Toxic interaction between acid yellow 23 and trypsin: Spectroscopic methods coupled with molecular docking

Acid yellow 23 (AY23) is a pervasive azo dye used in many fields which is potentially harmful to the environment and human health. This paper studied the toxic effects of AY23 on trypsin by spectroscopic and molecular docking methods. The addition of AY23 effectively quenched the intrinsic fluorescence of trypsin via static quenching with association constants of K₂₉₀,K = 3.67 × 10⁵ L mol^?1 and K₃₁₀,K = 1.83 × 10⁵ L mol^?1. The calculated thermodynamic parameters conformed that AY23 binds to trypsin predominantly via electrostatic forces with one binding site. Conformational investigations indicated the skeletal structure of trypsin unfolded and the microenvironment of tryptophan changed with the addition of AY23. Molecular docking study showed that AY23 interacted with the His 57 and Lys 224 residue of trypsin and led to the inhibition of enzyme activity. This study offers a more comprehensive picture of AY23–trypsin interaction and indicates their interaction may perform toxic effects within the organism. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:360–367, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21430     

腺病毒介导的nm23-H1对人结直肠癌裸鼠移植作用的实验研究

目的 探讨Ad-GFP-nm23-Hl对人高转移结直肠癌移植瘤的抑制作用,为临床肿瘤基因治疗提供一定的理论依据.方法 将15只人结直肠癌移植瘤裸鼠随机分为高剂量组(1010 PFU/ml Ad-GFP-nm23-Hl)、低剂量组(109 PFU/ml Ad-GFP-nm23-Hl)、阴性对照组(PBS).均采用瘤体注射给药,连续给药4 d,每2 d测一次肿瘤体积.观察10 d后取瘤块称重和组织病理学检查.结果 Ad-GFP-nm23-Hl高剂量组移植瘤体积较其他各组明显缩小(P<0.05),瘤体积抑制率和瘤重抑制率分别为76%和66%. 结论 Ad-GFP-nm23-Hl对人高转移结直肠癌移植瘤有抑制作用,并存在剂量依赖性.     

In Vivo Function of Hsp90 Is Dependent on ATP Binding and ATP Hydrolysis

Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH₂-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH₂-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysis–dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone.     

Nucleolar Disruption Ensures Nuclear Accumulation of p21 upon DNA Damage

p21^cip1 is a protein with a dual function in oncogenesis depending mainly on its intracellular localization: tumor suppressor in the nucleus and oncogenic in the cytoplasm. After DNA damage, p21^cip1 increases and accumulates in the nucleus to ensure cell cycle arrest. We show here that the nuclear accumulation of p21^cip1 is not only a consequence of its increased levels but to a DNA damage cellular response, which is ataxia telangiectasia and Rad3 related (ATR)/ataxia telangiectasia mutated (ATM) and p53 independent. Furthermore, after DNA damage, p21^cip1 not only accumulates in the nucleoplasm but also in the disrupted nucleolus. Inside the nucleolus, it is found in spherical structures, which are not a protrusion of the nucleoplasm. The steady‐state distribution of p21^cip1 in the nucleolus resulted from a highly dynamic equilibrium between nucleoplasmic and nucleolar p21^cip1 and correlated with the inhibition of p21^cip1 nuclear export. Most interestingly, inhibition of ribosomal export after expressing a dominant‐negative mutant of nucleophosmin induced p21^cip1 accumulation in the nucleus and the nucleolus in the absence of DNA damage. This proved the existence of a nucleolar export route to the cytoplasm for p21^cip1 in control conditions that would be inhibited upon DNA damage leading to nuclear and nucleolar accumulation of p21^cip1.     

Purification and characterization of OXA-23 from Acinetobacter baumannii

Although the existence of bla_OXA-23 is reported in various parts of the world, the product of bla_OXA-23 gene, OXA-23, has not been purified and its kinetic properties are not known. In this study, OXA-23 of Acinetobacter baumannii isolated from Kocaeli University intensive care unit was characterized after purification using recombinant methods. Preliminary results showed that conventional protein purification methods were not effective for purification of OXA-23. Therefore, OXA-23 was fused to maltose-binding protein of Escherichia coli, the fused protein was expressed and purified to homogeneity. Kinetic properties of the pure protein were then studied with substrates e.g., imipenem, meropenem, cefepime, ceftazidime, ampicilline, piperacillin, penicillin G, and nitrocefin. Also clavulanic acid, tazobactam, and sulbactam concentrations that inhibit 50% of OXA-23 enzyme activity were calculated. Modelling of OXA-23 revealed its ionic surface structure, conformation in the fused form and its topology allowing us to make predictions for OXA-23 substrate specificity.     

