NTR domains of NTRC contributing significantly to this interaction, as demonstrated by using the NTR and thioredoxin modules of the enzyme expressed separately. The presence of the thioredoxin domain seems to prevent the interaction of NTRC with thioredoxin x. PMID:25448674

  15. Association between PPAR-γ2 Pro12Ala polymorphism and obesity: a meta-analysis.

    PubMed

    Yao, Ying-Shui; Li, Jie; Jin, Yue-Long; Chen, Yan; He, Lian-Ping

    2015-06-01

    The peroxisome proliferator-activated receptor-γ2 (PPAR-γ2) gene has been reported in the pathogeny of obesity. However, the results have been inconsistent. The purpose of this meta-analysis was to acquire a more accurate assessment of the association between PPAR-γ2 Pro12Ala polymorphism and obesity. PubMed, Wan Fang (Chinese) databases, Chinese Biomedical Medical databases, and Chinese National Knowledge Infrastructure were searched to identify eligible studies. Finally, 25 studies (6491 cases and 8242 controls) were enrolled in this meta-analysis. The effect summary odds ratio (OR) with 95 % confidence interval (CI) was applied. Random-effects or fixed-effects model was performed based on the heterogeneity. STATA 12.0 was applied for this meta-analysis. The combined results showed that PPAR-γ Pro12Ala polymorphism was associated with the obesity risk (Ala vs. Pro: OR = 1.55, 95 % CI 1.34-1.80; Pro/Ala vs. Pro/Pro: OR = 1.54, 95 % CI 1.31-1.82; Ala/Ala + Pro/Ala vs. Pro/Pro: OR = 1.61, 95 % CI 1.36-1.90). Subgroup analysis by ethnicity showed that there were significant associations between PPAR-γ Pro12Ala polymorphism and obesity risk in Caucasians, Asians, and Mixed population. Subgroup analysis by obesity's cutoff points showed that the associations were found among the patients with the cutoff point of BMI ≥24 and BMI ≥30 but not among the patients with the cutoff point of BMI ≥95th percentile. These results suggested that PPAR-γ Pro12Ala polymorphism might be a risk factor for obesity susceptibility.

  16. CysK2 from Mycobacterium tuberculosis Is an O-Phospho-l-Serine-Dependent S-Sulfocysteine Synthase

    PubMed Central

    Steiner, Eva Maria; Böth, Dominic; Lössl, Philip; Vilaplana, Francisco; Schnell, Robert

    2014-01-01

    Mycobacterium tuberculosis is dependent on cysteine biosynthesis, and reduced sulfur compounds such as mycothiol synthesized from cysteine serve in first-line defense mechanisms against oxidative stress imposed by macrophages. Two biosynthetic routes to l-cysteine, each with its own specific cysteine synthase (CysK1 and CysM), have been described in M. tuberculosis, but the function of a third putative sulfhydrylase in this pathogen, CysK2, has remained elusive. We present biochemical and biophysical evidence that CysK2 is an S-sulfocysteine synthase, utilizing O-phosphoserine (OPS) and thiosulfate as substrates. The enzyme uses a mechanism via a central aminoacrylate intermediate that is similar to that of other members of this pyridoxal phosphate-dependent enzyme family. The apparent second-order rate of the first half-reaction with OPS was determined as kmax/Ks = (3.97 × 103) ± 619 M−1 s−1, which compares well to the OPS-specific mycobacterial cysteine synthase CysM with a kmax/Ks of (1.34 × 103) ± 48.2. Notably, CysK2 does not utilize thiocarboxylated CysO as a sulfur donor but accepts thiosulfate and sulfide as donor substrates. The specificity constant kcat/Km for thiosulfate is 40-fold higher than for sulfide, suggesting an annotation as S-sulfocysteine synthase. Mycobacterial CysK2 thus provides a third metabolic route to cysteine, either directly using sulfide as donor or indirectly via S-sulfocysteine. Hypothetically, S-sulfocysteine could also act as a signaling molecule triggering additional responses in redox defense in the pathogen upon exposure to reactive oxygen species during dormancy. PMID:25022854

  17. Gold Nanowire Forests for SERS Detection**

    PubMed Central

    La Porta, Andrea; Grzelczak, Marek; Liz-Marzán, Luis M

    2014-01-01

    Simple wet chemistry has been applied to control the vertical growth of gold nanowires on a glass substrate. As a consequence, the longitudinal localized surface plasmon band position can be tuned from 656 to 1477 nm in a few minutes by simply controlling the growth rate and time. This allowed us to select the optimum conditions for maximum electromagnetic enhancement and performance in surface enhanced Raman scattering (SERS) detection. SERS measurements confirmed the uniform and reproducible distribution of the nanowires on the substrate, with the subsequent high reproducibility of hot spot formation. Detection of malachite green in water and of 1-naphthalenethiol from the gas phase are demonstrated as proof-of-concept applications of these three-dimensional SERS substrates. PMID:25478310

  18. Development of a widefield SERS imaging endoscope

    NASA Astrophysics Data System (ADS)

    McVeigh, Patrick Z.; Mallia, Rupananda J.; Veilleux, Israel; Wilson, Brian C.

    2012-02-01

    We report on the design and testing of a prototype widefield surface enhanced Raman scattering (SERS) imaging system based on a fiber optic bronchoscope using bandpass filters for Raman signal selection. The SERS contrast agents employed consist of gold nanoparticles encoded with a Raman-active dye and made specific for lung adenocarcinoma tissue through the use of an anti-epidermal growth factor receptor (EGFR) antibody. By exploiting the extremely narrow SERS spectral peaks we demonstrate a facile method of background fluorescence rejection that can be implemented at sub-video rates. The system has been tested on in-vivo tissues and performance metrics, including the maximum tissue penetration and minimum detectable nanoparticle quantity have been determined in a standardized fashion.

  19. Rationally designed substrates for SERS biosensing

    NASA Astrophysics Data System (ADS)

    Yan, Bo

    The large electromagnetic field enhancement provided by nanostructured noble metal surfaces forms the foundation for a series of enabling optical analytical techniques, such as surface enhanced Raman spectroscopy (SERS), surface enhanced IR absorption spectroscopy (SEIRA), surface enhanced fluorescent microscopy (SEF), to name only a few. Critical sensing applications have, however, other substrate requirements than mere peak signal enhancement. The substrate needs to be reliable, provide reproducible signal enhancements, and be amenable to a combination with microfluidic chips or other integrated sensor platforms. These needs motivate the development of engineerable SERS substrate "chips" with defined near- and far-field responses. In this dissertation, two types of rationally designed SERS substrates - nanoparticle cluster arrays (NCAs) and SERS stamp - will be introduced and characterized. NCAs were fabricated through a newly developed template guided self-assembly fabrication approach, in which chemically synthesized nanoparticles are integrated into predefined patterns using a hybrid top-down/bottom-up approach. Since this method relies on chemically defined building blocks, it can overcome the resolution limit of conventional lithographical methods and facilitates higher structural complexity. NCAs sustain near-field interactions within individual clusters as well as between entire neighboring clusters and create a multi-scale cascaded E-field enhancement throughout the entire array. SERS stamps were generated using an oblique angle metal deposition on a lithographically defined piston. When mounted on a nanopositioning stage, the SERS stamps were enabled to contact biological surfaces with pristine nanostructured metal surfaces for a label-free spectroscopic characterization. The developed engineered substrates were applied and tested in critical sensing applications, including the ultra-trace detection of explosive vapors, the rapid discrimination of

  20. Electride Mediated Surface Enhanced Raman Scattering (SERS)

    NASA Technical Reports Server (NTRS)

    Anderson, Mark S. (Inventor)

    2016-01-01

    An electride may provide surface enhanced Raman scattering (SERS). The electride, a compound where the electrons serve as anions, may be a ceramic electride, such as a conductive ceramic derived from mayenite, or an organic electride, for example. The textured electride surface or electride particles may strongly enhance the Raman scattering of organic or other Raman active analytes. This may also provide a sensitive method for monitoring the chemistry and electronic environment at the electride surface. The results are evidence of a new class of polariton (i.e., a surface electride-polariton resonance mechanism) that is analogous to the surface plasmon-polariton resonance that mediates conventional SERS.

  1. [Computational analysis of a cys-loop ligand gated ion channel from the green alga Chlamydomonas reinhardtii].

    PubMed

    Mukherjee, Ashutosh

    2015-01-01

    Plants possess several neurotransmitters with well-known physiological roles. Currently only receptors for glutamate were reported to be found in plants, while receptors for acetylcholine, serotonin and GABA have not yet been reported. In animals, these neurotransmitters act via one class of ligand binding ion channels called Cys-loop receptors which play a major role in fast synaptic transmission. They show the presence of two domains namely Neurotransmitter-gated ion-channel ligand-binding domain (Pfam: PF02931) and Neurotransmitter-gated transmembrane domain (Pfam: PF02932). Cys-loop receptors are also known in prokaryotes. No cys-loop receptor has been characterized from plants yet. In this study, the Ensembl plants database was searched for proteins with these two domains in the sequenced plant genomes, what resulted in only one protein (LIC1) from the alga Chlamydomonas reinhardtii. BLAST and profile HMM searches against the pdb structure database showed that this protein is related to animal and prokaryotic cys-loop receptors, although the cysteine residues characteristic of the cys-loop are absent. Physico-chemical and sequence analysis indicate that LIC1 is an anionic receptor. A model of this protein was generated using homology modeling based on a nicotinic acetylcholine receptor of Torpedo marmorata. The characteristic extracellular domain (ECD) and transmembrane domain (TMD) are well structured but the intercellular region is poorly formed. This is the first report on a detailed characterization of a cys-loop receptor from the plant kingdom. PMID:26510602

  2. Molecular cloning and analysis of the regulation of cys-14/sup +/, a structural gene of the sulfur regulatory circuit of Neurospora crassa

    SciTech Connect

    Ketter, J.S.; Marzluf, G.A.

    1988-04-01

    The cys-14/sup +/ gene encodes sulfate permease II, which is primarily expressed in mycelia. cys-14/sup +/ is one of a set of sulfur-related structural genes under the control of cys-3/sup +/ and scon/sup +/, the regulatory genes of the sulfur control circuit. The authors cloned cys-14/sup +/ from a cosmid library of Neurospora crassa DNA. A restriction fragment length polymorphism analysis showed that this clone maps to the region of chromosome IV corresponding to the cys-14/sup +/ locus. Northern blot analyses were used to examine the regulated expression of the cys-14/sup +/ gene. In the wild type, a 3-kilobase cysc-14/sup +/ transcript was highly expressed under sulfur-derepressing conditions but completely absent during sulfur repression. A cys-3 mutant, which cannot synthesize any of the sulfur-controlled enzymes, including sulfate permease II, did not possess any cys-14/sup +/ transcript under wither condition. A cys-3 temperature-sensitive revertant completely lacked any cys-14/sup +/ mRNA at the conditional temperature but expressed the cys-14/sup +/ transcript upon derepression at the permissive temperature. Mutation of a second sulfur regulatory gene, scon/sup c/, causes the expression of sulfur-related enzymes in a constitutive fashion; the scon/sup c/ mutant showed a corresponding constitutive expression of cys-14/sup +/ mRNA, such that it was present even in cells subjected to sulfur repression conditions. These results show that the cys-14/sup +/ gene is regulated through the modulation of message content by the cys-3/sup +/ and scon/sup c/ control genes in response to the sulfur levels of the cells.

  3. New SERS Substrates For Polycyclic Aromatic Hydrocarbon (PAH) Detection: Towards Quantitative SERS Sensors For Environmental Analysis

    SciTech Connect

    Peron, O.; Rinnert, E.; Compere, C.; Toury, T.; Lamy de la Chapelle, M.

    2010-08-06

    In the investigation of chemical pollutions, such as PAHs (Polycyclic Aromatic Hydrocarbons) at low concentration in aqueous medium, surface-enhanced Raman scattering (SERS) stands for an alternative to the inherent low cross-section of normal Raman scattering. Indeed, SERS is a very sensitive spectroscopic technique due to the excitation of the surface plasmon modes of the nanostructured metallic film.

  4. Molecular characterization of antithrombin III (ATIII) variants using polymerase chain reaction. Identification of the ATIII Charleville as an Ala 384 Pro mutation.

    PubMed Central

    Molho-Sabatier, P; Aiach, M; Gaillard, I; Fiessinger, J N; Fischer, A M; Chadeuf, G; Clauser, E

    1989-01-01

    The genes of seven structural mutants of antithrombin III (ATIII), presenting either defective serine protease reactivity or abnormal heparin binding, were analyzed. The polymerase chain reaction (PCR) was used to amplify the corresponding gene exon and the mutation was identified by either dot blot analysis using a battery of allele-specific oligonucleotide probes or sequencing. Variants Paris and Paris 2 were identified as Arg 47 Cys mutations, and Clichy, Clichy 2, and Franconville were found to be Pro 41 Leu mutations. All five are heparin binding-site variants. ATIII Avranches is an Arg 393 His mutation and ATIII Charleville is an Ala 384 Pro mutation. These two mutations impair the reactive site of the molecule. ATIII Charleville is a new mutation of the reactive center, as predicted by previous biochemical data. The position of this new mutation, together with the other previously described mutations of the reactive center, sheds light on the molecular function of this site in inhibiting thrombin. Finally, genomic amplification by PCR is a powerful technique for the fast identification of antithrombin III mutations and their homozygous/heterozygous status, and should be useful for predicting thrombotic risk. Images PMID:2794060

  5. The detection of dust around NN Ser

    NASA Astrophysics Data System (ADS)

    Hardy, Adam; Schreiber, Matthias R.; Parsons, Steven G.; Caceres, Claudio; Brinkworth, Carolyn; Veras, Dimitri; Gänsicke, Boris T.; Marsh, Thomas R.; Cieza, Lucas

    2016-07-01

    Eclipse timing variations observed from the post-common-envelope binary (PCEB) NN Ser offer strong evidence in favour of circumbinary planets existing around PCEBs. If real, these planets may be accompanied by a disc of dust. We here present the ALMA detection of flux at 1.3 mm from NN Ser, which is likely due to thermal emission from a dust disc of mass ˜0.8 ± 0.2 M⊕. We performed simulations of the history of NN Ser to determine possible origins of this dust, and conclude that the most likely origin is, in fact, common-envelope material which was not expelled from the system and instead formed a circumbinary disc. These discs have been predicted by theory but previously remained undetected. While the presence of this dust does not prove the existence of planets around NN Ser, it adds credibility to the possibility of planets forming from common-envelope material in a `second-generation' scenario.

  6. Plasmonic SERS biosensing nanochips for DNA detection.

    PubMed

    Ngo, Hoan T; Wang, Hsin-Neng; Fales, Andrew M; Vo-Dinh, Tuan

    2016-03-01

    The development of rapid, cost-effective DNA detection methods for molecular diagnostics at the point-of-care (POC) has been receiving increasing interest. This article reviews several DNA detection techniques based on plasmonic-active nanochip platforms developed in our laboratory over the last 5 years, including the molecular sentinel-on-chip (MSC), the multiplex MSC, and the inverse molecular sentinel-on-chip (iMS-on-Chip). DNA probes were used as the recognition elements, and surface-enhanced Raman scattering (SERS) was used as the signal detection method. Sensing mechanisms were based on hybridization of target sequences and DNA probes, resulting in a distance change between SERS reporters and the nanochip's plasmonic-active surface. As the field intensity of the surface plasmon decays exponentially as a function of distance, the distance change in turn affects SERS signal intensity, thus indicating the presence and capture of the target sequences. Our techniques were single-step DNA detection techniques. Target sequences were detected by simple delivery of sample solutions onto DNA probe-functionalized nanochips and measuring the SERS signal after appropriate incubation times. Target sequence labeling or washing to remove unreacted components was not required, making the techniques simple, easy-to-use, and cost-effective. The usefulness of the nanochip platform-based techniques for medical diagnostics was illustrated by the detection of host genetic biomarkers for respiratory viral infection and of the dengue virus gene.

  7. SERS gene probe for DNA diagnostics

    NASA Astrophysics Data System (ADS)

    Stokes, David L.; Allain, Leonardo R.; Isola, Narayana R.; Vo-Dinh, Tuan

    2003-07-01

    We describe the development of a surface-enhanced Raman scattering gene (SERGen) probe technology for rapid screening for diseases and pathogens through DNA hybridization assays. The technology combines the use of gene probes labeled with SERS-active markers, and nanostructured metallic platforms for inducing the SERS effect. As a result, SERGen-based methods can offer the spectral selectivity and sensitivity of SERS as well as the molecular specificity of DNA sequence hybridization. Furthermore, these new probe s preclude the use of radioactive labels. As illustrated herein, SERGen probes have been used as primers in polymerase chain reaction (PCR) amplifications of specific DNA sequences, hence further boosting the sensitivity of the technology. We also describe several approaches to developing SERS-active DNA assay platforms, addressing the challenges of making the SERGen technology accessible and practical for clinical settings. The usefulness of the SERGen approach has been demonstrated in the detection of HIV, BRCA1 breast cancer, and BAX genes. There is great potential for the use of numerous SERGen probes for multiplexed detection of multiple biological targets.

  8. Plasmonic SERS biosensing nanochips for DNA detection.

    PubMed

    Ngo, Hoan T; Wang, Hsin-Neng; Fales, Andrew M; Vo-Dinh, Tuan

    2016-03-01

    The development of rapid, cost-effective DNA detection methods for molecular diagnostics at the point-of-care (POC) has been receiving increasing interest. This article reviews several DNA detection techniques based on plasmonic-active nanochip platforms developed in our laboratory over the last 5 years, including the molecular sentinel-on-chip (MSC), the multiplex MSC, and the inverse molecular sentinel-on-chip (iMS-on-Chip). DNA probes were used as the recognition elements, and surface-enhanced Raman scattering (SERS) was used as the signal detection method. Sensing mechanisms were based on hybridization of target sequences and DNA probes, resulting in a distance change between SERS reporters and the nanochip's plasmonic-active surface. As the field intensity of the surface plasmon decays exponentially as a function of distance, the distance change in turn affects SERS signal intensity, thus indicating the presence and capture of the target sequences. Our techniques were single-step DNA detection techniques. Target sequences were detected by simple delivery of sample solutions onto DNA probe-functionalized nanochips and measuring the SERS signal after appropriate incubation times. Target sequence labeling or washing to remove unreacted components was not required, making the techniques simple, easy-to-use, and cost-effective. The usefulness of the nanochip platform-based techniques for medical diagnostics was illustrated by the detection of host genetic biomarkers for respiratory viral infection and of the dengue virus gene. PMID:26547189

  9. Conversion of the agent-oriented domain-specific language ALAS into JavaScript

    NASA Astrophysics Data System (ADS)

    Sredojević, Dejan; Vidaković, Milan; Okanović, Dušan; Mitrović, Dejan; Ivanović, Mirjana

    2016-06-01

    This paper shows generation of JavaScript code from code written in agent-oriented domain-specific language ALAS. ALAS is an agent-oriented domain-specific language for writing software agents that are executed within XJAF middleware. Since the agents can be executed on various platforms, they must be converted into a language of the target platform. We also try to utilize existing tools and technologies to make the whole conversion process as simple as possible, as well as faster and more efficient. We use the Xtext framework that is compatible with Java to implement ALAS infrastructure - editor and code generator. Since Xtext supports Java, generation of Java code from ALAS code is straightforward. To generate a JavaScript code that will be executed within the target JavaScript XJAF implementation, Google Web Toolkit (GWT) is used.

  10. Bases in the anticodon loop of tRNA(Ala)(GGC) prevent misreading.

    PubMed

    Murakami, Hiroshi; Ohta, Atsushi; Suga, Hiroaki

    2009-04-01

    The bases at positions 32 and 38 in the tRNA anticodon loop are known to have a specific conservation depending upon the anticodon triplets. Here we report that evolutionarily conserved pairs of bases at positions 32 and 38 in tRNA(Ala)(GGC) prevent misreading of a near-cognate valine codon, GUC. The tRNA(Ala)(GGC) molecules with the conserved A32-U38 and C32-G38 pairs do not read GUC, whereas those with three representative nonconserved pairs, U32-U38, U32-A38 and C32-A38, direct the misincorporation of alanine at this valine codon into the peptide chain. Overexpression of the nonconserved tRNA(Ala)(GGC) in Escherichia coli is toxic and prevents cell growth. These results suggested that the bases at positions 32 and 38 in tRNA(Ala)(GGC) evolved to preserve the fidelity of the cognate codon reading.

  11. Latest results of 5-ALA-based fluorescence diagnosis and other medical disciplines

    NASA Astrophysics Data System (ADS)

    Baumgartner, Reinhold

    1999-02-01

    Preclinical and clinical studies on 5-aminolevulinic acid (5- ALA) induced Protoporphyrin IX (PPIX) are performed in various departments now following promising clinical results for the detection of bladder cancer in urology. This paper provides an overview on the progress of 5-ALA assisted fluorescence diagnosis in urology, pulmonology, neurosurgery, gynecology and ENT coordinated by the Laser Research Laboratory of the Ludwig-Maximilians-University in Munich. 5-ALA can be applied either topically or systematically to induce an intracellular accumulation of fluorescing PPIX. With appropriate dosage of 5-ALA, malignant tissue can be stained selectively, and irradiation with violet light excites a bright red fluorescence of the tumor visible with naked eyes. Optical properties of the tissue tend to hamper the precise identification and demarcation of suspect areas in fluorescence images. Multicolor remission and fluorescence imaging, therefore, should improve tumor localization in future.

  12. New developments in fluorescence detection of ALA-induced protoporphyrin IX for cancer localization

    NASA Astrophysics Data System (ADS)

    Stepp, Herbert G.; Baumgartner, Reinhold; Betz, Christian; Bise, Karl; Brand, P.; Gamarra, Fernando; Haeussinger, Karl; Hillemanns, Peter; Huber, Rudolf M.; Knuechel, Ruth; Kriegmair, M.; Leunig, Andreas; Pichler, J.; Rick, Kai; Schulz, H.; Stanzel, F.; Stocker, Susanne; Wagner, Simon; Weigandt, H.

    1997-12-01

    After the very promising clinical results for the detection of bladder cancer in urology, preclinical and clinical studies on aminolevulinic acid (5-ALA) induced protoporphyrin IX (PPIX) are preformed in various disciplines now. This paper provides a brief overview of the progress on 5-ALA assisted fluorescence diagnosis in urology, pulmonology, neurosurgery, gynecology and ENT performed in collaboration with the Laser Research Laboratory at the Department of Urology of the Ludwig-Maximilians-University in Munich. Five-ALA can be applied either topically or systemically to induce an intracellular accumulation of fluorescing PPIX. With appropriate dosage of 5-ALA, malignant tissue can be stained selectively, and irradiation with violet light excites a bright red fluorescence of the tumor. Optical properties of the tissue tend to hamper the precise identification and demarcation of suspect areas in fluorescence images. Multicolor remission and fluorescence imaging, therefore, seems to be indispensable for a reliable tumor localization.

  13. Characterization of plant sulfiredoxin and role of sulphinic form of 2-Cys peroxiredoxin

    PubMed Central

    Iglesias-Baena, Iván; Barranco-Medina, Sergio; Lázaro-Payo, Alfonso; López-Jaramillo, Francisco Javier; Sevilla, Francisca; Lázaro, Juan-José

    2010-01-01

    The antioxidant function of 2-Cys peroxiredoxin (Prx) involves the oxidation of its conserved peroxidatic cysteine to sulphenic acid that is recycled by a reductor agent. In conditions of oxidative stress, the peroxidatic cysteine can be overoxidized to sulphinic acid inactivating the Prx. An enzyme recently discovered, named sulfiredoxin (Srx), reduces the sulphinic 2-Cys Prx (Prx-SO2H). To explore the physiological functions of Srx in plants we have cloned, expressed and purified to homogeneity a Srx from Arabidopsis thaliana (AtSrx), as well as five variants by site-directed mutagenesis on amino acids involved in its activity. The activity of sulfiredoxin, determined by a new method, is dependent on the concentration of the sulphinic form of Prx and the conserved Srx is capable of regenerating the functionality of both pea and Arabidopsis Prx-SO2H. Molecular modelling of AtSrx and the facts that the R28Q variant shows a partial inactivation, that the activity of the E76A variant is equivalent to that of the native enzyme and that the double mutation R28Q/E76A abolishes the enzymatic activity suggests that the pair His100-Glu76 may be involved in the activation of C72 in the absence of R28. The knock-out mutant plants without Srx or 2-Cys Prx exhibited phenotypical differences under growth conditions of 16 h light, probably due to the signalling role of the sulphinic form of Prx. These mutants showed more susceptibility to oxidative stress than wild-type plants. This work presents the first systematic biochemical characterization of the Srx/Prx system from plants and contributes to a better understanding of its physiological function. PMID:20176891

  14. Overexpression of Hypo-Phosphorylated IκBβ at Ser313 Protects the Heart against Sepsis

    PubMed Central

    Liu, Ying-Ying; Wang, Li; Luo, Peng-Fei; Xia, Zhao-Fan

    2016-01-01

    IκBβis an inhibitor of nuclear factor kappa B(NF-κB) and participates in the cardiac response to sepsis. However, the role of the hypo-phosphorylated form of IκBβ at Ser313, which can be detected during sepsis, is unknown. Here, we examined the effects of IκBβ with a mutation at Ser313→Ala313 on cardiac damage induced by sepsis. Transgenic (Tg) mice were generated to overexpress IκBβ, in which Ser-313 is replaced with alanine ubiquitously, in order to mimic the hypo-phosphorylated form of IκBβ. Survival analysis showed that Tg mice exhibited decreased inflammatory cytokine levels and decreased rates of mortality in comparison to wild type (WT) mice, after sepsis in a cecal-ligation and puncture model (CLP). Compared to WT septic mice, sepsis in Tg mice resulted in improved cardiac functions, lower levels of troponin I and decreased rates of cardiomyocyte apoptosis, compared to WT mice. The increased formation of autophagicvacuoles detected with electron microscopy demonstrated the enhancement of cardiac autophagy. This phenomenon was further confirmed by the differential expression of genes related to autophagy, such as LC3, Atg5, Beclin-1, and p62. The increased expression of Cathepsin L(Ctsl), a specific marker for mitochondrial stress response, may be associated with the beneficial effects of the hypo-phosphorylated form of IκBβ. Our observations suggest that the hypo-phosphorylated form of IκBβ at Ser313 is beneficial to the heart in sepsis through inhibition of apoptosisand enhancement of autophagy in mutated IκBβ transgenic mice. PMID:27508931

  15. Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring*

    PubMed Central

    Li, He; Grigoryan, Hasmik; Funk, William E.; Lu, Sixin Samantha; Rose, Sherri; Williams, Evan R.; Rappaport, Stephen M.

    2011-01-01

    A method is described for profiling putative adducts (or other unknown covalent modifications) at the Cys34 locus of human serum albumin (HSA), which represents the preferred reaction site for small electrophilic species in human serum. By comparing profiles of putative HSA-Cys34 adducts across populations of interest it is theoretically possible to explore environmental causes of degenerative diseases and cancer caused by both exogenous and endogenous chemicals. We report a novel application of selected-reaction-monitoring (SRM) mass spectrometry, termed fixed-step SRM (FS-SRM), that allows detection of essentially all HSA-Cys34 modifications over a specified range of mass increases (added masses). After tryptic digestion, HSA-Cys34 adducts are contained in the third largest peptide (T3), which contains 21 amino acids and an average mass of 2433.87 Da. The FS-SRM method does not require that exact masses of T3 adducts be known in advance but rather uses a theoretical list of T3-adduct m/z values separated by a fixed increment of 1.5. In terms of added masses, each triply charged parent ion represents a bin of ±2.3 Da between 9.1 Da and 351.1 Da. Synthetic T3 adducts were used to optimize FS-SRM and to establish screening rules based upon selected b- and y-series fragment ions. An isotopically labeled T3 adduct is added to protein digests to facilitate quantification of putative adducts. We used FS-SRM to generate putative adduct profiles from six archived specimens of HSA that had been pooled by gender, race, and smoking status. An average of 66 putative adduct hits (out of a possible 77) were detected in these samples. Putative adducts covered a wide range of concentrations, were most abundant in the mass range below 100 Da, and were more abundant in smokers than in nonsmokers. With minor modifications, the FS-SRM methodology can be applied to other nucleophilic sites and proteins. PMID:21193536

  16. First principles predictions of intrinsic defects in aluminum arsenide, AlAs : numerical supplement.

    SciTech Connect

    Schultz, Peter Andrew

    2012-04-01

    This Report presents numerical tables summarizing properties of intrinsic defects in aluminum arsenide, AlAs, as computed by density functional theory. This Report serves as a numerical supplement to the results published in: P.A. Schultz, 'First principles predictions of intrinsic defects in Aluminum Arsenide, AlAs', Materials Research Society Symposia Proceedings 1370 (2011; SAND2011-2436C), and intended for use as reference tables for a defect physics package in device models.

  17. Treating cutaneous squamous cell carcinoma using ALA PLGA nanoparticle-mediated photodynamic therapy in a mouse model

    NASA Astrophysics Data System (ADS)

    Wang, Xiaojie; Shi, Lei; Tu, Qingfeng; Wang, Hongwei; Zhang, Haiyan; Wang, Peiru; Zhang, Linglin; Huang, Zheng; Wang, Xiuli; Zhao, Feng; Luan, Hansen

    2015-03-01

    Background: Squamous cell carcinoma (SCC) is a common skin cancer and its treatment is still difficult. The aim of this study was to evaluate the effectiveness of nanoparticle (NP)-assisted ALA delivery for topical photodynamic therapy (PDT) of cutaneous SCC. Methods: UV-induced cutaneous SCCs were established in hairless mice. ALA loaded polylactic-co-glycolic acid (PLGA) NPs were prepared and characterized. The kinetics of ALA PLGA NPs-induced protoporphyrin IX (PpIX) fluorescence in SCCs, therapeutic efficacy of ALA NP-mediated PDT, and immune responses were examined. Results: PLGA NPs could enhance PpIX production in SCC. ALA PLGA NP mediated topical PDT was more effective than free ALA of the same concentration in treating cutaneous SCC. Conclusion: PLGA NPs provide a promising strategy for delivering ALA in topical PDT of cutaneous SCC.

  18. Whole bladder wall photodynamic therapy using 5-ALA: an experimental study in pigs

    NASA Astrophysics Data System (ADS)

    van Staveren, Hugo J.; Beek, Johan F.; Verlaan, Cess W.; Edixhoven, Annie; Saarnak, Anne E.; Sterenborg, Dick; de Reijke, Theo M.; de la Riviere, Guy B.; Thomsen, Sharon L.; van Gemert, Martin J. C.; Star, Willem M.

    1996-01-01

    The agent 5-aminolevulinic acid (5-ALA) can be an alternative drug in whole bladder wall (WBW) photodynamic therapy (PDT), as its good tumor selectivity and the short time skin photosensitivity after systemic administration are advantageous for clinical use. To determine the maximum drug and light doses for reversible normal tissue damage, a pre-clinical study was performed using an in vivo normal piglet bladder model. First, the kinetics of PpIX production in 2 pigs was determined in vitro after oral administration of 75 and 150 mg/kg ALA respectively. The concentration of PpIX in plasma, and erythrocytes was determined by reversed phase high-performance liquid chromatography (HPLC) and the maximum was reached at approximately equals 5 hours after the administration of ALA. This provided a guideline for the optimum interval between ALA administration and light application. Next, various ALA doses were either administered orally or instilled in the bladder and different light doses were applied. Bladder biopsies were taken at regular intervals and normal tissue damage was investigated histologically. Reversible tissue damage was obtained using 60 mg/kg of 5-ALA in combination with a light dose of 100 J cm-2 (non-scattered plus scattered 630 nm wavelength light) in the case of oral administration. In the case of intravesical instillation, a drug dose of 2.5 gram and a light dose of 100 J cm-2 are still too high to obtain reversible tissue damage.

  19. Topical application of ALA PDT for the treatment of moderate to severe acne vulgaris

    NASA Astrophysics Data System (ADS)

    Wang, Xiu-Li; Wang, Hong-Wei; Zhang, Ling-Lin; Su, Lina; Guo, Ming-Xia; Huang, Zheng

    2009-06-01

    Objectives: To evaluate the effectiveness of topical 5-aminolevulinic acid (ALA)- medicated photodynamic therapy (ALA PDT) for the treatment of moderate to severe acne vulgaris. Methods: Sixteen Chinese patients with moderate to severe facial acne were treated with 1-3 courses of ALA PDT. ALA cream (3%) was freshly prepared and applied to acne lesions for 3-4 h. The lesions were irradiated by a 635 nm diode laser at dose levels of 60 - 80 J/cm2 at 100 mW/cm2. Clinical assessments were conducted before and after treatment up to 3 months. Results: All patents showed response to ALA PDT. Complete clearance was seen in 10 patients (62.5%) and partial clearance in 6 patients (37.5%). One case showed recurrence after complete clearance at 2 months and another two showed recurrence after complete clearance at 3 months. However, the number of new lesions were significantly reduced. Adverse effects were minimal. Conclusions: The results of this preliminary clinical study is encouraging. ALA PDT is a simple, safe and useful therapeutic option for the treatment of moderate to severe acne. Further studies to evaluate the treatment with a larger number of patients and for a longer period of follow-up are needed.

  20. ALA-based photodynamic therapy in epithelial tumors: in vivo and in vitro models

    NASA Astrophysics Data System (ADS)

    Casas, Adriana; Fukuda, Haydee; Batlle, Alcira

    2000-03-01

    PDT shows considerable potential as a treatment modality for superficial tumors. PDT is based on the accumulation of a photosensitizer in the target tissue. Subsequent illumination with light of an appropriate wavelength provokes a photochemical reaction that results in tumor destruction. Aminolevulinic acid (ALA) is a porphyrin precursor, and its administration result in the endogenous production of phototoxic porphyrins, which has been exploited for PDT. We assessed PDT efficacy employing both in vivo and in vitro models. We used papillomas, keratoacanthomas and in situ carcinomas chemically induced in the skin of SENCAR mice. Using ALA lotion and cream formulations, the maximal amount of porphyrin accumulation in papillomas was 5.52 (mu) g/g tissue. An energy of 150 of J/cm2 was delivered by a copper-dye laser tuned at 630 nm. Microscopically, we found several signs of tissue destruction, more markedly in the upper strata of the in situ carcinomas. Papillomas, characterized by hyperkeratinization, were resistant to PDT. In our in vitro studies, we used an epithelial adenocarcinoma cell line. We tested ALA and its hexyl and methyl derivatives with the aim of increasing porphyrin synthesis. We found that hexyl-ALA was the best compound. When cultures incubated 3 hours in 0.6 mM ALA and 0.1 mM hexyl-ALA respectively were irradiated with 3 J/cm2 only 5 percent of cells survived.

  1. The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants.

    PubMed

    Liang, Zhe; Demko, Viktor; Wilson, Robert C; Johnson, Kenneth A; Ahmad, Rafi; Perroud, Pierre-François; Quatrano, Ralph; Zhao, Sen; Shalchian-Tabrizi, Kamran; Otegui, Marisa S; Olsen, Odd-Arne; Johansen, Wenche

    2013-09-01

    DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML-CysPc-C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc-C2L domains of land plant calpains form a separate sub-clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1-like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1-3 mutant using CysPc-C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc-C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1-3 mutant phenotype. In contrast, neither the CysPc-C2L domains from M. viride nor chimeric animal-plant calpains complement this mutant. Co-evolution analysis identified differences in the interactions between the CysPc-C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1-3 complementation assay, we show that four conserved amino acid residues of two Ca²⁺-binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1. PMID:23663131

  2. The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants.

    PubMed

    Liang, Zhe; Demko, Viktor; Wilson, Robert C; Johnson, Kenneth A; Ahmad, Rafi; Perroud, Pierre-François; Quatrano, Ralph; Zhao, Sen; Shalchian-Tabrizi, Kamran; Otegui, Marisa S; Olsen, Odd-Arne; Johansen, Wenche

    2013-09-01

    DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML-CysPc-C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc-C2L domains of land plant calpains form a separate sub-clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1-like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1-3 mutant using CysPc-C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc-C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1-3 mutant phenotype. In contrast, neither the CysPc-C2L domains from M. viride nor chimeric animal-plant calpains complement this mutant. Co-evolution analysis identified differences in the interactions between the CysPc-C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1-3 complementation assay, we show that four conserved amino acid residues of two Ca²⁺-binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.

  3. Antiproliferative effect of T/Tn specific Artocarpus lakoocha agglutinin (ALA) on human leukemic cells (Jurkat, U937, K562) and their imaging by QD-ALA nanoconjugate.

    PubMed

    Chatterjee, Urmimala; Bose, Partha Pratim; Dey, Sharmistha; Singh, Tej P; Chatterjee, Bishnu P

    2008-11-01

    T/Tn specificity of Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha (Moraceae) fruit and a heterodimer (16 kD and 12 kD) of molecular mass 28 kD, was further confirmed by SPR analysis using T/Tn glycan containing mammalian glycoproteins. N-terminal amino acid sequence analysis of ALA showed homology at 15, 19-21, 24-27, and 29 residues with other lectin members of Moraceae family viz., Artocarpus integrifolia (jacalin) lectin, Artocarpus hirsuta lectin, and Maclura pomifera agglutinin. It is mitogenic to human PBMC and the maximum proliferation was observed at 1 ng/ml. It showed an antiproliferative effect on leukemic cells, with the highest effect toward Jurkat cells (IC(50) 13.15 ng/ml). Synthesized CdS quantum dot-ALA nanoconjugate was employed to detect the expression of T/Tn glycans on Jurkat, U937, and K562 leukemic cells surfaces as well as normal lymphocytes by fluorescence microscopy. No green fluorescence was observed with normal lymphocytes indicating that T/Tn determinants, which are recognized as human tumor associated structures were cryptic on normal lymphocyte surfaces, whereas intense green fluorescent dots appeared during imaging of leukemic cells, where such determinants were present in unmasked form. The above results indicated that QD-ALA nanoconjugate is an efficient fluorescent marker for identification of leukemic cell lines that gives rise to high quality images.

  4. The Thr- and Ala-rich hyperactive antifreeze protein from inchworm folds as a flat silk-like β-helix.

    PubMed

    Lin, Feng-Hsu; Davies, Peter L; Graham, Laurie A

    2011-05-31

    Inchworm larvae of the pale beauty geometer moth, Campaea perlata, exhibit strong (6.4 °C) freezing point depression activity, indicating the presence of hyperactive antifreeze proteins (AFPs). We have purified two novel Thr- and Ala-rich AFPs from the larvae as small (∼3.5 kDa) and large (∼8.3 kDa) variants and have cloned the cDNA sequences encoding both. They have no homology to known sequences in current BLAST databases. However, these proteins and the newly characterized AFP from the Rhagium inquisitor beetle both contain stretches rich in alternating Thr and Ala residues. On the basis of these repeats, as well as the discontinuities between them, a detailed structural model is proposed for the 8.3 kDa variant. This 88-residue protein is organized into an extended parallel-stranded β-helix with seven strands connected by classic β-turns. The alternating β-strands form two β-sheets with a thin core composed of interdigitating Ala and Ser residues, similar to the thin hydrophobic core proposed for some silks. The putative ice-binding face of the protein has a 4 × 5 regular array of Thr residues and is remarkably flat. In this regard, it resembles the nonhomologous Thr-rich AFPs from other moths and some beetles, which contain two longer rows of Thr in contrast to the five shorter rows in the inchworm protein. Like that of some other hyperactive AFPs, the spacing between these ice-binding Thr residues is a close match to the spacing of oxygen atoms on several planes of ice. PMID:21486083

  5. Functional and Biochemical Characterization of Alvinella pompejana Cys-Loop Receptor Homologues

    PubMed Central

    Debaveye, Sarah; Brams, Marijke; Pardon, Els; Willegems, Katrien; Bertrand, Daniel; Steyaert, Jan; Efremov, Rouslan; Ulens, Chris

    2016-01-01

    Cys-loop receptors are membrane spanning ligand-gated ion channels involved in fast excitatory and inhibitory neurotransmission. Three-dimensional structures of these ion channels, determined by X-ray crystallography or electron microscopy, have revealed valuable information regarding the molecular mechanisms underlying ligand recognition, channel gating and ion conductance. To extend and validate the current insights, we here present promising candidates for further structural studies. We report the biochemical and functional characterization of Cys-loop receptor homologues identified in the proteome of Alvinella pompejana, an extremophilic, polychaete annelid found in hydrothermal vents at the bottom of the Pacific Ocean. Seven homologues were selected, named Alpo1-7. Five of them, Alpo2-6, were unidentified prior to this study. Two-electrode voltage clamp experiments revealed that wild type Alpo5 and Alpo6, both sharing remarkably high sequence identity with human glycine receptor α subunits, are anion-selective channels that can be activated by glycine, GABA and taurine. Furthermore, upon expression in insect cells fluorescence size-exclusion chromatography experiments indicated that four homologues, Alpo1, Alpo4, Alpo6 and Alpo7, can be extracted out of the membrane by a wide variety of detergents while maintaining their oligomeric state. Finally, large-scale purification efforts of Alpo1, Alpo4 and Alpo6 resulted in milligram amounts of biochemically stable and monodisperse protein. Overall, our results establish the evolutionary conservation of glycine receptors in annelids and pave the way for future structural studies. PMID:26999666

  6. Aminoacylation of an unusual tRNA(Cys) from an extreme halophile.

    PubMed

    Evilia, Caryn; Ming, Xiaotian; DasSarma, Shiladitya; Hou, Ya-Ming

    2003-07-01

    The extreme halophile Halobacterium species NRC-1 overcomes external near-saturating salt concentrations by accumulating intracellular salts comparable to those of the medium. This raises the fundamental question of how halophiles can maintain the specificity of protein-nucleic acid interactions that are particularly sensitive to high salts in mesophiles. Here we address the specificity of the essential aminoacylation reaction of the halophile, by focusing on molecular recognition of tRNA(Cys) by the cognate cysteinyl-tRNA synthetase. Despite the high salt environments of the aminoacylation reaction, and despite an unusual structure of the tRNA with an exceptionally large dihydrouridine loop, we show that aminoacylation of the tRNA proceeds with a catalytic efficiency similar to that of its mesophilic counterparts. This is manifested by an essentially identical K(m) for tRNA to those of the mesophiles, and by recognition of the same nucleotide determinants that are conserved in evolution. Interestingly, aminoacylation of the halophile tRNA(Cys) is more closely related to that of bacteria than eukarya by placing a strong emphasis on features of the tRNA tertiary core. This suggests an adaptation to the highly negatively charged tRNA sugar-phosphate backbone groups that are the key elements of the tertiary core.

  7. Functional and Biochemical Characterization of Alvinella pompejana Cys-Loop Receptor Homologues.

    PubMed

    Wijckmans, Eveline; Nys, Mieke; Debaveye, Sarah; Brams, Marijke; Pardon, Els; Willegems, Katrien; Bertrand, Daniel; Steyaert, Jan; Efremov, Rouslan; Ulens, Chris

    2016-01-01

    Cys-loop receptors are membrane spanning ligand-gated ion channels involved in fast excitatory and inhibitory neurotransmission. Three-dimensional structures of these ion channels, determined by X-ray crystallography or electron microscopy, have revealed valuable information regarding the molecular mechanisms underlying ligand recognition, channel gating and ion conductance. To extend and validate the current insights, we here present promising candidates for further structural studies. We report the biochemical and functional characterization of Cys-loop receptor homologues identified in the proteome of Alvinella pompejana, an extremophilic, polychaete annelid found in hydrothermal vents at the bottom of the Pacific Ocean. Seven homologues were selected, named Alpo1-7. Five of them, Alpo2-6, were unidentified prior to this study. Two-electrode voltage clamp experiments revealed that wild type Alpo5 and Alpo6, both sharing remarkably high sequence identity with human glycine receptor α subunits, are anion-selective channels that can be activated by glycine, GABA and taurine. Furthermore, upon expression in insect cells fluorescence size-exclusion chromatography experiments indicated that four homologues, Alpo1, Alpo4, Alpo6 and Alpo7, can be extracted out of the membrane by a wide variety of detergents while maintaining their oligomeric state. Finally, large-scale purification efforts of Alpo1, Alpo4 and Alpo6 resulted in milligram amounts of biochemically stable and monodisperse protein. Overall, our results establish the evolutionary conservation of glycine receptors in annelids and pave the way for future structural studies. PMID:26999666

  8. Binding of Ni2+ and Cu2+ ions to peptides with a Cys-His motif.

    PubMed

    Kulon, Kinga; Valensin, Daniela; Kamysz, Wojciech; Nadolny, Rafał; Gaggelli, Elena; Valensin, Gianni; Kozłowski, Henryk

    2008-10-21

    Waglerin I is a 22 amino acid snake venom toxin. Its three fragments (GGKPDLRPCHP-NH2, PCHYIPRPKPR-NH2, PCHPPCHYIPR-NH2), due to the presence of two Cys and His residues, are potentially very attractive ligands for transition metal ions. The main aim of this work was to establish the impact of these two adjacent residues on Ni2+ ion binding, especially because this kind of motif is very common in nature, and the study of low molecular weight models could be helpful in understanding larger systems. In this work waglerin fragments and their N-protected analogues were studied with Ni2+ (and Cu2+ for peptides with disulfide bridges) ions using combined potentiometric and spectroscopic measurements (UV-Vis, CD, EPR and NMR). In all peptides, except PCHPPCHYIPR-NH2 with a disulfide bridge, the Cys-His motif was found to be crucial for the coordination of Ni2+ ions. In the case of the N-unprotected analogues, the N-terminal amino group participates in the coordination as well. PMID:18827939

  9. The enzymology of alanine aminotransferase (AlaAT) isoforms from Hordeum vulgare and other organisms, and the HvAlaAT crystal structure.

    PubMed

    Duff, Stephen M G; Rydel, Timothy J; McClerren, Amanda L; Zhang, Wenlan; Li, Jimmy Y; Sturman, Eric J; Halls, Coralie; Chen, Songyang; Zeng, Jiamin; Peng, Jiexin; Kretzler, Crystal N; Evdokimov, Artem

    2012-12-01

    In this paper we describe the expression, purification, kinetics and biophysical characterization of alanine aminotransferase (AlaAT) from the barley plant (Hordeum vulgare). This dimeric PLP-dependent enzyme is a pivotal element of several key metabolic pathways from nitrogen assimilation to carbon metabolism, and its introduction into transgenic plants results in increased yield. The enzyme exhibits a bi-bi ping-pong reaction mechanism with a K(m) for alanine, 2-oxoglutarate, glutamate and pyruvate of 3.8, 0.3, 0.8 and 0.2 mM, respectively. Barley AlaAT catalyzes the forward (alanine-forming) reaction with a k(cat) of 25.6 s(-1), the reverse (glutamate-forming) reaction with k(cat) of 12.1 s(-1) and an equilibrium constant of ~0.5. The enzyme is also able to utilize aspartate and oxaloacetate with ~10% efficiency as compared to the native substrates, which makes it much more specific than related bacterial/archaeal enzymes (that also have lower K(m) values). We have crystallized barley AlaAT in complex with PLP and l-cycloserine and solved the structure of this complex at 2.7 Å resolution. This is the first example of a plant AlaAT structure, and it reveals a canonical aminotransferase fold similar to structures of the Thermotoga maritima, Pyrococcus furiosus, and human enzymes. This structure bridges our structural understanding of AlaAT mechanism between three kingdoms of life and allows us to shed some light on the specifics of the catalysis performed by these proteins. PMID:22750542

  10. The enzymology of alanine aminotransferase (AlaAT) isoforms from Hordeum vulgare and other organisms, and the HvAlaAT crystal structure.

    PubMed

    Duff, Stephen M G; Rydel, Timothy J; McClerren, Amanda L; Zhang, Wenlan; Li, Jimmy Y; Sturman, Eric J; Halls, Coralie; Chen, Songyang; Zeng, Jiamin; Peng, Jiexin; Kretzler, Crystal N; Evdokimov, Artem

    2012-12-01

    In this paper we describe the expression, purification, kinetics and biophysical characterization of alanine aminotransferase (AlaAT) from the barley plant (Hordeum vulgare). This dimeric PLP-dependent enzyme is a pivotal element of several key metabolic pathways from nitrogen assimilation to carbon metabolism, and its introduction into transgenic plants results in increased yield. The enzyme exhibits a bi-bi ping-pong reaction mechanism with a K(m) for alanine, 2-oxoglutarate, glutamate and pyruvate of 3.8, 0.3, 0.8 and 0.2 mM, respectively. Barley AlaAT catalyzes the forward (alanine-forming) reaction with a k(cat) of 25.6 s(-1), the reverse (glutamate-forming) reaction with k(cat) of 12.1 s(-1) and an equilibrium constant of ~0.5. The enzyme is also able to utilize aspartate and oxaloacetate with ~10% efficiency as compared to the native substrates, which makes it much more specific than related bacterial/archaeal enzymes (that also have lower K(m) values). We have crystallized barley AlaAT in complex with PLP and l-cycloserine and solved the structure of this complex at 2.7 Å resolution. This is the first example of a plant AlaAT structure, and it reveals a canonical aminotransferase fold similar to structures of the Thermotoga maritima, Pyrococcus furiosus, and human enzymes. This structure bridges our structural understanding of AlaAT mechanism between three kingdoms of life and allows us to shed some light on the specifics of the catalysis performed by these proteins.

  11. Alpha linolenic acid (ALA) from Rosa canina, sacha inchi and chia oils may increase ALA accretion and its conversion into n-3 LCPUFA in diverse tissues of the rat.

    PubMed

    Valenzuela B, Rodrigo; Barrera R, Cynthia; González-Astorga, Marcela; Sanhueza C, Julio; Valenzuela B, Alfonso

    2014-07-25

    Alpha-linolenic acid (ALA) is an essential n-3 PUFA; its n-3 LCPUFA derivatives EPA and DHA, which have diverse beneficial effects, are scarce in the human diet. In recent years nontraditional vegetable oils rich in ALA (up to 45%) have been developed as new alternatives to increase ALA consumption. This work evaluated the accretion of ALA, EPA and DHA into the phospholipids extracted from erythrocytes, liver, kidney, small intestine, heart, quadriceps and the brain in rats fed sunflower (SFO), canola (CO), Rosa canina (RCO), sacha inchi (Plukenetia volubilis, SIO) and chia (Salvia hispánica, ChO) oils. Five experimental groups (n = 12 per group) were fed for 21 days with SFO (1% ALA), CO (10% ALA), RCO (33% ALA), SIO (49% ALA), and ChO (64% ALA). SIO and ChO allowed higher ALA accretion in all tissues, except the brain, and a reduction in the content of arachidonic acid in all tissues except the brain. EPA was increased in erythrocytes, liver, kidney, small intestine, heart and quadriceps, but not in the brain. DHA was increased in the liver, small intestine and brain tissues. Our results demonstrate that ALA, when provided in significant amounts, can be converted into n-3 LCPUFA, mostly DHA in the liver and brain. It is suggested that oils rich in ALA, such as SIO and ChO, are good sources for obtaining higher tissue levels of ALA, also allowing its selective conversion into n-3 LCPUFA in some tissues of the rat. PMID:24855655

  12. Functionalized paper SERS (P-SERS) substrates for selective targeting of analytes in complex samples

    NASA Astrophysics Data System (ADS)

    Yu, Wei W.; Hoppmann, Eric P.

    2015-05-01

    Surface enhanced Raman spectroscopy (SERS) requires the analyte molecule to be close to the plasmonic surface in order to generate SERS enhancement. This limitation restricts the practical application of SERS to molecules that possess functional groups that interact strongly with gold or silver surfaces. Moreover, the identification of target analytes in a complex sample matrix is made even more difficult when interferents compete with the target for binding to the plasmonic surface, resulting in overlapping spectral signatures. In this work, we report a strategy to functionalize inkjet printed P-SERS substrates by strategically placing supramolecular structures (such as nucleic acid aptamers) onto the gold nanoparticles. This promotes the selective interaction of target molecules with the plasmonic surface, leading to improved sensor performance.

  13. Periodic nanostructures for SERS-active substrate

    NASA Astrophysics Data System (ADS)

    Zou, Wenlong; Cai, Zhijian; Wu, Jianhong

    2015-08-01

    Raman spectroscopy has become a very important tool in many fields, including chemistry, material, environmental protecting and etc. While Raman scattering is a weak process, it is often hampered in background noise. Since surface enhanced Raman scattering (SERS) was observed for the first time in 1974, surface enhanced Raman spectroscopy has developed more and more rapidly. Periodic nanostructures have the advantage of reliable and reproducible Raman signal relative to the irregular nanostructures. In this paper, holographic gratings coated by metal are proposed to fabricate SERS-active substrate. The excitation light excite on the gratings with proper grating constant that will lead to plasmon resonant phenomena, which enhance local electric field for Raman scattering. The electric field enhancement of sinusoidal, rectangular and triangular gratings is simulated using a commercially available software finite-difference-time-domain (FDTD). Period, groove depth, duty cycle and coated metal thickness of gratings affecting the electric field enhancement are also discussed. The optimum grating structure for SERS-active substrate is obtained in theory.

  14. Nano-Enabled SERS Reporting Photosensitizers

    PubMed Central

    Farhadi, Arash; Roxin, Áron; Wilson, Brian C.; Zheng, Gang

    2015-01-01

    To impart effective cellular damage via photodynamic therapy (PDT), it is vital to deliver the appropriate light dose and photosensitizer concentration, and to monitor the PDT dose delivered at the site of interest. In vivo monitoring of photosensitizers has in large part relied on their fluorescence emission. Palladium-containing photosensitizers have shown promising clinical results by demonstrating near full conversion of light to PDT activity at the cost of having undetectable fluorescence. We demonstrate that, through the coupling of plasmonic nanoparticles with palladium-photosensitizers, surface-enhanced Raman scattering (SERS) provides both reporting and monitoring capability to otherwise quiescent molecules. Nano-enabled SERS reporting of photosensitizers allows for the decoupling of the therapeutic and imaging mechanisms so that both phenomena can be optimized independently. Most importantly, the design enables the use of the same laser wavelength to stimulate both the PDT and imaging features, opening the potential for real-time dosimetry of photosensitizer concentration and PDT dose delivery by SERS monitoring. PMID:25767614

  15. A water-soluble and retrievable ruthenium-based probe for colorimetric recognition of Hg(II) and Cys

    NASA Astrophysics Data System (ADS)

    Cui, Yali; Hao, Yuanqiang; Zhang, Yintang; Liu, Baoxia; Zhu, Xu; Qu, Peng; Li, Deliang; Xu, Maotian

    2016-08-01

    A new ruthenium-based complex 1 [(bis(4,4‧-dimethylphosphonic-2,2‧-bipyridine) dithiocyanato ruthenium (II))] was developed as a colorimetric probe for the detection of Hg(II) and Cys (Cysteine). The obtained compound 1 can give interconversional color changes upon the alternating addition of Hg(II) and Cys in 100% aqueous solution. The specific coordination between NCS groups with Hg(II) can lead to the formation of 1-Hg2 + complex, which can induce a remarkable spectral changes of probe 1. Afterwards the formed 1-Hg2 + complex can act as effective colorimetric sensor for Cys. Owing to the stronger binding affinity of sulfhydryl group to Hg2 +, Cys can extract Hg2 + from 1-Hg2 + complex resulting in the release of 1 and the revival of absorption profile of the probe 1. By introducing the hydrophilic phosphonic acid groups, the proposed probe exhibited excellent water solubility. The limits of detection (LODs) of the assay for Hg2 + and Cys are calculated to be 15 nM and 200 nM, respectively.

  16. The 'Pro-Drug' RibCys Decreases The Mutagenicity of High LET Radiation in Cultured Mammalian Cells

    NASA Technical Reports Server (NTRS)

    Lenarczyk, M.; Ueno, A.; Vannais, D. B.; Kraemer, S.; Kronenberg, A.; Roberts, J. C.; Tatsumi, K.; Hei, T. K.; Waldren, C. A.

    2000-01-01

    We have initiated studies aimed at reducing the mutational effects of high LET radiation such as Fe-56 ions and C-12 ions with certain drugs. The mutagenicity of high LET (143 keV/micrometer) Fe-56 or C-12 ions (LET = 100 keV/micrometer) was quantified at the CD59 locus of human-hamster hybrid AL cells. RibCys [2,S)-D-ribo-(1',2',3',4'- Tetrahydroxybutyl)-thiazolidine-4(R)-ca riboxylic acid], formed by condensation of L-cysteine with D-ribose, is designed so that the sulfhydryl amino acid L-cysteine is released intracellularly via nonenzymatic ring opening and hydrolysis leading to increased levels of glutathione (GSH). RibCys (4 or 10 mM), present during irradiation and a few hours post-irradiation, significantly decreased the yield of CD59(-) mutants induced by radiation. RibCys did not affect the clonogenic survival of irradiated cells, nor was it mutagenic itself. These results, together with the minimal side effects reported in mice and pigs, indicate that RibCys may be useful, perhaps even when used prophylactically, in reducing the load of mutations created by high LET radiation in astronauts or other exposed individuals. RibCys is an attractive drug that may reduce the risk of carcinogenesis in people exposed to high LET radiation.

  17. Phosphorylation of Tau at Thr212, Thr231, and Ser262 Combined Causes Neurodegeneration*

    PubMed Central

    Alonso, Alejandra D.; Di Clerico, John; Li, Bin; Corbo, Christopher P.; Alaniz, Maria E.; Grundke-Iqbal, Inge; Iqbal, Khalid

    2010-01-01

    Abnormal hyperphosphorylation of the microtubule-associated protein Tau is a hallmark of Alzheimer disease and related diseases called tauopathies. As yet, the exact mechanism by which this pathology causes neurodegeneration is not understood. The present study provides direct evidence that Tau abnormal hyperphosphorylation causes its aggregation, breakdown of the microtubule network, and cell death and identifies phosphorylation sites involved in neurotoxicity. We generated pseudophosphorylated Tau proteins by mutating Ser/Thr to Glu and, as controls, to Ala. These mutations involved one, two, or three pathological phosphorylation sites by site-directed mutagenesis using as backbones the wild type or FTDP-17 mutant R406W Tau. Pseudophosphorylated and corresponding control Tau proteins were expressed transiently in PC12 and CHO cells. We found that a single phosphorylation site alone had little influence on the biological activity of Tau, except Thr212, which, upon mutation to Glu in the R406W background, induced Tau aggregation in cells, suggesting phosphorylation at this site along with a modification on the C-terminal of the protein facilitates self-assembly of Tau. The expression of R406W Tau pseudophosphorylated at Thr212, Thr231, and Ser262 triggered caspase-3 activation in as much as 85% of the transfected cells, whereas the corresponding value for wild type pseudophosphorylated Tau was 30%. Cells transfected with pseudophosphorylated Tau became TUNEL-positive. PMID:20663882

  18. Crystallization of tholeiitic basalt in Alae Lava Lake, Hawaii

    USGS Publications Warehouse

    Peck, D.L.; Wright, T.L.; Moore, J.G.

    1966-01-01

    The eruption of Kilauea Volcano August 21-23, 1963, left 600,000 cubic meters of basaltic lava in a lava lake as much as 15 meters deep in Alae pit crater. Field studies of the lake began August 27 and include repeated core drilling, measurements of temperature in the crust and melt, and precise level surveys of the lake surface. The last interstitial melt in the lake solidified late in September 1964; by mid August 1965 the maximum temperature was 690??C at a depth of 11.5 meters. Pumice air-quenched from about 1140??C contains only 5 percent crystals - clinopyroxene, cuhedral olivine (Fo 80), and a trace of plagioclase, (An 70). Drill cores taken from the zone of crystallization in the lake show that olivine continued crystallizing to about 1070??C; below that it reacts with the melt, becoming corroded and mantled by pyroxene and plagioclase. Below 1070??C, pyroxene and plagioclase crystallized at a constant ratio. Ilmenite first appeared at about 1070??C and was joined by magnetite at about 1050??C; both increased rapidly in abundance to 1000??C. Apatite first appeared as minute needles in interstitial glass at 1000??C. Both the abundance and index of refraction of glass quenched from melt decreased nearly linearly with falling temperature. At 1070??C the quenched lava contains about 65 percent dark-brown glass with an index of 1.61; at 980??C it contains about 8 percent colorless glass with an index of 1.49. Below 980??C, the percentage of glass remained constant. Progressive crystallization forced exsolution of gases from the melt fraction; these formed vesicles and angular pores, causing expansion of the crystallizing lava and lifting the surface of the central part of the lake an average of 19.5 cm. The solidified basalt underwent pneumatolitic alteration, including deposition of cristobalite at 800??C, reddish alteration of olivine at 700??C, tarnishing of ilmenite at 550??C, deposition of anhydrite at 250??C, and deposition of native sulfur at 100??C

  19. PPARgamma Pro12Ala polymorphism in HIV-1-infected patients with HAART-related lipodystrophy.

    PubMed

    Saumoy, Maria; Veloso, Sergi; Alonso-Villaverde, Carlos; Domingo, Pere; Chacón, Matilde R; Miranda, Merce; Aragonès, Gerard; Gutiérrez, Maria Mar; Viladés, Consuelo; Peraire, Joaquim; Sirvent, Joan-Josep; López-Dupla, Miguel; Aguilar, Carmen; Richart, Cristóbal; Vidal, Francesc

    2009-09-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma) is involved in obesity and in some components of the metabolic syndrome in unselected population. To determine whether PPARgamma genetic variants are associated with the risk of developing lipodystrophy and its associated metabolic disturbances in HIV-1-infected patients treated with HAART and to assess PPARgamma mRNA expression in subcutaneous adipose tissue (SAT). The study group comprised 278 patients infected with HIV-1 and treated with antiretroviral drugs (139 with lipodystrophy and 139 without) and 105 uninfected controls (UC). The PPARgamma Pro12Ala (C%>G) single nucleotide polymorphism (SNP) was assessed using PCR-RFLPs on white cell DNA. PPARgamma mRNA expression in SAT was assessed in 38 patients (25 with lipodystrophy and 13 without) and in 21 UC by real-time PCR. Statistical analysis was based on Student's T tests, Chi(2) tests, Spearman's correlations tests and logistic regression tests. PPARgamma Pro12Ala genotype distribution and allele frequencies were non-significantly different between both HIV-1-infected categories, lipodystrophy vs non-lipodystrophy (p=0.9 and p=0.87, respectively). Lipodystrophic patients harbouring the rare X/Ala genotype (Ala/Ala plus Pro/Ala) had significantly greater plasma total and LDL cholesterol levels compared with carriers of the common Pro/Pro genotype (p=0.029 and p=0.016, respectively) at univariate analyses. At multivariate analyses these associations were no longer significant. There was a near-significant decreased SAT PPARgamma mRNA expression in patients with lipodystrophy compared to UC (p=0.054). PPARgamma Pro12Ala SNP has no effect on the risk of developing lipodystrophy in HIV-1-infected patients treated with HAART. PPARgamma mRNA SAT expression appears decreased in lipodystrophy.

  20. Harnessing cellular differentiation to improve ALA-based photodynamic therapy in an artificial skin model

    NASA Astrophysics Data System (ADS)

    Maytin, Edward; Anand, Sanjay; Sato, Nobuyuki; Mack, Judith; Ortel, Bernhard

    2005-04-01

    During ALA-based photodynamic therapy (PDT), a pro-drug (aminolevulinic acid; ALA) is taken up by tumor cells and metabolically converted to a photosensitizing intermediate (protoporphyrin IX; PpIX). ALA-based PDT, while an emerging treatment modality, remains suboptimal for most cancers (e.g. squamous cell carcinoma of the skin). Many treatment failures may be largely due to insufficient conversion of ALA to PpIX within cells. We discovered a novel way to increase the conversion of ALA to PpIX, by administering agents that can drive terminal differentiation (i.e., accelerate cellular maturation). Terminally-differentiated epithelial cells show higher levels of intracellular PpIX, apparently via increased levels of a rate-limiting enzyme, coproporphyrinogen oxidase (CPO). To study these mechanisms in a three-dimensional tissue, we developed an organotypic model that mimics true epidermal physiology in a majority of respects. A line of rat epidermal keratinocytes (REKs), when grown in raft cultures, displays all the features of a fully-differentiated epidermis. Addition of ALA to the culture medium results in ALA uptake and PpIX synthesis, with subsequent death of keratinocytes upon exposure to blue light. Using this model, we can manipulate cellular differentiation via three different approaches. (1) Vitamin D, a hormone that enhances keratinocyte differentiation; (2) Hoxb13, a nuclear transcription factor that affects the genetically-controlled differentiation program of stratifying cells (3) Hyaluronan, an abundant extracellular matrix molecule that regulates epidermal differentiation. Because the raft cultures contain only a single cell type (no blood, fibroblasts, etc.) the effects of terminal differentiation upon CPO, PpIX, and keratinocyte cell death can be specifically defined.

  1. Combination therapies in adjuvant with topical ALA-mediated photodynamic therapy for DMBA-induced hamster buccal pouch premalignant lesions

    NASA Astrophysics Data System (ADS)

    Yang, Deng-Fu; Hsu, Yih-Chih

    2012-03-01

    In Taiwan, oral cancer has becomes the fastest growth male cancer disease due to the betel nut chewing habit combing with smoking and alcohol-drinking lifestyle of people. In order to eliminate the systemic phototoxic effect of 5-aminolevulinic acid (ALA), this study was designed to use a topical ALA-mediated PDT for treatment of DMBA-induced hamster buccal pouch precancerous lesions. DMBA was applied to one of the buccal pouches of hamsters thrice a week for 10 to 12 weeks. Cancerous lesions were induced and proven by histological examination. These DMBA-induced cancerous lesions were used for testing the efficacy of topical ALA-mediated PDT. Before PDT, fluorescence spectroscopy was used to determine when ALA reached its peak level in the lesional epithelial cells after topical application of ALA gel. We found that ALA reached its peak level in precancerous lesions about 2.5 hrs after topical application of ALA gel. The cancerous lesions in hamsters were then treated with topical ALA -mediated PDT with light exposure dose of 150 J/cm2 using LED 635 nm fiber-guided light device. Visual examination demonstrated that adjuvant topical ALA -mediated PDT group has shown better therapeutic results in compared to those of non-adjuvant topical ALA-mediated PDT group for DMBA-induced hamster buccal pouch precancerous lesions.

  2. Intensified oxidative and nitrosative stress following combined ALA-based photodynamic therapy and local hyperthermia in rat tumors.

    PubMed

    Frank, Juergen; Lambert, Christine; Biesalski, Hans Konrad; Thews, Oliver; Vaupel, Peter; Kelleher, Debra K

    2003-12-20

    Oxidative stress-related changes in tumors upon localized hyperthermia (HT), 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) and their combination (ALA+HT) were examined after the observation that the antitumor effects of ALA-PDT could be significantly enhanced upon simultaneous application of HT. Rats bearing s.c. DS-sarcomas (0.6-1.0 ml) on the hind foot dorsum were anesthetized and underwent one of the following treatments: (i) ALA-PDT (375 mg/kg 5-ALA i.v.); (ii) localized HT, 43 degrees C for 60 min; (iii) combined ALA-PDT and HT [=ALA+HT]. Appropriate control experiments were also performed. After treatment, tumors were excised and rapidly frozen for later analysis of nitrosative stress (protein nitration), apoptotic events (TUNEL, caspase activation, DNA and RNA fragmentation), expression of heat shock proteins (hsp70 and HO-1), glutathione (GSH) levels and glutathione peroxidase (GPx) activity. Protein nitration was found to increase upon treatment, being especially pronounced in the ALA+HT group, and could partially be related to areas surrounding microvessels. The extent of nitrosative stress also correlated well with the appearance of the markers of apoptosis and the inhibition of in vivo tumor growth as seen in a previous study. GSH levels decreased upon treatment, the reduction being most prominent in the ALA-PDT and ALA+HT groups. GPx activity, however, showed a significant decrease only in the ALA-PDT group. Whereas hsp70 expression increased upon HT, ALA-PDT caused a decrease, and these opposing effects were nullified with ALA+HT. The results obtained point to a number of cellular mechanisms-including effects on cellular defense mechanisms and an abrogation of the heat shock defense mechanism-that may interact to achieve the potentiated tumor response rate seen in vivo upon combined treatment. PMID:14601053

  3. Amino acids that confer transport of raffinose and maltose sugars in the raffinose permease (RafB) of Escherichia coli as implicated by spontaneous mutations at Val-35, Ser-138, Ser-139, Gly-389 and Ile-391.

    PubMed

    Van Camp, Bonnie M; Crow, Robert R; Peng, Yang; Varela, Manuel F

    2007-12-01

    In order to identify amino acid residues in the Escherichia coli raffinose-H(+) permease (RafB) that play a role in sugar selection and transport, we first incubated E. coli HS4006 containing plasmid pRU600 (expresses inducible raffinose permease and alpha-galactosidase) on maltose MacConkey indicator plates overnight. Initially, all colonies were white, indicating no fermentation of maltose. Upon further incubation, 100 mutants appeared red. pRU600 DNA was prepared from 55 mutants. Five mutants transferred the phenotype for fermentation of maltose (red). Plasmid DNA from five maltose-positive phenotype transformants was prepared and sequenced, revealing three distinct types of mutations. Two mutants exhibited Val-35-->Ala (MT1); one mutant had Ile-391-->Ser (MT2); and two mutants had Ser-138-->Asp, Ser-139-->Leu and Gly-389-->Ala (MT3). Transport studies of [(3)H]-maltose showed that cells harboring MT1, MT2 and MT3 had greater uptake (P

  4. ERK/Egr-1 signaling pathway is involved in CysLT2 receptor-mediated IL-8 production in HEK293 cells.

    PubMed

    Lin, Kana; Fang, Sanhua; Cai, Beilei; Huang, Xueqin; Zhang, Xiayan; Lu, Yunbi; Zhang, Weiping; Wei, Erqing

    2014-07-01

    The CysLT2 receptor is involved in myocardial ischemia/reperfusion injury, differentiation of colorectal cancers, bleomycin-induced pulmonary inflammation and fibrosis. However, the signal transduction of cysteinyl leukotriene receptor 2 (CysLT2) in inflammatory responses remains to be clarified. In HEK293 cells stably expressing hCysLT1, hCysLT2 and rGPR17, we determined the signaling pathways for interleukin-8 (IL-8) production after CysLT2 receptor activation. HEK293 cells were stably transfected with the recombinant plasmids of pcDNA3.1(+)-hCysLT1, pcDNA3.1(+)-hCysLT2 and pcDNA3.1-rGPR17. Leukotriene C4 (LTC4) and LTD4 were used as the agonists to induce IL-8 production and the related changes in signal molecules. We found that LTC4 and LTD4 significantly induced IL-8 promoter activation in the HEK293 cells stably expressing hCysLT2, but not in those expressing hCysLT1 and rGPR17. In hCysLT2-HEK293 cells, LTC4 induced elevation of intracellular calcium, ERK1/2 phosphorylation and Egr-1 expression, and stimulated IL-8 expression and release. These responses were blocked by the selective CysLT2 receptor antagonist HAMI3379. The ERK1/2 inhibitor U0126 inhibited Egr-1 and IL-8 expression as well as IL-8 release, but the JNK and p38 inhibitors did not have the inhibitory effects. Down-regulation of Egr-1 by RNA interference with its siRNA inhibited the LTC4-induced IL-8 expression and release. In conclusion, these findings indicate the ERK-Egr-1 pathway of CysLT2 receptors mediates IL-8 production induced by the pro-inflammatory mediators LTC4 and LTD4.

  5. An additional cysteine in a typical 2-Cys peroxiredoxin of Pseudomonas promotes functional switching between peroxidase and molecular chaperone.

    PubMed

    An, Byung Chull; Lee, Seung Sik; Jung, Hyun Suk; Kim, Jin Young; Lee, Yuno; Lee, Keun Woo; Lee, Sang Yeol; Tripathi, Bhumi Nath; Chung, Byung Yeoup

    2015-09-14

    Peroxiredoxins (Prx) have received considerable attention during recent years. This study demonstrates that two typical Pseudomonas-derived 2-Cys Prx proteins, PpPrx and PaPrx can alternatively function as a peroxidase and chaperone. The amino acid sequences of these two Prx proteins exhibit 93% homology, but PpPrx possesses an additional cysteine residue, Cys112, instead of the alanine found in PaPrx. PpPrx predominates with a high molecular weight (HMW) complex and chaperone activity, whereas PaPrx has mainly low molecular weight (LMW) structures and peroxidase activity. Mass spectrometry and structural analyses showed the involvement of Cys112 in the formation of an inter-disulfide bond, the instability of LMW structures, the formation of HMW complexes, and increased hydrophobicity leading to functional switching of Prx proteins between peroxidase and chaperone. PMID:26278368

  6. Translational Control of Host Gene Expression by a Cys-Motif Protein Encoded in a Bracovirus.

    PubMed

    Kim, Eunseong; Kim, Yonggyun

    2016-01-01

    Translational control is a strategy that various viruses use to manipulate their hosts to suppress acute antiviral response. Polydnaviruses, a group of insect double-stranded DNA viruses symbiotic to some endoparasitoid wasps, are divided into two genera: ichnovirus (IV) and bracovirus (BV). In IV, some Cys-motif genes are known as host translation-inhibitory factors (HTIF). The genome of endoparasitoid wasp Cotesia plutellae contains a Cys-motif gene (Cp-TSP13) homologous to an HTIF known as teratocyte-secretory protein 14 (TSP14) of Microplitis croceipes. Cp-TSP13 consists of 129 amino acid residues with a predicted molecular weight of 13.987 kDa and pI value of 7.928. Genomic DNA region encoding its open reading frame has three introns. Cp-TSP13 possesses six conserved cysteine residues as other Cys-motif genes functioning as HTIF. Cp-TSP13 was expressed in Plutella xylostella larvae parasitized by C. plutellae. C. plutellae bracovirus (CpBV) was purified and injected into non-parasitized P. xylostella that expressed Cp-TSP13. Cp-TSP13 was cloned into a eukaryotic expression vector and used to infect Sf9 cells to transiently express Cp-TSP13. The synthesized Cp-TSP13 protein was detected in culture broth. An overlaying experiment showed that the purified Cp-TSP13 entered hemocytes. It was localized in the cytosol. Recombinant Cp-TSP13 significantly inhibited protein synthesis of secretory proteins when it was added to in vitro cultured fat body. In addition, the recombinant Cp-TSP13 directly inhibited the translation of fat body mRNAs in in vitro translation assay using rabbit reticulocyte lysate. Moreover, the recombinant Cp-TSP13 significantly suppressed cellular immune responses by inhibiting hemocyte-spreading behavior. It also exhibited significant insecticidal activities by both injection and feeding routes. These results indicate that Cp-TSP13 is a viral HTIF. PMID:27598941

  7. Translational Control of Host Gene Expression by a Cys-Motif Protein Encoded in a Bracovirus

    PubMed Central

    Kim, Eunseong; Kim, Yonggyun

    2016-01-01

    Translational control is a strategy that various viruses use to manipulate their hosts to suppress acute antiviral response. Polydnaviruses, a group of insect double-stranded DNA viruses symbiotic to some endoparasitoid wasps, are divided into two genera: ichnovirus (IV) and bracovirus (BV). In IV, some Cys-motif genes are known as host translation-inhibitory factors (HTIF). The genome of endoparasitoid wasp Cotesia plutellae contains a Cys-motif gene (Cp-TSP13) homologous to an HTIF known as teratocyte-secretory protein 14 (TSP14) of Microplitis croceipes. Cp-TSP13 consists of 129 amino acid residues with a predicted molecular weight of 13.987 kDa and pI value of 7.928. Genomic DNA region encoding its open reading frame has three introns. Cp-TSP13 possesses six conserved cysteine residues as other Cys-motif genes functioning as HTIF. Cp-TSP13 was expressed in Plutella xylostella larvae parasitized by C. plutellae. C. plutellae bracovirus (CpBV) was purified and injected into non-parasitized P. xylostella that expressed Cp-TSP13. Cp-TSP13 was cloned into a eukaryotic expression vector and used to infect Sf9 cells to transiently express Cp-TSP13. The synthesized Cp-TSP13 protein was detected in culture broth. An overlaying experiment showed that the purified Cp-TSP13 entered hemocytes. It was localized in the cytosol. Recombinant Cp-TSP13 significantly inhibited protein synthesis of secretory proteins when it was added to in vitro cultured fat body. In addition, the recombinant Cp-TSP13 directly inhibited the translation of fat body mRNAs in in vitro translation assay using rabbit reticulocyte lysate. Moreover, the recombinant Cp-TSP13 significantly suppressed cellular immune responses by inhibiting hemocyte-spreading behavior. It also exhibited significant insecticidal activities by both injection and feeding routes. These results indicate that Cp-TSP13 is a viral HTIF. PMID:27598941

  8. Stimulation and oxidative catalytic inactivation of thermolysin by copper.Cys-Gly-His-Lys.

    PubMed

    Gokhale, Nikhil H; Bradford, Seth; Cowan, J A

    2007-09-01

    [Cu(2+).Cys-Gly-His-Lys] stimulates thermolysin (TLN) activity at low concentration (below 10 microM) and inhibits the enzyme at higher concentration, with binding affinities of 2.0 and 4.9 microM, respectively. The metal-free Cys-Gly-His-Lys peptide also stimulates TLN activity, with an apparent binding affinity of 2.2 microM. Coordination of copper through deprotonated imine nitrogens, the histidyl nitrogen, and the free N-terminal amino group is consistent with the characteristic absorption spectrum of a Cu(2+)-amino-terminal copper and nickel binding motif (lambda (max) approximately 525 nm). The lack of thiol coordination is suggested by both the absence of a thiol to Cu(2+) charge transfer band and electrochemical studies, since the electrode potential (vs. Ag/AgCl) 0.84 V (DeltaE = 92 mV) for the Cu(3+/2+) redox couple obtained for [Cu(2+).Cys-Gly-His-Lys] was found to be in close agreement with that of a related complex [Cu(2+).Lys-Gly-His-Lys](+) (0.84 V, DeltaE = 114 mV). The N-terminal cysteine appears to be available as a zinc-anchoring residue and plays a critical functional role since the [Cu(2+).Lys-Gly-His-Lys](+) homologue exhibits neither stimulation nor inhibition of TLN. Under oxidizing conditions (ascorbate/O(2)) the catalyst is shown to mediate the complete irreversible inactivation of TLN at concentrations where enzyme activity would otherwise be stimulated. The observed rate constant for inactivation of TLN activity was determined as k (obs) = 7.7 x 10(-2) min(-1), yielding a second-order rate constant of (7.7 +/- 0.9) x 10(4) M(-1) min(-1). Copper peptide mediated generation of reactive oxygen species that subsequently modify active-site residues is the most likely pathway for inactivation of TLN rather than cleavage of the peptide backbone. PMID:17618468

  9. How pH modulates the dimer-decamer interconversion of 2-Cys peroxiredoxins from the Prx1 subfamily.

    PubMed

    Morais, Mariana A B; Giuseppe, Priscila O; Souza, Tatiana A C B; Alegria, Thiago G P; Oliveira, Marcos A; Netto, Luis E S; Murakami, Mario T

    2015-03-27

    2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H2O2 and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His(113)) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp(76) from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H2O2 can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H2O2 and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress.

  10. Tomato cystatin SlCYS8 as a stabilizing fusion partner for human serpin expression in plants.

    PubMed

    Sainsbury, Frank; Varennes-Jutras, Philippe; Goulet, Marie-Claire; D'Aoust, Marc-André; Michaud, Dominique

    2013-12-01

    Studies have reported the usefulness of fusion proteins to bolster recombinant protein yields in plants. Here, we assess the potential of tomato SlCYS8, a Cys protease inhibitor of the cystatin protein superfamily, as a stabilizing fusion partner for human alpha-1-antichymotrypsin (α1ACT) targeted to the plant cell secretory pathway. Using the model expression platform Nicotiana benthamiana, we show that the cystatin imparts a strong stabilizing effect when expressed as a translational fusion with α1ACT, allowing impressive accumulation yields of over 2 mg/g of fresh weight tissue for the human serpin, a 25-fold improvement on the yield of α1ACT expressed alone. Natural and synthetic peptide linkers inserted between SlCYS8 and α1ACT have differential effects on protease inhibitory potency of the two protein partners in vitro. They also have a differential impact on the yield of α1ACT, dependent on the extent to which the hybrid protein may remain intact in the plant cell environment. The stabilizing effect of SlCYS8 does not involve Cys protease inhibition and can be partly reproduced in the cytosol, where peptide linkers are less susceptible to degradation. The effect of SlCYS8 on α1ACT yields could be explained by: (i) an improved translation of the human protein coding sequence; and/or (ii) an overall stabilization of its tertiary structure preventing proteolytic degradation and/or polymerization. These findings suggest the potential of plant cystatins as stabilizing fusion partners for recombinant proteins in plant systems. They also underline the need for an empirical assessment of peptide linker functions in plant cell environments.

  11. How pH modulates the dimer-decamer interconversion of 2-Cys peroxiredoxins from the Prx1 subfamily.

    PubMed

    Morais, Mariana A B; Giuseppe, Priscila O; Souza, Tatiana A C B; Alegria, Thiago G P; Oliveira, Marcos A; Netto, Luis E S; Murakami, Mario T

    2015-03-27

    2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H2O2 and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His(113)) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp(76) from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H2O2 can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H2O2 and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress. PMID:25666622

  12. How pH Modulates the Dimer-Decamer Interconversion of 2-Cys Peroxiredoxins from the Prx1 Subfamily*

    PubMed Central

    Morais, Mariana A. B.; Giuseppe, Priscila O.; Souza, Tatiana A. C. B.; Alegria, Thiago G. P.; Oliveira, Marcos A.; Netto, Luis E. S.; Murakami, Mario T.

    2015-01-01

    2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H2O2 and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His113) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp76 from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H2O2 can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H2O2 and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress. PMID:25666622

  13. Influence of ALA54THR polymorphism of fatty acid-binding protein 2 on obesity and cardiovascular risk factors.

    PubMed

    de Luis, D A; Sagrado, M G; Aller, R; Izaola, O; Conde, R

    2007-11-01

    A transition of G to A at codon 54 of FABP2 results in an amino acid substitution (Ala54 to Thr54). This polymorphism was associated with some cardiovascular risk factors. The aim of our study was to investigate the influence of Thr54 polymorphism in the FABP2 gene on obesity anthropometric parameters and cardiovascular risk factors. A population of 226 obesity (body mass index >30) nondiabetic outpatients were analyzed. An indirect calorimetry, tetrapolar electrical bioimpedance, blood pressure, a serial assessment of nutritional intake with 3 days of written food records, and biochemical analysis (lipid profile, adipocytokines, insulin, CRP, and lipoprotein-a) were performed. The statistical analysis was performed for the combined ALA54/THR54 and THR54/THR54 as a mutant group and wild type ALA54/ALA54 as a second group. Two-hundred and twenty-six patients gave informed consent and were enrolled in the study. The mean age was 44.2+/-16 years and the mean BMI 35.1+/-5.1, with 63 males (28.3%) and 163 females (71.7%). One-hundred and thirteen patients (50%) had the genotype ALA54/ALA54 (wild group) and 113 (50%) patients had the genotype ALA54/THR54 (91 patients, 40.2%) or THR54/THR54 (22 patients, 9.8%) (mutant group). The ANOVA analysis of the three groups ( ALA54/THR54, THR54/THR54 and ALA54/ALA54) shows a higher levels of fat mass in Thr54/Thr54 group (45.6+/-14.6 kg) than Ala54/Ala54 (37.5+/-11.2 kg: p<0.05), without differences with Ala54/Thr54 group (41.2+/-13.5 kg). CRP, IL-6, and lipoprotein-a were higher in mutant group ( ALA54/THR54, THR54/THR54) than in wild group ( ALA54/ALA54). The novel finding of this study is the association of the Thr54/Ala54 and Thr54/Thr54 FABP2 phenotypes with higher levels of C reactive protein, IL6, and lipoprotein-a. Further studies are needed to explain the role of this polymorphism in different populations.

  14. Flourescence analysis of ALA-induced Protoporphyrin IX in psoriatic plaque

    NASA Astrophysics Data System (ADS)

    Stringer, Mark R.; Robinson, Dominic J.; Collins, P.

    1996-01-01

    The success reported for the treatment of superficial skin carcinomas by photodynamic therapy (PDT), following topical application of 5-aminolaevulinic acid (ALA), has therapeutic implications for the treatment of other skin disorders. This presentation describes the accumulation of the photosensitizing agent protoporphyrin IX (PpIX) in areas of psoriatic plaque, by monitoring the fluorescence emission induced by low-intensity laser excitation at 488 nm. We present the results from 15 patients, with a total of 42 plaques. These results show that PpIX fluorescence increases in intensity within the 6 hour period following application of ALA, which implies there is a potential for PDT. The emission is localized to the area of ALA application and the effect of occlusion appears insignificant. Also, the rate of increase, and maximum intensity of fluorescence emission, is not directly related to the applied quantity of ALA. The variability of the fluorescence intensity is as great between plaques at different sites on the same patient as between different patients. We also present measurements of the depletion in intensity of fluorescence emission during PDT treatment, using white light, at an irradiance of 25 mW cm-2, that is a consequence of the molecular photo-oxidation of PpIX. The use of fluorescence measurements in predicting the therapeutic effect of treating plaque psoriasis by ALA-PDT is discussed.

  15. Supplementation of milled chia seeds increases plasma ALA and EPA in postmenopausal women.

    PubMed

    Jin, Fuxia; Nieman, David C; Sha, Wei; Xie, Guoxiang; Qiu, Yunping; Jia, Wei

    2012-06-01

    Ten postmenopausal women (age 55.6 ± 0.8 years, BMI 24.6 ± 1.1 kg/m²) ingested 25 g/day milled chia seed during a 7-week period, with six plasma samples collected for measurement of α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA). Subjects operated as their own controls with overnight fasted blood samples taken at baseline (average of two samples), and then after 1, 2, 3, 5, and 7 weeks supplementation. Plasma ALA increased significantly after one week supplementation and was 138 % above baseline levels by the end of the study (overall time effect, P < 0.001). EPA increased 30 % above baseline (overall time effect, P = 0.019) and was correlated across time with ALA (r = 0.84, P = 0.02). No significant change in plasma DPA levels was measured (overall time effect, P = 0.067). Plasma DHA decreased slightly by the end of the study (overall time effect, P = 0.030) and was not correlated with change in ALA. In conclusion, ingestion of 25 g/day milled chia seeds for seven weeks by postmenopausal women resulted in significant increases in plasma ALA and EPA but not DPA and DHA.

  16. Simplified and optimized multispectral imaging for 5-ALA-based fluorescence diagnosis of malignant lesions

    PubMed Central

    Minamikawa, Takeo; Matsuo, Hisataka; Kato, Yoshiyuki; Harada, Yoshinori; Otsuji, Eigo; Yanagisawa, Akio; Tanaka, Hideo; Takamatsu, Tetsuro

    2016-01-01

    5-aminolevulinic acid (5-ALA)-based fluorescence diagnosis is now clinically applied for accurate and ultrarapid diagnosis of malignant lesions such as lymph node metastasis during surgery. 5-ALA-based diagnosis evaluates fluorescence intensity of a fluorescent metabolite of 5-ALA, protoporphyrin IX (PPIX); however, the fluorescence of PPIX is often affected by autofluorescence of tissue chromophores, such as collagen and flavins. In this study, we demonstrated PPIX fluorescence estimation with autofluorescence elimination for 5-ALA-based fluorescence diagnosis of malignant lesions by simplified and optimized multispectral imaging. We computationally optimized observation wavelength regions for the estimation of PPIX fluorescence in terms of minimizing prediction error of PPIX fluorescence intensity in the presence of typical chromophores, collagen and flavins. By using the fluorescence intensities of the optimized wavelength regions, we verified quantitative detection of PPIX fluorescence by using chemical mixtures of PPIX, flavins, and collagen. Furthermore, we demonstrated detection capability by using metastatic and non-metastatic lymph nodes of colorectal cancer patients. These results suggest the potential and usefulness of the background-free estimation method of PPIX fluorescence for 5-ALA-based fluorescence diagnosis of malignant lesions, and we expect this method to be beneficial for intraoperative and rapid cancer diagnosis. PMID:27149301

  17. Protective Effect of ALA in Crushed Optic Nerve Cat Retinal Ganglion Cells Using a New Marker RBPMS

    PubMed Central

    Wang, Yanling; Wang, Wenyao; Liu, Jessica; Huang, Xin; Liu, Ruixing; Xia, Huika; Brecha, Nicholas C.; Pu, Mingliang; Gao, Jie

    2016-01-01

    In this study we first sought to determine whether RNA-binding protein with multiple splicing (RBPMS) can serve as a specific marker for cat retina ganglion cells (RGCs) using retrograde labeling and immunohistochemistry staining. RBPM was then used as an RGC marker to study RGC survival after optic nerve crush (ONC) and alpha-lipoic acid (ALA) treatment in cats. ALA treatment yielded a peak density of RBPMS-alpha cells within the peak isodensity zone (>60/mm2) which did not differ from ONC retinas. The area within the zone was significantly enlarged (control: 2.3%, ONC: 0.06%, ONC+ALA: 0.1%). As for the 10-21/mm2 zone, ALA treatment resulted in a significant increase in area (control: 34.5%, ONC: 12.1%, ONC+ALA: 35.9%). ALA can alleviate crush-induced RGC injury. PMID:27504635

  18. Protective Effect of ALA in Crushed Optic Nerve Cat Retinal Ganglion Cells Using a New Marker RBPMS.

    PubMed

    Wang, Yanling; Wang, Wenyao; Liu, Jessica; Huang, Xin; Liu, Ruixing; Xia, Huika; Brecha, Nicholas C; Pu, Mingliang; Gao, Jie

    2016-01-01

    In this study we first sought to determine whether RNA-binding protein with multiple splicing (RBPMS) can serve as a specific marker for cat retina ganglion cells (RGCs) using retrograde labeling and immunohistochemistry staining. RBPM was then used as an RGC marker to study RGC survival after optic nerve crush (ONC) and alpha-lipoic acid (ALA) treatment in cats. ALA treatment yielded a peak density of RBPMS-alpha cells within the peak isodensity zone (>60/mm2) which did not differ from ONC retinas. The area within the zone was significantly enlarged (control: 2.3%, ONC: 0.06%, ONC+ALA: 0.1%). As for the 10-21/mm2 zone, ALA treatment resulted in a significant increase in area (control: 34.5%, ONC: 12.1%, ONC+ALA: 35.9%). ALA can alleviate crush-induced RGC injury. PMID:27504635

  19. Isolation and characterization of two peptides with prolactin release-inhibiting activity from porcine hypothalami.

    PubMed Central

    Schally, A V; Guoth, J G; Redding, T W; Groot, K; Rodriguez, H; Szonyi, E; Stults, J; Nikolics, K

    1991-01-01

    Two peptides with in vitro prolactin release-inhibiting activity were purified from stalk median eminence (SME) fragments of 20,000 pig hypothalami. Monolayer cultures of rat anterior pituitary cells were incubated with aliquots of chromatographic fractions and the inhibition of release of prolactin in vitro was measured by RIA in order to monitor the purification. The hypothalamic tissue extract was separated into 11 fractions by high-performance aqueous size-exclusion chromatography with one fraction showing a 4-fold increase in prolactin release-inhibiting factor (PIF) activity. This material was further purified by semipreparative reversed-phase (RP) HPLC. This process resulted in the separation of two distinct fractions that showed high PIF activity. These were further purified by semipreparative and analytical RP-HPLC to apparent homogeneity as judged by the UV absorbance profiles. Neither of the two peptides showed cross-reactivity with gonadotropin releasing hormone-associated peptide or with somatostatin-14 antibodies. Protein sequence analysis revealed that one of the PIF peptides was Trp-Cys-Leu-Glu-Ser-Ser-Gln-Cys-Gln-Asp-Leu-Ser-Thr-Glu-Ser-Asn-Leu-Leu- Ala-Cys - Ile-Arg-Ala-Cys-Lys-Pro, identical to residues 27-52 of the N-terminal region of the proopiomelanocortin (POMC) precursor (corresponding to amino acids 1-26 of the 16-kDa fragment). The sequence of the other PIF was Ala-Ser-Asp-Arg-Ser-Asn-Ala-Thr-Leu-Leu-Asp-Gly-Pro-Ser-Gly-Ala-Leu-Leu- Leu-Arg - Leu-Val-Gln-Leu-Ala-Gly-Ala-Pro-Glu-Pro-Ala-Glu-Pro-Ala-Gln-Pro-Gly-Val- Tyr, representing residues 109-147 of the vasopressin-neurophysin precursor. Synthetic peptides corresponding to the N-terminal region of POMC had significant PIF activity in vitro. PMID:2023899

  20. Identification of the reactive cysteine residue (Cys227) in human carbonyl reductase.

    PubMed

    Tinguely, J N; Wermuth, B

    1999-02-01

    Carbonyl reductase is highly susceptible to inactivation by organomercurials suggesting the presence of a reactive cysteine residue in, or close to, the active site. This residue is also close to a site which binds glutathione. Structurally, carbonyl reductase belongs to the short-chain dehydrogenase/reductase family and contains five cysteine residues, none of which is conserved within the family. In order to identify the reactive residue and investigate its possible role in glutathione binding, alanine was substituted for each cysteine residue of human carbonyl reductase by site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli and purified to homogeneity. Four of the five mutants (C26A, C122A C150A and C226A) exhibited wild-type-like enzyme activity, although K(m) values of C226A for three structurally different substrates were increased threefold to 10-fold. The fifth mutant, C227A, showed a 10-15-fold decrease in kcat and a threefold to 40-fold increase in K(m), resulting in a 30-500-fold drop in kcat/K(m). NaCl (300 mM) increased the activity of C227A 16-fold, whereas the activity of the wild-type enzyme was only doubled. Substitution of serine rather than alanine for Cys227 similarly affected the kinetic constants with the exception that NaCl did not activate the enzyme. Both C227A and C227S mutants were insensitive to inactivation by 4-hydroxymercuribenzoate. Unlike the parent carbonyl compounds, the glutathione adducts of menadione and prostaglandin A1 were better substrates for the C227A and C227S mutants than the wild-type enzyme. Conversely, the binding of free glutathione to both mutants was reduced. Our findings indicate that Cys227 is the reactive residue and suggest that it is involved in the binding of both substrate and glutathione. PMID:10091578

  1. Cys-loop ligand-gated chloride channels in dorsal unpaired median neurons of Locusta migratoria.

    PubMed

    Janssen, Daniel; Derst, Christian; Rigo, Jean-Michel; Van Kerkhove, Emmy

    2010-05-01

    In insects, inhibitory neurotransmission is generally associated with members of the cys-loop ligand-gated anion channels, such as the glutamate-gated chloride channel (GluCl), the GABA-gated chloride channels (GABACl), and the histamine-gated chloride channels (HisCl). These ionotropic receptors are considered established target sites for the development of insecticides, and therefore it is necessary to obtain a better insight in their distribution, structure, and functional properties. Here, by combining electrophysiology and molecular biology techniques, we identified and characterized GluCl, GABACl, and HisCl in dorsal unpaired median (DUM) neurons of Locust migratoria. In whole cell patch-clamp recordings, application of glutamate, GABA, or histamine induced rapidly activating ionic currents. GluCls were sensitive to ibotenic acid and blocked by picrotoxin and fipronil. The pharmacological profile of the L. migratoria GABACl fitted neither the vertebrate GABA(A) nor GABA(C) receptor and was similar to the properties of the cloned Drosophila melanogaster GABA receptor subunit (Rdl). The expression of Rdl-like subunit-containing GABA receptors was shown at the molecular level using RT-PCR. Sequencing analysis indicated that the orthologous GABACl of D. melanogaster CG10357-A is expressed in DUM neurons of L. migratoria. Histamine-induced currents exhibited a fast onset and desensitized completely on continuous application of histamine. In conclusion, within the DUM neurons of L. migratoria, we identified three different cys-loop ligand-gated anion channels that use GABA, glutamate, or histamine as their neurotransmitter. PMID:20200125

  2. Light Fractionation Significantly Increases the Efficacy of Photodynamic Therapy Using BF-200 ALA in Normal Mouse Skin

    PubMed Central

    de Bruijn, Henriëtte S.; Brooks, Sander; van der Ploeg-van den Heuvel, Angélique; ten Hagen, Timo L. M.; de Haas, Ellen R. M.; Robinson, Dominic J.

    2016-01-01

    Background Light fractionation significantly increases the efficacy of 5-aminolevulinic acid (ALA) based photodynamic therapy (PDT) using the nano-emulsion based gel formulation BF-200. PDT using BF-200 ALA has recently been clinically approved and is under investigation in several phase III trials for the treatment of actinic keratosis. This study is the first to compare BF-200 ALA with ALA in preclinical models. Results In hairless mouse skin there is no difference in the temporal and spatial distribution of protoporphyrin IX determined by superficial imaging and fluorescence microscopy in frozen sections. In the skin-fold chamber model, BF-200 ALA leads to more PpIX fluorescence at depth in the skin compared to ALA suggesting an enhanced penetration of BF-200 ALA. Light fractionated PDT after BF-200 ALA application results in significantly more visual skin damage following PDT compared to a single illumination. Both ALA formulations show the same visual skin damage, rate of photobleaching and change in vascular volume immediately after PDT. Fluorescence immunohistochemical imaging shows loss of VE-cadherin in the vasculature at day 1 post PDT which is greater after BF-200 ALA compared to ALA and more profound after light fractionation compared to a single illumination. Discussion The present study illustrates the clinical potential of light fractionated PDT using BF-200 ALA for enhancing PDT efficacy in (pre-) malignant skin conditions such as basal cell carcinoma and vulval intraepithelial neoplasia and its application in other lesion such as cervical intraepithelial neoplasia and oral squamous cell carcinoma where current approaches have limited efficacy. PMID:26872051

  3. Assessment of ALA-induced PpIX production in porcine skin pretreated with microneedles.

    PubMed

    Rodrigues, Phamilla Gracielli Sousa; Campos de Menezes, Priscila Fernanda; Fujita, Alessandra Keiko Lima; Escobar, André; Barboza de Nardi, Andrigo; Kurachi, Cristina; Bagnato, Vanderlei S

    2015-09-01

    Photodynamic therapy (PDT) is used for skin treatments of premalignant and cancer lesions and recognized as a non-invasive technique that combines tissue photosensitization and subsequent exposure to light to induce cell death. However, it is limited to the treatment of superficial lesions, mainly due to the low cream penetration. Therefore, the improvement of transdermal distribution of aminolevulinic acid (ALA) is needed. In this study, the kinetics and homogeneity of production of ALA-induced PpIX after the skin pre-treatment with microneedles rollers of 0.5, 1.0 and 1.5 mm length were investigated. An improvement in homogeneity and production of PpIX was shown in a porcine model. Widefield fluorescence imaging three hours after the topical application of ALA-cream in the combined treatment with microeedles rollers. PMID:25319567

  4. Kinetic study of delta-Ala induced porphyrins in mice using photoacoustic and fluorescence spectroscopies.

    PubMed

    Stolik, Suren; Tomás, Sergio A; Ramón-Gallegos, Eva; Sánchez, Feliciano

    2002-11-01

    The production of delta-aminolevulinic acid (ALA)-induced porphyrins in mice skin and blood was studied by photoacoustic and fluorescence spectroscopies. Mice were intraperitoneally administered with 30 mg/kg of ALA. The abdominal skin was subsequently excised at specific times within an 8-h interval and its absorption spectrum obtained by photoacoustics. The highest porphyrins concentration in skin, determined from the optical absorption of the Soret band at 410 nm, was found to occur nearly 2 h after ALA administration, but a first peak was also observed at approximately 15 min. Our hypothesis that the first peak represents the porphyrins content in blood vessels within the skin, whereas the second peak corresponds to porphyrins production in skin tissue, was confirmed by analysing the evolution of protoporphyrin IX content in plasma extracted intracardiacally. By finally applying phase resolved photoacoustic spectroscopy, we were able to evaluate the mean depth at which porphyrins are generated.

  5. Conformations of Gly(n)H+ and Ala(n)H+ peptides in the gas phase.

    PubMed Central

    Hudgins, R R; Mao, Y; Ratner, M A; Jarrold, M F

    1999-01-01

    High-resolution ion mobility measurements and molecular dynamics simulations have been used to probe the conformations of protonated polyglycine and polyalanine (Gly(n)H and Ala(n)H+, n = 3-20) in the gas phase. The measured collision integrals for both the polyglycine and the polyalanine peptides are consistent with a self-solvated globule conformation, where the peptide chain wraps around and solvates the charge located on the terminal amine. The conformations of the small peptides are governed entirely by self-solvation, whereas the larger ones have additional backbone hydrogen bonds. Helical conformations, which are stable for neutral Alan peptides, were not observed in the experiments. Molecular dynamics simulations for Ala(n)H+ peptides suggest that the charge destabilizes the helix, although several of the low energy conformations found in the simulations for the larger Ala(n)H+ peptides have small helical regions. PMID:10049339

  6. Hollow plasmonic antennas for broadband SERS spectroscopy.

    PubMed

    Messina, Gabriele C; Malerba, Mario; Zilio, Pierfrancesco; Miele, Ermanno; Dipalo, Michele; Ferrara, Lorenzo; De Angelis, Francesco

    2015-01-01

    The chemical environment of cells is an extremely complex and multifaceted system that includes many types of proteins, lipids, nucleic acids and various other components. With the final aim of studying these components in detail, we have developed multiband plasmonic antennas, which are suitable for highly sensitive surface enhanced Raman spectroscopy (SERS) and are activated by a wide range of excitation wavelengths. The three-dimensional hollow nanoantennas were produced on an optical resist by a secondary electron lithography approach, generated by fast ion-beam milling on the polymer and then covered with silver in order to obtain plasmonic functionalities. The optical properties of these structures have been studied through finite element analysis simulations that demonstrated the presence of broadband absorption and multiband enhancement due to the unusual geometry of the antennas. The enhancement was confirmed by SERS measurements, which showed a large enhancement of the vibrational features both in the case of resonant excitation and out-of-resonance excitation. Such characteristics indicate that these structures are potential candidates for plasmonic enhancers in multifunctional opto-electronic biosensors.

  7. Time- and concentration-dependent reactivity of Cys, Hcy, and GSH on the Diels-Alder-grafted 1,3,5-tris conjugate of calix[6]arene to bring selectivity for Cys: spectroscopy, microscopy, and its reactivity in cells.

    PubMed

    Mummidivarapu, V V Sreenivasu; Yarramala, Deepthi S; Kondaveeti, Karuna Kumar; Rao, Chebrolu P

    2014-11-01

    Herein we report the synthesis and characterization of 7-oxanorbornadiene (OND)-appended 1,3,5-tris conjugate of calix[6]arene (L2). L2 has been shown to exhibit selective reactivity toward cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH) under stoichiometric conditions. The selectivity of L2 is attributed to the steric crowding of three Diels-Alder centers possessing OND units present on the calix[6]arene platform, while a control molecular system possessing only one such unit without the calix[6]arene platform (L1) does not show any selectivity toward Cys. While L2 exhibited spherical particles, its reactivity with Cys resulted in flowerlike morphological features, as revealed by scanning electron microscopy. However, the reaction with GSH did not result in any such morphological features, a result that is in agreement with that observed from fluorescence studies in solution. L2 has been shown to react with Cys present in HeLa and Jurkat E6 cells by fluorescence microscopy.

  8. Photodynamic therapy (PDT) and photodiagnosis (PD) using endogenous photosensitization induced by 5-aminolevulinic acid (ALA): current clinical and development status

    NASA Astrophysics Data System (ADS)

    Marcus, Stuart L.; Sobel, Russel S.; Golub, Allyn L.; Carroll, Ronald L.; Lundahl, Scott L.; Shulman, D. Geoffrey

    1996-04-01

    Exogenous provision of ALA to many tissues results in the accumulation of sufficient quantities of the endogenous photosensitizer protoporphyrin IX, (PpIX), to produce a photodynamic effect. Therefore, ALA may be considered the only current PDT agent in clinical development which is a biochemical precursor of a photosensitizer. Topical ALA application, followed by exposure to activating light (ALA PDT), has been reported effective for the treatment of a variety of dermatologic diseases including cutaneous T-cell lymphoma, superficial basal cell carcinoma, Bowen's disease, and actinic (solar) keratoses, and is also being examined for treatment of acne and hirsutism. PpIX induced by ALA application also may serve as a fluorescence detection marker for photodiagnosis (PD) of malignant and pre- malignant conditions of the urinary bladder and other organs. Local internal application of ALA has also been used for selective endometrial ablation in animal model systems and is beginning to be examined in human clinical studies. Systemic, oral administration of ALA has been used for ALA PDT of superficial head and neck cancer, various gastrointestinal cancers, and the condition known as Barrett's esophagus. This brief paper reviews the current clinical and development status of ALA PDT.

  9. ALA PDT for high grade dysplasia in Barrett's oesophagus: review of a decade's experience

    NASA Astrophysics Data System (ADS)

    Bown, Stephen G.; Mackenzie, Gary D.; Dunn, Jason M.; Thorpe, Sally M.; Lovat, Laurence B.

    2009-06-01

    We have been investigating PDT with 5 aminolaevulinic acid (ALA) for the treatment of high grade dysplasia (HGD) in Barrett's oesophagus (BO) for over a decade. This drug has inherent advantages over porfimer sodium (Photofrin), the current approved photosensitiser in the UK and USA, which causes strictures in 18-50% and light sensitivity for up to three months. ALA has a lower rate of oesophageal strictures due to its preferential activity in the mucosa, sparing the underlying muscle, and patients are only light sensitive for 1-2 days. Within a randomised controlled trial, we demonstrated that an ALA dose of 60mg/kg activated by 1000J/cm red laser light is the most effective. Using these values we achieved complete reversal of HGD at 1 year in 89% of 27 patients. A randomised controlled trial of ALA vs porfimer sodium PDT for HGD is currently under way with end points of efficacy and safety. 50 of 66 patients have been recruited. Preliminary data suggest ALA PDT is safer with a trend to higher efficacy. Late relapse can occur in 20% of patients. New prognostic markers, in particular aneuploidy, are helping us to identify and target patients at risk of late relapse. Furthermore optical biopsy techniques such as elastic scattering spectroscopy (ESS) may allow detection of nuclear abnormalities in vivo and enable us to target areas of interest whilst reducing sampling error. PDT faces new challenges for the treatment of HGD in BO, with the recent introduction of balloon based radiofrequency ablation. This technique appears simpler and as effective as PDT, but follow up is currently short and long term safety data is lacking. In our experience ALA PDT is currently the most effective minimally invasive treatment for HGD in BO. This work was undertaken at UCLH/UCL who received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding scheme.

  10. Contribution of Ser386 and Ser396 to activation of interferon regulatory factor 3.

    PubMed

    Chen, Weijun; Srinath, Hema; Lam, Suvana S; Schiffer, Celia A; Royer, William E; Lin, Kai

    2008-05-30

    IRF-3, a member of the interferon regulatory factor (IRF) family of transcription factors, functions in innate immune defense against viral infection. Upon infection, host cell IRF-3 is activated by phosphorylation at its seven C-terminal Ser/Thr residues: (385)SSLENTVDLHISNSHPLSLTS(405). This phosphoactivation triggers IRF-3 to react with the coactivators, CREB-binding protein (CBP)/p300, to form a complex that activates target genes in the nucleus. However, the role of each phosphorylation site for IRF-3 phosphoactivation remains unresolved. To address this issue, all seven Ser/Thr potential phosphorylation sites were screened by mutational studies, size-exclusion chromatography, and isothermal titration calorimetry. Using purified proteins, we show that CBP (amino acid residues 2067-2112) interacts directly with IRF-3 (173-427) and six of its single-site mutants to form heterodimers, but when CBP interacts with IRF-3 S396D, oligomerization is evident. CBP also interacts in vitro with IRF-3 double-site mutants to form different levels of oligomerization. Among all the single-site mutants, IRF-3 S396D showed the strongest binding to CBP. Although IRF-3 S386D alone did not interact as strongly with CBP as did other mutants, it strengthened the interaction and oligomerization of IRF-3 S396D with CBP. In contrast, IRF-3 S385D weakened the interaction and oligomerization of IRF-3 S396D and S386/396D with CBP. Thus, it appears that Ser385 and Ser386 serve antagonistic functions in regulating IRF-3 phosphoactivation. These results indicate that Ser386 and Ser396 are critical for IRF-3 activation, and support a phosphorylation-oligomerization model for IRF-3 activation.

  11. Penetrance of Hemochromatosis in HFE Genotypes Resulting in p.Cys282Tyr and p.[Cys282Tyr];[His63Asp] in the eMERGE Network

    PubMed Central

    Gallego, Carlos J.; Burt, Amber; Sundaresan, Agnes S.; Ye, Zi; Shaw, Christopher; Crosslin, David R.; Crane, Paul K.; Fullerton, S. Malia; Hansen, Kris; Carrell, David; Kuivaniemi, Helena; Derr, Kimberly; de Andrade, Mariza; McCarty, Catherine A.; Kitchner, Terrie E.; Ragon, Brittany K.; Stallings, Sarah C.; Papa, Gabriella; Bochenek, Joseph; Smith, Maureen E.; Aufox, Sharon A.; Pacheco, Jennifer A.; Patel, Vaibhav; Friesema, Elisha M.; Erwin, Angelika Ludtke; Gottesman, Omri; Gerhard, Glenn S.; Ritchie, Marylyn; Motulsky, Arno G.; Kullo, Iftikhar J.; Larson, Eric B.; Tromp, Gerard; Brilliant, Murray H.; Bottinger, Erwin; Denny, Joshua C.; Roden, Dan M.; Williams, Marc S.; Jarvik, Gail P.

    2015-01-01

    Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder associated with pathogenic HFE variants, most commonly those resulting in p.Cys282Tyr and p.His63Asp. Recommendations on returning incidental findings of HFE variants in individuals undergoing genome-scale sequencing should be informed by penetrance estimates of HH in unselected samples. We used the eMERGE Network, a multicenter cohort with genotype data linked to electronic medical records, to estimate the diagnostic rate and clinical penetrance of HH in 98 individuals homozygous for the variant coding for HFE p.Cys282Tyr and 397 compound heterozygotes with variants resulting in p.[His63Asp];[Cys282Tyr]. The diagnostic rate of HH in males was 24.4% for p.Cys282Tyr homozygotes and 3.5% for compound heterozygotes (p < 0.001); in females, it was 14.0% for p.Cys282Tyr homozygotes and 2.3% for compound heterozygotes (p < 0.001). Only males showed differences across genotypes in transferrin saturation levels (100% of homozygotes versus 37.5% of compound heterozygotes with transferrin saturation > 50%; p = 0.003), serum ferritin levels (77.8% versus 33.3% with serum ferritin > 300 ng/ml; p = 0.006), and diabetes (44.7% versus 28.0%; p = 0.03). No differences were found in the prevalence of heart disease, arthritis, or liver disease, except for the rate of liver biopsy (10.9% versus 1.8% [p = 0.013] in males; 9.1% versus 2% [p = 0.035] in females). Given the higher rate of HH diagnosis than in prior studies, the high penetrance of iron overload, and the frequency of at-risk genotypes, in addition to other suggested actionable adult-onset genetic conditions, opportunistic screening should be considered for p.[Cys282Tyr];[Cys282Tyr] individuals with existing genomic data. PMID:26365338

  12. Penetrance of Hemochromatosis in HFE Genotypes Resulting in p.Cys282Tyr and p.[Cys282Tyr];[His63Asp] in the eMERGE Network.

    PubMed

    Gallego, Carlos J; Burt, Amber; Sundaresan, Agnes S; Ye, Zi; Shaw, Christopher; Crosslin, David R; Crane, Paul K; Fullerton, S Malia; Hansen, Kris; Carrell, David; Kuivaniemi, Helena; Derr, Kimberly; de Andrade, Mariza; McCarty, Catherine A; Kitchner, Terrie E; Ragon, Brittany K; Stallings, Sarah C; Papa, Gabriella; Bochenek, Joseph; Smith, Maureen E; Aufox, Sharon A; Pacheco, Jennifer A; Patel, Vaibhav; Friesema, Elisha M; Erwin, Angelika Ludtke; Gottesman, Omri; Gerhard, Glenn S; Ritchie, Marylyn; Motulsky, Arno G; Kullo, Iftikhar J; Larson, Eric B; Tromp, Gerard; Brilliant, Murray H; Bottinger, Erwin; Denny, Joshua C; Roden, Dan M; Williams, Marc S; Jarvik, Gail P

    2015-10-01

    Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder associated with pathogenic HFE variants, most commonly those resulting in p.Cys282Tyr and p.His63Asp. Recommendations on returning incidental findings of HFE variants in individuals undergoing genome-scale sequencing should be informed by penetrance estimates of HH in unselected samples. We used the eMERGE Network, a multicenter cohort with genotype data linked to electronic medical records, to estimate the diagnostic rate and clinical penetrance of HH in 98 individuals homozygous for the variant coding for HFE p.Cys282Tyr and 397 compound heterozygotes with variants resulting in p.[His63Asp];[Cys282Tyr]. The diagnostic rate of HH in males was 24.4% for p.Cys282Tyr homozygotes and 3.5% for compound heterozygotes (p < 0.001); in females, it was 14.0% for p.Cys282Tyr homozygotes and 2.3% for compound heterozygotes (p < 0.001). Only males showed differences across genotypes in transferrin saturation levels (100% of homozygotes versus 37.5% of compound heterozygotes with transferrin saturation > 50%; p = 0.003), serum ferritin levels (77.8% versus 33.3% with serum ferritin > 300 ng/ml; p = 0.006), and diabetes (44.7% versus 28.0%; p = 0.03). No differences were found in the prevalence of heart disease, arthritis, or liver disease, except for the rate of liver biopsy (10.9% versus 1.8% [p = 0.013] in males; 9.1% versus 2% [p = 0.035] in females). Given the higher rate of HH diagnosis than in prior studies, the high penetrance of iron overload, and the frequency of at-risk genotypes, in addition to other suggested actionable adult-onset genetic conditions, opportunistic screening should be considered for p.[Cys282Tyr];[Cys282Tyr] individuals with existing genomic data. PMID:26365338

  13. Penetrance of Hemochromatosis in HFE Genotypes Resulting in p.Cys282Tyr and p.[Cys282Tyr];[His63Asp] in the eMERGE Network.

    PubMed

    Gallego, Carlos J; Burt, Amber; Sundaresan, Agnes S; Ye, Zi; Shaw, Christopher; Crosslin, David R; Crane, Paul K; Fullerton, S Malia; Hansen, Kris; Carrell, David; Kuivaniemi, Helena; Derr, Kimberly; de Andrade, Mariza; McCarty, Catherine A; Kitchner, Terrie E; Ragon, Brittany K; Stallings, Sarah C; Papa, Gabriella; Bochenek, Joseph; Smith, Maureen E; Aufox, Sharon A; Pacheco, Jennifer A; Patel, Vaibhav; Friesema, Elisha M; Erwin, Angelika Ludtke; Gottesman, Omri; Gerhard, Glenn S; Ritchie, Marylyn; Motulsky, Arno G; Kullo, Iftikhar J; Larson, Eric B; Tromp, Gerard; Brilliant, Murray H; Bottinger, Erwin; Denny, Joshua C; Roden, Dan M; Williams, Marc S; Jarvik, Gail P

    2015-10-01

    Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder associated with pathogenic HFE variants, most commonly those resulting in p.Cys282Tyr and p.His63Asp. Recommendations on returning incidental findings of HFE variants in individuals undergoing genome-scale sequencing should be informed by penetrance estimates of HH in unselected samples. We used the eMERGE Network, a multicenter cohort with genotype data linked to electronic medical records, to estimate the diagnostic rate and clinical penetrance of HH in 98 individuals homozygous for the variant coding for HFE p.Cys282Tyr and 397 compound heterozygotes with variants resulting in p.[His63Asp];[Cys282Tyr]. The diagnostic rate of HH in males was 24.4% for p.Cys282Tyr homozygotes and 3.5% for compound heterozygotes (p < 0.001); in females, it was 14.0% for p.Cys282Tyr homozygotes and 2.3% for compound heterozygotes (p < 0.001). Only males showed differences across genotypes in transferrin saturation levels (100% of homozygotes versus 37.5% of compound heterozygotes with transferrin saturation > 50%; p = 0.003), serum ferritin levels (77.8% versus 33.3% with serum ferritin > 300 ng/ml; p = 0.006), and diabetes (44.7% versus 28.0%; p = 0.03). No differences were found in the prevalence of heart disease, arthritis, or liver disease, except for the rate of liver biopsy (10.9% versus 1.8% [p = 0.013] in males; 9.1% versus 2% [p = 0.035] in females). Given the higher rate of HH diagnosis than in prior studies, the high penetrance of iron overload, and the frequency of at-risk genotypes, in addition to other suggested actionable adult-onset genetic conditions, opportunistic screening should be considered for p.[Cys282Tyr];[Cys282Tyr] individuals with existing genomic data.

  14. Neurotransmitter transporter family including SLC6A6 and SLC6A13 contributes to the 5-aminolevulinic acid (ALA)-induced accumulation of protoporphyrin IX and photodamage, through uptake of ALA by cancerous cells.

    PubMed

    Tran, Tai Tien; Mu, Anfeng; Adachi, Yuka; Adachi, Yasushi; Taketani, Shigeru

    2014-01-01

    δ-Aminolevulinic acid (ALA)-induced protoporphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy (PDT). To clarify the mechanisms of ALA uptake by tumor cells, we have examined the ALA-induced accumulation of protoporphyrin by the treatment of colon cancer DLD-1 and epithelial cancer HeLa cells with γ-aminobutyric acid (GABA)-related compounds. When the cells were treated with GABA, taurine and β-alanine, the level of protoporphyrin was decreased, suggesting that plasma membrane transporters involved in the transport of neurotransmitters contribute to the uptake of ALA. By transfection with neurotransmitter transporters SLC6A6, SLC6A8 and SLC6A13 cDNA, the ALA- and ALA methylester-dependent accumulation of protoporphyrin markedly increased in HEK293T cells, dependent on an increase in the uptake of ALA. When ALA-treated cells were exposed to white light, the extent of photodamage increased in SLC6A6- and SLC6A13-expressing cells. Conversely, knockdown of SLC6A6 or SLC6A13 with siRNAs in DLD-1 and HeLa cells decreased the ALA-induced accumulation. The expression of SLC6A6 and SLC6A13 was found in some cancer cell lines. Immunohistochemical studies revealed that the presence of these transporters was elevated in colon cancerous cells. These results indicated that neurotransmitter transporters including SLC6A6 and SLC6A13 mediate the uptake of ALA and can play roles in the enhancement of ALA-induced accumulation of protoporphyrin in cancerous cells.

  15. Expression, purification and preliminary crystallographic analysis of Mycobacterium tuberculosis CysQ, a phosphatase involved in sulfur metabolism

    SciTech Connect

    Erickson, Anna I.; Sarsam, Reta D.; Fisher, Andrew J.

    2014-05-10

    The cysQ gene from Mycobacterium tuberculosis was cloned and the expressed protein, a 3′-phosphoadenosine-5′’-phosphatase, was purified and crystallized. X-ray diffraction data were collected to 1.7 Å resolution.

  16. Proton uptake and pKa changes in the uncoupled Asn139Cys variant of cytochrome c oxidase.

    PubMed

    Johansson, Ann-Louise; Carlsson, Jens; Högbom, Martin; Hosler, Jonathan P; Gennis, Robert B; Brzezinski, Peter

    2013-02-01

    Cytochrome c oxidase (CytcO) is a membrane-bound enzyme that links electron transfer from cytochrome c to O(2) to proton pumping across the membrane. Protons are transferred through specific pathways that connect the protein surface with the catalytic site as well as the proton input with the proton output sides. Results from earlier studies have shown that one site within the so-called D proton pathway, Asn139, located ~10 Å from the protein surface, is particularly sensitive to mutations that uncouple the O(2) reduction reaction from the proton pumping activity. For example, none of the Asn139Asp (charged) or Asn139Thr (neutral) mutant CytcOs pump protons, although the proton-uptake rates are unaffected. Here, we have investigated the Asn139Cys and Asn139Cys/Asp132Asn mutant CytcOs. In contrast to other structural variants investigated to date, the Cys side chain may be either neutral or negatively charged in the experimentally accessible pH range. The data show that the Asn139Cys and Asn139Asp mutations result in the same changes of the kinetic and thermodynamic parameters associated with the proton transfer. The similarity is not due to introduction of charge at position 139, but rather introduction of a protonatable group that modulates the proton connectivity around this position. These results illuminate the mechanism by which CytcO couples electron transfer to proton pumping.

  17. Binuclear Cu(A) Formation in Biosynthetic Models of Cu(A) in Azurin Proceeds via a Novel Cu(Cys)2His Mononuclear Copper Intermediate.

    PubMed

    Chakraborty, Saumen; Polen, Michael J; Chacón, Kelly N; Wilson, Tiffany D; Yu, Yang; Reed, Julian; Nilges, Mark J; Blackburn, Ninian J; Lu, Yi

    2015-10-01

    Cu(A) is a binuclear electron transfer (ET) center found in cytochrome c oxidases (CcOs), nitrous oxide reductases (N₂ORs), and nitric oxide reductase (NOR). In these proteins, the Cu(A) centers facilitate efficient ET (kET > 10⁴s⁻¹) under low thermodynamic driving forces (10-90 mV). While the structure and functional properties of Cu(A) are well understood, a detailed mechanism of the incorporation of copper into the protein and the identity of the intermediates formed during the Cu(A) maturation process are still lacking. Previous studies of the Cu(A) assembly mechanism in vitro using a biosynthetic model Cu(A) center in azurin (Cu(A)Az) identified a novel intermediate X (Ix) during reconstitution of the binuclear site. However, because of the instability of Ix and the coexistence of other Cu centers, such as Cu(A)' and type 1 copper centers, the identity of this intermediate could not be established. Here, we report the mechanism of Cu(A) assembly using variants of Glu114XCuAAz (X = Gly, Ala, Leu, or Gln), the backbone carbonyl of which acts as a ligand to the Cu(A) site, with a major focus on characterization of the novel intermediate Ix. We show that Cu(A) assembly in these variants proceeds through several types of Cu centers, such as mononuclear red type 2 Cu, the novel intermediate Ix, and blue type 1 Cu. Our results show that the backbone flexibility of the Glu114 residue is an important factor in determining the rates of T2Cu → Ix formation, suggesting that Cu(A) formation is facilitated by swinging of the ligand loop, which internalizes the T2Cu capture complex to the protein interior. The kinetic data further suggest that the nature of the Glu114 side chain influences the time scales on which these intermediates are formed, the wavelengths of the absorption peaks, and how cleanly one intermediate is converted to another. Through careful understanding of these mechanisms and optimization of the conditions, we have obtained Ix in ∼80

  18. Glacier lake outburst floods caused by glacier shrinkage: case study of Ala-Archa valley, Kyrgyz Ala Too, northern Tian Shan, Kyrgyzstan

    NASA Astrophysics Data System (ADS)

    Petrakov, D.; Erochin, S. A.; Harbor, J.; Ivanov, M.; Rogozhina, I.; Stroeven, A. P.; Usubaliev, R.

    2012-12-01

    Changes in glacier extent and runoff in Central Asia increase socio-economic stress and may result in political conflict between donors of freshwater (Kyrgyzstan, Tajikistan) and recipients of freshwater (Uzbekistan, China). Glaciers in the Pamir and Tian Shan regions have experienced an unprecedented downwasting due to regional climate changes over the past decades. This is because air temperature increases are in some areas accompanied by a decrease in precipitation. Such conditions have already resulted in a reduction of glacier runoff, especially in the northern and western Tian Shan, and an increase of the number and area of glacial lakes in Kyrgyzstan. Even though glacial lakes in the mountains are in general relatively small and located far from densely populated areas, their outbursts often produce destructive debris flows. Such debris flows are especially common in Kyrgyzstan because of its steep river channels and abundance of Holocene and Quaternary glacier deposits that can be remobilized. The glacial lake outburst flood (GLOF) in the Shakhimardan river catchment in 1999, for example, resulted in 100 fatalities in Uzbekistan, and the GLOF from the Zyndan glacial lake led to substantial economic losses in 2009. According to the latest inventory, there are more than 350 glacial lakes in Kyrgyzstan of which about 70 occur in the Kyrgyz Ala Too. The Ala-Archa valley is among the most important glacierized catchments in Kyrgyzstan. Despite the presence of a relatively small glacier-covered area of 36 km2, the Ala-Archa river is of critical importance to the Bishkek area, its agriculture, and its population which currently exceeds one million. GLOFs are therefore a threat to both numerous settlements of touristic value in the Ala-Archa headwaters and to Bishkek. The Teztor lake in the Adygene catchment of the Ala-Archa river system experienced an outburst during 1988 and 2005. On the early morning of July 31, 2012, this lake began draining through a dam

  19. Development of a three-dimensional CysLT1 (LTD4) antagonist model with an incorporated amino acid residue from the receptor.

    PubMed

    Zwaagstra, M E; Schoenmakers, S H; Nederkoorn, P H; Gelens, E; Timmerman, H; Zhang, M Q

    1998-04-23

    This paper describes the molecular modeling of leukotriene CysLT1 (or LTD4) receptor antagonists. Several different structural classes of CysLT1 antagonists were superimposed onto the new and highly rigid CysLT1 antagonist 8-carboxy-3'-[2-(2-quinolinyl)ethenyl]flavone (1, VUF 5017) to generate a common pharmacophoric arrangement. On the basis of known structure-activity relationships of CysLT1 antagonists, the quinoline nitrogen (or a bioisosteric equivalent thereof) and an acidic function were taken as the matching points. In order to optimize the fitting of acidic moieties of all antagonists, an arginine residue from the receptor was proposed as the interaction site for the acidic moieties. Incorporation of this amino acid residue into the model revealed additional interactions between the guanidine group and the nitrogen atoms of quinoline-containing CysLT1 antagonists. In some cases, the arginine may even interact with pi-clouds of phenyl residues of CysLT1 antagonists. The alignment of Montelukast (MK-476) suggests the presence of an additional pocket in the binding site for CysLT1 antagonists. The derived model should be useful for a better understanding of the molecular recognition of the leukotriene CysLT1 receptor.

  20. Repeated Superovulation via PMSG/hCG Administration Induces 2-Cys Peroxiredoxins Expression and Overoxidation in the Reproductive Tracts of Female Mice.

    PubMed

    Park, Sun-Ji; Kim, Tae-Shin; Kim, Jin-Man; Chang, Kyu-Tae; Lee, Hyun-Shik; Lee, Dong-Seok

    2015-12-01

    Superovulation induced by exogenous gonadotropin treatment (PMSG/hCG) increases the number of available oocytes in humans and animals. However, Superovulatory PMSG/hCG treatment is known to affect maternal environment, and these effects may result from PMSG/hCG treatment-induced oxidative stress. 2-Cys peroxiredoxins (2-Cys Prxs) act as antioxidant enzymes that protect cells from oxidative stress induced by various exogenous stimuli. Therefore, the objective of this study was to test the hypothesis that repeated PMSG/hCG treatment induces 2-Cys Prx expression and overoxidation in the reproductive tracts of female mice. Immunohistochemistry and western blotting analyses further demonstrated that, after PMSG/hCG treatment, the protein expression levels of 2-Cys Prxs increased most significantly in the ovaries, while that of Prx1 was most affected by PMSG/hCG stimulation in all tissues of the female reproductive tract. Repeated PMSG/hCG treatment eventually leads to 2-Cys Prxs overoxidation in all reproductive organs of female mice, and the abundance of the 2-Cys Prxs-SO2/3 proteins reported here supports the hypothesis that repeated superovulation induces strong oxidative stress and damage to the female reproductive tract. Our data suggest that excessive oxidative stress caused by repeated PMSG/hCG stimulation increases 2-Cys Prxs expression and overoxidation in the female reproductive organs. Intracellular 2-Cys Prx therefore plays an important role in maintaining the reproductive organ environment of female mice upon exogenous gonadotropin treatment.

  1. Repeated Superovulation via PMSG/hCG Administration Induces 2-Cys Peroxiredoxins Expression and Overoxidation in the Reproductive Tracts of Female Mice

    PubMed Central

    Park, Sun-Ji; Kim, Tae-Shin; Kim, Jin-Man; Chang, Kyu-Tae; Lee, Hyun-Shik; Lee, Dong-Seok

    2015-01-01

    Superovulation induced by exogenous gonadotropin treatment (PMSG/hCG) increases the number of available oocytes in humans and animals. However, Superovulatory PMSG/hCG treatment is known to affect maternal environment, and these effects may result from PMSG/hCG treatment-induced oxidative stress. 2-Cys peroxiredoxins (2-Cys Prxs) act as antioxidant enzymes that protect cells from oxidative stress induced by various exogenous stimuli. Therefore, the objective of this study was to test the hypothesis that repeated PMSG/hCG treatment induces 2-Cys Prx expression and overoxidation in the reproductive tracts of female mice. Immunohistochemistry and western blotting analyses further demonstrated that, after PMSG/hCG treatment, the protein expression levels of 2-Cys Prxs increased most significantly in the ovaries, while that of Prx1 was most affected by PMSG/hCG stimulation in all tissues of the female reproductive tract. Repeated PMSG/hCG treatment eventually leads to 2-Cys Prxs overoxidation in all reproductive organs of female mice, and the abundance of the 2-Cys Prxs-SO2/3 proteins reported here supports the hypothesis that repeated superovulation induces strong oxidative stress and damage to the female reproductive tract. Our data suggest that excessive oxidative stress caused by repeated PMSG/hCG stimulation increases 2-Cys Prxs expression and overoxidation in the female reproductive organs. Intracellular 2-Cys Prx therefore plays an important role in maintaining the reproductive organ environment of female mice upon exogenous gonadotropin treatment. PMID:26486164

  2. Feasibility of colloidal silver SERS for rapid bacterial screening

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrate-reduced silver colloids have been used extensively for surface-enhanced Raman scattering (SERS) study and are commonly characterized by UV-visible spectroscopy. In this work, relative standard deviation (RSD) of SERS spectra from silver colloidal suspensions and ratios of SERS peaks from sma...

  3. Optimization of SERS Tag Intensity, Binding Footprint, and Emittance

    PubMed Central

    2015-01-01

    Nanoparticle surface enhanced Raman scattering (SERS) tags have attracted interest as labels for use in a variety of applications, including biomolecular assays. An obstacle to progress in this area is a lack of standardized approaches to compare the brightness of different SERS tags within and between laboratories. Here we present an approach based on binding of SERS tags to beads with known binding capacities that allows evaluation of the average intensity, the relative binding footprint of particles in a SERS tag preparation, and the size-normalized intensity or emittance. We tested this on four different SERS tag compositions and show that aggregated gold nanorods produce SERS tags that are 2–4 times brighter than relatively more monodisperse nanorods, but that the aggregated nanorods are also correspondingly larger, which may negate the intensity if steric hindrance limits the number of tags bound to a target. By contrast, SERS tags prepared from smaller gold nanorods coated with a silver shell produce SERS tags that are 2–3 times brighter, on a size-normalized basis, than the Au nanorod-based tags, resulting in labels with improved performance in SERS-based image and flow cytometry assays. SERS tags based on red-resonant Ag plates showed similarly bright signals and small footprint. This approach to evaluating SERS tag brightness is general, uses readily available reagents and instruments, and should be suitable for interlab comparisons of SERS tag brightness. PMID:24892497

  4. Pro-oxidant effect of ALA is implicated in mitochondrial dysfunction of HepG2 cells.

    PubMed

    Laafi, Jihane; Homedan, Chadi; Jacques, Caroline; Gueguen, Naig; Schmitt, Caroline; Puy, Hervé; Reynier, Pascal; Carmen Martinez, Maria; Malthièry, Yves

    2014-11-01

    Heme biosynthesis begins in the mitochondrion with the formation of delta-aminolevulinic acid (ALA). In acute intermittent porphyria, hereditary tyrosinemia type I and lead poisoning patients, ALA is accumulated in plasma and in organs, especially the liver. These diseases are also associated with neuromuscular dysfunction and increased incidence of hepatocellular carcinoma. Many studies suggest that this damage may originate from ALA-induced oxidative stress following its accumulation. Using the MnSOD as an oxidative stress marker, we showed here that ALA treatment of cultured cells induced ROS production, increasing with ALA concentration. The mitochondrial energetic function of ALA-treated HepG2 cells was further explored. Mitochondrial respiration and ATP content were reduced compared to control cells. For the 300 μM treatment, ALA induced a mitochondrial mass decrease and a mitochondrial network imbalance although neither necrosis nor apoptosis were observed. The up regulation of PGC-1, Tfam and ND5 genes was also found; these genes encode mitochondrial proteins involved in mitochondrial biogenesis activation and OXPHOS function. We propose that ALA may constitute an internal bioenergetic signal, which initiates a coordinated upregulation of respiratory genes, which ultimately drives mitochondrial metabolic adaptation within cells. The addition of an antioxidant, Manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), resulted in improvement of maximal respiratory chain capacity with 300 μM ALA. Our results suggest that mitochondria, an ALA-production site, are more sensitive to pro-oxidant effect of ALA, and may be directly involved in pathophysiology of patients with inherited or acquired porphyria.

  5. ALA Office for Intellectual Freedom: Who We Are and How We Help Librarians

    ERIC Educational Resources Information Center

    Pekoll, Kristin

    2015-01-01

    The American Library Association's (ALA's) Office for Intellectual Freedom (OIF) strives to educate librarians and the public about the nature and importance of intellectual freedom in libraries, and it will celebrate its fiftieth anniversary in 2017. Libraries are a forum for information and ideas (under the First Amendment), and librarians are…

  6. Studying Online: Student Motivations and Experiences in ALA-Accredited LIS Programs

    ERIC Educational Resources Information Center

    Oguz, Fatih; Chu, Clara M.; Chow, Anthony S.

    2015-01-01

    This paper presents a large scale study of online MLIS students (n = 910), who completed at least one online course and were enrolled in 36 of the 58 ALA-accredited MLIS programs in Canada and the United States. The results indicate that the typical student is female, White, lives in an urban setting, and is in her mid-30s. Online students were…

  7. "LJ" Report "Anaheim, ALA 2008": Amid the Fantasy, Doses of Reality

    ERIC Educational Resources Information Center

    Blumenstein, Lynn; Berry, John; Fialkoff, Francine; Fox, Bette-Lee; Hadro, Josh; Horrocks, Norman; Oder, Norman; Roncevic, Mirela

    2008-01-01

    If the resort city of Anaheim, California, home of Disneyland and its "imagineers," marked a departure from the urban reality of the typical American Library Association (ALA) annual conference, it was impossible, at this 2008 meeting, to avoid urgent library issues. How do libraries maintain their value and cultural presence as users turn to the…

  8. Notes from the Teenage Underground: Opinionated Teen Panelist Hits ALA New Orleans.

    ERIC Educational Resources Information Center

    Christian, Jill

    2000-01-01

    Describes the American Library Association (ALA) Convention in New Orleans from a teen's perspective, particularly the Intellectual Freedom Teen Panel. Topics include barriers to information, including Internet filters; censoring and the First Amendment; and general impressions of the convention, including meeting authors. Sidebar includes notes…

  9. X-linked macrocytic dyserythropoietic anemia in females with an ALAS2 mutation.

    PubMed

    Sankaran, Vijay G; Ulirsch, Jacob C; Tchaikovskii, Vassili; Ludwig, Leif S; Wakabayashi, Aoi; Kadirvel, Senkottuvelan; Lindsley, R Coleman; Bejar, Rafael; Shi, Jiahai; Lovitch, Scott B; Bishop, David F; Steensma, David P

    2015-04-01

    Macrocytic anemia with abnormal erythropoiesis is a common feature of megaloblastic anemias, congenital dyserythropoietic anemias, and myelodysplastic syndromes. Here, we characterized a family with multiple female individuals who have macrocytic anemia. The proband was noted to have dyserythropoiesis and iron overload. After an extensive diagnostic evaluation that did not provide insight into the cause of the disease, whole-exome sequencing of multiple family members revealed the presence of a mutation in the X chromosomal gene ALAS2, which encodes 5'-aminolevulinate synthase 2, in the affected females. We determined that this mutation (Y365C) impairs binding of the essential cofactor pyridoxal 5'-phosphate to ALAS2, resulting in destabilization of the enzyme and consequent loss of function. X inactivation was not highly skewed in wbc from the affected individuals. In contrast, and consistent with the severity of the ALAS2 mutation, there was a complete skewing toward expression of the WT allele in mRNA from reticulocytes that could be recapitulated in primary erythroid cultures. Together, the results of the X inactivation and mRNA studies illustrate how this X-linked dominant mutation in ALAS2 can perturb normal erythropoiesis through cell-nonautonomous effects. Moreover, our findings highlight the value of whole-exome sequencing in diagnostically challenging cases for the identification of disease etiology and extension of the known phenotypic spectrum of disease. PMID:25705881

  10. Hey, Small Spender: An Insider's Guide to Navigating ALA's Chicago Conference on the Cheap

    ERIC Educational Resources Information Center

    School Library Journal, 2009

    2009-01-01

    This article presents an insider's guide to navigating the American Library Association's (ALA) annual conference in Chicago on July 9-15. As for the extracurricular activities, Chicago has a lot to offer. This article provides tips from the arts and entertainment bible "Time Out Chicago" on where to go and what to do (on a limited budget) while…

  11. Preservice Legal Education for Academic Librarians within ALA-Accredited Degree Programs

    ERIC Educational Resources Information Center

    Cross, William M.; Edwards, Phillip M.

    2011-01-01

    In order to explore the current state of legal education for graduates of LIS programs, we present the results of an examination of the curricula and faculty composition at all 57 institutions that offer ALA-accredited graduate degrees. Concluding that, even under the best circumstances, many students graduate with a limited understanding of legal…

  12. ALA-PDT mediated DC vaccine for skin squamous cell carcinoma

    NASA Astrophysics Data System (ADS)

    Ji, Jie; Fan, Zhixia; Zhou, Feifan; Wang, Xiaojie; Shi, Lei; Zhang, Haiyan; Wang, Peiru; Yang, Degang; Zhang, Linglin; Wang, Xiuli; Chen, Wei R.

    2015-03-01

    Dendritic cell (DC) based vaccine has emerged as a promising immunotherapy for cancers. However, most DC vaccines so far have only achieved limited success in cancer treatment. Photodynamic therapy (PDT), an established cancer treatment strategy, can cause immunogenic apoptosis to induce an effective antitumor immune response. In this study, we developed a DC-based cancer vaccine using immunogenic apoptotic tumor cells induced by 5-aminolevulinic acid (ALA) mediated PDT. The maturation of DCs induced by PDT-treated apoptotic cells was evaluated. The anti-tumor immunity of ALA-PDT-DC vaccine was tested with mouse model. We observed the maturations of DCs potentiated by ALA-PDT treated tumor cells, including phenotypic maturation (upregulation of surface expression of MHC-II, DC80, and CD86), and functional maturation (enhanced capability to secret INF-Υ and IL-12). ALA-PDT-DC vaccine mediated by apoptotic cells provided protection against tumor in mice, far stronger than that of DC vaccine obtained from freeze/thaw treated tumor cells. Our results indicate that immunogenic apoptotic tumor cells can be more effective in enhancing DC-based cancer vaccine, which could improve the clinical application of PDT- DC vaccines.

  13. ALAS: Achievement for Latinos through Academic Success. What Works Clearinghouse Intervention Report

    ERIC Educational Resources Information Center

    What Works Clearinghouse, 2006

    2006-01-01

    "ALAS," an acronym for "Achievement for Latinos through Academic Success" that means "wings" in Spanish, is a middle school (or junior high school) intervention designed to address student, school, family, and community factors that affect dropping out. Each student is assigned a counselor who monitors attendance, behavior, and academic…

  14. Shifting with the Paradigm: LJ's Picks & Pans for ALA in Disneyland

    ERIC Educational Resources Information Center

    Berry, John N., III

    2008-01-01

    The feelings of librarians planning for the American Library Association (ALA) conference at Disneyland (aka Anaheim, California, June 26-July 2) range from moderate pleasure to dread. Some remember the joys and difficulties of Orlando, especially the exorbitant cab fares and mediocre restaurants. Others quail at screaming kids and tourists in…

  15. ALA-based fluorescent diagnosis of malignant oral lesions in the presence of bacterial porphyrin formation

    NASA Astrophysics Data System (ADS)

    Schleier, P.; Berndt, A.; Zinner, K.; Zenk, W.; Dietel, W.; Pfister, W.

    2006-02-01

    The aminolevulinic acid (5-ALA) -based fluorescence diagnosis has been found to be promising for an early detection and demarcation of superficial oral squamous cell carcinomas (OSCC). This method has previously demonstrated high sensitivity, however this clinical trial showed a specificity of approximately 62 %. This specificity was mainly restricted by tumor detection in the oral cavity in the presence of bacteria. After topical ALA application in the mouth of patients with previously diagnosed OSSC, red fluorescent areas were observed which did not correlate to confirm histological findings. Swabs and plaque samples were taken from 44 patients and cultivated microbiologically. Fluorescence was investigated (OMA-system) from 32 different bacteria strains found naturally in the oral cavity. After ALA incubation, 30 of 32 strains were found to synthesize fluorescent porphyrins, mainly Protoporphyrin IX. Also multiple fluorescent spectra were obtained having peak wavelengths of 636 nm and around 618 nm - 620 nm indicating synthesis of different porphyrins, such as the lipophylic Protoporphyrin IX (PpIX) and hydrophylic porphyrins (water soluble porphyrins, wsp). Of the 32 fluorescent bacterial strains, 18 produced wsp, often in combination with PpIX, and 5 produced solely wsp. These results clarify that ALA-based fluorescence diagnosis without consideration or suppression of bacteria fluorescence may lead to false-positive findings. It is necessary to suppress bacteria fluorescence with suitable antiseptics before starting the procedure. In this study, when specific antiseptic pre-treatment was performed bacterial associated fluorescence was significantly reduced.

  16. Disruption of the Blood–Brain Barrier Following ALA-Mediated Photodynamic Therapy

    PubMed Central

    Hirschberg, Henry; Uzal, Francisco A.; Chighvinadze, David; Zhang, Michelle J.; Peng, Qian; Madsen, Steen J.

    2009-01-01

    Background and Objective Photodynamic therapy (PDT) is a local antineoplastic treatment with the potential for tumor cell specificity. PDT using either hematoporphyrin derivatives or 5-aminolevulinic acid (ALA) has been reported to induce brain edema indicating disruption of the blood–brain barrier (BBB). We have evaluated the ability of ALA-mediated PDT to open the BBB in rats. This will permit access of chemotherapeutic agents to brain tumor cells remaining in the resection cavity wall, but limit their penetration into normal brain remote from the site of illumination. Study Design/Materials and Methods ALA-PDT was performed on non-tumor bearing inbred Fischer rats at increasing fluence levels. Contrast T1-weighted high field (3 T) magnetic resonance imaging (MRI) scans were used to monitor the degree of BBB disruption which could be inferred from the intensity and volume of the contrast agent visualized. Results PDT at increasing fluence levels between 9 and 26 J demonstrated an increasing contrast flow rate. A similar increased contrast volume was observed with increasing fluence rates. The BBB was found to be disrupted 2 hours following PDT and 80–100% restored 72 hours later at the lowest fluence level. No effect on the BBB was observed if 26 J of light was given in the absence of ALA. Conclusion ALA-PDT was highly effective in opening the BBB in a localized region of the brain. The degradation of the BBB was temporary in nature at fluence levels of 9 J, opening rapidly following treatment and significantly restored during the next 72 hours. No signs of tissue damage were seen on histological sections at this fluence level. However, higher fluences did demonstrate permanent tissue changes localized in the immediate vicinity of the light source. PMID:18798293

  17. Genetic background modulates phenotypes of serotonin transporter Ala56 knock-in mice

    PubMed Central

    2013-01-01

    Background Previously, we identified multiple, rare serotonin (5-HT) transporter (SERT) variants in children with autism spectrum disorder (ASD). Although in our study the SERT Ala56 variant was over-transmitted to ASD probands, it was also seen in some unaffected individuals, suggesting that associated ASD risk is influenced by the epistatic effects of other genetic variation. Subsequently, we established that mice expressing the SERT Ala56 variant on a 129S6/S4 genetic background display multiple biochemical, physiological and behavioral changes, including hyperserotonemia, altered 5-HT receptor sensitivity, and altered social, communication, and repetitive behavior. Here we explore the effects of genetic background on SERT Ala56 knock-in phenotypes. Methods To explore the effects of genetic background, we backcrossed SERT Ala56 mice on the 129 background into a C57BL/6 (B6) background to achieve congenic B6 SERT Ala56 mice, and assessed autism-relevant behavior, including sociability, ultrasonic vocalizations, and repetitive behavior in the home cage, as well as serotonergic phenotypes, including whole blood serotonin levels and serotonin receptor sensitivity. Results One consistent phenotype between the two strains was performance in the tube test for dominance, where mutant mice displayed a greater tendency to withdraw from a social encounter in a narrow tube as compared to wildtype littermate controls. On the B6 background, mutant pup ultrasonic vocalizations were significantly increased, in contrast to decreased vocalizations seen previously on the 129 background. Several phenotypes seen on the 129 background were reduced or absent when the mutation was placed on the B6 background, including hyperserotonemia, 5-HT receptor hypersensivity, and repetitive behavior. Conclusions Our findings provide a cogent example of how epistatic interactions can modulate the impact of functional genetic variation and suggest that some aspects of social behavior may be

  18. Optimization of Influencing Factors on Biomass Accumulation and 5-Aminolevulinic Acid (ALA) Yield in Rhodobacter sphaeroides Wastewater Treatment.

    PubMed

    Liu, Shuli; Li, Xiangkun; Zhang, Guangming; Zhang, Jie

    2015-11-01

    This study aimed to optimize four factors affecting biomass accumulation and 5-aminolevulinic acid (ALA) yield together with pollutants removal in Rhodobacter sphaeroides wastewater treatment. Results showed that it was feasible to produce biomass and ALA in R. sphaeroides wastewater treatment. Microaerobic, 1,000-3,000 lux, and pH 7.0 were optimal conditions for the highest ALA yield of 4.5 ± 0.5 mg/g-biomass. Under these conditions, COD removal and biomass production rate were 93.3 ± 0.9% and 31.8 ± 0.5 mg/l/h, respectively. In addition, trace elements Fe(2+), Mg(2+), Ni(2+), and Zn(2+) further improved the ALA yield, COD removal, and biomass production rate. Specifically, the highest ALA yield (12.5 ± 0.6 mg/g-biomass) was achieved with Fe(2+) addition.

  19. Expression, purification and characterization of an atypical 2-Cys peroxiredoxin from the silkworm, Bombyx mori.

    PubMed

    Zhang, L; Lu, Z

    2015-04-01

    Peroxiredoxins (Prxs) play important roles in protecting organisms against damage caused by reactive oxygen species (ROS). In this study, we cloned a cDNA of Bombyx mori peroxiredoxin 5 (BmPrx5), which contained a 565-bp open reading frame for a 188-residue protein. Sequence analysis indicated that BmPrx5 belongs to the atypical 2-Cys peroxiredoxin family. Recombinant BmPrx5 purified from Escherichia coli showed antioxidant activity that removes H2 O2 and protects DNA from oxidative damage. Quantitative real-time PCR showed that the level of BmPrx5 mRNA in haemocytes increased early and decreased by 24 h after injection of H2 O2 whereas, in the fat body, the transcript level decreased at 6 h and increased at 12 h. Pseudomonas aeruginosa and Staphylococcus aureus infection resulted in higher levels of H2 O2 in the haemolymph and of BmPrx5 mRNA in haemocytes at 8 h postinfection. These data suggest that BmPrx5 acts as an antioxidant enzyme to protect the silkworm from oxidative damage induced by bacterial infection. Further study is needed to elucidate the exact role of BmPrx5 in the silkworm immune system.

  20. Cys34-PEGylated Human Serum Albumin for Drug Binding and Delivery

    PubMed Central

    Mehtala, Jonathan G.; Kulczar, Chris; Lavan, Monika; Knipp, Gregory; Wei, Alexander

    2015-01-01

    Polyethylene glycol (PEG) derivatives were conjugated onto the Cys-34 residue of human serum albumin (HSA) to determine their effects on the solubilization, permeation, and cytotoxic activity of hydrophobic drugs such as paclitaxel (PTX). PEG(C34)HSA conjugates were prepared on a multigram scale by treating native HSA (n-HSA) with 5- or 20-kDa mPEG-maleimide, resulting in up to 77% conversion of the mono-PEGylated adduct. Nanoparticle tracking analysis of PEG(C34)HSA formulations in phosphate buffer revealed an increase in nanosized aggregates relative to n-HSA, both in the absence and presence of PTX. Cell viability studies conducted with MCF-7 breast cancer cells indicated that PTX cytotoxicity was enhanced by PEG(C34)HSA when mixed at 10:1 mole ratios, up to a two-fold increase in potency relative to n-HSA. The PEG(C34)HSA conjugates were also evaluated as PTX carriers across monolayers of HUVEC and hCMEC/D3 cells, and found to have nearly identical permeation profiles as n-HSA. PMID:25918947

  1. Systematic analysis of reactivities and fragmentation of glutathione and its isomer GluCysGly.

    PubMed

    Feng, Shan; Zheng, Xiaoyan; Wang, Dong; Gong, Yiyi; Wang, Qingtao; Deng, Haiteng

    2014-09-18

    Glutathione (GSH) is the most abundant tripeptide in human cells and plays an important role in protecting cells' integrity against oxidative stress. GSH has an unusual amide linkage formed between the γ-carboxylic group of the glutamic acid in its side-chain and the amine group of cysteine residue. In the present study, we have compared reactivities of GSH to its isomer GluCysGly (ECG), which has a regular amide bond formed between the α-carboxylic group of glutamic acid and the amine group of cysteine residue. The fragmentation pattern of GSH ions in the gas phase is different from that of ECG ions, showing that the loss of H2O is the major dissociation pathway in ECG fragmentation. This is consistent with the dissociation pathway predicted by density functional calculation. Formation of GSSG from oxidation of GSH is faster than that of ECG disulfide, and the gas phase fragmentation pattern of GSSG is different from that of ECG disulfide. GSH and ECG display similar rates in nucleophilic aromatic substitution when reacting with 1-chloro-2,4-dinitrobenzene (CDNB). However, in the presence of glutathione S-transferases (GST), substitution of CDNB by GSH is 10 times faster than that by ECG. GSH and ECG also show differences in clustering patterns in the gas phase. Taken together, our results shed light on understanding effects of unique boding structure in GSH on its stability and reactivities.

  2. A Novel Family of Cys-Rich Membrane Proteins Mediates Cadmium Resistance in Arabidopsis1

    PubMed Central

    Song, Won-Yong; Martinoia, Enrico; Lee, Joohyun; Kim, Dongwoo; Kim, Do-Young; Vogt, Esther; Shim, Donghwan; Choi, Kwan Sam; Hwang, Inhwan; Lee, Youngsook

    2004-01-01

    Cadmium (Cd) is a widespread pollutant that is toxic to plant growth. However, only a few genes that contribute to Cd resistance in plants have been identified. To identify additional Cd(II) resistance genes, we screened an Arabidopsis cDNA library using a yeast (Saccharomyces cerevisiae) expression system employing the Cd(II)-sensitive yeast mutant ycf1. This screening process yielded a small Cys-rich membrane protein (Arabidopsis plant cadmium resistance, AtPcrs). Database searches revealed that there are nine close homologs in Arabidopsis. Homologs were also found in other plants. Four of the five homologs that were tested also increased resistance to Cd(II) when expressed in ycf1. AtPcr1 localizes at the plasma membrane in both yeast and Arabidopsis. Arabidopsis plants overexpressing AtPcr1 exhibited increased Cd(II) resistance, whereas antisense plants that showed reduced AtPcr1 expression were more sensitive to Cd(II). AtPcr1 overexpression reduced Cd uptake by yeast cells and also reduced the Cd contents of both yeast and Arabidopsis protoplasts treated with Cd. Thus, it appears that the Pcr family members may play an important role in the Cd resistance of plants. PMID:15181212

  3. An improved predictive recognition model for Cys2-His2 zinc finger proteins

    PubMed Central

    Gupta, Ankit; Christensen, Ryan G.; Bell, Heather A.; Goodwin, Mathew; Patel, Ronak Y.; Pandey, Manishi; Enuameh, Metewo Selase; Rayla, Amy L.; Zhu, Cong; Thibodeau-Beganny, Stacey; Brodsky, Michael H.; Joung, J. Keith; Wolfe, Scot A.; Stormo, Gary D.

    2014-01-01

    Cys2-His2 zinc finger proteins (ZFPs) are the largest family of transcription factors in higher metazoans. They also represent the most diverse family with regards to the composition of their recognition sequences. Although there are a number of ZFPs with characterized DNA-binding preferences, the specificity of the vast majority of ZFPs is unknown and cannot be directly inferred by homology due to the diversity of recognition residues present within individual fingers. Given the large number of unique zinc fingers and assemblies present across eukaryotes, a comprehensive predictive recognition model that could accurately estimate the DNA-binding specificity of any ZFP based on its amino acid sequence would have great utility. Toward this goal, we have used the DNA-binding specificities of 678 two-finger modules from both natural and artificial sources to construct a random forest-based predictive model for ZFP recognition. We find that our recognition model outperforms previously described determinant-based recognition models for ZFPs, and can successfully estimate the specificity of naturally occurring ZFPs with previously defined specificities. PMID:24523353

  4. The interaction of DNA with multi-Cys2His2 zinc finger proteins

    NASA Astrophysics Data System (ADS)

    Liu, Lei; Heermann, Dieter W.

    2015-02-01

    The multi-Cys2His2 (mC2H2) zinc finger protein, like CTCF, plays a central role in the three-dimensional organization of chromatin and gene regulation. The interaction between DNA and mC2H2 zinc finger proteins becomes crucial to better understand how CTCF dynamically shapes the chromatin structure. Here, we study a coarse-grained model of the mC2H2 zinc finger proteins in complexes with DNA, and in particular, we study how a mC2H2 zinc finger protein binds to and searches for its target DNA loci. On the basis of coarse-grained molecular dynamics simulations, we present several interesting kinetic conformational properties of the proteins, such as the rotation-coupled sliding, the asymmetrical roles of different zinc fingers and the partial binding partial dangling mode. In addition, two kinds of studied mC2H2 zinc finger proteins, of CG-rich and AT-rich binding motif each, were able to recognize their target sites and slide away from their non-target sites, which shows a proper sequence specificity in our model and the derived force field for mC2H2-DNA interaction. A further application to CTCF shows that the protein binds to a specific DNA duplex only with its central zinc fingers. The zinc finger domains of CTCF asymmetrically bend the DNA, but do not form a DNA loop alone in our simulations.

  5. Expression, purification and characterization of an atypical 2-Cys peroxiredoxin from the silkworm, Bombyx mori.

    PubMed

    Zhang, L; Lu, Z

    2015-04-01

    Peroxiredoxins (Prxs) play important roles in protecting organisms against damage caused by reactive oxygen species (ROS). In this study, we cloned a cDNA of Bombyx mori peroxiredoxin 5 (BmPrx5), which contained a 565-bp open reading frame for a 188-residue protein. Sequence analysis indicated that BmPrx5 belongs to the atypical 2-Cys peroxiredoxin family. Recombinant BmPrx5 purified from Escherichia coli showed antioxidant activity that removes H2 O2 and protects DNA from oxidative damage. Quantitative real-time PCR showed that the level of BmPrx5 mRNA in haemocytes increased early and decreased by 24 h after injection of H2 O2 whereas, in the fat body, the transcript level decreased at 6 h and increased at 12 h. Pseudomonas aeruginosa and Staphylococcus aureus infection resulted in higher levels of H2 O2 in the haemolymph and of BmPrx5 mRNA in haemocytes at 8 h postinfection. These data suggest that BmPrx5 acts as an antioxidant enzyme to protect the silkworm from oxidative damage induced by bacterial infection. Further study is needed to elucidate the exact role of BmPrx5 in the silkworm immune system. PMID:25512182

  6. Structure of a (Cys3His) zinc ribbon, a ubiquitous motif in archaeal and eucaryal transcription.

    PubMed

    Chen, H T; Legault, P; Glushka, J; Omichinski, J G; Scott, R A

    2000-09-01

    Transcription factor IIB (TFIIB) is an essential component in the formation of the transcription initiation complex in eucaryal and archaeal transcription. TFIIB interacts with a promoter complex containing the TATA-binding protein (TBP) to facilitate interaction with RNA polymerase II (RNA pol II) and the associated transcription factor IIF (TFIIF). TFIIB contains a zinc-binding motif near the N-terminus that is directly involved in the interaction with RNA pol II/TFIIF and plays a crucial role in selecting the transcription initiation site. The solution structure of the N-terminal residues 2-59 of human TFIIB was determined by multidimensional NMR spectroscopy. The structure consists of a nearly tetrahedral Zn(Cys)3(His)1 site confined by type I and "rubredoxin" turns, three antiparallel beta-strands, and disordered loops. The structure is similar to the reported zinc-ribbon motifs in several transcription-related proteins from archaea and eucarya, including Pyrococcus furiosus transcription factor B (PfTFB), human and yeast transcription factor IIS (TFIIS), and Thermococcus celer RNA polymerase II subunit M (TcRPOM). The zinc-ribbon structure of TFIIB, in conjunction with the biochemical analyses, suggests that residues on the beta-sheet are involved in the interaction with RNA pol II/TFIIF, while the zinc-binding site may increase the stability of the beta-sheet. PMID:11045620

  7. Structure of a (Cys3His) zinc ribbon, a ubiquitous motif in archaeal and eucaryal transcription.

    PubMed Central

    Chen, H. T.; Legault, P.; Glushka, J.; Omichinski, J. G.; Scott, R. A.

    2000-01-01

    Transcription factor IIB (TFIIB) is an essential component in the formation of the transcription initiation complex in eucaryal and archaeal transcription. TFIIB interacts with a promoter complex containing the TATA-binding protein (TBP) to facilitate interaction with RNA polymerase II (RNA pol II) and the associated transcription factor IIF (TFIIF). TFIIB contains a zinc-binding motif near the N-terminus that is directly involved in the interaction with RNA pol II/TFIIF and plays a crucial role in selecting the transcription initiation site. The solution structure of the N-terminal residues 2-59 of human TFIIB was determined by multidimensional NMR spectroscopy. The structure consists of a nearly tetrahedral Zn(Cys)3(His)1 site confined by type I and "rubredoxin" turns, three antiparallel beta-strands, and disordered loops. The structure is similar to the reported zinc-ribbon motifs in several transcription-related proteins from archaea and eucarya, including Pyrococcus furiosus transcription factor B (PfTFB), human and yeast transcription factor IIS (TFIIS), and Thermococcus celer RNA polymerase II subunit M (TcRPOM). The zinc-ribbon structure of TFIIB, in conjunction with the biochemical analyses, suggests that residues on the beta-sheet are involved in the interaction with RNA pol II/TFIIF, while the zinc-binding site may increase the stability of the beta-sheet. PMID:11045620

  8. Conduction-band minimum of (GaAs) sub 1 /(AlAs) sub 1 superlattices: Relationship to X minimum of AlAs

    SciTech Connect

    Ge, W.; Schmidt, W.D.; Sturge, M.D. ); Pfeiffer, L.N.; West, K.W. )

    1991-08-15

    We show that the conduction-band minimum in a (GaAs){sub 1}/(AlAs){sub 1} superlattice derives from the {ital X}{sub {ital x},}{ital y} minimum of bulk AlAs, not from {ital L} of GaAs as predicted by many theoretical calculations. This is shown by the sign of the shift in the low-temperature photoluminescence under (001) stress, by the relative magnitudes of the shifts under (100) and (110) stress, by the phonon sidebands, and by the observation of a splitting under (100) but not under (110) stress.

  9. Aβ-affected pathogenic induction of S-nitrosylation of OGT and identification of Cys-NO linkage triplet.

    PubMed

    Ryu, In-Hyun; Lee, Ki-Young; Do, Su-Il

    2016-05-01

    Mechanistic link of protein hypo-O-GlcNAcylation to the pathogenesis of Alzheimer's disease (AD) remains unclear. Here, we found that S-nitrosylation of O-linked N-acetylglucosaminyltransferase (SNO-OGT) was induced by β-amyloid peptide (Aβ) exposure to SK-N-MC and SK-N-SH human neuroblastoma cells. Subsequently, Aβ-induced SNO-OGT led to protein hypo-O-GlcNAcylation globally including tau hypo-O-GlcNAcylation. Our results support that underlying mechanism for induction of SNO-OGT comprises the concerted action of Aβ-triggered Ca2+ entry into cells and nNOS-catalyzed NO production. Intriguingly, OGT was found to be associated with nNOS and its association was enhanced during Aβ treatment. In parallel with SNO-OGT-mediated tau hypo-O-GlcNAcylation, Aβ led to SNO-Akt-mediated GSK3β activation for tau phosphorylation, suggesting that tau hyperphosphorylation is established by synergistic connection between SNO-OGT and GSK3β activation. We also observed that Aβ-neurotoxicity including both reactive oxygen species (ROS) production and cell death was amplified with DON treatment, whereas it was restored by PUGNAc treatment, GlcNH2 treatment or OGT overexpression. Early time-course Aβ-monitoring assay revealed that premaintained hyper-O-GlcNAcylation inside cells blocked not only Aβ-triggered Ca2+ entry into cells but also induction of SNO-OGT and SNO-Akt. Together, these findings suggest that induction of SNO-OGT by Aβ exposure is a pathogenic mechanism to cause cellular hypo-O-GlcNAcylation by which Aβ neurotoxicity is executed, and conversely, hyper-O-GlcNAcylation within cells can defend against Aβ neurotoxicity. Furthermore, our Cys mapping demonstrates that cysteine-nitric oxide (Cys-NO) linkages in SNO-OGT occur at triple Cys845, Cys921, and Cys965 residues in C-terminal catalytic domain (C-CAT), suggesting that Cys-NO linkage triplet in SNO-OGT is associated with null OGT activity. PMID:26854602

  10. Flexible and mechanical strain resistant large area SERS active substrates

    NASA Astrophysics Data System (ADS)

    Singh, J. P.; Chu, Hsiaoyun; Abell, Justin; Tripp, Ralph A.; Zhao, Yiping

    2012-05-01

    We report a cost effective and facile way to synthesize flexible, uniform, and large area surface enhanced Raman scattering (SERS) substrates using an oblique angle deposition (OAD) technique. The flexible SERS substrates consist of 1 μm long, tilted silver nanocolumnar films deposited on flexible polydimethylsiloxane (PDMS) and polyethylene terephthalate (PET) sheets using OAD. The SERS enhancement activity of these flexible substrates was determined using 10-5 M trans-1,2-bis(4-pyridyl) ethylene (BPE) Raman probe molecules. The in situ SERS measurements on these flexible substrates under mechanical (tensile/bending) strain conditions were performed. Our results show that flexible SERS substrates can withstand a tensile strain (ε) value as high as 30% without losing SERS performance, whereas the similar bending strain decreases the SERS performance by about 13%. A cyclic tensile loading test on flexible PDMS SERS substrates at a pre-specified tensile strain (ε) value of 10% shows that the SERS intensity remains almost constant for more than 100 cycles. These disposable and flexible SERS substrates can be integrated with biological substances and offer a novel and practical method to facilitate biosensing applications.

  11. Unique Gold Nanoparticle Aggregates as a Highly Active SERS Substrate

    SciTech Connect

    Schwartzberg, A M; Grant, C D; Wolcott, A; Talley, C E; Huser, T R; Bogomolni, R; Zhang, J Z

    2004-04-06

    A unique gold nanoparticle aggregate (GNA) system has been shown to be an excellent substrate for surface-enhanced Raman scattering (SERS) applications. Rhodamine 6G (R6G), a common molecule used for testing SERS activity on silver, but generally difficult to detect on gold substrates, has been found to readily bind to the GNA and exhibit strong SERS activity due to the unique surface chemistry afforded by sulfur species on the surface. This GNA system has yielded a large SERS enhancement of 10{sup 7}-10{sup 9} in bulk solution for R6G, on par with or greater than any previously reported gold SERS substrate. SERS activity has also been successfully demonstrated for several biological molecules including adenine, L-cysteine, L-lysine, and L-histidine for the first time on a gold SERS substrate, showing the potential of this GNA as a convenient and powerful SERS substrate for biomolecular detection. In addition, SERS spectrum of R6G on single aggregates has been measured. We have shown that the special surface properties of the GNA, in conjunction with strong near IR absorption, make it useful for SERS analysis of a wide variety of molecules.

  12. Hydrogen Sulfide Targets the Cys320/Cys529 Motif in Kv4.2 to Inhibit the Ito Potassium Channels in Cardiomyocytes and Regularizes Fatal Arrhythmia in Myocardial Infarction

    PubMed Central

    Ma, Shan-Feng; Luo, Yan; Ding, Ying-Jiong; Chen, Ying; Pu, Shi-Xin; Wu, Hang-Jing; Wang, Zhong-Feng; Tao, Bei-Bei; Wang, Wen-Wei

    2015-01-01

    Abstract Aims: The mechanisms underlying numerous biological roles of hydrogen sulfide (H2S) remain largely unknown. We have previously reported an inhibitory role of H2S in the L-type calcium channels in cardiomyocytes. This prompts us to examine the mechanisms underlying the potential regulation of H2S on the ion channels. Results: H2S showed a novel inhibitory effect on Ito potassium channels, and this effect was blocked by mutation at the Cys320 and/or Cys529 residues of the Kv4.2 subunit. H2S broke the disulfide bridge between a pair of oxidized cysteine residues; however, it did not modify single cysteine residues. H2S extended action potential duration in epicardial myocytes and regularized fatal arrhythmia in a rat model of myocardial infarction. H2S treatment significantly increased survival by ∼1.4-fold in the critical 2-h time window after myocardial infarction with a protection against ventricular premature beats and fatal arrhythmia. However, H2S did not change the function of other ion channels, including IK1 and INa. Innovation and Conclusion: H2S targets the Cys320/Cys529 motif in Kv4.2 to regulate the Ito potassium channels. H2S also shows a potent regularizing effect against fatal arrhythmia in a rat model of myocardial infarction. The study provides the first piece of evidence for the role of H2S in regulating Ito potassium channels and also the specific motif in an ion channel labile for H2S regulation. Antioxid. Redox Signal. 23, 129–147. PMID:25756524

  13. Ser262 determines the chloride-dependent colour tuning of a new halorhodopsin from Haloquadratum walsbyi

    PubMed Central

    Fu, Hsu-Yuan; Chang, Yung-Ning; Jheng, Ming-Jin; Yang, Chii-Shen

    2012-01-01

    Light is an important environmental signal for all organisms on earth because it is essential for physiological signalling and the regulation of most biological systems. Halophiles found in salt-saturated ponds encode various archaeal rhodopsins and thereby harvest various wavelengths of light either for ion transportation or as sensory mediators. HR (halorhodopsin), one of the microbial rhodopsins, senses yellow light and transports chloride or other halides into the cytoplasm to maintain the osmotic balance during cell growth, and it exists almost ubiquitously in all known halobacteria. To date, only two HRs, isolated from HsHR (Halobacterium salinarum HR) and NpHR (Natronomonas pharaonis HR), have been characterized. In the present study, two new HRs, HmHR (Haloarcula marismortui HR) and HwHR (Haloquadratum walsbyi HR), were functionally overexpressed in Escherichia coli, and the maximum absorbance (λmax) of the purified proteins, the light-driven chloride uptake and the chloride-binding affinity were measured. The results showed them to have similar properties to two HRs reported previously. However, the λmax of HwHR is extremely consistent in a wide range of salt/chloride concentrations, which had not been observed previously. A structural-based sequence alignment identified a single serine residue at 262 in HwHR, which is typically a conserved alanine in all other known HRs. A Ser262 to alanine replacement in HwHR eliminated the chloride-independent colour tuning, whereas an Ala246 to serine mutagenesis in HsHR transformed it to have chloride-independent colour tuning similar to that of HwHR. Thus Ser262 is a key residue for the mechanism of chloride-dependent colour tuning in HwHR. PMID:22716305

  14. Phosphorylation of MeCP2 at Ser421 contributes to chronic antidepressant action

    PubMed Central

    Hutchinson, Ashley N.; Deng, Jay V.; Cohen, Sonia; West, Anne E.

    2012-01-01

    Although tricyclic antidepressants rapidly activate monoaminergic neurotransmission, these drugs must be administered chronically to alleviate symptoms of depression. This observation suggests that molecular mechanisms downstream of monoamine receptor activation, which include the induction of gene transcription, underlie chronic antidepressant-induced changes in behavior. Here we show that methyl-CpG binding protein 2 (MeCP2) regulates behavioral responses to chronic antidepressant treatment. Imipramine administration induces phosphorylation of MeCP2 at Ser421 (pMeCP2) selectively in the nucleus accumbens and the lateral habenula, two brain regions important for depressive-like behaviors. To test the role of pMeCP2 in depressive-like behaviors, we utilized male mice that bear a germline mutation knocked into the X-linked Mecp2 locus that changes Ser421 to a nonphosphorylatable Ala residue (S421A). MeCP2 S421A knockin (KI) mice showed increased immobility in forced swim and tail suspension tests compared with their wildtype (WT) littermates. However immobility of both MeCP2 WT and KI mice in forced swim was reduced by acute administration of imipramine, demonstrating that loss of pMeCP2 does not impair acute pharmacological sensitivity to this drug. Following chronic social defeat stress, chronic administration of imipramine significantly improved social interaction in the MeCP2 WT mice. By contrast, the MeCP2 KI mice did not respond to chronic imipramine administration. These data suggest novel roles for pMeCP2 in the sensitivity to stressful stimuli and demonstrate that pMeCP2 is required for the effects of chronic imipramine on depressive-like behaviors induced by chronic social defeat stress. PMID:23055506

  15. A 1-Cys Peroxiredoxin from a Thermophilic Archaeon Moonlights as a Molecular Chaperone to Protect Protein and DNA against Stress-Induced Damage

    PubMed Central

    Pham, Bang Phuong; Kwak, Jae Myeong; Xuan, Yuan Hu; Cheong, Gang-Won

    2015-01-01

    Peroxiredoxins (Prxs) act against hydrogen peroxide (H2O2), organic peroxides, and peroxynitrite. Thermococcus kodakaraensis KOD1, an anaerobic archaeon, contains many antioxidant proteins, including three Prxs (Tk0537, Tk0815, and Tk1055). Only Tk0537 has been found to be induced in response to heat, osmotic, and oxidative stress. Tk0537 was found to belong to a 1-Cys Prx6 subfamily based on sequence analysis and was named 1-Cys TkPrx. Using gel filtration chromatography, electron microscopy, and blue-native polyacrylamide gel electrophoresis, we observed that 1-Cys TkPrx exhibits oligomeric forms with reduced peroxide reductase activity as well as decameric and dodecameric forms that can act as molecular chaperones by protecting both proteins and DNA from oxidative stress. Mutational analysis showed that a cysteine residue at the N-terminus (Cys46) was responsible for the peroxide reductase activity, and cysteine residues at the C-terminus (Cys205 and Cys211) were important for oligomerization. Based on our results, we propose that interconversion between different oligomers is important for regulating the different functions of 1-Cys TkPrx. PMID:25933432

  16. Free-thiol Cys331 exposed during activation process is critical for native tetramer structure of cathepsin C (dipeptidyl peptidase I)

    PubMed Central

    Horn, Martin; Baudyš, Miroslav; Voburka, Zdeněk; Kluh, Ivan; Vondrášek, Jiří; Mareš, Michael

    2002-01-01

    The mature bovine cathepsin C (CC) molecule is composed of four identical monomers, each proteolytically processed into three chains. Five intrachain disulfides and three nonpaired cysteine residues per monomer were identified. Beside catalytic Cys234 in the active site, free-thiol Cys331 and Cys424 were characterized. Cys424 can be classified as inaccessible buried residue. Selective modification of Cys331 results in dissociation of native CC tetramer into dimers. The 3D homology-based model of the CC catalytic core suggests that Cys331 becomes exposed as the activation peptide is removed during procathepsin C activation. The model further shows that exposed Cys331 is surrounded by a surface hydrophobic cluster, unique to CC, forming a dimer–dimer interaction interface. Substrate/inhibitor recognition of the active site in the CC dimer differs significantly from that in the native tetramer. Taken together, a mechanism is proposed that assumes that the CC tetramer formation results in a site-specific occlusion of endopeptidase-like active site cleft of each CC monomeric unit. Thus, tetramerization provides for the structural basis of the dipeptidyl peptidase activity of CC through a substrate access-limiting mechanism different from those found in homologous monomeric exopeptidases cathepsin H and B. In conclusion, the mechanism of tetramer formation as well as specific posttranslational processing segregates CC in the family of papain proteases. PMID:11910036

  17. Transient Transcriptional Regulation of the CYS-C1 Gene and Cyanide Accumulation upon Pathogen Infection in the Plant Immune Response1[C][W

    PubMed Central

    García, Irene; Rosas, Tábata; Bejarano, Eduardo R.; Gotor, Cecilia; Romero, Luis C.

    2013-01-01

    Cyanide is produced concomitantly with ethylene biosynthesis. Arabidopsis (Arabidopsis thaliana) detoxifies cyanide primarily through the enzyme β-cyanoalanine synthase, mainly by the mitochondrial CYS-C1. CYS-C1 loss of function is not toxic for the plant and leads to an increased level of cyanide in cys-c1 mutants as well as a root hairless phenotype. The classification of genes differentially expressed in cys-c1 and wild-type plants reveals that the high endogenous cyanide content of the cys-c1 mutant is correlated with the biotic stress response. Cyanide accumulation and CYS-C1 gene expression are negatively correlated during compatible and incompatible plant-bacteria interactions. In addition, cys-c1 plants present an increased susceptibility to the necrotrophic fungus Botrytis cinerea and an increased tolerance to the biotrophic Pseudomonas syringae pv tomato DC3000 bacterium and Beet curly top virus. The cys-c1 mutation produces a reduction in respiration rate in leaves, an accumulation of reactive oxygen species, and an induction of the alternative oxidase AOX1a and pathogenesis-related PR1 expression. We hypothesize that cyanide, which is transiently accumulated during avirulent bacterial infection and constitutively accumulated in the cys-c1 mutant, uncouples the respiratory electron chain dependent on the cytochrome c oxidase, and this uncoupling induces the alternative oxidase activity and the accumulation of reactive oxygen species, which act by stimulating the salicylic acid-dependent signaling pathway of the plant immune system. PMID:23784464

  18. Transient transcriptional regulation of the CYS-C1 gene and cyanide accumulation upon pathogen infection in the plant immune response.

    PubMed

    García, Irene; Rosas, Tábata; Bejarano, Eduardo R; Gotor, Cecilia; Romero, Luis C

    2013-08-01

    Cyanide is produced concomitantly with ethylene biosynthesis. Arabidopsis (Arabidopsis thaliana) detoxifies cyanide primarily through the enzyme β-cyanoalanine synthase, mainly by the mitochondrial CYS-C1. CYS-C1 loss of function is not toxic for the plant and leads to an increased level of cyanide in cys-c1 mutants as well as a root hairless phenotype. The classification of genes differentially expressed in cys-c1 and wild-type plants reveals that the high endogenous cyanide content of the cys-c1 mutant is correlated with the biotic stress response. Cyanide accumulation and CYS-C1 gene expression are negatively correlated during compatible and incompatible plant-bacteria interactions. In addition, cys-c1 plants present an increased susceptibility to the necrotrophic fungus Botrytis cinerea and an increased tolerance to the biotrophic Pseudomonas syringae pv tomato DC3000 bacterium and Beet curly top virus. The cys-c1 mutation produces a reduction in respiration rate in leaves, an accumulation of reactive oxygen species, and an induction of the alternative oxidase AOX1a and pathogenesis-related PR1 expression. We hypothesize that cyanide, which is transiently accumulated during avirulent bacterial infection and constitutively accumulated in the cys-c1 mutant, uncouples the respiratory electron chain dependent on the cytochrome c oxidase, and this uncoupling induces the alternative oxidase activity and the accumulation of reactive oxygen species, which act by stimulating the salicylic acid-dependent signaling pathway of the plant immune system. PMID:23784464

  19. Metallopolymer-peptide conjugates: synthesis and self-assembly of polyferrocenylsilane graft and block copolymers containing a beta-sheet forming Gly-Ala-Gly-Ala tetrapeptide segment.

    PubMed

    Vandermeulen, Guido W M; Kim, Kyoung Taek; Wang, Zhuo; Manners, Ian

    2006-04-01

    We describe the synthesis and self-assembly of two beta-sheet forming metallopolymer-peptide conjugates. The ability of the oligotetrapeptide sequence Gly-Ala-Gly-Ala (GAGA) to form antiparallel beta-sheets was retained in PFS-b-AGAG (PFS = polyferrocenylsilane) and PFS-g-AGAG conjugates with block and graft architectures, respectively. In the solid state, DSC experiments suggest a phase separation between the peptide and PFS domains. In toluene, PFS-b-AGAG interestingly forms a fibrous network which consists of a core containing the self-assembled antiparallel beta-sheet peptide and a corona of organometallic PFS. The self-assembly of the peptide into antiparallel beta-sheets is the driving force for the fiber formation, whereas PFS prevents uncontrolled lateral aggregation of the fibers. The use of an oligopeptide to self-assemble an otherwise random coiled organometallic polymer may be a useful strategy to enhance nanostructure formation. In the cases described here, the conjugates may be used to create nanopatterned ceramics, and the redox properties of the resulting supramolecular aggregates are of significant interest. PMID:16602714

  20. Analysis of PGC-1{alpha} variants Gly482Ser and Thr612Met concerning their PPAR{gamma}2-coactivation function

    SciTech Connect

    Nitz, Inke . E-mail: initz@molnut.uni-kiel.de; Ewert, Agnes; Klapper, Maja; Doering, Frank

    2007-02-09

    Peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) is a cofactor involved in adaptive thermogenesis, fatty acid oxidation, and gluconeogenesis. Dysfunctions of this protein are likely to contribute to the development of obesity and the metabolic syndrome. This is in part but not definitely confirmed by results of population studies. The aim of this study was to investigate if common genetic variants rs8192678 (Gly482Ser) and rs3736265 (Thr612Met) in the PGC-1{alpha} gene lead to a functional consequence in cofactor activity using peroxisome proliferator-activated receptor-{gamma} 2 (PPAR{gamma}2) as interacting transcription factor. Reporter gene assays in HepG2 cells with wildtype and mutant proteins of both PGC1{alpha} and PPAR{gamma}2 (Pro12Ala, rs1801282) using the acyl-CoA-binding protein (ACBP) promoter showed no difference in coactivator activity. This is First study implicating that the Gly482Ser and Thr612Met polymorphisms in PGC-1{alpha} and Pro12Ala polymorphism in PPAR{gamma}2 do not affect the functional integrity of these proteins.

  1. Involvement of the Cys-Tyr cofactor on iron binding in the active site of human cysteine dioxygenase.

    PubMed

    Arjune, Sita; Schwarz, Guenter; Belaidi, Abdel A

    2015-01-01

    Sulfur metabolism has gained increasing medical interest over the last years. In particular, cysteine dioxygenase (CDO) has been recognized as a potential marker in oncology due to its altered gene expression in various cancer types. Human CDO is a non-heme iron-dependent enzyme, which catalyzes the irreversible oxidation of cysteine to cysteine sulfinic acid, which is further metabolized to taurine or pyruvate and sulfate. Several studies have reported a unique post-translational modification of human CDO consisting of a cross-link between cysteine 93 and tyrosine 157 (Cys-Tyr), which increases catalytic efficiency in a substrate-dependent manner. However, the reaction mechanism by which the Cys-Tyr cofactor increases catalytic efficiency remains unclear. In this study, steady-state kinetics were determined for wild type CDO and two different variants being either impaired or saturated with the Cys-Tyr cofactor. Cofactor formation in CDO resulted in an approximately fivefold increase in k cat and tenfold increase in k cat/K m over the cofactor-free CDO variant. Furthermore, iron titration experiments revealed an 18-fold decrease in K d of iron upon cross-link formation. This finding suggests a structural role of the Cys-Tyr cofactor in coordinating the ferrous iron in the active site of CDO in accordance with the previously postulated reaction mechanism of human CDO. Finally, we identified product-based inhibition and α-ketoglutarate and glutarate as CDO inhibitors using a simplified well plate-based activity assay. This assay can be used for high-throughput identification of additional inhibitors, which may contribute to understand the functional importance of CDO in sulfur amino acid metabolism and related diseases.

  2. Phosphorylation of pRB at Ser612 by Chk1/2 leads to a complex between pRB and E2F-1 after DNA damage.

    PubMed

    Inoue, Yasumichi; Kitagawa, Masatoshi; Taya, Yoichi

    2007-04-18

    The retinoblastoma tumor suppressor protein (pRB) plays a critical role in the control of cell proliferation and in the DNA damage checkpoints. pRB inhibits cell cycle progression through interactions with the E2F family of transcription factors. Here, we report that DNA damage induced not only the dephosphorylation of pRB at Cdk phosphorylation sites and the binding of pRB to E2F-1, but also the phosphorylation of pRB at Ser612. Phosphorylation of pRB at Ser612 enhanced the formation of a complex between pRB and E2F-1. Substitution of Ser612 with Ala decreased pRB-E2F-1 binding and the transcriptional repression activity. Until now, Ser612 of pRB has been thought to be phosphorylated by Cdk2. However, the phosphorylation of pRB at Ser612 was conducted by Chk1/2 after DNA damage, and inhibition of ATM-Chk1/2 activity suppressed the phosphorylation of Ser612 and the binding of pRB to E2F-1. These results suggest that Ser612 is phosphorylated by Chk1/2 after DNA damage, leading to the formation of pRB-E2F-1. This is the first report that pRB is phosphorylated in vivo by a kinase other than Cdk.

  3. Preclinical Development of a Subcutaneous ALAS1 RNAi Therapeutic for Treatment of Hepatic Porphyrias Using Circulating RNA Quantification

    PubMed Central

    Chan, Amy; Liebow, Abigail; Yasuda, Makiko; Gan, Lin; Racie, Tim; Maier, Martin; Kuchimanchi, Satya; Foster, Don; Milstein, Stuart; Charisse, Klaus; Sehgal, Alfica; Manoharan, Muthiah; Meyers, Rachel; Fitzgerald, Kevin; Simon, Amy; Desnick, Robert J; Querbes, William

    2015-01-01

    The acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1) by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs) following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology. PMID:26528940

  4. Preclinical Development of a Subcutaneous ALAS1 RNAi Therapeutic for Treatment of Hepatic Porphyrias Using Circulating RNA Quantification.

    PubMed

    Chan, Amy; Liebow, Abigail; Yasuda, Makiko; Gan, Lin; Racie, Tim; Maier, Martin; Kuchimanchi, Satya; Foster, Don; Milstein, Stuart; Charisse, Klaus; Sehgal, Alfica; Manoharan, Muthiah; Meyers, Rachel; Fitzgerald, Kevin; Simon, Amy; Desnick, Robert J; Querbes, William

    2015-01-01

    The acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1) by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs) following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology. PMID:26528940

  5. Live-cell SERS endoscopy using plasmonic nanowire waveguides.

    PubMed

    Lu, Gang; De Keersmaecker, Herlinde; Su, Liang; Kenens, Bart; Rocha, Susana; Fron, Eduard; Chen, Chang; Van Dorpe, Pol; Mizuno, Hideaki; Hofkens, Johan; Hutchison, James A; Uji-i, Hiroshi

    2014-08-13

    Live-cell surface-enhanced Raman spectroscopy (SERS) endoscopy is developed by using plasmonic nanowire waveguides as endoscopic probes. It is demonstrated that the probe insertion does not stress the cell. Opposed to conventional SERS endoscopy, with excitation at the hotspot within the cell, the remote excitation method yields low-background SERS spectra from specific cell compartments with minimal associated photodamage. PMID:24866811

  6. Chemical monitors based on Surface-Enhanced Raman Scattering (SERS)

    SciTech Connect

    Vo-Dinh, T.; Alarie, J.P.; Sutherland, W.S.; Stokes, D.L.; Miller, G.H.

    1992-12-31

    This paper presents an overview of the development of chemical monitors using the Surface-Enhanced Raman Scattering (SERS) technique. The SERS effect is based on recent experimental observations, which have indicated enhancement of the Raman scattering efficiency by factors up to 10{sup 8} when a compound is adsorbed on rough metallic surfaces having submicron protrusions. The focus of our research efforts is on the development of SERS-active sensors and instrumentation capable of field analysis and remote sensing.

  7. The structure of reduced tryparedoxin peroxidase reveals a decamer and insight into reactivity of 2Cys-peroxiredoxins.

    PubMed

    Alphey, M S; Bond, C S; Tetaud, E; Fairlamb, A H; Hunter, W N

    2000-07-21

    Tryparedoxin peroxidase (TryP) is a recently discovered 2Cys-peroxiredoxin involved in defence against oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata TryP, in the reduced state, has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises a decamer with 52 symmetry, ten chloride ions with 23 water molecules and has been refined, using data to 3.2 A resolution (1 A=0.1 nm), to an R-factor and R(free) of 27.3 and 28.6 %, respectively. Secondary structure topology places TryP along with tryparedoxin and glutathione peroxidase in a distinct subgroup of the thioredoxin super-family. The molecular details at the active site support ideas about the enzyme mechanism and comparisons with an oxidised 2Cys-peroxiredoxin reveal structural alterations induced by the change in oxidation state. These include a difference in quaternary structure from dimer (oxidised form) to decamer (reduced form). The 2Cys-peroxiredoxin assembly may prevent indiscriminate oligomerisation, localise ten peroxidase active sites and contribute to both the specificity of reduction by the redox partner tryparedoxin and attraction of peroxides into the active site.

  8. ALA Inhibits ABA-induced Stomatal Closure via Reducing H2O2 and Ca(2+) Levels in Guard Cells.

    PubMed

    An, Yuyan; Liu, Longbo; Chen, Linghui; Wang, Liangju

    2016-01-01

    5-Aminolevulinic acid (ALA), a newly proved natural plant growth regulator, is well known to improve plant photosynthesis under both normal and stressful conditions. However, its underlying mechanism remains largely unknown. Stomatal closure is one of the major limiting factors for photosynthesis and abscisic acid (ABA) is the most important hormone in provoking stomatal closing. Here, we showed that ALA significantly inhibited ABA-induced stomatal closure using wild-type and ALA-overproducing transgenic Arabidopsis (YHem1). We found that ALA decreased ABA-induced H2O2 and cytosolic Ca(2+) accumulation in guard cells with stomatal bioassay, laser-scanning confocal microscopy and pharmacological methods. The inhibitory effect of ALA on ABA-induced stomatal closure was similar to that of AsA (an important reducing substrate for H2O2 removal), CAT (a H2O2-scavenging enzyme), DPI (an inhibitor of the H2O2-generating NADPH oxidase), EGTA (a Ca-chelating agent), and AlCl3 (an inhibitor of calcium channel). Furthermore, ALA inhibited exogenous H2O2- or Ca(2+)-induced stomatal closure. Taken together, we conclude that ALA inhibits ABA-induced stomatal closure via reducing H2O2, probably by scavenging, and Ca(2+) levels in guard cells. Moreover, the inhibitive effect of ALA on ABA-induced stomatal closure was further confirmed in the whole plant. Finally, we demonstrated that ALA inhibits stomatal closing, but significantly improves plant drought tolerance. Our results provide valuable information for the promotion of plant production and development of a sustainable low-carbon society.

  9. ALA Inhibits ABA-induced Stomatal Closure via Reducing H2O2 and Ca(2+) Levels in Guard Cells.

    PubMed

    An, Yuyan; Liu, Longbo; Chen, Linghui; Wang, Liangju

    2016-01-01

    5-Aminolevulinic acid (ALA), a newly proved natural plant growth regulator, is well known to improve plant photosynthesis under both normal and stressful conditions. However, its underlying mechanism remains largely unknown. Stomatal closure is one of the major limiting factors for photosynthesis and abscisic acid (ABA) is the most important hormone in provoking stomatal closing. Here, we showed that ALA significantly inhibited ABA-induced stomatal closure using wild-type and ALA-overproducing transgenic Arabidopsis (YHem1). We found that ALA decreased ABA-induced H2O2 and cytosolic Ca(2+) accumulation in guard cells with stomatal bioassay, laser-scanning confocal microscopy and pharmacological methods. The inhibitory effect of ALA on ABA-induced stomatal closure was similar to that of AsA (an important reducing substrate for H2O2 removal), CAT (a H2O2-scavenging enzyme), DPI (an inhibitor of the H2O2-generating NADPH oxidase), EGTA (a Ca-chelating agent), and AlCl3 (an inhibitor of calcium channel). Furthermore, ALA inhibited exogenous H2O2- or Ca(2+)-induced stomatal closure. Taken together, we conclude that ALA inhibits ABA-induced stomatal closure via reducing H2O2, probably by scavenging, and Ca(2+) levels in guard cells. Moreover, the inhibitive effect of ALA on ABA-induced stomatal closure was further confirmed in the whole plant. Finally, we demonstrated that ALA inhibits stomatal closing, but significantly improves plant drought tolerance. Our results provide valuable information for the promotion of plant production and development of a sustainable low-carbon society. PMID:27148309

  10. ALA Inhibits ABA-induced Stomatal Closure via Reducing H2O2 and Ca2+ Levels in Guard Cells

    PubMed Central

    An, Yuyan; Liu, Longbo; Chen, Linghui; Wang, Liangju

    2016-01-01

    5-Aminolevulinic acid (ALA), a newly proved natural plant growth regulator, is well known to improve plant photosynthesis under both normal and stressful conditions. However, its underlying mechanism remains largely unknown. Stomatal closure is one of the major limiting factors for photosynthesis and abscisic acid (ABA) is the most important hormone in provoking stomatal closing. Here, we showed that ALA significantly inhibited ABA-induced stomatal closure using wild-type and ALA-overproducing transgenic Arabidopsis (YHem1). We found that ALA decreased ABA-induced H2O2 and cytosolic Ca2+ accumulation in guard cells with stomatal bioassay, laser-scanning confocal microscopy and pharmacological methods. The inhibitory effect of ALA on ABA-induced stomatal closure was similar to that of AsA (an important reducing substrate for H2O2 removal), CAT (a H2O2-scavenging enzyme), DPI (an inhibitor of the H2O2-generating NADPH oxidase), EGTA (a Ca-chelating agent), and AlCl3 (an inhibitor of calcium channel). Furthermore, ALA inhibited exogenous H2O2- or Ca2+-induced stomatal closure. Taken together, we conclude that ALA inhibits ABA-induced stomatal closure via reducing H2O2, probably by scavenging, and Ca2+ levels in guard cells. Moreover, the inhibitive effect of ALA on ABA-induced stomatal closure was further confirmed in the whole plant. Finally, we demonstrated that ALA inhibits stomatal closing, but significantly improves plant drought tolerance. Our results provide valuable information for the promotion of plant production and development of a sustainable low-carbon society. PMID:27148309

  11. Bioenabled SERS substrates for food safety and drinking water monitoring

    NASA Astrophysics Data System (ADS)

    Yang, Jing; Rorrer, Gregory L.; Wang, Alan X.

    2015-05-01

    We present low-cost bioenabled surface-enhanced Raman scattering (SERS) substrates that can be massively produced in sustainable and eco-friendly methods with significant commercial potentials for the detection of food contamination and drinking water pollution. The sensors are based on diatom frustules with integrated plasmonic nanoparticles. The ultra-high sensitivity of the SERS substrates comes from the coupling between the diatom frustules and Ag nanoparticles to achieve dramatically increased local optical field to enhance the light-matter interactions for SERS sensing. We successfully applied the bioenabled SERS substrates to detect melamine in milk and aromatic compounds in water with sensitivity down to 1μg/L.

  12. Photodynamic therapy (ALA-PDT) in the treatment of pathological states of the cornea

    NASA Astrophysics Data System (ADS)

    Switka-Wieclawska, Iwona; Kecik, Tadeusz; Kwasny, Miroslaw; Graczyk, Alfreda

    2003-10-01

    Each year an increasing amount of research is published on the use of photodynamic therapy in medicine. The most recent research has focused mostly on the use of photosensitizer called vertoporphyrin (Visudyne) is the treatment of subretinal neovascularization in age-related macular degeneration (AMD) or myopia, following a substantial amount of ophthalmology research mostly experimental on the application of the method in diagnosis and treatment of some eye tumors. In the Department of Ophthalmology of Polish Medical University in Warsaw, PDT was used as supplementary method in a selected group of patients with chronic virus ulcer of the cornea and keratopathies. During the treatment 5-aminolevulinic acid (5-ALA) was applied in ointment form as a photosensitizer activated with light wave of 633 nm. It appears, on the basis of the results obtained, that photodynamic therapy (ALA-PDT) may become in the future a valuable supplement to the methods being used at the present treating pathological states of the cornea.

  13. Endoscopic fluorescence of gastrointestinal neoplasia after sensitization with 5-aminolaevulinic acid (ALA) or Photofrin

    NASA Astrophysics Data System (ADS)

    Messmann, Helmut; Mlkvy, Peter; Montan, Sune; Wang-Nordman, Ingrid; Nilsson, Annika M.; Svanberg, Katarina; Svanberg, Sune; MacRobert, Alexander J.; Bown, Stephen G.

    1995-03-01

    Fluorescence after photosensitization has the potential to identify lesions not visible on conventional endoscopy. We assessed 12 patients at high risk of or with established GI cancers (u ulcerative colitis, 1 colon polyp, 2 familial polyposis with duodenal polyps, 2 early oesophageal cancers). Fluorescence images (excitation 390 nm) were recorded with endoscopic equipment and additional spot measurements (optical multichannel analyzer). Patients were given 10 - 60 mg/kg ALA orally or 2 mg/kg Photofrin i.v. 60 mg/kg ALA gave high levels of PP IX (proto-porphyrin IX) in all areas, but 10 - 15 mg/kg resulted in selectivity in macroscopically inflamed colon. Photofrin gave oesophageal tumors selectivity at 4 and 48 hours. Photofrin patients subsequently had PDT. Photobleaching was documented in 3. We conclude that these techniques have potential as `optical biopsy tools' and for screening for early neoplastic changes.

  14. Photodynamic diagnosis following intravesical instillation of aminolevulinic acid (ALA): first clinical experiences in urology

    NASA Astrophysics Data System (ADS)

    Baumgartner, Reinhold; Kriegmair, M.; Stepp, Herbert G.; Lumper, W.; Heil, Peter; Riesenberg, Rainer; Stocker, Susanne; Hofstetter, Alfons G.

    1993-06-01

    Delta Aminolevulinic acid (ALA), a precursor of Protoporphyrin IX (PP IX) in hem biosynthesis has been topically applied in urinary bladders in order to study its potential as fluorescent tumor marker. Preclinical experiments have been performed on chemically induced tumors in rats, revealing a ratio of PP IX-fluorescence intensity up to 20:1 in tumors as compared to healthy urothelium. Synthesis of PP IX has been stimulated in 56 patients by intravesical instillation of a pH-neutral ALA-solution. After an incubation time of two to four hours strong red fluorescence was endoscopically observed even in tiny superficial tumors. Brightness and contrast allows visualization of early stage urothelial diseases with naked eyes and without the necessity suppressing background fluorescence or violet excitation light.

  15. Interaction between Pro12Ala polymorphism of PPARγ2 and diet on adiposity phenotypes.

    PubMed

    Lapice, Emanuela; Vaccaro, Olga

    2014-12-01

    The aim of this report is to perform a systematic review and qualitative synthesis of the literature to address whether, and to what extent, diet modulates the effects of the Pro12Ala polymorphism of peroxisome proliferator-activated receptor gamma 2 (PPARγ2) on body weight and other measures of adiposity. A systematic search of the literature was conducted, wherein both observational and experimental studies of adults were reviewed. Overall, the results of the observational studies show little consistency. Methodological differences in their design, conduct and analysis may largely account for the apparently discrepant findings. This notwithstanding, the main picture that emerges is that the energy content and composition of the diet may affect BMI, body composition and metabolic parameters in Ala allele carriers more than in Pro/Pro homozygotes. In most studies, carriers of the Ala allele with an obesogenic lifestyle (i.e. high-energy, high-carbohydrate and, to some extent, high-fat diets) are more obese than Pro homozygotes. Well-designed intervention studies with a sufficiently large sample size consistently show that carriers of the Ala allele are more prone to weight loss when exposed to a healthy lifestyle; however, these individuals do not seem to retain these benefits when returning to a sedentary lifestyle and inadequate dieting behaviours. Some key questions in this area of research have emerged. Carefully designed and adequately powered studies are needed, particularly involving the development and validation of standardized tools for the assessment of dietary exposure, including the use of biomarkers, to move the field forward. PMID:25342491

  16. Nuclear transcription factors: a new approach to enhancing cellular responses to ALA-mediated photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Maytin, Edward V.; Anand, Sanjay; Sato, Nobuyuki; Moore, Brian; Mack, Judith; Gasbarre, Christopher; Keevey, Samantha; Ortel, Bernhard; Sinha, Alok; Khachemoune, Amor

    2006-02-01

    Photodynamic therapy (PDT) using aminolevulinic acid (ALA) relies upon the uptake of ALA into cancer cells, where it is converted into a porphyrin intermediate, protoporphyrin IX (PpIX) that is highly photosensitizing. For large or resistant tumors, however, ALA/PDT is often not completely effective due to inadequate PpIX levels. Therefore, new approaches to enhance the intracellular production of PpIX are sought. Here, we describe a general approach to improve intracellular PpIX accumulation via manipulations that increase the expression of an enzyme, coproporphyrinogen oxidase (CPO), that is rate-determining for PpIX production. We show that nuclear hormones that promote terminal differentiation, e.g. vitamin D or androgens, can also increase the accumulation of PpIX and the amount of killing of the target cells upon exposure to light. These hormones bind to intracellular hormone receptors that translocate to the nucleus, where they act as transcription factors to increase the expression of target genes. We have found that several other transcription factors associated with terminal differentiation, including members of the CCAAT enhancer binding (C/EBP) family, and a homeobox protein named Hoxb13, are also capable of enhancing PpIX accumulation. These latter transcription factors appear to interact directly with the CPO gene promoter, resulting in enhanced CPO transcriptional activity. Our data in several different cell systems, including epithelial cells of the skin and prostate cancer cells, indicate that enhancement of CPO expression and PpIX accumulation represents a viable new approach toward improving the efficacy of ALA/PDT.

  17. Co-Circulation of the Rare CPV-2c with Unique Gln370Arg Substitution, New CPV-2b with Unique Thr440Ala Substitution, and New CPV-2a with High Prevalence and Variation in Heilongjiang Province, Northeast China

    PubMed Central

    Geng, Yufei; Guo, Donghua; Li, Chunqiu; Wang, Enyu; Wei, Shan; Wang, Zhihui; Yao, Shuang; Zhao, Xiwen; Su, Mingjun; Wang, Xinyu; Wang, Jianfa; Wu, Rui; Feng, Li; Sun, Dongbo

    2015-01-01

    To trace evolution of canine parvovirus-2 (CPV-2), a total of 201 stool samples were collected from dogs with diarrhea in Heilongjiang province of northeast China from May 2014 to April 2015. The presence of CPV-2 in the samples was determined by PCR amplification of the VP2 gene (568 bp) of CPV-2. The results revealed that 95 samples (47.26%) were positive for CPV-2, and they showed 98.8%–100% nucleotide identity and 97.6%–100% amino acid identity. Of 95 CPV-2-positive samples, types new2a (Ser297Ala), new2b (Ser297Ala), and 2c accounted for 64.21%, 21.05%, and 14.74%, respectively. The positive rate of CPV-2 and the distribution of the new2a, new2b and 2c types exhibited differences among regions, seasons, and ages. Immunized dogs accounted for 48.42% of 95 CPV-2-positive samples. Coinfections with canine coronavirus, canine kobuvirus, and canine bocavirus were identified. Phylogenetic analysis revealed that the identified new2a, new2b, and CPV-2c strains in our study exhibited a close relationship with most of the CPV-2 strains from China; type new2a strains exhibited high variability, forming three subgroups; type new2b and CPV-2c strains formed one group with reference strains from China. Of 95 CPV-2 strains, Tyr324Ile and Thr440Ala substitutions accounted for 100% and 64.21%, respectively; all type new2b strains exhibited the Thr440Ala substitution, while the unique Gln370Arg substitution was found in all type 2c strains. Recombination analysis using entire VP2 gene indicated possible recombination events between the identified CPV-2 strains and reference strains from China. Our data revealed the co-circulation of new CPV-2a, new CPV-2b, and rare CPV-2c, as well as potential recombination events among Chinese CPV-2 strains. PMID:26348721

  18. Structure and receptor-binding activity of insulin from a holostean fish, the bowfin (Amia calva).

    PubMed Central

    Conlon, J M; Youson, J H; Whittaker, J

    1991-01-01

    The holostean fishes are the extant representatives of the primitive ray-finned fishes from which the present-day teleosts may have evolved. The primary structure of insulin from a holostean fish, the bowfin (Amia calva), was established as: A-chain: Gly-Ile-Val-Glu-Gln-Cys-Cys-Leu-Lys-Pro-Cys-Thr-Ile-Tyr-Glu-Met-Glu- Lys-Tyr-Cys-Asn B-chain: Ala-Ala-Ser-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Phe-Leu- Val-Cys-Gly-Glu-Ser-Gly-Phe-Phe-Tyr-Asn-Pro-Asn-Lys-Ser This amino acid sequence contains several substitutions (methionine at A16, phenylalanine at B16 and serine at B22) at sites that have been strongly conserved in other vertebrate species and that may be expected to influence biological activity. Consistent with this prediction, bowfin insulin was approx. 14-fold less potent than pig insulin in inhibiting the binding of [125I-Tyr-A14](human insulin) to transfected mouse NIH 3T3 cells expressing the human insulin receptor. PMID:2039477

  19. Characterization of a TK6-Bcl-xL gly-159-ala Human Lymphoblast Clone

    SciTech Connect

    Chyall, L.: Gauny, S.; Kronenberg, A.

    2006-01-01

    TK6 cells are a well-characterized human B-lymphoblast cell line derived from WIL-2 cells. A derivative of the TK6 cell line that was stably transfected to express a mutated form of the anti-apoptotic protein Bcl-xL (TK6-Bcl-xL gly-159- ala clone #38) is compared with the parent cell line. Four parameters were evaluated for each cell line: growth under normal conditions, plating efficiency, and frequency of spontaneous mutation to 6‑thioguanine resistance (hypoxanthine phosphoribosyl transferase locus) or trifluorothymidine resistance (thymidine kinase locus). We conclude that the mutated Bcl-xL protein did not affect growth under normal conditions, plating efficiency or spontaneous mutation frequencies at the thymidine kinase (TK) locus. Results at the hypoxanthine phosphoribosyl transferase (HPRT) locus were inconclusive. A mutant fraction for TK6‑Bcl-xL gly-159-ala clone #38 cells exposed to 150cGy of 160kVp x-rays was also calculated. Exposure to x-irradiation increased the mutant fraction of TK6‑Bcl-xL gly-159-ala clone #38 cells.

  20. Establishment of treatment parameters for ALA-PDT of plaque psoriasis

    NASA Astrophysics Data System (ADS)

    Stringer, Mark R.; Robinson, Dominic J.; Collins, P.

    1996-12-01

    We report an investigation into the use of photodynamic therapy (PDT), following topically applied 5-aminolaevulinic acid (ALA), as a treatment for plaque psoriasis. Treatment was performed 4 hours post-ALA, using white light doses of 2 - 16 J cm-2 delivered at 10 - 40 mW cm-2. The fluorescence emission of protoporphyrin IX was used as an indicator of the relative concentration of photosensitizer within each plaque before, during, and after therapy. Results show that the rate of sensitizer photo- oxidation is proportional to both pre-treatment fluorescence intensity and surface irradiance, consistent with a rate- equation analysis. A correlation of fluorescence measurements with clinical response of plaques indicates that the effectiveness of PDT is dominated by the level of PpIX at the onset of treatment, and is much less dependent upon light dose. Using these findings we have established a PDT treatment protocol that involves the delivery of 8 J cm-2 of white light, at a rate of 15 mW cm-2. The possibility of ALA-PDT being established as the therapy of choice is discussed.

  1. Challenges for Community-Based Forest Management in the KoloAla Site Manompana

    NASA Astrophysics Data System (ADS)

    Urech, Zora Lea; Sorg, Jean-Pierre; Felber, Hans Rudolph

    2013-03-01

    Following the IUCN 5th World Congress on Protected Areas in 2003, the then-President of Madagascar decided to increase the area of Madagascar's protected areas from 1.7 to 6 million ha. To combine the aims of protection and timber production, a new concept was developed through the establishment of community-based forest management (CBFM) sites, called KoloAla. However, experience shows that similar management transfers to communities in Madagascar have only been successful in a very few cases. We aimed to explore the success to be expected of this new approach in the particular case of the Manompana corridor at Madagascar's eastern coast. In a first step, the readiness of the corridor's resource users for CBFM has been analysed according to the seven resource users' attributes developed by Ostrom that predict an effective self-organized resource management. In a second step, we explored how KoloAla addresses known challenges of Madagascar's CBFM. Analyses lead in a rather sober conclusion. Although KoloAla attempts to address the goals of poverty alleviation, biodiversity conservation and timber production under a single umbrella, it does so in a rather non-innovative way. Challenges with regard to the state's environmental governance, agricultural inefficiency and thus deforestation remain unsolved.

  2. High-resolution solution structure of the double Cys2His2 zinc finger from the human enhancer binding protein MBP-1.

    PubMed

    Omichinski, J G; Clore, G M; Robien, M; Sakaguchi, K; Appella, E; Gronenborn, A M

    1992-04-28

    The high-resolution three-dimensional structure of a synthetic 57-residue peptide comprising the double zinc finger of the human enhancer binding protein MBP-1 has been determined in solution by nuclear magnetic resonance spectroscopy on the basis of 1280 experimental restraints. A total of 30 simulated annealing structures were calculated. The backbone atomic root-mean-square distributions about the mean coordinate positions are 0.32 and 0.33 A for the N- and C-terminal fingers, respectively, and the corresponding values for all atoms, excluding disordered surface side chains, are 0.36 and 0.40 A. Each finger comprises an irregular antiparallel sheet and a helix, with the zinc tetrahedrally coordinated to two cysteines and two histidines. The overall structure is nonglobular in nature, and the angle between the long axes of the helices is 47 +/- 5 degrees. The long axis of the antiparallel sheet in the N-terminal finger is approximately parallel to that of the helix in the C-terminal finger. Comparison of this structure with the X-ray structure of the Zif-268 triple finger complexed with DNA indicates that the relative orientation of the individual zinc fingers is clearly distinct in the two cases. This difference can be attributed to the presence of a long Lys side chain in the C-terminal finger of MBP-1 at position 40, instead of a short Ala or Ser side chain at the equivalent position in Zif-268. This finding suggests that different contacts may be involved in the binding of the zinc fingers of MBP-1 and Zif-268 to DNA, consistent with the findings from methylation interference experiments that the two fingers of MBP-1 contact 10 base pairs, while the three fingers of Zif-268 contact only 9 base pairs. PMID:1567844

  3. The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion.

    PubMed

    Jervis, Adrian J; Crack, Jason C; White, Gaye; Artymiuk, Peter J; Cheesman, Myles R; Thomson, Andrew J; Le Brun, Nick E; Green, Jeffrey

    2009-03-24

    Fumarate and nitrate reduction regulatory (FNR) proteins are bacterial transcription factors that coordinate the switch between aerobic and anaerobic metabolism. In the absence of O(2), FNR binds a [4Fe-4S](2+) cluster (ligated by Cys-20, 23, 29, 122) promoting the formation of a transcriptionally active dimer. In the presence of O(2), FNR is converted into a monomeric, non-DNA-binding form containing a [2Fe-2S](2+) cluster. The reaction of the [4Fe-4S](2+) cluster with O(2) has been shown to proceed via a 2-step process, an O(2)-dependent 1-electron oxidation to yield a [3Fe-4S](+) intermediate with release of 1 Fe(2+) ion, followed by spontaneous rearrangement to the [2Fe-2S](2+) form with release of 1 Fe(3+) and 2 S(2-) ions. Here, we show that replacement of Ser-24 by Arg, His, Phe, Trp, or Tyr enhances aerobic activity of FNR in vivo. The FNR-S24F protein incorporates a [4Fe-4S](2+) cluster with spectroscopic properties similar to those of FNR. However, the substitution enhances the stability of the [4Fe-4S](2+) cluster in the presence of O(2). Kinetic analysis shows that both steps 1 and 2 are slower for FNR-S24F than for FNR. A molecular model suggests that step 1 of the FNR-S24F iron-sulfur cluster reaction with O(2) is inhibited by shielding of the iron ligand Cys-23, suggesting that Cys-23 or the cluster iron bound to it is a primary site of O(2) interaction. These data lead to a simple model of the FNR switch with physiological implications for the ability of FNR proteins to operate over different ranges of in vivo O(2) concentrations. PMID:19261852

  4. AMP-activated protein kinase (AMPK) cross-talks with canonical Wnt signaling via phosphorylation of {beta}-catenin at Ser 552

    SciTech Connect

    Zhao, Junxing; Yue, Wanfu; Zhu, Mei J.; Sreejayan, Nair; Du, Min

    2010-04-23

    AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism; its activity is regulated by a plethora of physiological conditions, exercises and many anti-diabetic drugs. Recent studies show that AMPK involves in cell differentiation but the underlying mechanism remains undefined. Wingless Int-1 (Wnt)/{beta}-catenin signaling pathway regulates the differentiation of mesenchymal stem cells through enhancing {beta}-catenin/T-cell transcription factor 1 (TCF) mediated transcription. The objective of this study was to determine whether AMPK cross-talks with Wnt/{beta}-catenin signaling through phosphorylation of {beta}-catenin. C3H10T1/2 mesenchymal cells were used. Chemical inhibition of AMPK and the expression of a dominant negative AMPK decreased phosphorylation of {beta}-catenin at Ser 552. The {beta}-catenin/TCF mediated transcription was correlated with AMPK activity. In vitro, pure AMPK phosphorylated {beta}-catenin at Ser 552 and the mutation of Ser 552 to Ala prevented such phosphorylation, which was further confirmed using [{gamma}-{sup 32}P]ATP autoradiography. In conclusion, AMPK phosphorylates {beta}-catenin at Ser 552, which stabilizes {beta}-catenin, enhances {beta}-catenin/TCF mediated transcription, expanding AMPK from regulation of energy metabolism to cell differentiation and development via cross-talking with the Wnt/{beta}-catenin signaling pathway.

  5. The feeding route (enteral or parenteral) affects the plasma response of the dipetide Ala-Gln and the amino acids glutamine, citrulline and arginine, with the administration of Ala-Gln in preoperative patients.

    PubMed

    Melis, Gerdien C; Boelens, Petra G; van der Sijp, Joost R M; Popovici, Theodora; De Bandt, Jean-Pascal; Cynober, Luc; van Leeuwen, Paul A M

    2005-07-01

    Enhancement of depressed plasma concentrations of glutamine and arginine is associated with better clinical outcome. Supplementation of glutamine might be a way to provide the patient with glutamine, and also arginine, because glutamine provides the kidney with citrulline, from which the kidney produces arginine when plasma levels of arginine are low. The aim of the present study was to investigate the parenteral and enteral response of the administered dipeptide Ala-Gln, glutamine, citrulline and arginine. Therefore, seven patients received 20 g Ala-Gln, administered over 4 h, parenterally or enterally, on two separate occasions. Arterial blood samples were taken before and during the administration of Ala-Gln. ANOVA and a paired t test were used to test differences (P<0.05). Ala-Gln was undetectable with enteral administration, whereas Ala-Gln remained stable at a plasma concentration of 268 micromol/l throughout parenteral infusion and rapidly decreased towards zero after infusion was stopped. The highest level of glutamine was observed with parenteral infusion of the dipeptide, although enteral infusion also significantly increased plasma levels of glutamine. The highest plasma response of citrulline was observed with the enteral administration of the dipeptide, although parenteral administration also increased plasma levels of citrulline. Plasma arginine increased significantly with parenteral infusion, but not with enteral administration of Ala-Gln. In conclusion, administrations of Ala-Gln, parenteral or enteral, resulted in an increased plasma glutamine response, as compared with baseline. Interestingly, in spite of the high availability of citrulline with enteral administration of the dipeptide, only parenteral infusion of Ala-Gln increased plasma arginine concentration.

  6. (Ala)(4)-X-(Ala)4 as a model system for the optimization of the χ1 and χ2 amino acid side-chain dihedral empirical force field parameters.

    PubMed

    Shim, Jihyun; Zhu, Xiao; Best, Robert B; MacKerell, Alexander D

    2013-03-15

    Amino acid side-chain fluctuations play an essential role in the structure and function of proteins. Accordingly, in theoretical studies of proteins, it is important to have an accurate description of their conformational properties. Recently, new side-chain torsion parameters were introduced into the CHARMM and Amber additive force fields and evaluated based on the conformational properties of the individual side-chains using protein simulations in explicit solvent. While effective for validation, molecular dynamics simulations of proteins must be extended into the microsecond regime to obtain full convergence of the side-chain conformations, limiting their use for force field optimization. To address this, we systematically test the utility of explicit solvent simulations of (Ala)(4)-X-(Ala)(4) peptides, where X represents the amino acids, as model systems for the optimization of χ(1) and χ(2) side-chain parameters. The effect of (Ala)(4)-X-(Ala)(4) backbone conformation was tested by constraining the backbone in the α-helical, C5, C7(eq), and PPII conformations and performing exhaustive sampling using Hamiltonian replica exchange simulations. Rotamer distributions from protein and the (Ala)(4)-X-(Ala)(4) simulations showed the highest correlation for the C7(eq) and PPII conformations, although agreement was the best for the α-helical conformation for Asn. Hydrogen bond analysis indicates the utility of the C7(eq) and PPII conformations to be due to specific side-chain-backbone hydrogen bonds not being oversampled, thereby allowing sampling of a range of side-chain conformations consistent with the distributions occurring in full proteins. It is anticipated that the (Ala)(4)-X-(Ala)(4) model system will allow for iterative force field optimization targeting condensed-phase conformational distributions of side-chains.

  7. IR, Raman and SERS studies of methyl salicylate

    NASA Astrophysics Data System (ADS)

    Varghese, Hema Tresa; Yohannan Panicker, C.; Philip, Daizy; Mannekutla, James R.; Inamdar, S. R.

    2007-04-01

    The IR and Raman spectra of methyl salicylate (MS) were recorded and analysed. Surface enhanced Raman scattering (SERS) spectrum was recorded in silver colloid. The vibrational wave numbers of the compound have been computed using the Hartree-Fock/6-31G * basis and compared with the experimental values. SERS studies suggest a flat orientation of the molecule at the metal surface.

  8. Performance and Flow Dynamics Studies of Polymeric Optofluidic SERS Sensors

    NASA Astrophysics Data System (ADS)

    Uusitalo, S.; Hiltunen, J.; Karioja, P.; Siitonen, S.; Kontturi, V.; Myllylä, R.; Kinnunen, M.; Meglinski, I.

    2015-09-01

    We present a polymer-based optofluidic surface enhanced Raman scattering chip for biomolecule detection, serving as a disposable sensor choice with cost-effective production. The SERS substrate is fabricated by using industrial roll-to-roll UV-nanoimprinting equipment and integrated with adhesive-based polymeric microfluidics. The functioning of the SERS detection on-chip is confirmed and the effect of the polymer lid on the obtainable Raman spectra is analysed. Rhodamine 6G is used as a model analyte to demonstrate continuous flow measurements on a planar SERS substrate in a microchannel. The relation between the temporal response of the sensors and sample flow dynamics is studied with varied flow velocities, using SERS and fluorescence detection. The response time of the surface-dependent SERS signal is longer than the response time of the fluorescence signal of the bulk flow. This observation revealed the effect of convection on the temporal SERS responses at 25 μl/min to 1000 µl/min flow velocities. The diffusion of analyte molecules from the bulk concentration into the sensing surface induces about a 40-second lag time in the SERS detection. This lag time, and its rising trend with slower flow velocities, has to be taken into account in future trials of the optofluidic SERS sensor, with active analyte binding on the sensing surface.

  9. SERS as analytical tool for detection of bacteria

    NASA Astrophysics Data System (ADS)

    Cialla, Dana; Rösch, Petra; Möller, Robert; Popp, Jürgen

    2007-07-01

    The detection of single bacteria should be improved by lowering the acquisition time via the application of SERS (surface enhanced Raman spectroscopy). Nano structured colloids or surfaces consisting of gold or silver can be used as SERS active substrates. However, for biological applications mostly gold is used as SERS active substrate since silver is toxic for bacterial cells. Furthermore, the application of gold as a SERS-active substrate allows the usage of Raman excitation wavelengths in the red part of the electromagnetic spectrum. For the SERS investigations on bacteria different colloids (purchased and self prepared, preaggregated and non-aggregated) are chosen as SERS active substrates. The application of different gold colloids under gently mixing conditions to prevent the bacterial damage allowed the recording of reproducible SERS spectra of bacteria. The SERS spectra of B. pumilus are dominated by contributions of ingredients of the outer cell wall, e.g. the peptidoglycan layer. SEM images of the coated bacteria demonstrate the incomplete adsorption most probably due to variations within the binding affinities between different outer cell components and the gold colloids.

  10. 6-Shogaol has anti-amyloidogenic activity and ameliorates Alzheimer's disease via CysLT1R-mediated inhibition of cathepsin B.

    PubMed

    Na, Ji-Young; Song, Kibbeum; Lee, Ju-Woon; Kim, Sokho; Kwon, Jungkee

    2016-08-12

    Although 6-shogaol, a constituent of ginger, has been reported to have anti-inflammatory and anti-oxidant effects on neuronal cells, the effects of 6-shogaol on Alzheimer's disease (AD) have not yet been investigated. Here we aimed to determine whether 6-shogaol exerts neuroprotective effects against AD. Specifically, we investigated the effects of 6-shogaol on the cysteinyl leukotriene 1 receptor (CysLT1R), a major factor in AD pathogenesis. Moreover, we clarified the relationship between CysLT1R and cathepsin B, a cysteine protease. We used in vitro and in vivo models to determine whether 6-shogaol inhibits CysLT1R/cathepsin B in an amyloid-beta (Aβ; 1-42)-induced model of neurotoxicity. We first confirmed that CysLT1R and cathepsin B are upregulated by Aβ (1-42) and that CysLT1R activation induces cathepsin B. In contrast, we found that 6-shogaol-mediated inhibition of CysLT1R downregulates cathepsin B in both in vitro and in vivo models. Furthermore, we found that 6-shogaol-mediated inhibition of CysLT1R/cathepsin B reduces Aβ deposition in the brain and ameliorates behavioral deficits in APPSw/PS1-dE9 Tg mice. Our results indicate that 6-shogaol is a CysLT1R/cathepsin B inhibitor and is a novel potential therapeutic agent for the treatment of various neurodegenerative diseases, including AD. PMID:27286707

  11. Fluorescence photobleaching of ALA and ALA-heptyl ester induced protoporphyrin IX during photodynamic therapy of normal hairless mouse skin: a comparison of two light sources and different illumination schemes.

    PubMed

    Pudroma, Xiao; Juzeniene, Asta; Ma, Li-Wei; Iani, Vladimir; Moan, Johan

    2011-01-01

    This study investigated photobleaching of protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) and ALA-heptyl ester during superficial photodynamic therapy (PDT) in normal skin of the female BALB/c-nu/nu athymic mouse. We examined the effects of two light sources (laser and broadband lamp) and two different illumination schemes (fractionated light and continuous irradiation) on the kinetics of photobleaching. Our results show that light exposure (0-30 minutes, 10 mW/cm2) of wavelengths of approximately 420 nm (blue light) and 635 nm (red light) induced time-dependent PpIX photobleaching for mouse skin of 2% ALA and ALA-heptyl ester. Blue light (10 mW/cm2) caused more rapid PpIX photobleaching than did red light (100 mW/cm2), which is attributed to stronger absorption at 407 nm than at 632 nm for PpIX. In the case of light fractionation, fractionated light induced faster photobleaching compared with continuous light exposure after topical application of 2% ALA and ALA-heptyl ester in vivo. These have been suggested to allow reoxygenation of the irradiated tissue, with a consequent enhancement of singlet oxygen production in the second and subsequent fractions.

  12. Short-term supplementation of low-dose gamma-linolenic acid (GLA), alpha-linolenic acid (ALA), or GLA plus ALA does not augment LCP omega 3 status of Dutch vegans to an appreciable extent.

    PubMed

    Fokkema, M R; Brouwer, D A; Hasperhoven, M B; Martini, I A; Muskiet, F A

    2000-11-01

    Vegans do not consume meat and fish and have therefore low intakes of long chain polyunsaturated fatty acids (LCP). They may consequently have little negative feedback inhibition from dietary LCP on conversion of alpha -linolenic acid (ALA) to the LCP omega 3 eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. We investigated whether supplementation of nine apparently healthy vegans with 2.01 g ALA (4 ml linseed oil), 1.17 g gamma-linolenic acid (GLA) (6 ml borage oil) or their combination increases the LCP omega 3 contents of erythrocytes (RBC) and platelets (PLT), and of plasma phospholipids (PL), cholesterol esters (CE) and triglycerides (TG). The supplements changed the dietary LA/ALA ratio (in g/g) from about 13.7 (baseline) to 6.8 (linseed oil), 14.3 (borage oil) and 6.4 (linseed + borage oil), respectively. ALA or GLA given as single supplements did not increase LCP omega 3 status, but their combination augmented LCP omega 3 (in CE) and EPA (in fasting TG) to a statistically significant, but nevertheless negligible, extent. We conclude that negative feedback inhibition by dietary LCP, if any, does not play an important role in the inability to augment notably DHA status by dietary ALA. The reach of a DHA plateau already at low dietary ALA intakes suggests that dietary DHA causes a non-functional DHA surplus, or is, alternatively, important for maintaining DHA status at a functionally relevant level. PMID:11090255

  13. SERS-based viral fingerprinting: current capabilities and challenges

    NASA Astrophysics Data System (ADS)

    Driskell, J. D.; Abell, J. L.; Dluhy, R. A.; Zhao, Y.-P.; Tripp, R. A.

    2010-04-01

    Silver nanorod array substrates are fabricated by oblique angle deposition and characterized for optimal SERS performance. Using UV-visible-NIR spectrophotometry we show that the nanorods have a transverse surface plasmon resonance mode at ~357 nm and a broad absorbance spanning 600-800 nm when excited along the longitudinal direction. We demonstrate that SERS enhancement is optimized using an excitation wavelength of 633 or 785 nm. The large area uniformity in SERS signal (<10% variation) and reproducibility among preparations (<15% variation) provides a unique opportunity for SERS-based whole-organism fingerprinting. Egg prepared avian influenza virus and clinical sputum samples of human influenza virus were investigated to demonstrate SERS-based detection of a virus in a complex sample matrix and to assess the effect of different background matrices on the detection of similar viruses.

  14. Solution NMR evidence for a cis Tyr-Ala peptide group in the structure of [Pro93Ala] bovine pancreatic ribonuclease A.

    PubMed Central

    Xiong, Y.; Juminaga, D.; Swapna, G. V.; Wedemeyer, W. J.; Scheraga, H. A.; Montelione, G. T.

    2000-01-01

    Proline peptide group isomerization can result in kinetic barriers in protein folding. In particular, the cis proline peptide conformation at Tyr92-Pro93 of bovine pancreatic ribonuclease A (RNase A) has been proposed to be crucial for chain folding initiation. Mutation of this proline-93 to alanine results in an RNase A molecule, P93A, that exhibits unfolding/refolding kinetics consistent with a cis Tyr92-Ala93 peptide group conformation in the folded structure (Dodge RW, Scheraga HA, 1996, Biochemistry 35:1548-1559). Here, we describe the analysis of backbone proton resonance assignments for P93A together with nuclear Overhauser effect data that provide spectroscopic evidence for a type VI beta-bend conformation with a cis Tyr92-Ala93 peptide group in the folded structure. This is in contrast to the reported X-ray crystal structure of [Pro93Gly]-RNase A (Schultz LW, Hargraves SR, Klink TA, Raines RT, 1998, Protein Sci 7:1620-1625), in which Tyr92-Gly93 forms a type-II beta-bend with a trans peptide group conformation. While a glycine residue at position 93 accommodates a type-II bend (with a positive value of phi93), RNase A molecules with either proline or alanine residues at this position appear to require a cis peptide group with a type-VI beta-bend for proper folding. These results support the view that a cis Pro93 conformation is crucial for proper folding of wild-type RNase A. PMID:10716195

  15. Delta-aminolevulinic acid dehydratase (ALAD) polymorphism in lead exposed Bangladeshi children and its effect on urinary aminolevulinic acid (ALA)

    SciTech Connect

    Tasmin, Saira; Furusawa, Hana; Ahmad, Sk. Akhtar; Watanabe, Chiho

    2015-01-15

    Background and objective: Lead has long been recognized as a harmful environmental pollutant. People in developing countries like Bangladesh still have a higher risk of lead exposure. Previous research has suggested that the delta-aminolevulinic acid dehydratase (ALAD) genotype can modify lead toxicity and individual susceptibility. As children are more susceptible to lead-induced toxicity, this study investigated whether the ALAD genotype influenced urinary excretion of delta-aminolevulinic acid (U-ALA) among children exposed to environmental lead in Bangladesh. Methods: Subjects were elementary schoolchildren from a semi-urban industrialized area in Bangladesh. A total of 222 children were studied. Blood and urine were collected to determine ALAD genotypes, blood lead levels and urinary aminolevulinic acid (U-ALA). Results: The mean BPb level was 9.7 µg/dl for the study children. BPb was significantly positively correlated with hemoglobin (p<0.01). In total, allele frequency for ALAD 1 and 2 was 0.83 and 0.17 respectively. The mean U-ALA concentration was lower in ALAD1-2/2-2 carriers than ALAD1-1 carriers for boys (p=0.001). But for girls, U-ALA did not differ significantly by genotype (p=0.26). When U-ALA was compared by genotype at the same exposure level in a multiple linear regression analysis, boys who were ALAD1-2/2-2 carriers still had a lower level of U-ALA compared to ALAD1-1carriers. Conclusion: This study provides information about the influence of ALAD polymorphism and its association with U-ALA in Bangladeshi children. Our results indicate that the ALAD1-2/2-2 genotype may have a protective effect in terms of U-ALA for environmentally lead exposed boys. - Highlights: • High blood lead level for the environmentally exposed schoolchildren. • BPb was significantly correlated with U-ALA and Hb. • Effect of ALAD genotype on U-ALA is differed by sex. • Lower U-ALA in ALAD2 than ALAD1 carriers only for boys at same exposure.

  16. The Association between RAD23B Ala249Val Polymorphism and Cancer Susceptibility: Evidence from a Meta-Analysis

    PubMed Central

    Ying, Xiaojiang; Song, Junmin; Zhang, Ruoxin; Li, Zhen; Chen, Hongliang; Ye, Pingjiang; Shen, Yi; Pan, Weihuo; Chen, Zhiliang

    2014-01-01

    Background A number of studies have investigated associations of genetic variation in RAD23B Ala249Val (rs1805329 C>T) with cancer susceptibility; however, the findings are inconsistent. We performed a meta-analysis to acquire a more precise estimation of the relationship. Method We searched literatures from PubMed, Embase and Web of Science. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the association between Ala249Val polymorphism and cancer risk. Results A total of 23 studies consisting of 10837 cases and 13971 controls were included in this meta-analysis. Overall, no significant associations were found between RAD23B Ala249Val polymorphism and cancer risk (Val/Val vs. Ala/Ala: OR = 0.97, 95% CI = 0.75–1.25; Ala/Val vs. Ala/Ala: OR = 1.08, 95% CI = 0.96–1.22; recessive model: OR = 0.93, 95% CI = 0.76–1.14 and dominant model: OR = 1.07, 95% CI = 0.94–1.20). We did not find any significant associations in the further stratification analyses by cancer type, ethnicity and source of control. Conclusions Despite some limitations, this meta-analysis indicates that it is unlikely that the RAD23B 249Val/Val polymorphism may contribute to the individual susceptibility to cancer risk. However, further advanced designed studies with larger sample size and different ethnicities should be conducted to confirm our results. PMID:24643114

  17. 5-ALA Fluorescence in Native Pituitary Adenoma Cell Lines: Resection Control and Basis for Photodynamic Therapy (PDT)?

    PubMed Central

    Poeschke, Stephan; Greve, Burkhard; Prevedello, Daniel; Santacroce, Antonio; Stummer, Walter; Senner, Volker

    2016-01-01

    Objective: Pituitary adenomas (PA), especially invasive ones, are often not completely resectable. Usage of 5-aminolevulinic acid (5-ALA) for fluorescence guided surgery could improve the rate of total resection and, additionally, open the doors for photodynamic therapy (PDT) in case of unresectable or partially resected PAs. The aim of this study was to investigate the uptake of 5-ALA and the effect of 5-ALA based PDT in cell lines. Methods: GH3 and AtT-20 cell lines were incubated with different concentrations of 5-ALA, protoporphyrin IX (PPIX) fluorescence was measured by flow cytometry and fluorescencespectrometry. WST-1 assays were performed to determine the surviving fraction of cells after PDT. PPIX fluorescence intensities and PDT effect of the pituitary adenoma cells were compared to U373MG, a well-known glioblastoma cell line. Results: Both cell lines showed a 5-ALA dependent intracellular PPIX fluorescence. Significant differences after 24hrs of incubation were observed in AtT-20 cells in comparison to GH3. Regardless of the incubation or metabolism time, there was a proliferation inhibiting effect after PDT, with no statistical significance. Conclusion: Since GH3 cells showed a heterogenous uptake of 5-ALA in the flow cytometry profile, but not constantly high concentrations they might have a 5-ALA efflux mechanism, which still needs to be determined. In the case of AtT-20, the cells might need a longer time for the uptake due to their size or slow metabolism. We showed that the different cell lines have different uptake and metabolism mechanisms, which needs to be further investigated. The general uptake of 5-ALA allows the possibility of resection control and PDT for pituitary adenomas. But, the role of PDT for unresectable pituitary adenomas deserves further investigations. PMID:27583461

  18. Immobilization of ALA-Zn(II) Coordination Polymer Pro-photosensitizers on Magnetite Colloidal Supraparticles for Target Photodynamic Therapy of Bladder Cancer.

    PubMed

    Tan, Jing; Sun, Chuanyu; Xu, Ke; Wang, Changchun; Guo, Jia

    2015-12-16

    5-Aminolevulinic acid (ALA) is a widely used photodynamic therapy (PDT) prodrug in the clinic. It can be metalized to the photosensitizer PpIX, which produces toxic singlet oxygen to kill cancer cells upon visible light irradiation. Herein, a core/shell-structured vehicle is designed to comprise magnetite colloidal supraparticles (MCSPs) as cores and ALA-Zn(II) coordination polymers as shells (Fe3O4@ALA-Zn(II) ) for target pro-photosensitizer delivery. The coordination polymers with 2D layered structures are locally deposited on the MCSPs by the complexation of the ALA and Zn(II) ions, and are readily controlled by varying the feed precursors and reaction temperatures. The maximum conjugated ALA amount is up to 17%. The Fe3O4@ALA-Zn(II) microspheres exhibit pH-sensitive release of ALA in acidic environment and rapid magnetic responsiveness. Cytotoxicity results demonstrate that Fe3O4@ALA-Zn(II) shows a significant inhibitory effect to T24 cells and is nontoxic to 293T normal cells as exposed to the 630 nm visible light for a very short time, which may due to the selective accumulation of ALA-induced PpIX in T24 cancer cells. Compared to the ALA used alone, the coordination polymer form is more efficient because of the bioactivity of incorporated Zn ions despite underlying the same apoptosis mechanism as ALA agent.

  19. Distinct Roles of Ser-764 and Lys-773 at the N Terminus of von Willebrand Factor in Complex Assembly with Coagulation Factor VIII*

    PubMed Central

    Castro-Núñez, Lydia; Bloem, Esther; Boon-Spijker, Mariëtte G.; van der Zwaan, Carmen; van den Biggelaar, Maartje; Mertens, Koen; Meijer, Alexander B.

    2013-01-01

    Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII, which does not bind VWF. Nano-LC-MS analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or activated FVIII. However, Lys-773 of peptide Ser-766–Leu-774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser-764–Arg-782, which comprises the first 19 amino acid residues of mature VWF, showed a differential modification of both Lys-773 and the α-amino group of Ser-764. To verify the role of Lys-773 and the N-terminal Ser-764 in FVIII binding, we employed VWF variants in which either Lys-773 or Ser-764 was replaced with Ala. Surface plasmon resonance analysis and competition studies revealed that VWF(K773A) exhibited reduced binding to FVIII and the FVIII light chain, which harbors the VWF-binding site. In contrast, VWF(S764A) revealed more effective binding to FVIII and the FVIII light chain compared with WT VWF. The results of our study show that the N terminus of VWF is critical for the interaction with FVIII and that Ser-764 and Lys-773 have opposite roles in the binding mechanism. PMID:23168412

  20. Distinct roles of Ser-764 and Lys-773 at the N terminus of von Willebrand factor in complex assembly with coagulation factor VIII.

    PubMed

    Castro-Núñez, Lydia; Bloem, Esther; Boon-Spijker, Mariëtte G; van der Zwaan, Carmen; van den Biggelaar, Maartje; Mertens, Koen; Meijer, Alexander B

    2013-01-01

    Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII, which does not bind VWF. Nano-LC-MS analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or activated FVIII. However, Lys-773 of peptide Ser-766-Leu-774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser-764-Arg-782, which comprises the first 19 amino acid residues of mature VWF, showed a differential modification of both Lys-773 and the α-amino group of Ser-764. To verify the role of Lys-773 and the N-terminal Ser-764 in FVIII binding, we employed VWF variants in which either Lys-773 or Ser-764 was replaced with Ala. Surface plasmon resonance analysis and competition studies revealed that VWF(K773A) exhibited reduced binding to FVIII and the FVIII light chain, which harbors the VWF-binding site. In contrast, VWF(S764A) revealed more effective binding to FVIII and the FVIII light chain compared with WT VWF. The results of our study show that the N terminus of VWF is critical for the interaction with FVIII and that Ser-764 and Lys-773 have opposite roles in the binding mechanism. PMID:23168412

  1. Biogenesis of phycobiliproteins: II. CpcS-I and CpcU comprise the heterodimeric bilin lyase that attaches phycocyanobilin to CYS-82 OF beta-phycocyanin and CYS-81 of allophycocyanin subunits in Synechococcus sp. PCC 7002.

    PubMed

    Saunée, Nicolle A; Williams, Shervonda R; Bryant, Donald A; Schluchter, Wendy M

    2008-03-21

    The Synechococcus sp. PCC 7002 genome encodes three genes, denoted cpcS-I, cpcU, cpcV, with sequence similarity to cpeS. CpcS-I copurified with His(6)-tagged (HT) CpcU as a heterodimer, CpcSU. When CpcSU was assayed for bilin lyase activity in vitro with phycocyanobilin (PCB) and apophycocyanin, the reaction product had an absorbance maximum of 622 nm and was highly fluorescent (lambda(max) = 643 nm). In control reactions with PCB and apophycocyanin, the products had absorption maxima at 635 nm and very low fluorescence yields, indicating they contained the more oxidized mesobiliverdin (Arciero, D. M., Bryant, D. A., and Glazer, A. N. (1988) J. Biol. Chem. 263, 18343-18349). Tryptic peptide mapping showed that the CpcSU-dependent reaction product had one major PCB-containing peptide that contained the PCB binding site Cys-82. The CpcSU lyase was also tested with recombinant apoHT-allophycocyanin (aporHT-AP) and PCB in vitro. AporHT-AP formed an ApcA/ApcB heterodimer with an apparent mass of approximately 27 kDa. When aporHT-AP was incubated with PCB and CpcSU, the product had an absorbance maximum of 614 nm and a fluorescence emission maximum at 636 nm, the expected maxima for monomeric holo-AP. When no enzyme or CpcS-I or CpcU was added alone, the products had absorbance maxima between 645 and 647 nm and were not fluorescent. When these reaction products were analyzed by gel electrophoresis and zinc-enhanced fluorescence emission, only the reaction products from CpcSU had PCB attached to both AP subunits. Therefore, CpcSU is the bilin lyase-responsible for attachment of PCB to Cys-82 of CpcB and Cys-81 of ApcA and ApcB.

  2. The activity of TcCYS4 modified by variations in pH and temperature can affect symptoms of witches' broom disease of cocoa, caused by the fungus Moniliophthora perniciosa.

    PubMed

    Freitas, Ana Camila Oliveira; Souza, Cristiane Ferreira; Monzani, Paulo Sérgio; Garcia, Wanius; de Almeida, Alex Alan Furtado; Costa, Marcio Gilberto Cardoso; Pirovani, Carlos Priminho

    2015-01-01

    The phytocystatins regulate various physiological processes in plants, including responses to biotic and abiotic stresses, mainly because they act as inhibitors of cysteine proteases. In this study, we have analyzed four cystatins from Theobroma cacao L. previously identified in ESTs libraries of the interaction with the fungus Moniliophthora perniciosa and named TcCYS1, TcCYS2, TcCYS3 and TcCYS4. The recombinant cystatins were purified and subjected to the heat treatment, at different temperatures, and their thermostabilities were monitored using their ability to inhibit papain protease. TcCYS1 was sensitive to temperatures above 50°C, while TcCYS2, TcCYS3, and TcCYS4 were thermostable. TcCYS4 presented a decrease of inhibitory activity when it was treated at temperatures between 60 and 70°C, with the greater decrease occurring at 65°C. Analyses by native gel electrophoresis and size-exclusion chromatography showed that TcCYS4 forms oligomers at temperatures between 60 and 70°C, condition where reduction of inhibitory activity was observed. TcCYS4 oligomers remain stable for up to 20 days after heat treatment and are undone after treatment at 80°C. TcCYS4 presented approximately 90% of inhibitory activity at pH values between 5 and 9. This protein treated at temperatures above 45°C and pH 5 presented reduced inhibitory activity against papain, suggesting that the pH 5 enhances the formation of TcCYS4 oligomers. A variation in the titratable acidity was observed in tissues of T. cacao during the symptoms of witches' broom disease. Our findings suggest that the oligomerization of TcCYS4, favored by variations in pH, is an endergonic process. We speculate that this process can be involved in the development of the symptoms of witches' broom disease in cocoa.

  3. Comparsion of light dose on topical ALA-mediated photodynamic therapy for DMBA-induced hamster buccal pouch premalignant lesions

    NASA Astrophysics Data System (ADS)

    Yang, Deng-Fu; Tseng, Meng-Ke; Liu, Chung-Ji; Hsu, Yih-Chih

    2012-03-01

    Oral cancer has becomes the most prominent male cancer disease due to the local betel nut chewing habit combing with smoking and alcohol-drinking lifestyle. In order to minimize the systemic phototoxic effect of 5-aminolevulinic acid (ALA), this study was designed to use a topical ALA-mediated PDT for treatment of DMBA-induced hamster buccal pouch cancerous lesions. DMBA was applied to one of the buccal pouches of hamsters thrice a week for 8 to 10 weeks. Precancerous lesions were induced and proven by histological examination. These DMBA-induced cancerous lesions were used for testing the efficacy of topical ALA -mediated PDT. We found that ALA reached its peak level in cancerous lesions about 2.5 hrs after topical application of ALA gel. The precancerous lesions in hamsters were then treated with topical ALA -mediated PDT with light exposure dose of 75 and 100 J/cm2 using LED 635 nm Wonderlight device. It is suggesting that optimization of the given light dose is critical to the success of PDT results.

  4. Preparation and SERS Study of Silver Microstructures with Dendritic Shape.

    PubMed

    Zhang, Xiao-tong; Zhao, Chun-liu; Zhou, Yu-meng; Dong, Qian-min; Lang, Ting-ting; Jin, Shang-zhong

    2015-10-01

    In the surface ehanced Raman scattering (SERS) technology, not only to improve the making process of SERS substrates, to be fast and easily, but also to enhance the SERS enhance factor, an easy replacement reaction between zinc and silver nitrate solution has been adopted to prepare silver micro-structures SERS-active substrate. The silver micro-substrates have many advantages. These substrates have good stability, well preservation, an easy making method and a fast making process. The surface profile of the silver microparticles is investigated by scanning electron microscope (SEM). The silver microstructures are dendritic shape in a symmetrical fashion with symmetrical distribution. When the time of the replace reaction is 40, 50 and 60 s, respectively, the average lengths of "trunks" in the silver dendritic microsubstractes are about 3, 5 and 10 μm, and the lengths of the "branches" are about 700 nm, 2 μm and 3 μm, respectively. The result shows that the longer time the replacement reaction takes, the longer lengths of the "trunks" and "branches" in the silver dendritic microsubstractes become. With the time of replace reaction increasing, the "trunk" and "branch" in the silver dendritic microsubstractes grow longer and a large amount of nano-level "leaves" grow out from the "branches" of the silver dendritic microsubstractes, so the silver micro size dendrates have nano level structure on surface. In order to investigate the SERS-active substrates application in SERS, a Fourier transform Raman spectrograph with a 1 064 nm laser wavelength is used to measure the SERS spectra. And good SERS spectra have been obtained by using dendritic silver microsubstrates on the silicon chip as a SERS substract, and Rhodamine 6G (R6G) as a molecule probe. It is found that the silver micro-substrates have good Raman characteristics. And comparing these SERS spectra, it gets the conclution that the spectra with best SERS enhance effect are obtained when R6G is obsorbed on the

  5. Increasing the hydrolysis constant of the reactive site upon introduction of an engineered Cys¹⁴-Cys³⁹ bond into the ovomucoid third domain from silver pheasant.

    PubMed

    Hemmi, Hikaru; Kumazaki, Takashi; Kojima, Shuichi; Yoshida, Takuya; Ohkubo, Tadayasu; Yokosawa, Hideyoshi; Miura, Kin-Ichiro; Kobayashi, Yuji

    2011-08-01

    P14C/N39C is the disulfide variant of the ovomucoid third domain from silver pheasant (OMSVP3) introducing an engineered Cys¹⁴-Cys³⁹ bond near the reactive site on the basis of the sequence homology between OMSVP3 and ascidian trypsin inhibitor. This variant exhibits a narrower inhibitory specificity. We have examined the effects of introducing a Cys¹⁴-Cys³⁹ bond into the flexible N-terminal loop of OMSVP3 on the thermodynamics of the reactive site peptide bond hydrolysis, as well as the thermal stability of reactive site intact inhibitors. P14C/N39C can be selectively cleaved by Streptomyces griseus protease B at the reactive site of OMSVP3 to form a reactive site modified inhibitor. The conversion rate of intact to modified P14C/N39C is much faster than that for wild type under any pH condition. The pH-independent hydrolysis constant (K(hyd) °) is estimated to be approximately 5.5 for P14C/N39C, which is higher than the value of 1.6 for natural OMSVP3. The reactive site modified form of P14C/N39C is thermodynamically more stable than the intact one. Thermal denaturation experiments using intact inhibitors show that the temperature at the midpoint of unfolding at pH 2.0 is 59 °C for P14C/N39C and 58 °C for wild type. There have been no examples, except P14C/N39C, where introducing an engineered disulfide causes a significant increase in K(hyd) °, but has no effect on the thermal stability. The site-specific disulfide introduction into the flexible N-terminal loop of natural Kazal-type inhibitors would be useful to further characterize the thermodynamics of the reactive site peptide bond hydrolysis.

  6. Silver Nanorods Wrapped with Ultrathin Al2O3 Layers Exhibiting Excellent SERS Sensitivity and Outstanding SERS Stability

    PubMed Central

    Ma, Lingwei; Huang, Yu; Hou, Mengjing; Xie, Zheng; Zhang, Zhengjun

    2015-01-01

    Silver nanostructures have been considered as promising substrates for surface-enhanced Raman scattering (SERS) with extremely high sensitivity. The applications, however, are hindered by the facts that their morphology can be easily destroyed due to the low melting points (~100 °C) and their surfaces are readily oxidized/sulfured in air, thus losing the SERS activity. It was found that wrapping Ag nanorods with an ultrathin (~1.5 nm) but dense and amorphous Al2O3 layer by low-temperature atomic layer deposition (ALD) could make the nanorods robust in morphology up to 400 °C, and passivate completely their surfaces to stabilize the SERS activity in air, without decreasing much the SERS sensitivity. This simple strategy holds great potentials to generate highly robust and stable SERS substrates for real applications. PMID:26264281

  7. Quantitative SERS sensors for environmental analysis of naphthalene.

    PubMed

    Péron, O; Rinnert, E; Toury, T; Lamy de la Chapelle, M; Compère, C

    2011-03-01

    In the investigation of chemical pollutants, such as PAHs (Polycyclic Aromatic Hydrocarbons) at low concentration in aqueous medium, Surface-Enhanced Raman Scattering (SERS) stands for an alternative to the inherent low cross-section of normal Raman scattering. Indeed, SERS is a very sensitive spectroscopic technique due to the excitation of the surface plasmon modes of the nanostructured metallic film. The surface of quartz substrates was coated with a hydrophobic film obtained by silanization and subsequently reacted with polystyrene (PS) beads coated with gold nanoparticles. The hydrophobic surface of the SERS substrates pre-concentrates non-polar molecules such as naphthalene. Under laser excitation, the SERS-active substrates allow the detection and the identification of the target molecules localized close to the gold nanoparticles. The morphology of the SERS substrates based on polystyrene beads surrounded by gold nanoparticles was characterized by scanning electron microscopy (SEM). Furthermore, the Raman fingerprint of the polystyrene stands for an internal spectral reference. To this extent, an innovative method to detect and to quantify organic molecules, as naphthalene in the range of 1 to 20 ppm, in aqueous media was carried out. Such SERS-active substrates tend towards an application as quantitative SERS sensors for the environmental analysis of naphthalene. PMID:21165476

  8. Carbon-Dot/Silver-Nanoparticle Flexible SERS-Active Films.

    PubMed

    Bhunia, Susanta Kumar; Zeiri, Leila; Manna, Joydeb; Nandi, Sukhendu; Jelinek, Raz

    2016-09-28

    Development of effective platforms for surface enhanced Raman scattering (SERS) sensing has mostly focused on fabrication of colloidal metal surfaces and tuning of their surface morphologies, designed to create "hot spots" in which plasmonic fields yield enhanced SERS signals. We fabricated distinctive SERS-active flexible films comprising polydimethylsiloxane (PDMS) embedding carbon dots (C-dots) and coated with silver nano-particles (Ag NPs). We show that the polymer-associated Ag NPs and C-dots intimately affected the physical properties of each other. In particular, the C-dot-Ag-NP-polymer films exhibited SERS properties upon deposition of versatile targets, both conventional SERS-active dyes as well as bacterial samples. We show that the SERS response was correlated to the formation C-dots within the polymer film and the physical proximity between the C-dots and Ag NPs, indicating that coupling between the plasmonic fields of the Ag NPs and C-dots' excitons constituted a prominent factor in the SERS properties.

  9. Both thioredoxin 2 and glutaredoxin 2 contribute to the reduction of the mitochondrial 2-Cys peroxiredoxin Prx3.

    PubMed

    Hanschmann, Eva-Maria; Lönn, Maria Elisabet; Schütte, Lena Dorothee; Funke, Maria; Godoy, José R; Eitner, Susanne; Hudemann, Christoph; Lillig, Christopher Horst

    2010-12-24

    The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (K(m), 23.8 μmol·liter(-1); V(max), 1.2 μmol·(mg·min)(-1)) similar to Trx2 (K(m), 11.2 μmol·liter(-1); V(max), 1.1 μmol·(mg·min)(-1)). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms. PMID:20929858

  10. Reactivity of Cys4 zinc finger domains with gold(III) complexes: insights into the formation of "gold fingers".

    PubMed

    Jacques, Aurélie; Lebrun, Colette; Casini, Angela; Kieffer, Isabelle; Proux, Olivier; Latour, Jean-Marc; Sénèque, Olivier

    2015-04-20

    Gold(I) complexes such as auranofin or aurothiomalate have been used as therapeutic agents for the treatment of rheumatoid arthritis for several decades. Several gold(I) and gold(III) complexes have also shown in vitro anticancer properties against human cancer cell lines, including cell lines resistant to cisplatin. Because of the thiophilicity of gold, cysteine-containing proteins appear as likely targets for gold complexes. Among them, zinc finger proteins have attracted attention and, recently, gold(I) and gold(III) complexes have been shown to inhibit poly(adenosine diphosphate ribose)polymerase-1 (PARP-1), which is an essential protein involved in DNA repair and in cancer resistance to chemotherapies. In this Article, we characterize the reactivity of the gold(III) complex [Au(III)(terpy)Cl]Cl2 (Auterpy) with a model of Zn(Cys)4 "zinc ribbon" zinc finger by a combination of absorption spectroscopy, circular dichroism, mass spectrometry, high-performance liquid chromatography analysis, and X-ray absorption spectroscopy. We show that the Zn(Cys)4 site of Zn·LZR is rapidly oxidized by Auterpy to form a disulfide bond. The Zn(2+) ion is released, and the two remaining cysteines coordinate the Au(+) ion that is produced during the redox reaction. Subsequent oxidation of these cysteines can take place in conditions of excess gold(III) complex. In the presence of excess free thiols mimicking the presence of glutathione in cells, mixing of the zinc finger model and gold(III) complex yields a different product: complex (Au(I))2·LZR with two Au(+) ions bound to cysteines is formed. Thus, on the basis of detailed speciation and kinetic measurements, we demonstrate herein that the destruction of Zn(Cys)4 zinc fingers by gold(III) complexes to achieve the formation of "gold fingers" is worth consideration, either directly or mediated by reducing agents.

  11. Evaluation of acid-labile S-protecting groups to prevent Cys racemization in Fmoc solid-phase peptide synthesis

    PubMed Central

    Hibino, Hajime; Miki, Yasuyoshi; Nishiuchi, Yuji

    2014-01-01

    Phosphonium and uronium salt-based reagents enable efficient and effective coupling reactions and are indispensable in peptide chemistry, especially in machine-assisted SPPS. However, after the activating and coupling steps with these reagents in the presence of tertiary amines, Fmoc derivatives of Cys are known to be considerably racemized during their incorporation. To avoid this side reaction, a coupling method mediated by phosphonium/uronium reagents with a weaker base, such as 2,4,6-trimethylpyridine, than the ordinarily used DIEA or that by carbodiimide has been recommended. However, these methods are appreciably inferior to the standard protocol applied for SPPS, that is, a 1 min preactivation procedure of coupling with phosphonium or uronium reagents/DIEA in DMF, in terms of coupling efficiency, and also the former method cannot reduce racemization of Cys(Trt) to an acceptable level (<1.0%) even when the preactivation procedure is omitted. Here, the 4,4′-dimethoxydiphenylmethyl and 4-methoxybenzyloxymethyl groups were demonstrated to be acid-labile S-protecting groups that can suppress racemization of Cys to an acceptable level (<1.0%) when the respective Fmoc derivatives are incorporated via the standard SPPS protocol of phosphonium or uronium reagents with the aid of DIEA in DMF. Furthermore, these protecting groups significantly reduced the rate of racemization compared to the Trt group even in the case of microwave-assisted SPPS performed at a high temperature. © 2013 The Authors. European Peptide Society published by John Wiley & Sons, Ltd. PMID:24357151

  12. Targeting of histone acetyltransferase p300 by cyclopentenone prostaglandin Δ(12)-PGJ(2) through covalent binding to Cys(1438).

    PubMed

    Ravindra, Kodihalli C; Narayan, Vivek; Lushington, Gerald H; Peterson, Blake R; Prabhu, K Sandeep

    2012-02-20

    Inhibitors of histone acetyltransferases (HATs) are perceived to treat diseases like cancer, neurodegeneration, and AIDS. On the basis of previous studies, we hypothesized that Cys(1438) in the substrate binding site could be targeted by Δ(12)-prostaglandin J(2) (Δ(12)-PGJ(2)), a cyclopentenone prostaglandin (CyPG) derived from PGD(2). We demonstrate here the ability of CyPGs to inhibit p300 HAT-dependent acetylation of histone H3. A cell-based assay system clearly showed that the α,β-unsaturation in the cyclopentenone ring of Δ(12)-PGJ(2) was crucial for the inhibitory activity, while the 9,10-dihydro-15-deoxy-Δ(12,14)-PGJ(2), which lacks the electrophilic carbon (at carbon 9), was ineffective. Molecular docking studies suggested that Δ(12)-PGJ(2) places the electrophilic carbon in the cyclopentenone ring well within the vicinity of Cys(1438) of p300 to form a covalent Michael adduct. Site-directed mutagenesis of the p300 HAT domain, peptide competition assay involving p300 wild type and mutant peptides, followed by mass spectrometric analysis confirmed the covalent interaction of Δ(12)-PGJ(2) with Cys(1438). Using biotinylated derivatives of Δ(12)-PGJ(2) and 9,10-dihydro-15-deoxy-Δ(12,14)-PGJ(2), we demonstrate the covalent interaction of Δ(12)-PGJ(2) with the p300 HAT domain, but not the latter. In agreement with the in vitro filter binding assay, CyPGs were also found to inhibit H3 histone acetylation in cell-based assays. In addition, Δ(12)-PGJ(2) also inhibited the acetylation of the HIV-1 Tat by recombinant p300 in in vitro assays. This study demonstrates, for the first time, that Δ(12)-PGJ(2) inhibits p300 through Michael addition, where α,β-unsaturated carbonyl function is absolutely required for the inhibitory activity.

  13. Nanoimprint lithography of Al nanovoids for deep-UV SERS.

    PubMed

    Ding, Tao; Sigle, Daniel O; Herrmann, Lars O; Wolverson, Daniel; Baumberg, Jeremy J

    2014-10-22

    Deep-ultraviolet surface-enhanced Raman scattering (UV-SERS) is a promising technique for bioimaging and detection because many biological molecules possess UV absorption lines leading to strongly resonant Raman scattering. Here, Al nanovoid substrates are developed by combining nanoimprint lithography of etched polymer/silica opal films with electron beam evaporation, to give a high-performance sensing platform for UV-SERS. Enhancement by more than 3 orders of magnitude in the UV-SERS performance was obtained from the DNA base adenine, matching well the UV plasmonic optical signatures and simulations, demonstrating its suitability for biodetection. PMID:25291629

  14. Optimization of a multi-well array SERS chip

    NASA Astrophysics Data System (ADS)

    Abell, J. L.; Driskell, J. D.; Dluhy, R. A.; Tripp, R. A.; Zhao, Y.-P.

    2009-05-01

    SERS-active substrates are fabricated by oblique angle deposition and patterned by a polymer-molding technique to provide a uniform array for high throughput biosensing and multiplexing. Using a conventional SERS-active molecule, 1,2-Bis(4-pyridyl)ethylene (BPE), we show that this device provides a uniform Raman signal enhancement from well to well. The patterning technique employed in this study demonstrates a flexibility allowing for patterning control and customization, and performance optimization of the substrate. Avian influenza is analyzed to demonstrate the ability of this multi-well patterned SERS substrate for biosensing.

  15. Preparation of SERS-active substrates using thermal inkjet technology

    NASA Astrophysics Data System (ADS)

    Fierro-Mercado, P.; Renteria-Beleño, B.; Hernández-Rivera, S. P.

    2012-11-01

    Highly sensitive SERS substrates capable of detecting less than a hundred molecules were prepared via TIJ technology. Films were prepared by printing silver nanoparticles (Ag NP) suspensions on quartz and other surfaces. Prepared substrates were characterized by UV-Vis spectrophotometry. Morphological evolution of films was monitored by atomic force microscopy. Inhomogeneous coverage of Ag NP was obtained for a single deposition, while a more uniform distribution of Ag NP was obtained when the number of depositions increased. SERS performance of the prepared SERS substrates was evaluated using p-aminobenzenethiol as a probe molecule. An estimated enhancement factor of 9.0 × 1012 was obtained.

  16. Nanoimprint Lithography of Al Nanovoids for Deep-UV SERS

    PubMed Central

    2014-01-01

    Deep-ultraviolet surface-enhanced Raman scattering (UV-SERS) is a promising technique for bioimaging and detection because many biological molecules possess UV absorption lines leading to strongly resonant Raman scattering. Here, Al nanovoid substrates are developed by combining nanoimprint lithography of etched polymer/silica opal films with electron beam evaporation, to give a high-performance sensing platform for UV-SERS. Enhancement by more than 3 orders of magnitude in the UV-SERS performance was obtained from the DNA base adenine, matching well the UV plasmonic optical signatures and simulations, demonstrating its suitability for biodetection. PMID:25291629

  17. Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site.

    PubMed Central

    Neuschäfer-Rube, Frank; Hermosilla, Ricardo; Rehwald, Mathias; Rönnstrand, Lars; Schülein, Ralf; Wernstedt, Christer; Püschel, Gerhard Paul

    2004-01-01

    hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a G(s)-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C-terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced beta-arrestin complex formation, which is stabilized by phosphorylation. Subsequently beta-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist-induced phosphorylation, interaction with beta-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370-382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389-Ser-390-Thr-391-Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of beta-arrestin1. However, phosphorylation greatly enhanced the stability of the beta-arrestin1-receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both beta-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary. PMID:14709160

  18. A new glucose-6-phosphate dehydrogenase variant, G6PD Orissa (44 Ala{yields}Gly), is the major polymorphic variant in tribal populations in India

    SciTech Connect

    Kaeda, J.S.; Bautista, J.M.; Stevens, D.

    1995-12-01

    Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is usually found at high frequencies in areas of the world where malaria has been epidemic. The frequency and genetic basis of G6PD deficiency have been studied in Africa, around the Mediterranean, and in the Far East, but little such information is available about the situation in India. To determine the extent of heterogeneity of G6PD, we have studied several different Indian populations by screening for G6PD deficiency, followed by molecular analysis of deficient alleles. The frequency of G6PD deficiency varies between 3% and 15% in different tribal and urban groups. Remarkably, a previously unreported deficient variant, G6PD Orissa (44 Ala{yields}Gly), is responsible for most of the G6PD deficiency in tribal Indian populations but is not found in urban populations, where most of the G6PD deficiency is due to the G6PD Mediterranean (188 Ser{yields}Phe) variant. The K{sup NADP}{sub m} of G6PD Orissa is fivefold higher than that of the normal enzyme. This may be due to the fact that the alanine residue that is replaced by glycine is part of a putative coenzyme-binding site. 37 refs., 2 figs., 3 tabs.

  19. Fluorescence imaging and spectroscopy of ALA-induced protoporphyrin IX preferentially accumulated in tumor tissue

    NASA Astrophysics Data System (ADS)

    Stepp, Herbert G.; Baumgartner, Reinhold; Beyer, Wolfgang; Knuechel, Ruth; Koerner, T. O.; Kriegmair, M.; Rick, Kai; Steinbach, Pia; Hofstetter, Alfons G.

    1995-12-01

    In a clinical pilot study performed on 104 patients suffering from bladder cancer it could be shown that intravesical instillation of a solution of 5-aminolevulinic acid (5-ALA) induces a tumorselective accumulation of Protoporphyrin IX (PPIX). Malignant lesions could be detected with a sensitivity of 97% and a specificity of 67%. The Kr+-laser as excitation light source could successfully be replaced by a filtered short arc Xe-lamp. Its emission wavelength band (375 nm - 440 nm) leads to an efficiency of 58% for PPIX- excitation compared to the laser. Two-hundred-sixty mW of output power at the distal end of a slightly modified cystoscope could be obtained. This is sufficient for recording fluorescence images with a target integrating color CCD-camera. Red fluorescence and blue remitted light are displayed simultaneously. Standard white light observation is possible with the same instrumentation. Pharmacokinetic measurements were performed on 18 patients after different routes of 5-ALA application (oral, inhalation and intravesical instillation). PPIX-fluorescence measurements were made on the skin and on the blood plasma. Pharmacokinetic of 5-ALA could be performed on blood plasma. Endoscopical florescence spectroscopy showed the high fluorescence contrast between tumor and normal tissue with a mean value of 10.7. Forthcoming clinical multicenter studies require an objective measure of the fluorescence intensity. Monte Carlo computer simulations showed that artifacts due to observation geometry and varying absorption can largely be reduced by ratioing fluorescence (red channel of camera) to remission (blue channel). Real time image ratioing provides false color images with a reliable fluorescence information.

  20. Imiquimod immunotherapy and ALA photodynamic therapy combination for the treatment of genital bowenoid papulosis

    NASA Astrophysics Data System (ADS)

    Wang, Xiu-Li; Wang, Hong-Wei; Guo, Ming-Xia; Huang, Zheng

    2007-02-01

    To investigate the feasibility and efficacy of combination of imiquimod immunotherapy and 5- aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) for the treatment of genital bowenoid papulosis (BP). A total of twenty seven BP patients were randomized into two groups: (I) fifteen patients (12 male and 3 female, age 22-56 years old) were treated with topical application of 5% imiquimod cream (three times a week) and ALA-PDT (100 J/cm2 at 100 mW/cm2, once a week) for 1-4 times in one week interval. (II) Twelve patients (6 male and 6 female, age 29-58 years old) were treated with CO II laser vaporization as a control. Patients were followed up for 3 to 12 months. Results: In combined therapy group, 60% (9/15) patients showed complete remission and only one recurred (11.1%) during follow up. Local side effects included mild erythema, edema, erosion and burning and/or stinging sensation. No systemic side effect was found. In CO II laser vaporization group, 83.3% (10/12) patients showed complete remission. However, recurrence occurred in 6 patients (60.0%). Local side effects included mild to moderate edema, erosion, ulceration, delayed healing, prolonged pain and scarring. The difference of recurrence rate between two groups was statistically significant (P < 0.05). Topical application of imiquimod cream and ALA-PDT is safe, effective and associated with low recurrence and less side effect. Its true clinical value needs to be further investigated by a long-term follow-up of large scale trial.

  1. IL6R Variation Asp358Ala Is a Potential Modifier of Lung Function in Asthma

    PubMed Central

    Hawkins, Gregory A; Robinson, Mac B; Hastie, Annette T; Li, Xingnan; Li, Huashi; Moore, Wendy C; Howard, Timothy D; Busse, William W.; Erzurum, Serpil C.; Wenzel, Sally E.; Peters, Stephen P; Meyers, Deborah A; Bleecker, Eugene R

    2012-01-01

    Background The IL6R SNP rs4129267 has recently been identified as an asthma susceptibility locus in subjects of European ancestry but has not been characterized with respect to asthma severity. The SNP rs4129267 is in linkage disequilibrium (r2=1) with the IL6R coding SNP rs2228145 (Asp358Ala). This IL6R coding change increases IL6 receptor shedding and promotes IL6 transsignaling. Objectives To evaluate the IL6R SNP rs2228145 with respect to asthma severity phenotypes. Methods The IL6R SNP rs2228145 was evaluated in subjects of European ancestry with asthma from the Severe Asthma Research Program (SARP). Lung function associations were replicated in the Collaborative Study on the Genetics of Asthma (CSGA) cohort. Serum soluble IL6 receptor (sIL6R) levels were measured in subjects from SARP. Immunohistochemistry was used to qualitatively evaluate IL6R protein expression in BAL cells and endobronchial biopsies. Results The minor C allele of IL6R SNP rs2228145 was associated with lower ppFEV1 in the SARP cohort (p=0.005), the CSGA cohort (0.008), and in combined cohort analysis (p=0.003). Additional associations with ppFVC, FEV1/FVC, and PC20 were observed. The rs2228145 C allele (Ala358) was more frequent in severe asthma phenotypic clusters. Elevated serum sIL6R was associated with lower ppFEV1 (p=0.02) and lower ppFVC (p=0.008) (N=146). IL6R protein expression was observed in BAL macrophages, airway epithelium, vascular endothelium, and airway smooth muscle. Conclusions The IL6R coding SNP rs2228145 (Asp358Ala) is a potential modifier of lung function in asthma and may identify subjects at risk for more severe asthma. IL6 transsignaling may have a pathogenic role in the lung. PMID:22554704

  2. Phonon-drag thermopower in anisotropic AlAs quantum wells

    SciTech Connect

    Lehmann, Dietmar; Tsaousidou, Margarita; Kubakaddi, Shrishail

    2013-12-04

    In the present work we have developed a generalized theory of phonon-drag thermopower Ŝ{sup g} for a highly anisotropic two-dimensional electron gas. For electrons confined in AlAs quantum wells we calculate Ŝ{sup g} as function of temperature. We show that Ŝ{sup g} exhibits a strong anisotropic behavior depending on valley occupancy which can be tuned by well width and strain. Also a great enhancement of Ŝ{sup g} is observed compared to GaAs quantum wells.

  3. The peptide Z-Aib-Aib-Aib-L-Ala-OtBu.

    PubMed

    Gessmann, Renate; Brückner, Hans; Petratos, Kyriacos

    2014-04-01

    The title peptide, N-benzyloxycarbonyl-α-aminoisobutyryl-α-aminoisobutyryl-α-aminoisobutyryl-L-alanine tert-butyl ester or Z-Aib-Aib-Aib-L-Ala-OtBu (Aib is α-aminoisobutyric acid, Z is benzyloxycarbonyl and OtBu indicates the tert-butyl ester), C27H42N4O7, is a left-handed helix with a right-handed conformation in the fourth residue, which is the only chiral residue. There are two 4→1 intramolecular hydrogen bonds in the structure. In the lattice, molecules are hydrogen bonded to form columns along the c axis.

  4. In vivo imaging of transplanted islets with 64Cu-DO3A-VS-Cys40-Exendin-4 by targeting GLP-1 receptor.

    PubMed

    Wu, Zhanhong; Todorov, Ivan; Li, Lin; Bading, James R; Li, Zibo; Nair, Indu; Ishiyama, Kohei; Colcher, David; Conti, Peter E; Fraser, Scott E; Shively, John E; Kandeel, Fouad

    2011-08-17

    Glucagon-like peptide 1 receptor (GLP-1R) is highly expressed in pancreatic islets, especially on β-cells. Therefore, a properly labeled ligand that binds to GLP-1R could be used for in vivo pancreatic islet imaging. Because native GLP-1 is degraded rapidly by dipeptidyl peptidase-IV (DPP-IV), a more stable agonist of GLP-1 such as Exendin-4 is a preferred imaging agent. In this study, DO3A-VS-Cys(40)-Exendin-4 was prepared through the conjugation of DO3A-VS with Cys(40)-Exendin-4. The in vitro binding affinity of DO3A-VS-Cys(40)-Exendin-4 was evaluated in INS-1 cells, which overexpress GLP-1R. After (64)Cu labeling, biodistribution studies and microPET imaging of (64)Cu-DO3A-VS-Cys(40)-Exendin-4 were performed on both subcutaneous INS-1 tumors and islet transplantation models. The subcutaneous INS-1 tumor was clearly visualized with microPET imaging after the injection of (64)Cu-DO3A-VS-Cys(40)-Exendin-4. GLP-1R positive organs, such as pancreas and lung, showed high uptake. Tumor uptake was saturable, reduced dramatically by a 20-fold excess of unlabeled Exendin-4. In the intraportal islet transplantation models, (64)Cu-DO3A-VS-Cys(40)-Exendin-4 demonstrated almost two times higher uptake compared with normal mice. (64)Cu-DO3A-VS-Cys(40)-Exendin-4 demonstrated persistent and specific uptake in the mouse pancreas, the subcutaneous insulinoma mouse model, and the intraportal human islet transplantation mouse model. This novel PET probe may be suitable for in vivo pancreatic islets imaging in the human.

  5. Microwave synthesis of Au nanoparticles as promising SERS substrates

    NASA Astrophysics Data System (ADS)

    Wang, Lan; Feng, Shangyuan; Liu, Nenrong; Lei, Jinping; Lin, Hongxin; Sun, Liqing; Chen, Rong

    2012-11-01

    A novel method for rapidly synthesized Au colloidal under microwave irradiation was present in this paper. Size of the Au nanoparticles varied from 10 nm to 60 nm along with varying mol fractions by chloroauric acid solution reduced with sodium citrate. The prepared Au nanoparticles were characterized by transmission electron microscope (TEM) and ultraviolet-visible (UV-Vis) spectrophotometer. It is found that the nanoparticle size and shape are highly dependent on the reaction time and the molar ratios of the reducing agent. By the SERS measurements of R6G, 4-MBA and Crystal violet, this Au colloid is shown to be an excellent SERS substrate with good stability. As the fabrication process of this SERS substrate is simple and inexpensive, this method may be used in large-scale preparation of substrates that can serve as an ideal SERS substrate in biomedical application.

  6. Bioenabled SERS Substrates for Food Safety and Drinking Water Monitoring

    PubMed Central

    Yang, Jing; Rorrer, Gregory L.; Wang, Alan X.

    2016-01-01

    We present low-cost bioenabled surface-enhanced Raman scattering (SERS) substrates that can be massively produced in sustainable and eco-friendly methods with significant commercial potentials for the detection of food contamination and drinking water pollution. The sensors are based on diatom frustules with integrated plasmonic nanoparticles. The ultra-high sensitivity of the SERS substrates comes from the coupling between the diatom frustules and Ag nanoparticles to achieve dramatically increased local optical field to enhance the light-matter interactions for SERS sensing. We successfully applied the bioenabled SERS substrates to detect melamine in milk and aromatic compounds in water with sensitivity down to 1μg/L. PMID:26900205

  7. Determination of nicotine by surface-enhanced Raman scattering (SERS)

    SciTech Connect

    Barber, T.E.; List, M.S.; Haas, J.W. III; Wachter, E.A. )

    1994-11-01

    The analytical application of surface-enhanced Raman spectroscopy (SERS) to the determination of nicotine is demonstrated. A simple spectroelectrochemical method using a copper or silver electrode as the SERS substrate has been developed, consisting of three steps: polishing a working electrode to a mirror finish; roughening the electrode in an electrolyte solution; and, finally, depositing the nicotine analyte onto the roughened electrode after immersion in a sample solution. During the reduction cycle, a large enhancement in nicotine Raman scattering is observed at the electrode surface. The intensity of the SERS signal on a silver electrode is linear with concentration from 10 to 900 ppb, with an estimated detection limit of 7 ppb. The total analysis time per sample is approximately five minutes. This procedure has been used to analyze the extract from a cigarette side-stream smoke sample (environmental tobacco smoke); the SERS results agree well with those of conventional gas chromatographic analysis.

  8. Principal component analysis based methodology to distinguish protein SERS spectra

    NASA Astrophysics Data System (ADS)

    Das, G.; Gentile, F.; Coluccio, M. L.; Perri, A. M.; Nicastri, A.; Mecarini, F.; Cojoc, G.; Candeloro, P.; Liberale, C.; De Angelis, F.; Di Fabrizio, E.

    2011-05-01

    Surface-enhanced Raman scattering (SERS) substrates were fabricated using electro-plating and e-beam lithography techniques. Nano-structures were obtained comprising regular arrays of gold nanoaggregates with a diameter of 80 nm and a mutual distance between the aggregates (gap) ranging from 10 to 30 nm. The nanopatterned SERS substrate enabled to have better control and reproducibility on the generation of plasmon polaritons (PPs). SERS measurements were performed for various proteins, namely bovine serum albumin (BSA), myoglobin, ferritin, lysozyme, RNase-B, α-casein, α-lactalbumin and trypsin. Principal component analysis (PCA) was used to organize and classify the proteins on the basis of their secondary structure. Cluster analysis proved that the error committed in the classification was of about 14%. In the paper, it was clearly shown that the combined use of SERS measurements and PCA analysis is effective in categorizing the proteins on the basis of secondary structure.

  9. SERS diagnostic platforms, methods and systems microarrays, biosensors and biochips

    DOEpatents

    Vo-Dinh, Tuan

    2007-09-11

    A Raman integrated sensor system for the detection of targets including biotargets includes at least one sampling platform, at least one receptor probe disposed on the sampling platform, and an integrated circuit detector system communicably connected to the receptor. The sampling platform is preferably a Raman active surface-enhanced scattering (SERS) platform, wherein the Raman sensor is a SERS sensor. The receptors can include at least one protein receptor and at least one nucleic acid receptor.

  10. Nanostructured plasmonic substrates for use as SERS sensors

    NASA Astrophysics Data System (ADS)

    Jeon, Tae Yoon; Kim, Dong Jae; Park, Sung-Gyu; Kim, Shin-Hyun; Kim, Dong-Ho

    2016-08-01

    Plasmonic nanostructures strongly localize electric fields on their surfaces via the collective oscillations of conducting electrons under stimulation by incident light at a certain wavelength. Molecules adsorbed onto the surfaces of plasmonic structures experience a strongly enhanced electric field due to the localized surface plasmon resonance (LSPR), which amplifies the Raman scattering signal obtained from these adsorbed molecules. This phenomenon is referred to as surface-enhanced Raman scattering (SERS). Because Raman spectra serve as molecular fingerprints, SERS has been intensively studied for its ability to facilely detect molecules and provide a chemical analysis of a solution. Further enhancements in the Raman intensity and therefore higher sensitivity in SERS-based molecular analysis have been achieved by designing plasmonic nanostructures with a controlled size, shape, composition, and arrangement. This review paper focuses on the current state of the art in the fabrication of SERS-active substrates and their use as chemical and biosensors. Starting with a brief description of the basic principles underlying LSPR and SERS, we discuss three distinct nanofabrication methods, including the bottom-up assembly of nanoparticles, top-down nanolithography, and lithography-free random nanoarray formation. Finally, typical applications of SERS-based sensors are discussed, along with their perspectives and challenges.

  11. Lethal course of meconium ileus in preterm twins revealing a novel cystic fibrosis mutation (p.Cys524Tyr)

    PubMed Central

    2014-01-01

    Background In term newborns meconium ileus is frequently associated with cystic fibrosis. Reports on meconium ileus in preterm infants being diagnosed with cystic fibrosis early after birth are very scarce. Associations between genotype and phenotype in cystic fibrosis and its particular comorbidities have been reported. Case presentation Two extremely preterm twin infants (26 weeks of gestation) born from a Malaysian mother and a Caucasian father were presented with typical signs of meconium ileus. Despite immediate surgery both displayed a unique and finally lethal course. Mutation analysis revealed a novel, probably pathogenic cystic fibrosis mutation, p.Cys524Tyr. The novel mutation might explain the severity of disease next to typical sequelae of prematurity. Conclusion Preterm neonates with meconium ileus have to be evaluated for cystic fibrosis beyond ethnical boundaries, but may take devastating clinical courses despite early treatment. The novel, potentially pathogenic CF mutation p.Cys524Tyr might be associated with severe meconium ileus in neonates. Disease-modifying loci are important targets for intestinal comorbidity of cystic fibrosis. PMID:24433235

  12. Challenging a paradigm: theoretical calculations of the protonation state of the Cys25-His159 catalytic diad in free papain.

    PubMed

    Shokhen, Michael; Khazanov, Netaly; Albeck, Amnon

    2009-12-01

    A central mechanistic paradigm of cysteine proteases is that the His-Cys catalytic diad forms an ion-pair NH(+)/S(-) already in the catalytically active free enzyme. Most molecular modeling studies of cysteine proteases refer to this paradigm as their starting point. Nevertheless, several recent kinetics and X-ray crystallography studies of viral and bacterial cysteine proteases depart from the ion-pair mechanism, suggesting general base catalysis. We challenge the postulate of the ion-pair formation in free papain. Applying our QM/SCRF(VS) molecular modeling approach, we analyzed all protonation states of the catalytic diad in free papain and its SMe derivative, comparing the predicted and experimental pK(a) data. We conclude that the His-Cys catalytic diad in free papain is fully protonated, NH(+)/SH. The experimental pK(a) = 8.62 of His159 imidazole in free papain, obtained by NMR-controlled titration and originally interpreted as the NH(+)/S(-) <==> N/S(-) NH(+)/S(-) <==> N/S(-) equilibrium, is now assigned to the NH(+)/SH <==> N/SH NH(+)/SH <==> N/SH equilibrium.

  13. Highly efficient and selective enrichment of glycopeptides using easily synthesized magG/PDA/Au/l-Cys composites.

    PubMed

    Wu, Runqing; Li, Lanting; Deng, Chunhui

    2016-05-01

    Highly selective and efficient enrichment of glycopeptides from complex biological samples is necessary. In this study, novel zwitterionic hydrophilic polydopamine-coated magnetic graphene composites (magG/PDA/Au/l-Cys) were synthesized and applied to the enrichment of glycopeptides. The size, morphology, and composition of magG/PDA/Au/l-Cys composites were investigated by transmission electron microscopy, scanning electron microscopy, FT-infrared spectroscopy, and X-ray diffraction. The composites possessed a number of desirable characteristics, including good biocompatibility easy separation property and excellent hydrophilicity. By virtue of the features contributed by different ingredients, the prepared composites demonstrated superior performance for glycopeptide enrichment with high sensitivity (0.1 fmol), efficiency, selectivity (1:100), and repeatability (at least ten times). In addition, the composites were successfully applied to the enrichment of glycopeptides from human serum and 40 unique N-glycosylation peptides from 31 different N-linked glycoproteins were identified. The superior hydrophilic material is of great potential for the analysis of glycoproteins.

  14. Point mutations in KAL1 and the mitochondrial gene MT-tRNA(cys) synergize to produce Kallmann syndrome phenotype.

    PubMed

    Wang, Fei; Huang, Guo-Dong; Tian, Hui; Zhong, Ying-Bin; Shi, Hui-Juan; Li, Zheng; Zhang, Xian-Sheng; Wang, Han; Sun, Fei

    2015-01-01

    Kallmann syndrome (KS) is an inherited developmental disorder defined as the association of hypogonadotropic hypogonadism and anosmia or hyposmia. KS has been shown to be a genetically heterogeneous disease with different modes of inheritance. However, variants in any of the causative genes identified so far are only found in approximately one third of KS patients, thus indicating that other genes or pathways remain to be discovered. Here, we report a large Han Chinese family with inherited KS which harbors two novel variants, KAL1 c.146G>T (p.Cys49Phe) and mitochondrial tRNA(cys) (m.5800A>G). Although two variants can't exert obvious effects on the migration of GnRH neurons, they show the synergistic effect, which can account for the occurrence of the disorder in this family. Furthermore, the disturbance of the mitochondrial cysteinyl-tRNA pathway can significantly affect the migration of GnRH cells in vitro and in vivo by influencing the chemomigration function of anosmin-1. Our work highlights a new mode of inheritance underlay the genetic etiology of KS and provide valuable clues to understand the disease development. PMID:26278626

  15. Importance of thioredoxin in the proteolysis of an immunoglobulin G as antigen by lysosomal Cys-proteases

    PubMed Central

    KERBLAT, I; DROUET, C; CHESNE, S; MARCHE, P N

    1999-01-01

    For disulphide-bonded antigens, reduction has been postulated to be a prerequisite for proteolytic antigen processing, with subsequent production of major histocompatibility complex (MHC) class II binding fragments. The murine monoclonal immunoglobulin G (IgG) CE25/B7 was used as a multimeric antigen in a human model. Native IgG is highly resistant to proteolysis and has been previously found to be partially reduced at early steps of cell processing to become a suitable substrate for endopeptidases. The role of the oxidoreductase thioredoxin (Trx) was assessed in the reduction of the IgG by cleavage of H–L and H–H disulphide bonds. Recombinant human Trx (rTrx) has been assayed in a proteolytic in vitro system on IgG using endosomal and lysosomal subcellular fractions from B lymphoblastoid cells. rTrx is required in a dose-dependent manner for development of efficient proteolysis, catalysed by thiol-dependent Cys-proteases, such as cathepsin B. We demonstrated that cathepsin B activity was stimulated by the addition of rTrx. Thus, we propose that Trx-dependent IgG proteolysis occurred, on the one hand by means of the unfolding of the IgG after disulphide reduction, becoming a substrate of lysosomal proteases, and on the other hand by Cys-proteases such as cathepsin B that are fully active upon the regeneration of their activity by hydrogen donors. PMID:10447715

  16. Modulating the Copper-Sulfur Interaction in Type 1 Blue Copper Azurin by Replacing Cys112 with Nonproteinogenic Homocysteine

    PubMed Central

    Clark, Kevin M; Yu, Yang; Blackburn, Ninian; Lu, Yi

    2014-01-01

    The Cu-SCys interaction is known to play a dominant role in defining the type 1 (T1) blue copper center with respect to both its electronic structure and electron transfer function. Despite this importance, its role has yet to be probed by mutagenesis studies without dramatic change of its T1 copper character. We herein report replacement of the conserved Cys112 in azurin with the nonproteinogenic amino acid homocysteine. Based on electronic absorption, electron paramagnetic resonance, and extended x-ray absorption fine structural spectroscopic studies, this variant displays typical type 1 copper site features. Surprisingly, instead of increasing the strength of the Cu-sulfur interaction by the introduction of the extra methylene group, the Cys112Hcy azurin showed a decrease in the covalent interaction between SHcy and Cu(II) when compared with the WT SCys-Cu(II) interaction. This is likely due to geometric adjustment of the center that resulted in the copper ion moving out of the trigonal plane defined by two histidines and one Hcy and closer to Met121. These structural changes resulted in an increase of reduction potential by 35 mV, consistent with lower Cu-S covalency. These results suggest that the Cu-SCys interaction is close to being optimal in native blue copper protein. It also demonstrates the power of using nonproteinogenic amino acids in addressing important issues in bioinorganic chemistry. PMID:24707355

  17. Multimodal approach to explore the pathogenicity of BARD1, ARG 658 CYS, and ILE 738 VAL mutants.

    PubMed

    Choudhary, Rajan Kumar; Vikrant; Siddiqui, Quadir M; Thapa, Pankaj S; Raikundalia, Sweta; Gadewal, Nikhil; Kumar, Nachimuthu Senthil; Hosur, M V; Varma, Ashok K

    2016-07-01

    BARD1-BRCA1 complex plays an important role in DNA damage repair, apoptosis, chromatin remodeling, and other important processes required for cell survival. BRCA1 and BARD1 heterodimer possess E3 ligase activity and is involved in genome maintenance, by functioning in surveillance for DNA damage, thereby regulating multiple pathways including tumor suppression. BRCT domains are evolutionary conserved domains present in different proteins such as BRCA1, BARD1, XRCC, and MDC1 regulating damage response and cell-cycle control through protein-protein interactions. Nonetheless, the role of BARD1BRCT in the recruitment of DNA repair mechanism and structural integrity with BRCA1 complex is still implicit. To explicate the role of BARD1BRCT in the DNA repair mechanism, in silico, in vitro, and biophysical approach were applied to characterize BARD1 BRCT wild-type and Arg658Cys and Ile738Val mutants. However, no drastic secondary and tertiary structural changes in the mutant proteins were observed. Thermal and chemical denaturation studies revealed that mutants Arg658Cys and Ile738Val have a decrease in Tm and ∆G than the wild type. In silico studies of BARD1 BRCT (568-777) and mutant protein indicate loss in structural compactness on the Ile738Val mutant. Comparative studies of wild-type and mutants will thus be helpful in understanding the basic role of BARD1BRCT in DNA damage repair.

  18. A Zn(II)2Cys6 DNA binding protein regulates the sirodesmin PL biosynthetic gene cluster in Leptosphaeria maculans

    PubMed Central

    Fox, Ellen M.; Gardiner, Donald M.; Keller, Nancy P.; Howlett, Barbara J.

    2008-01-01

    A gene, sirZ, encoding a Zn(II)2Cys6 DNA binding protein is present in a cluster of genes responsible for the biosynthesis of the epipolythiodioxopiperazine (ETP) toxin, sirodesmin PL in the ascomycete plant pathogen, Leptosphaeria maculans. RNA-mediated silencing of sirZ gives rise to transformants that produce only residual amounts of sirodesmin PL and display a decrease in the transcription of several sirodesmin PL biosynthetic genes. This indicates that SirZ is a major regulator of this gene cluster. Proteins similar to SirZ are encoded in the gliotoxin biosynthetic gene cluster of Aspergillus fumigatus (gliZ) and in an ETP-like cluster in Penicillium lilacinoechinulatum (PlgliZ). Despite its high level of sequence similarity to gliZ, PlgliZ is unable to complement the gliotoxin-deficiency of a mutant of gliZ in A. fumigatus. Putative binding sites for these regulatory proteins in the promoters of genes in these clusters were predicted using bioinformatic analysis. These sites are similar to those commonly bound by other proteins with Zn(II)2Cys6 DNA binding domains. PMID:18023597

  19. A hereditary bleeding disorder resulting from a premature stop codon in thrombomodulin (p.Cys537Stop).

    PubMed

    Langdown, Jonathan; Luddington, Roger J; Huntington, James A; Baglin, Trevor P

    2014-09-18

    In this study, we describe a novel thrombomodulin (TM) mutation (c.1611C>A) that codes for a change from cysteine 537 to a premature stop codon (p.Cys537Stop). Three members of a family with a history of posttraumatic bleeding were identified to be heterozygous for this TM mutation. All coagulation screening tests, coagulation factor assays, and platelet function test results were within normal limits. However, the endogenous thrombin potential was markedly reduced at low-tissue factor concentration, and failure to correct with normal plasma indicated the presence of a coagulation inhibitor. Plasma TM levels were highly elevated (433-845 ng/ml, normal range 2-8 ng/ml, equating to 5 to 10 nM), and the addition of exogenous protein C further decreased thrombin generation. The mutation, p.Cys537Stop, results in a truncation within the carboxyl-terminal transmembrane helix. We predict that as a consequence of the truncation, the variant TM is shed from the endothelial surface into the blood plasma. This would promote systemic protein C activation and early cessation of thrombin generation within a developing hemostatic clot, thereby explaining the phenotype of posttraumatic bleeding observed within this family. PMID:25049278

  20. The Arabidopsis SUPERMAN protein is able to specifically bind DNA through its single Cys2-His2 zinc finger motif.

    PubMed

    Dathan, Nina; Zaccaro, Laura; Esposito, Sabrina; Isernia, Carla; Omichinski, James G; Riccio, Andrea; Pedone, Carlo; Di Blasio, Benedetto; Fattorusso, Roberto; Pedone, Paolo V

    2002-11-15

    The Arabidopsis SUPERMAN (SUP) gene has been shown to be important in maintaining the boundary between stamens and carpels, and is presumed to act by regulating cell proliferation. In this work, we show that the SUP protein, which contains a single Cys2-His2 zinc finger domain including the QALGGH sequence, highly conserved in the plant zinc finger proteins, binds DNA. Using a series of deletion mutants, it was determined that the minimal domain required for specific DNA binding (residues 15-78) includes the single zinc finger and two basic regions located on either side of this motif. Furthermore, amino acid substitutions in the zinc finger or in the basic regions, including a mutation that knocks out the function of the SUP protein in vivo (glycine 63 to aspartate), have been found to abolish the activity of the SUP DNA-binding domain. These results strongly suggest that the SUP protein functions in vivo by acting as a DNA-binding protein, likely involved in transcriptional regulation. The association of both an N-terminal and a C-terminal basic region with a single Cys2-His2 zinc finger represents a novel DNA-binding motif suggesting that the mechanism of DNA recognition adopted by the SUP protein is different from that described so far in other zinc finger proteins. PMID:12433998

  1. An experimentally tested scenario for the structural evolution of eukaryotic Cys2His2 zinc fingers from eubacterial ros homologs.

    PubMed

    Netti, Fortuna; Malgieri, Gaetano; Esposito, Sabrina; Palmieri, Maddalena; Baglivo, Ilaria; Isernia, Carla; Omichinski, James G; Pedone, Paolo V; Lartillot, Nicolas; Fattorusso, Roberto

    2013-07-01

    The exact evolutionary origin of the zinc finger (ZF) domain is unknown, as it is still not clear from which organisms it was first derived. However, the unique features of the ZF domains have made it very easy for evolution to tinker with them in a number of different manners, including their combination, variation of their number by unequal crossing-over or tandem duplication and tuning of their affinity for specific DNA sequence motifs through point substitutions. Classical Cys2His2 ZF domains as structurally autonomous motifs arranged in multiple copies are known only in eukaryotes. Nonetheless, a single prokaryotic Cys2His2 ZF domain has been identified in the transcriptional regulator Ros from Agrobacterium tumefaciens and recently characterized. The present work focuses on the evolution of the classical ZF domains with the goal of trying to determine whether eukaryotic ZFs have evolved from the prokaryotic Ros-like proteins. Our results, based on computational and experimental data, indicate that a single insertion of three amino acids in the short loop that separates the β-sheet from the α-helix of the Ros protein is sufficient to induce a structural transition from a Ros like to an eukaryotic-ZF like structure. This observation provides evidence for a structurally plausible and parsimonious scenario of fold evolution, giving a structural basis to the hypothesis of a horizontal gene transfer (HGT) from bacteria to eukaryotes. PMID:23576569

  2. AlaScan: A Graphical User Interface for Alanine Scanning Free-Energy Calculations.

    PubMed

    Ramadoss, Vijayaraj; Dehez, François; Chipot, Christophe

    2016-06-27

    Computation of the free-energy changes that underlie molecular recognition and association has gained significant importance due to its considerable potential in drug discovery. The massive increase of computational power in recent years substantiates the application of more accurate theoretical methods for the calculation of binding free energies. The impact of such advances is the application of parent approaches, like computational alanine scanning, to investigate in silico the effect of amino-acid replacement in protein-ligand and protein-protein complexes, or probe the thermostability of individual proteins. Because human effort represents a significant cost that precludes the routine use of this form of free-energy calculations, minimizing manual intervention constitutes a stringent prerequisite for any such systematic computation. With this objective in mind, we propose a new plug-in, referred to as AlaScan, developed within the popular visualization program VMD to automate the major steps in alanine-scanning calculations, employing free-energy perturbation as implemented in the widely used molecular dynamics code NAMD. The AlaScan plug-in can be utilized upstream, to prepare input files for selected alanine mutations. It can also be utilized downstream to perform the analysis of different alanine-scanning calculations and to report the free-energy estimates in a user-friendly graphical user interface, allowing favorable mutations to be identified at a glance. The plug-in also assists the end-user in assessing the reliability of the calculation through rapid visual inspection.

  3. The Simplified Aircraft-Based Paired Approach With the ALAS Alerting Algorithm

    NASA Technical Reports Server (NTRS)

    Perry, Raleigh B.; Madden, Michael M.; Torres-Pomales, Wilfredo; Butler, Ricky W.

    2013-01-01

    This paper presents the results of an investigation of a proposed concept for closely spaced parallel runways called the Simplified Aircraft-based Paired Approach (SAPA). This procedure depends upon a new alerting algorithm called the Adjacent Landing Alerting System (ALAS). This study used both low fidelity and high fidelity simulations to validate the SAPA procedure and test the performance of the new alerting algorithm. The low fidelity simulation enabled a determination of minimum approach distance for the worst case over millions of scenarios. The high fidelity simulation enabled an accurate determination of timings and minimum approach distance in the presence of realistic trajectories, communication latencies, and total system error for 108 test cases. The SAPA procedure and the ALAS alerting algorithm were applied to the 750-ft parallel spacing (e.g., SFO 28L/28R) approach problem. With the SAPA procedure as defined in this paper, this study concludes that a 750-ft application does not appear to be feasible, but preliminary results for 1000-ft parallel runways look promising.

  4. Ala-9Val polymorphism of Mn-SOD gene in sickle cell anemia.

    PubMed

    Sogut, S; Yonden, Z; Kaya, H; Oktar, S; Tutanc, M; Yilmaz, H R; Yigit, A; Ozcelik, N; Gali, E

    2011-01-01

    Oxidative stress may be contributory to the pathophysiology of the abnormalities that underlie the clinical course of sickle cell anemia. We looked for a possible genetic association between the functional polymorphism Ala-9Val in the human Mn-SOD gene and sickle cell anemia. One hundred and twenty-seven patients with sickle cell anemia and 127 healthy controls were recruited into the study. Alanine versus valine polymorphism in the signal peptide of the Mn-SOD gene was evaluated using a primer pair to amplify a 107-bp fragment followed by digestion with the restriction enzyme NgoMIV. In the sickle cell anemia patients, the frequency of Val/Val genotype was approximately 1.4-fold lower and that of Ala/Val was 1.3-fold higher compared to the controls. No significant difference in genotype frequencies was found between patients and controls (χ(2) = 4.561, d.f. = 2, P = 0.101). The Val-9 was the most common allele in patient and healthy subjects. No significant difference in allele frequencies was found between patients and controls (χ(2) = 1.496, d.f. = 1, P = 0.221). We conclude that the Mn-SOD gene polymorphism is not associated with sickle cell anemia. PMID:21574139

  5. Metal coordination and tyrosinase inhibition studies with Kojic-βAla-Kojic.

    PubMed

    Lachowicz, Joanna Izabela; Nurchi, Valeria Marina; Crisponi, Guido; Pelaez, Maria de Guadalupe Jaraquemada; Rescigno, Antonio; Stefanowicz, Piotr; Cal, Marta; Szewczuk, Zbigniew

    2015-10-01

    Kojic acid is a natural antifungal and antibacterial agent that has been extensively studied for its tyrosinase inhibitory and metal coordination properties. Tyrosinase is a metalloenzyme with two copper ions in the active site. It is widely accepted that the tyrosinase inhibitory activity of kojic acid is related to its ability to coordinate metals. Over the past five years, we have used kojic acid to synthesize new and efficient bis-kojic acid chelators of iron and aluminium. In parallel, we investigated whether the de novo designed ligands could interfere with proper tyrosinase functioning. The present study combines our experience with inhibition and coordination studies of the new ligand: Kojic-βAla-Kojic. Research aimed at the assembly of a new potent tyrosinase inhibitor was based on the well-known crystal structure of the enzyme. Two questions were whether two kojic acids could act better than one and to what extent the length and kind of linker could ameliorate metal coordination, and inhibitory activity. Our results show that Kojic-βAla-Kojic has high affinity for Fe(III), Al(III), Zn(II), and Cu(II) and strong tyrosinase inhibitory effect and it can be proposed for use in industrial and pharmaceutical applications.

  6. Spontaneous abortion and functional polymorphism (Val16Ala) in the manganese SOD gene.

    PubMed

    Eskafi Sabet, E; Salehi, Z; Khodayari, S; Sabouhi Zarafshan, S; Zahiri, Z

    2015-02-01

    Spontaneous abortion is the most common complication of early pregnancy. Genetic factors have been hypothesised to play a role in spontaneous abortion. Since it is possible that the balance of oxidants and antioxidants can be affected by different genetic variants, gene polymorphisms have been proposed as a susceptibility factor that increases the chance of miscarriage. Manganese superoxide dismutase is an important antioxidant enzyme encoded by manganese superoxide dismutase (MnSOD) gene. The aim of this experiment was to assess whether Val16Ala polymorphism of MnSOD gene is associated with miscarriage in northern Iran. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for genotyping. Statistical analyses were conducted using the χ(2)-test. The genetic distributions did not differ significantly between cases and controls, however slightly more Val/Val genotypes were found among the patients compared with control subjects (p = 0.059). No correlation was observed between susceptibility to abortion and MnSOD Val16Ala polymorphism. Larger population-based studies are needed for clarifying the relationship between abortion and MnSOD genotypes.

  7. Rapid detection of mutations by conformation sensitive gel electrophoresis: Application to the identification of three new mutations in the type II procollagen gene and a fourth family with the Arg{sub 519}{yields}Cys base substitution

    SciTech Connect

    Williams, C.J.; Rock, M.; McCarron, S.

    1994-09-01

    Conformation sensitive gel electrophoresis (CSGE) detects differences as small as a single base mismatch in DNA heteroduplexes of polymerase chain reaction (PCR) products. The altered migration of heteroduplexes versus homoduplexes is resolved in a polyacrylamide-based gel electrophoresis system. The technique was used here to detect conformational changes in the type II procollagen gene (COL2A1) in patients with growth plate defects. PCR products which displayed heteroduplex species were directly sequenced and all revealed either base substitutions or base deletions. Three of the base substitutions resulted in the identification of new mutations. These include a Gly{sub 691}{yields}Arg substitution in a proband with hypochondrogenesis, a Gly{sub 975}{yields}Ser base substitution in a family with late-onset spondyloepiphyseal dysplasia (SEDT) and precocious osteoarthritis (POA), and a Gly{sub 988}{yields}Arg mutation in another patient with hypochondrogenesis. A fourth substitution was found to be the fourth example of an Arg{sub 519}{yields}Cys point mutation in a family with SEDT and POA. All mutations were confirmed by restriction site analysis. These results illustrate the utility of the CSGE method for the rapid detection of mutations in PCR products without the need for special equipment, primers or sample preparation.

  8. Trace detection of herbicides by SERS technique, using SERS-active substrates fabricated from different silver nanostructures deposited on silicon

    NASA Astrophysics Data System (ADS)

    Cao Dao, Tran; Quynh Ngan Luong, Truc; Cao, Tuan Anh; Hai Nguyen, Ngoc; Kieu, Ngoc Minh; Thuy Luong, Thi; Le, Van Vu

    2015-09-01

    In this report we present the initial results of the use of different silver nanostructures deposited on silicon for trace detection of paraquat (a commonly used herbicide) using the surface-enhanced Raman scattering (SERS) effect. More specifically, the SERS-active substrates were fabricated from silver nanoparticles (AgNPs) deposited onto the flat surface of a silicon wafer (AgNPs@Si substrate), as well as on the surface of an obliquely aligned silicon nanowire (SiNW) array (AgNPs@SiNWs substrate), and from silver nanodendrites (AgNDs) deposited onto the flat surface of a silicon wafer (AgNDs@Si substrate). Results showed that with the change of the structure of the SERS-active substrate, higher levels of SERS enhancement have been achieved. Specifically, with the fabricated AgNDs@Si substrate, paraquat concentration as low as 1 ppm can be detected.

  9. Effects of Ala-Gln feeding strategies on growth, metabolism, and crowding stress resistance of juvenile Cyprinus carpio var. Jian.

    PubMed

    Chen, Xiu-Mei; Guo, Gui-Liang; Sun, Li; Yang, Qiu-Shi; Wang, Gui-Qin; Qin, Gui-Xin; Zhang, Dong-Ming

    2016-04-01

    The present study was conducted to evaluate the effects of different L-alanyl-l-glutamine (Ala-Gln) feeding strategies on the growth performance, metabolism and crowding stress resistance related parameters in juvenile Jian carp (Cyprinus carpio var. Jian) under crowded condition (80 g/L). Juvenile Jian carp (initial weight 26.1 ± 0.6 g) were distributed into five groups which fed with graded concentrations (0% or 1.0%) of Ala-Gln for eight weeks. Control group (I, 0/0) fed with control diet (0% Ala-Gln) throughout the feeding trial. The other four groups employed different control and experimental diet feeding strategies ranging from two weeks control diet fed and two weeks experimental diet (1% Ala-Gln) fed (II, 0/2) to eight weeks experimental diet fed (V, 4/4). Results revealed that Mean weight gain (MEG) under all different feeding strategies of Ala-Gln were significantly higher than that of the control group (p < 0.05), and MEG of group II (201.90%) was even higher than that of group IV (184.70%). Liver glycogen and blood total protein of groups II, III and V were significantly higher than that in groups I and IV (p < 0.05). The highest level of serum thyroxine (10.07 ng/ml), insulin-like growth factor-I (52.40 ng/ml) and insulin (9.73 μ IU/mL) were observed in group V. However, diet supplemented with Ala-Gln did not affect the levels of serum glucose, cortisol and catecholamine in fish. The mRNA expression of GR1a, GR1b and GR2 were also significantly changed in Ala-Gln supplementation groups compared with control group (p < 0.05). After fish intraperitoneally injected with virulent Aeromonas hydrophila, the fish survival rates were significantly increased in all Ala-Gln supplementation groups compared with control group (p < 0.05). Results from the present experiment showed the importance of dietary supplementation of Ala-Gln in benefaction of the growth performance, metabolism and crowding stress resistance in Jian carp breeding. The

  10. Effects of Toll-like receptor 2 agonist Pam(3)CysSK(4) on inflammation and brain damage in experimental pneumococcal meningitis.

    PubMed

    Sellner, Johann; Grandgirard, Denis; Gianinazzi, Christian; Landmann, Regine M; Leib, Stephen L

    2009-01-01

    TLR2 signaling participates in the pathogenesis of pneumococcal meningitis. In infant rats, the TLR2 agonist Pam(3)CysSK(4) was applied intracisternally (0.5 microg in 10 microl saline) alone or after induction of pneumococcal meningitis to investigate the effect of TLR2 activation on cerebrospinal fluid (CSF) inflammation and hippocampal apoptosis. A dose effect of Pam(3)CysSK(4) on apoptosis was investigated by intracisternal application of 0.5 microg in 10 microl saline and 40 microg in 20 microl saline. Pam(3)CysSK(4) neither induced apoptosis in sham-operated mice nor aggravated apoptosis in acute infection. However, Pam(3)CysSK(4) induced pleocytosis, TNF-alpha and MMP-9 in CSF in sham-infection but not during acute meningitis. We conclude that TLR2 signaling triggered by Pam(3)CysSK(4) at a dosage capable to induce a neuroinflammatory response does not induce hippocampal apoptosis in the infant rat model of experimental pneumococcal meningitis.

  11. CYS3, a hotspot of meiotic recombination in Saccharomyces cerevisiae. Effects of heterozygosity and mismatch repair functions on gene conversion and recombination intermediates.

    PubMed Central

    Vedel, M; Nicolas, A

    1999-01-01

    We have examined meiotic recombination at the CYS3 locus. Genetic analysis indicates that CYS3 is a hotspot of meiotic gene conversion, with a putative 5'-3' polarity gradient of conversion frequencies. This gradient is relieved in the presence of msh2 and pms1 mutations, indicating an involvement of mismatch repair functions in meiotic recombination. To investigate the role of mismatch repair proteins in meiotic recombination, we performed a physical analysis of meiotic DNA in wild-type and msh2 pms1 strains in the presence or absence of allelic differences at CYS3. Neither the mutations in CYS3 nor the absence of mismatch repair functions affects the frequency and distribution of nearby recombination-initiating DNA double-strand breaks (DSBs). Processing of DSBs is also similar in msh2 pms1 and wild-type strains. We conclude that mismatch repair functions do not control the distribution of meiotic gene conversion events at the initiating steps. In the MSH2 PMS1 background, strains heteroallelic for frameshift mutations in CYS3 exhibit a frequency of gene conversion greater than that observed for either marker alone. Physical analysis revealed no modification in the formation of DSBs, suggesting that this marker effect results from subsequent processing events that are not yet understood. PMID:10101154

  12. Identification, cloning and characterization of cysK, the gene encoding O-acetylserine (thiol)-lyase from Azospirillum brasilense, which is involved in tellurite resistance.

    PubMed

    Ramírez, Alberto; Castañeda, Miguel; Xiqui, María L; Sosa, Araceli; Baca, Beatriz E

    2006-08-01

    O-Acetylserine (thiol)-lyase (cysteine synthase) was purified from Azospirillum brasilense Sp7. After hydrolysis of the purified protein, amino acid sequences of five peptides were obtained, which permitted the cloning and sequencing of the cysK gene. The deduced amino acid sequence of cysteine synthase exhibited homology with several putative proteins from Alpha- and Gammaproteobacteria. Azospirillum brasilense Sp7 cysK exhibited 58% identity (72% similarity) with Escherichia coli K12 and Salmonella enterica serovar Typhimurium cysteine synthase proteins. An E. coli auxotroph lacking cysteine synthase loci could be complemented with A. brasilense Sp7 cysK. The 3.0-kb HindIII-EcoRI fragment bearing cysK contained two additional ORFs encoding a putative transcriptional regulator and dUTPase. Insertional disruption of the cysK gene did not produce a cysteine auxotroph, indicating that gene redundancy in the cysteine biosynthetic or other biosynthetic pathways exists in Azospirillum, as already described in other bacteria. Nitrogen fixation was not altered in the mutant strain as determined by acetylene reduction. However, this strain showed an eight-fold reduction in tellurite resistance as compared to the wild-type strain, which was only observed during growth in minimal medium. These data confirm earlier observations regarding the importance of cysteine metabolism in tellurite resistance.

  13. RNA polymerase II CTD phospho-sites Ser5 and Ser7 govern phosphate homeostasis in fission yeast.

    PubMed

    Schwer, Beate; Sanchez, Ana M; Shuman, Stewart

    2015-10-01

    Phosphorylation of the tandem YSPTSPS repeats of the RNA polymerase II CTD inscribes an informational code that orchestrates eukaryal mRNA synthesis. Here we interrogate the role of the CTD in phosphate homeostasis in fission yeast. Expression of Pho1 acid phosphatase, which is repressed during growth in phosphate-rich medium and induced by phosphate starvation, is governed strongly by CTD phosphorylation status, but not by CTD repeat length. Inability to place a Ser7-PO4 mark (as in S7A) results in constitutive derepression of Pho1 expression in phosphate-replete medium. In contrast, indelible installation of a Ser7-PO4 mimetic (as in S7E) hyper-represses Pho1 in phosphate-replete cells and inhibits Pho1 induction during starvation. Pho1 phosphatase is derepressed by ablation of the CTD Ser5-PO4 mark, achieved either by mutating Ser5 in all consensus heptads to alanine, or replacing all Pro6 residues with alanine. We find that Ser5 status is a tunable determinant of Pho1 regulation, i.e., serial decrements in the number of consensus Ser5 heptads from seven to two elicits a progressive increase in Pho1 expression in phosphate-replete medium. Pho1 is also derepressed by hypomorphic mutations of the CTD kinase Cdk9. Inactivation of the CTD phosphatase Ssu72 attenuates Pho1 induction in wild-type cells and blocks Pho1 derepression in S7A cells. These experiments implicate Ser5, Pro6, and Ser7 as component letters of a CTD coding "word" that transduces a repressive transcriptional signal via serine phosphorylation.

  14. Current methods for photodynamic therapy in the US: comparison of MAL/PDT and ALA/PDT.

    PubMed

    Lee, Peter K; Kloser, Andrew

    2013-08-01

    There is some debate regarding the rate of progression of actinic keratosis (AK) into squamous cell carcinoma (SCC).1-4 However, it is clear that treatment for AK lesions is warranted. Results from numerous studies with aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) photodynamic therapy (PDT) for the treatment of AKs, SCC, and Bowen's disease show high rates of clearance for these lesions. MAL/PDT provides similar efficacy to ALA/PDT with the benefits of shorter incubation times according to the approved FDA labeling, greater selectivity, reduced pain during and immediately following therapy, and fewer systemic side effects. Cosmetic outcomes are better with PDT than with cryosurgery or excisional surgery. A number of case reports show efficacy with ALA/PDT and MAL/PDT for acne, photorejuvenation, and other off-label indications. Side effects with PDT tend to be mild to moderate and transient in nature. Overall, ALA/PDT and MAL/PDT are effective for a variety of skin diseases and conditions. MAL/PDT provides some advantages over ALA/PDT.

  15. Comparative in vivo study of precursors of PpIX (ALA and MAL) used topically in photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Rego, Raquel F.; Inada, Natalia M.; Ferreira, Juliana; Araújo-Moreira, Fernando M.; Bagnato, Vanderlei S.

    2009-06-01

    The efficacy of Photodynamic Therapy (PDT) combined with aminolevulinic acid (ALA) or methyl aminolevulinate (MAL) in treatment of cancer has been studied for over ten years. However, there is no established dose for the topical use of these drugs in PDT. The purpose of this study was the comparison of induced PDT response of ALAsense (5-aminolevulinic acid - ALA) and Metvix (methyl aminolevulinate - MAL). Depth of necrosis induced by PDT was analyzed in normal liver of male Wistar rats, using different light doses and topical application of both PpIX precursors - ALA and MAL. PDT was performed with a diode laser at 630 nm with different doses of light (20, 50, 100 and 200 J/cm2), and intensity of 250 mW/cm2. Depth of necrosis analysis was used to calculate the threshold dose for each drug. The results showed that MAL-PDT presented a better response than ALA-PDT, mainly due to formulation differences. Moreover, the ability of the ALA PpIX production was more efficient.

  16. UVB-induced COX-2 expression requires histone H3 phosphorylation at Ser10 and Ser28

    PubMed Central

    Keum, Young-Sam; Kim, Hong-Gyum; Bode, Ann M.; Surh, Young-Joon; Dong, Zigang

    2012-01-01

    Cyclooxygenase-2 (COX-2) is an inducible enzyme that contributes to the generation of chronic inflammation in response to chemical carcinogens and environmental stresses, including ultraviolet B (UVB) irradiation. Although post-translational histone modifications are believed to play an important role in modulating transcriptional regulation of UVB-induced COX-2, the underlying biochemical mechanisms are completely unknown. Here, we show that UVB activates the p38 MAPK/MSK1 kinase cascade to phosphorylate histone H3 at Ser10 and Ser28, contributing to UVB-induced COX-2 expression. UVB has no effect on the global trimethylation level of histone H3 (H3K4me3, H3K9me3, and H3K27me3). We observed that selected mammalian 14-3-3 proteins bind to UVB-induced phosphorylated histone H3 (Ser10 and Ser28). In particular, 14-3-3ε is critical for recruiting MSK1 and Cdk9 to the chromatin and subsequently phosphorylating the C-terminal domain (CTD) of RNA polymerase II in the cox-2 promoter. We propose that histone H3 phosphorylation at Ser10 and Ser28 serve as critical switches to promote cox-2 gene expression by facilitating the recruitment of MSK1 and Cdk9 to the cox-2 promoter, thereby promoting RNA polymerase II phosphorylation. PMID:22391560

  17. Sensitive cylindrical SERS substrate array for rapid microanalysis of nucleobases.

    PubMed

    Rajapandiyan, Panneerselvam; Yang, Jyisy

    2012-12-01

    In this work, a cylindrical-substrate array for surface-enhanced Raman scattering (SERS) measurements was developed to enable analysis of nucleobases in a few microliters of liquid. To eliminate uncertainties associated with SERS detection of aqueous samples, a new type of cylindrical SERS substrate was designed to confine the aqueous sample at the tip of the SERS probe. Poly(methyl methacrylate) (PMMA) optical fibers in a series of different diameters were used as the basic substrate. A solution of poly(vinylidene fluoride)/dimethylformamide (PVDF/DMF) was used to coat the tip of each fiber to increase the surface roughness and facilitate adsorption of silver nanoparticles (AgNPs) for enhancing Raman signals. A chemical reduction method was used to form AgNPs in and on the PVDF coating layer. The reagents and reaction conditions were systematically examined with the aim of estimating the optimum parameters. Unlike the spreading of aqueous sample on most SERS substrates, particularly flat ones, the new SERS substrates showed enough hydrophobicity to restrict aqueous sample to the tip area, thus enabling quantitative analysis. The required volume of sample could be as low as 1 μL with no need for a drying step in the procedure. By aligning the cylindrical SERS substrates into a solid holder, an array of cylindrical substrates was produced for mass analysis of aqueous samples. This new substrate improves both reproducibility and sensitivity for detection in aqueous samples. The enhancement factor approaches 7 orders in magnitude with a relative standard error close to 8%. Using the optimized conditions, nucleobases of adenine, cytosine, thymine, and uracil could be detected with limits approaching a few hundreds nanomolar in only a few microliters of solution. PMID:23140099

  18. Effects of mutations in the {beta} subunit hinge domain on ATP synthase F{sub 1} sector rotation: Interaction between Ser 174 and Ile 163

    SciTech Connect

    Kashiwagi, Sachiko; Iwamoto-Kihara, Atsuko; Kojima, Masaki; Nonaka, Takamasa; Futai, Masamitsu Nakanishi-Matsui, Mayumi

    2008-01-11

    A complex of {gamma}, {epsilon}, and c subunits rotates in ATP synthase (F{sub o}F{sub 1}) coupling with proton transport. Replacement of {beta}Ser174 by Phe in {beta}-sheet4 of the {beta} subunit ({beta}S174F) caused slow {gamma} subunit revolution of the F{sub 1} sector, consistent with the decreased ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F1 sector. Stochastic fluctuation and a key domain of the {beta} subunit, J. Biol. Chem. 282 (2007) 20698-20704]. Modeling of the domain including {beta}-sheet4 and {alpha}-helixB predicted that the mutant {beta}Phe174 residue undergoes strong and weak hydrophobic interactions with {beta}Ile163 and {beta}Ile166, respectively. Supporting this prediction, the replacement of {beta}Ile163 in {alpha}-helixB by Ala partially suppressed the {beta}S174F mutation: in the double mutant, the revolution speed and ATPase activity recovered to about half of the levels in the wild-type. Replacement of {beta}Ile166 by Ala lowered the revolution speed and ATPase activity to the same levels as in {beta}S174F. Consistent with the weak hydrophobic interaction, {beta}Ile166 to Ala mutation did not suppress {beta}S174F. Importance of the hinge domain [phosphate-binding loop (P-loop)/{alpha}-helixB/loop/{beta}-sheet4, {beta}Phe148-{beta}Gly186] as to driving rotational catalysis is discussed.

  19. The sequential action of a dipeptidase and a beta-lyase is required for the release of the human body odorant 3-methyl-3-sulfanylhexan-1-ol from a secreted Cys-Gly-(S) conjugate by Corynebacteria.

    PubMed

    Emter, Roger; Natsch, Andreas

    2008-07-25

    Human axillary odor is formed by the action of Corynebacteria on odorless axilla secretions. Sulfanylalkanols, 3-methyl-3-sulfanylhexan-1-ol in particular, form one key class of the odoriferous compounds. A conjugate with the dipeptide Cys-Gly has been reported as the secreted precursor for 3-methyl-3-sulfanylhexan-1-ol. Here, we confirm the Cys-Gly-(S) conjugate as the major precursor of this odorant, with lower levels of the Cys-(S) conjugate being present in axilla secretions. The enzymatic release of 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate by the axilla isolate Corynebacterium Ax20 was thus investigated. Cellular extracts of Ax20 released 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate and from the Cys-(S) conjugate, whereas the previously isolated C-S lyase of this bacterial strain was only able to cleave the Cys-(S) conjugate. o-Phenanthroline blocked the release from the Cys-Gly-(S) conjugate but did not affect cleavage of the Cys-(S) conjugate, indicating that in a first step, a metal-dependent dipeptidase hydrolyzes the Cys-Gly bond. This enzyme was purified by four chromatographic steps and gel electrophoresis, and the partial amino acid sequence was determined. The corresponding gene was cloned and expressed in Escherichia coli. It codes for a novel dipeptidase with a high affinity toward the Cys-Gly-(S) conjugate of 3-methyl-3-sulfanylhexan-1-ol. Co-incubating either the synthetic Cys-Gly-(S) conjugate or fresh axilla secretions with both the C-S lyase and the novel dipeptidase did release 3-methyl-3-sulfanylhexan-1-ol, proving that the sequential action of these two enzymes from the skin bacterium Corynebacterium Ax20 does release the odorant from the key secreted precursor.

  20. Properties of myelin altered peptide ligand cyclo(87-99)(Ala91,Ala96)MBP87-99 render it a promising drug lead for immunotherapy of multiple sclerosis.

    PubMed

    Deraos, George; Rodi, Maria; Kalbacher, Hubert; Chatzantoni, Kokona; Karagiannis, Fotios; Synodinos, Loukas; Plotas, Panayiotis; Papalois, Apostolos; Dimisianos, Nikolaos; Papathanasopoulos, Panagiotis; Gatos, Dimitrios; Tselios, Theodore; Apostolopoulos, Vasso; Mouzaki, Athanasia; Matsoukas, John

    2015-08-28

    Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system, and it has been established that autoreactive T helper (Th) cells play a crucial role in its pathogenesis. Myelin basic protein (MBP) epitopes are major autoantigens in MS, and the sequence MBP87-99 is an immunodominant epitope. We have previously reported that MBP87-99 peptides with modifications at principal T-cell receptor (TCR) contact sites suppressed the induction of EAE symptoms in rats and SJL/J mice, diverted the immune response from Th1 to Th2 and generated antibodies that did not cross react with the native MBP protein. In this study, the linear and cyclic analogs of the MBP87-99 epitope, namely linear (Ala91,Ala96)MBP87-99 (P2) and cyclo(87-99)(Ala91,Ala96)MBP87-99 (P3), were evaluated for their binding to HLA-DR4, stability to lysosomal enzymes, their effect on cytokine secretion by peripheral blood mononuclear cells (PBMC) derived from MS patients or healthy subjects (controls), and their effect in rat EAE. P1 peptide (wild-type, MBP87-99) was used as control. P2 and P3 did not alter significantly the cytokine secretion by control PBMC, in contrast to P1 that induced moderate IL-10 production. In MS PBMC, P2 and P3 induced the production of IL-2 and IFN-γ, with a simultaneous decrease of IL-10, whereas P1 caused a reduction of IL-10 secretion only. The cellular response to P3 indicated that cyclization did not affect the critical TCR contact sites in MS PBMC. Interestingly, the cyclic P3 analog was found to be a stronger binder to HLA-DR4 compared to linear P2. Moreover, cyclic P3 was more stable to proteolysis compared to linear P2. Finally, both P2 and P3 suppressed EAE induced by an encephalitogenic guinea pig MBP74-85 epitope in Lewis rats whereas P1 failed to do so. In conclusion, cyclization of myelin altered peptide ligand (Ala91,Ala96)MBP87-99 improved binding affinity to HLA-DR4, resistance to proteolysis and antigen-specific immunomodulation

  1. Large area flexible SERS active substrates using engineered nanostructures

    NASA Astrophysics Data System (ADS)

    Chung, Aram J.; Huh, Yun Suk; Erickson, David

    2011-07-01

    Surface enhanced Raman scattering (SERS) is an analytical sensing method that provides label-free detection, molecularly specific information, and extremely high sensitivity. The Raman enhancement that makes this method attractive is mainly attributed to the local amplification of the incident electromagnetic field that occurs when a surface plasmon mode is excited at a metallic nanostructure. Here, we present a simple, cost effective method for creating flexible, large area SERS-active substrates using a new technique we call shadow mask assisted evaporation (SMAE). The advantage of large, flexible SERS substrates such as these is they have more area for multiplexing and can be incorporated into irregular surfaces such as clothing. We demonstrate the formation of four different types of nanostructure arrays (pillar, nib, ellipsoidal cylinder, and triangular tip) by controlling the evaporation angle, substrate rotation, and deposition rate of metals onto anodized alumina nanoporous membranes as large as 27 mm. In addition, we present experimental results showing how a hybrid structure comprising of gold nanospheres embedded in a silver nano-pillar structure can be used to obtain a 50× SERS enhancement over the raw nanoparticles themselves.Surface enhanced Raman scattering (SERS) is an analytical sensing method that provides label-free detection, molecularly specific information, and extremely high sensitivity. The Raman enhancement that makes this method attractive is mainly attributed to the local amplification of the incident electromagnetic field that occurs when a surface plasmon mode is excited at a metallic nanostructure. Here, we present a simple, cost effective method for creating flexible, large area SERS-active substrates using a new technique we call shadow mask assisted evaporation (SMAE). The advantage of large, flexible SERS substrates such as these is they have more area for multiplexing and can be incorporated into irregular surfaces such as

  2. Normal Raman and SERS spectroscopy of the vitamin E

    NASA Astrophysics Data System (ADS)

    Cai, Tiantian; Gu, Huaimin; Yuan, Xiaojuan; Liu, Fangfang

    2011-01-01

    In this study, surface-enhanced Raman scattering(SERS)spectra of vitamin E were obtained on colloidal silver(Ag). Alpha-(-) tocopherol which is the only form that is recognized to meet human requirements was selected to study. The analytes (±)- -tocopherol were dissolved in chloroform (CHCl3) and the silver colloid was poured into the compound. Silver colloid was reduced by hydroxylamine hydrochloride. The analytes were the supernatant after standing the mixture for the reason that chloroform have no signals in surface-enhanced Raman scattering in the Ag colloid, and it would not affect the determination of the (±)- -tocopherol. The Normal Raman and SERS spectrum of Vitamin E were contrastively studied to realize how the vitamin E stuck to the silver nanoparticles. The results show the fat-soluble substances can be analysed by SERS. The spectra indicate that the molecules are adsorbed on the surface through the COO- groups by the simultaneous involvement of a and -type coordination. These results suggest some important criteria for consideration in SERS measurements and also provide important insights into the problem of predicting SERS activities for different fat-soluble substances.

  3. Plasmonic crystal based solid substrate for biomedical application of SERS

    NASA Astrophysics Data System (ADS)

    Morasso, Carlo F.; Mehn, Dora; Picciolini, Silvia; Vanna, Renzo; Bedoni, Marzia; Gramatica, Furio; Pellacani, Paola; Frangolho, Ana; Marchesini, Gerardo; Valsesia, Andrea

    2014-02-01

    Surface Enhanced Raman Spectroscopy is a powerful analytical technique that combines the excellent chemical specificity of Raman spectroscopy with the good sensitivity provided by the enhancement of the signal observed when a molecule is located on (or very close to) the surface of suitable nanostructured metallic materials. The availability of cheap, reliable and easy to use SERS substrates would pave the road to the development of bioanalytical tests that can be used in clinical practice. SERS, in fact, is expected to provide not only higher sensitivity and specificity, but also the simultaneous and markedly improved detection of several targets at the same time with higher speed compared to the conventional analytical methods. Here, we present the SERS activity of 2-D plasmonic crystals made by polymeric pillars embedded in a gold matrix obtained through the combination of soft-lithography and plasma deposition techniques on a transparent substrates. The use of a transparent support material allowed us to perform SERS detection from support side opening the possibility to use these substrates in combination with microfluidic devices. In order to demonstrate the potentialities for bioanalytical applications, we used our SERS active gold surface to detect the oxidation product of apomorphine, a well-known drug molecule used in Parkinson's disease which has been demonstrated being difficult to study by traditional HPLC based approaches.

  4. Enhancing SERS by Means of Supramolecular Charge Transfer

    NASA Technical Reports Server (NTRS)

    Wong, Eric; Flood, Amar; Morales, Alfredo

    2009-01-01

    In a proposed method of sensing small quantities of molecules of interest, surface enhanced Raman scattering (SERS) spectroscopy would be further enhanced by means of intermolecular or supramolecular charge transfer. There is a very large potential market for sensors based on this method for rapid detection of chemical and biological hazards. In SERS, the Raman signals (vibrational spectra) of target molecules become enhanced by factors of the order of 108 when those molecules are in the vicinities of nanostructured substrate surfaces that have been engineered to have plasmon resonances that enhance local electric fields. SERS, as reported in several prior NASA Tech Briefs articles and elsewhere, has remained a research tool and has not yet been developed into a practical technique for sensing of target molecules: this is because the short range (5 to 20 nm) of the field enhancement necessitates engineering of receptor molecules to attract target molecules to the nanostructured substrate surfaces and to enable reliable identification of the target molecules in the presence of interferants. Intermolecular charge-transfer complexes have been used in fluorescence-, photoluminescence-, and electrochemistry-based techniques for sensing target molecules, but, until now, have not been considered for use in SERS-based sensing. The basic idea of the proposed method is to engineer receptor molecules that would be attached to nanostructured SERS substrates and that would interact with the target molecules to form receptor-target supramolecular charge-transfer complexes wherein the charge transfer could be photoexcited.

  5. Gold nanoparticles paper as a SERS bio-diagnostic platform.

    PubMed

    Ngo, Ying Hui; Then, Whui Lyn; Shen, Wei; Garnier, Gil

    2013-11-01

    Bioactive papers are usually challenged by four major limitations: sensitivity, selectivity, simplicity and strength (4S). Gold nanoparticles (AuNPs) treated paper has previously been demonstrated as a Surface Enhanced Raman Scattering (SERS) active substrate, capable of addressing the 4S issues. In this study, AuNPs on paper substrate were functionalized by a series of biomolecules to develop a generic SERS platform for antibody-antigen detection. The functionalization steps were performed by taking advantage of the high affinity association between Streptomyces avidinii-derived protein, streptavidin, and biotin. Streptavidin was firstly bound onto the AuNPs treated paper using biotinylated-thiol. Subsequently, desired biotinylated-antibody was bound onto the streptavidin. SERS spectra of each functionalization step were obtained to ensure specific adsorption of the bio-molecules. The binding interaction of the antibody with its specific antigen was detected using SERS. Shifts of Raman band associated with α-helix and β-sheet structures indicated structural modification of the antibody upon interaction with its antigen. Predominant tryptophan and tyrosine residue bands were also detected, confirming the presence of antigen. Reproducible spectral features were quantified as AuNP papers were subjected to different concentrations of antigen; the spectra intensity increased as a function of the antigen concentration. The retention of AuNPs on paper remained constant after all the consecutive washing and functionalization steps. The feasibility of AuNPs paper as a low-cost and generic SERS platform for bio-diagnostic applications was demonstrated.

  6. Combined SPR and SERS: Otto and Kretschmann configurations

    NASA Astrophysics Data System (ADS)

    Barchiesi, Dominique; Grosges, Thomas; Colas, Florent; Lamy de la Chapelle, Marc

    2015-11-01

    Combined surface enhanced Raman spectroscopy (SERS) and surface plasmon resonance (SPR) setup was proposed recently. The experimental setup requires a gold layer deposited on a glass substrate. However, due to the poor ability of sticking gold material on glass, a nanometric adhesion layer is used. In this paper we compare numerically both the SERS gain, and the SPR signal, for metallic and dielectric adhesion layers, for two substrates. We show that even if the dielectric materials can be considered as equivalent for the SPR signal, this is not the case for the SERS gain. In particular the dielectric adhesion layer reduces the sensitivity of the SERS gain to this parameter and therefore their use could be more suitable for the fabrication of the sensor. Moreover the higher the refractive index of substrate with regards to the adhesion layer is, the higher efficiency of setup is obtained, and therefore the Otto configuration seems to be more efficient than the Kretschmann one. Optimization of the thicknesses of the adhesion layer and of the gold layer can lead to a SERS gain greater than 103 without nanostructuring.

  7. The Spacecraft Emergency Response System (SERS) for Autonomous Mission Operations

    NASA Technical Reports Server (NTRS)

    Breed, Julia; Chu, Kai-Dee; Baker, Paul; Starr, Cynthia; Fox, Jeffrey; Baitinger, Mick

    1998-01-01

    Today, most mission operations are geared toward lowering cost through unmanned operations. 7-day/24-hour operations are reduced to either 5-day/8-hour operations or become totally autonomous, especially for deep-space missions. Proper and effective notification during a spacecraft emergency could mean success or failure for an entire mission. The Spacecraft Emergency Response System (SERS) is a tool designed for autonomous mission operations. The SERS automatically contacts on-call personnel as needed when crises occur, either on-board the spacecraft or within the automated ground systems. Plus, the SERS provides a group-ware solution to facilitate the work of the person(s) contacted. The SERS is independent of the spacecraft's automated ground system. It receives and catalogues reports for various ground system components in near real-time. Then, based on easily configurable parameters, the SERS determines whom, if anyone, should be alerted. Alerts may be issued via Sky-Tel 2-way pager, Telehony, or e-mail. The alerted personnel can then review and respond to the spacecraft anomalies through the Netscape Internet Web Browser, or directly review and respond from the Sky-Tel 2-way pager.

  8. Application of SERS Nanoparticles for Intracellular pH Measurements

    SciTech Connect

    Laurence, T; Talley, C; Colvin, M; Huser, T

    2004-10-21

    We present an alternative approach to optical probes that will ultimately allow us to measure chemical concentrations in microenvironments within cells and tissues. This approach is based on monitoring the surface-enhanced Raman scattering (SERS) response of functionalized metal nanoparticles (50-100 nm in diameter). SERS allows for the sensitive detection of changes in the state of chemical groups attached to individual nanoparticles and small clusters. Here, we present the development of a nanoscale pH meter. The pH response of these nanoprobes is tested in a cell-free medium, measuring the pH of the solution immediately surrounding the nanoparticles. Heterogeneities in the SERS signal, which can result from the formation of small nanoparticle clusters, are characterized using SERS correlation spectroscopy and single particle/cluster SERS spectroscopy. The response of the nanoscale pH meters is tested under a wide range of conditions to approach the complex environment encountered inside living cells and to optimize probe performance.

  9. Delta-aminolevulinic acid dehydratase activity (ALA-D) in red mullet (Mullus barbatus) from Mediterranean waters as biomarker of lead exposure.

    PubMed

    Fernández, B; Martínez-Gómez, C; Benedicto, J

    2015-05-01

    The enzyme delta-aminolevulinic acid dehydratase (ALA-D) has been investigated as biomarker of lead (Pb) exposure in red mullet (Mullus barbatus) from the Spanish continental shelf. Concentrations of Pb and Zn in muscle and organosomatic indices were also measured to explore causality. Blood ALA-D assay conditions were optimized; the optimum pH for this species has been set to 6.5. Results showed that ALA-D activity ranged from 3.2 to 16.9 nmol PBGmin(-1)mg(-1). No significant differences on ALA-D levels between genders have been detected. ALA-D Baseline level and Background Assessment Criteria (BAC) for this species have been set to 9.1 and 6.6 nmol PBGmin(-1)mg(-1), respectively. There have been detected significant differences on ALA-D activity levels among areas, though the markedly low levels of Pb measured in fish muscle seemed not to be able to produce a relevant suppression on ALA-D. In spite of this, a weak inverse relationship detected between ALA-D and Pb concentrations pointed out the potential of this biomarker in red mullet to reflect Pb bioavailability in marine environment. Nevertheless, subsequent research on ALA-D in marine fish species is recommended to be limited to areas where environmental Pb is effectively accumulated by fish.

  10. Deuterium NMR of Val1. (2-2H)Ala3. gramicidin A in oriented DMPC bilayers

    SciTech Connect

    Hing, A.W.; Adams, S.P.; Silbert, D.F.; Norberg, R.E. )

    1990-05-01

    Deuterium NMR is used to study the selectively labeled Val1...(2-2H)Ala3...gramicidin A molecule to investigate the structure and dynamics of the C alpha-2H bond in the Ala3 residue of gramicidin. Val1...(2-2H)Ala3...gramicidin A is synthesized, purified, and characterized and then incorporated into oriented bilayers of dimyristoylphosphatidylcholine sandwiched between glass coverslips. Phosphorus NMR line shapes obtained from this sample are consistent with the presence of the bilayer phase and indicate that no nonbilayer phases are present in significant amounts. Deuterium NMR line shapes obtained from this sample indicate that the motional axis of the gramicidin Ala3 residue is parallel to the coverslip normal, that the distribution of motional axis orientations has a width of 2 degrees, and that only one major conformational and dynamical state of the Ala3 C alpha-2H bond is observed on the NMR time scale. Furthermore, the Ala3 C alpha-2H bond angle relative to the motional axis is 19-20 degrees if fast axial rotation is assumed to be the only motion present but is less than or equal to 19-20 degrees in the absence of such an assumption. This result indicates that various double-stranded, helical dimer models are very unlikely to represent the structure of gramicidin in the sample studied but that the single-stranded, beta 6.3 helical dimer models are consistent with the experimental data. However, a definitive distinction between the left-handed, single-stranded, beta 6.3 helical dimer model and the right-handed, single-stranded, beta 6.3 helical dimer model cannot be made on the basis of the experimental data obtained in this study.

  11. Successful treatment of recalcitrant dissecting cellulitis of the scalp with ALA-PDT: case report and literature review.

    PubMed

    Liu, Ye; Ma, Ying; Xiang, Lei-Hong

    2013-12-01

    A case of refractory dissecting cellulitis of the scalp (DCS) in a forty-one-year-old Chinese female patient was treated with a total of 6 sessions of topical ALA-PDT at one week intervals. The patient tolerated and responded well to this new approach without any adverse events. This suggested that topical ALA-PDT could be an effective and safe alternative for DCS patients who were refractory to other conventional therapies. We also reviewed etiology, pathophysiology, natural history and treatment options for DCS. PMID:24284093

  12. In situ SERS monitoring of photochemistry within a nanojunction reactor.

    PubMed

    Taylor, Richard W; Coulston, Roger J; Biedermann, Frank; Mahajan, Sumeet; Baumberg, Jeremy J; Scherman, Oren A

    2013-01-01

    We demonstrate a powerful SERS-nanoreactor concept composed of self-assembled gold nanoparticles (AuNP) linked by the sub-nm macrocycle cucurbit[n]uril (CB[n]). The CB[n] functions simultaneously as a nanoscale reaction vessel, sequestering and templating a photoreaction within, and also as a powerful SERS-transducer through the large field enhancements generated within the nanojunctions that CB[n]s define. Through the enhanced Raman fingerprint, the real-time SERS-monitoring of a prototypical stilbene photoreaction is demonstrated. By choosing the appropriate CB[n] nanoreactor, selective photoisomerism or photodimerization is monitored in situ from within the AuNP-CB[n] nanogap.

  13. Rapid ratiometric biomarker detection with topically applied SERS nanoparticles

    PubMed Central

    Wang, Yu “Winston”; Khan, Altaz; Som, Madhura; Wang, Danni; Chen, Ye; Leigh, Steven Y.; Meza, Daphne; McVeigh, Patrick Z.; Wilson, Brian C.; Liu, Jonathan T.C.

    2014-01-01

    Multiplexed surface-enhanced Raman scattering (SERS) nanoparticles (NPs) offer the potential for rapid molecular phenotyping of tissues, thereby enabling accurate disease detection as well as patient stratification to guide personalized therapies or to monitor treatment outcomes. The clinical success of molecular diagnostics based on SERS NPs would be facilitated by the ability to accurately identify tissue biomarkers under time-constrained staining and detection conditions with a portable device. In vitro, ex vivo and in vivo experiments were performed to optimize the technology and protocols for the rapid detection (0.1-s integration time) of multiple cell-surface biomarkers with a miniature fiber-optic spectral-detection probe following a brief (5 min) topical application of SERS NPs on tissues. Furthermore, we demonstrate that the simultaneous detection and ratiometric quantification of targeted and nontargeted NPs allows for an unambiguous assessment of molecular expression that is insensitive to nonspecific variations in NP concentrations. PMID:25045721

  14. SERS-based pesticide detection by using nanofinger sensors

    NASA Astrophysics Data System (ADS)

    Kim, Ansoon; Barcelo, Steven J.; Li, Zhiyong

    2015-01-01

    Simple, sensitive, and rapid detection of trace levels of extensively used and highly toxic pesticides are in urgent demand for public health. Surface-enhanced Raman scattering (SERS)-based sensor was designed to achieve ultrasensitive and simple pesticide sensing. We developed a portable sensor system composed of high performance and reliable gold nanofinger sensor strips and a custom-built portable Raman spectrometer. Compared to the general procedure and previously reported studies that are limited to laboratory settings, our analytical method is simple, sensitive, rapid, and cost-effective. Based on the SERS results, the chemical interaction of two pesticides, chlorpyrifos (CPF) and thiabendazole (TBZ), with gold nanofingers was studied to determine a fingerprint for each pesticide. The portable SERS-sensor system was successfully demonstrated to detect CPF and TBZ pesticides within 15 min with a detection limit of 35 ppt in drinking water and 7 ppb on apple skin, respectively.

  15. Ala(0)-actagardine, a new lantibiotic from cultures of Actinoplanes liguriae ATCC 31048.

    PubMed

    Vértesy, L; Aretz, W; Bonnefoy, A; Ehlers, E; Kurz, M; Markus, A; Schiell, M; Vogel, M; Wink, J; Kogler, H

    1999-08-01

    The actagardine-producing strain Actinoplanes liguriae ATCC 31048, forms an additional lantibiotic when it is cultured on mannitol and soya meal. The new compound, Ala(0)-actagardine (1), has been isolated by solid-phase extraction followed by a two-step chromatographic separation. The molecular formula of 1 is C84H129N21O25S4. Its chemical structure was determined by 2D-NMR analysis and was further confirmed by an amino acid analysis, Edman degradation, and partial synthesis from actagardine. 1 exhibits a slightly higher biological activity than the parent compound actagardine. The synthetic analogs Lys(0)-actagardine (2) and Ile(0)-actagardine (3) demonstrate also antibacterial activities and emphasize the importance of the N-terminus for further derivatization. PMID:10580386

  16. Optimal control of AlAs oxidation via digital alloy heterostructure compositions

    NASA Astrophysics Data System (ADS)

    Suárez, I.; Almuneau, G.; Condé, M.; Arnoult, A.; Fontaine, C.

    2009-09-01

    A thorough study of wet thermal oxidation in AlAs/AlxGa1-xAs superlattices is presented. The results shown here demonstrate that the final oxidation depth can be finely tuned via the composition and thickness of AlxGa1-xAs into the digital alloy. A complete model of oxidation in these structures is proposed, relying on diffusion through the AlAs layer, its oxidation and an additional effect due to the AlxGa1-xAs intermediate barriers. This barrier contribution is shown to further improve the control of the oxidation rate, and thereby fabrication of sophisticated AlOx/GaAs integrated optoelectronic devices.

  17. Familial amyloid polyneuropathy (TTR ala 60) in north west Ireland: a clinical, genetic, and epidemiological study.

    PubMed Central

    Reilly, M M; Staunton, H; Harding, A E

    1995-01-01

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