Antigenic variation in mucilage secreted by members of the genus Symbiodinium (Dinophyceae)

Symbiodinium reside intracellularly in a complex symbiosome (host and symbiont‐derived) within cnidarian hosts in a specific host‐symbiont association. Symbiodinium is a diverse genus with variation greater than other dinoflagellate orders. In this paper, our investigation into specificity examines antigenic variation in the algal mucilage secretions at the host‐symbiont interface. Cultured Symbiodinium from a variety of clades were labeled with one of two antibodies to symbiont mucilage (PC3, developed using a clade B alga cultured from Aiptasia pallida; BF10, developed using a clade F alga cultured from Briareum sp.). The labeling was visualized with a fluorescent marker and examined with epifluorescence and confocal microscopy. PC3 antigen was found in cultured Symbiodinium from clades A and B, but not clades C, D, E and F. The correlation between labeling and clade may account for some of the specificity between host and symbiont in the field. Within clades A and B there was variation in the amount of label present. BF10 antigen was more specific and only found in cultures of the same cp23S‐rDNA strain the antibody was created against. These results indicate that the mucilage secretions do vary both qualitatively and quantitatively amongst Symbiodinium strains. Since the mucilage forms the host‐symbiont interface, variation in its molecular composition is likely to be the source of any signals involved in recognition and specificity.     

Phylogenetic study of Geitlerinema and Microcystis (Cyanobacteria) using PC‐IGS and 16S–23S ITS as markers: investigation of horizontal gene transfer

Selection of genes that have not been horizontally transferred for prokaryote phylogenetic inferences is regarded as a challenging task. The markers internal transcribed spacer of ribosomal genes (16S–23S ITS) and phycocyanin intergenic spacer (PC‐IGS), based on the operons of ribosomal and phycocyanin genes respectively, are among the most used markers in cyanobacteria. The region of the ribosomal genes has been considered stable, whereas the phycocyanin operon may have undergone horizontal transfer. To investigate the occurrence of horizontal transfer of PC‐IGS, phylogenetic trees of Geitlerinema and Microcystis strains were generated using PC‐IGS and 16S–23S ITS and compared. Phylogenetic trees based on the two markers were mostly congruent for Geitlerinema and Microcystis, indicating a common evolutionary history among ribosomal and phycocyanin genes with no evidence for horizontal transfer of PC‐IGS. Thus, PC‐IGS is a suitable marker, along with 16S–23S ITS for phylogenetic studies of cyanobacteria.     

Starch-related cytosolic heteroglycans in roots from Arabidopsis thaliana

Both photoautotrophic and heterotrophic plant cells are capable of accumulating starch inside the plastid. However, depending on the metabolic state of the respective cell the starch-related carbon fluxes are different. The vast majority of the transitory starch biosynthesis relies on the hexose phosphate pools derived from the reductive pentose phosphate cycle and, therefore, is restricted to ongoing photosynthesis. Transitory starch is usually degraded in the subsequent dark period and mainly results in the formation of neutral sugars, such as glucose and maltose, that both are exported into the cytosol. The cytosolic metabolism of the two carbohydrates includes reversible glucosyl transfer reactions to a heteroglycan that are mediated by two glucosyl transferases, DPE2 and PHS2 (or, in all other species, Pho2).In heterotrophic cells, accumulation of starch mostly depends on the long distance transport of reduced carbon compounds from source to sink organs and, therefore, includes as an essential step the import of carbohydrates from the cytosol into the starch forming plastids.In this communication, we focus on starch metabolism in heterotrophic tissues from Arabidopsis thaliana wild type plants (and in various starch-related mutants as well). By using hydroponically grown A. thaliana plants, we were able to analyse starch-related biochemical processes in leaves and roots from the same plants. Within the roots we determined starch levels and the morphology of native starch granules. Cytosolic and apoplastic heteroglycans were analysed in roots and compared with those from leaves of the same plants. A. thaliana mutants lacking functional enzymes either inside the plastid (such as phosphoglucomutase) or in the cytosol (disproportionating isoenzyme 2 or the phosphorylase isozyme, PHS2) were included in this study. In roots and leaves from the three mutants (and from the respective wild type organ as well), starch and heteroglycans as well as enzyme patterns were analysed.     

TIM23-mediated insertion of transmembrane α-helices into the mitochondrial inner membrane

While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) α-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems.