Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in Its Dissociation from Chromatin and the Nuclear Matrix

Protein-tyrosine phosphorylation regulates a wide variety of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine kinases induce global nuclear structure changes, which we called chromatin structural changes. However, the mechanisms are not fully understood. In this study we identify protein kinase A anchoring protein 8 (AKAP8/AKAP95), which associates with chromatin and the nuclear matrix, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of AKAP8 is induced by several tyrosine kinases, such as Src, Fyn, and c-Abl but not Syk. Nucleus-targeted Lyn and c-Src strongly dissociate AKAP8 from chromatin and the nuclear matrix in a kinase activity-dependent manner. The levels of tyrosine phosphorylation of AKAP8 are decreased by substitution of multiple tyrosine residues on AKAP8 into phenylalanine. Importantly, the phenylalanine mutations of AKAP8 inhibit its dissociation from nuclear structures, suggesting that the association/dissociation of AKAP8 with/from nuclear structures is regulated by its tyrosine phosphorylation. Furthermore, the phenylalanine mutations of AKAP8 suppress the levels of nuclear tyrosine kinase-induced chromatin structural changes. In contrast, AKAP8 knockdown increases the levels of chromatin structural changes. Intriguingly, stimulation with hydrogen peroxide induces chromatin structural changes accompanied by the dissociation of AKAP8 from nuclear structures. These results suggest that AKAP8 is involved in the regulation of chromatin structural changes through nuclear tyrosine phosphorylation.     

Arthrobacter pokkalii sp nov,a Novel Plant Associated Actinobacterium with Plant Beneficial Properties,Isolated from Saline Tolerant Pokkali Rice,Kerala, India

A novel yellow colony-forming bacterium, strain P3B162^T was isolated from the pokkali rice rhizosphere from Kerala, India, as part of a project study aimed at isolating plant growth beneficial rhizobacteria from saline tolerant pokkali rice and functionally evaluate their abilities to promote plant growth under saline conditions. The novel strain P3B162^T possesses plant growth beneficial traits such as positive growth on 1-aminocyclopropane-1-carboxylic acid (ACC), production of indole acetic acid (IAA) and siderophore. In addition, it also showed important phenotypic characters such as ability to form biofilm and utilization of various components of plant root exudates (sugars, amino acids and organic acids), clearly indicating its lifestyle as a plant rhizosphere associated bacterium. Taxonomically, the novel strain P3B162^T was affiliated to the genus Arthrobacter based on the collective results of phenotypic, genotypic and chemotaxonomic analyses. Moreover, molecular analysis using 16S rRNA gene showed Arthrobacter globiformis NBRC 12137^T, Arthrobacter pascens DSM 20545^T and Arthrobacter liuii DSXY973^T as the closely related phylogenetic neighbours, showing more than 98% 16S rRNA similarity values, whereas the recA gene analysis displayed Arthrobacter liuii JCM 19864^T as the nearest neighbour with 94.7% sequence similarity and only 91.7% to Arthrobacter globiformis LMG 3813^T and 88.7% to Arthrobacter pascens LMG 16255^T. However, the DNA-DNA hybridization values between strain P3B162^T, Arthrobacter globiformis LMG 3813^T, Arthrobacter pascens LMG 16255^T and Arthrobacter liuii JCM 19864^T was below 50%. In addition, the novel strain P3B162^T can be distinguished from its closely related type strains by several phenotypic characters such as colony pigment, tolerance to NaCl, motility, reduction of nitrate, hydrolysis of DNA, acid from sucrose, cell wall sugars and cell wall peptidoglycan structure. In conclusion, the combined results of this study support the classification of strain P3B162^T as a novel Arthrobacter species and we propose Arthrobacter pokkalii sp.nov.as its name. The type strain is P3B162^T (= KCTC 29498^T = MTCC 12358^T).     

6-formylpterin, a xanthine oxidase inhibitor, intracellularly generates reactive oxygen species involved in apoptosis and cell proliferation

The chemical property of 6-formylpterin and its biological functions were examined. Polarographic studies revealed that 6-formylpterin reacted with NAD(P)H and consumed oxygen. In contrast, other conjugated pterins, such as biopterin and neopterin, showed no consumption of oxygen. The production analysis using high-performance liquid chromatography documented that 6-formylpterin catalyzes the conversion from NADH to NAD. Electroparamagnetic resonance spin trapping experiments demonstrated that this reaction is accompanied with the generation of reactive oxygen species (ROS), superoxide anion and hydrogen peroxide. When 6-formylpterin was administered to HL-60 cells, intracellular ROS generation was observed and apoptosis was induced. In contrast, other conjugated pterins induced neither intracellular ROS generation nor apoptosis in HL-60 cells. The intracellular ROS generation by 6-formylpterin was observed in other cells, such as PanC-1 cells and Jurkat cells. 6-formylpterin suppressed cell proliferation in PanC-1 cells and inhibited Fas-mediated apoptosis in Jurkat cells. These findings indicate that, among conjugated pterins, 6-formylpterin has the unique property to transfer electron from NAD(P)H to oxygen and that the property brings about intracellular ROS generation, which exerts various biological functions such as induction of apoptosis, suppression of cell proliferation, and inhibition of Fas-mediated apoptosis.     

Two kinds of chlorocatechol 1,2-dioxygenase from 2,4-dichlorophenoxyacetate-degrading Sphingomonas sp. strain TFD44

Two kinds of chlorocatechol 1,2-dioxygenase (CCD), TfdC and TfdC2 were detected in Sphingomonas sp. strain TFD44. These two CCDs could be simultaneously synthesized in TFD44 during its growth with 2,4-D as the sole carbon and energy sources. The apparent subunit molecular masses of TfdC and TfdC2 estimated by SDS-PAGE analysis were 33.8 and 33.1 kDa, respectively. The genes encoding the two CCDs were cloned and expressed in Escherichia coli. The two purified CCDs showed broad substrate specificities but had different specificity patterns. TfdC showed the highest specificity constant for 3-chlorocatechol and TfdC2 showed the highest specificity constant for 3,5-dichlorocatechol. The substrate specificity difference seemed to correlate with the alternation of amino acid supposed to be involved in the interaction with substrates. Whereas phylogenetic analysis indicated that the CCDs of Sphingomonas constitute a distinctive group among Gram-negative bacteria, TfdC and TfdC2 of TFD44 have divergently evolved in terms of their substrate specificity.     

Immunocytochemical studies on the pituitary pars distalis of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus

Summary Immunocytochemical studies were performed to describe the characteristics of cell types and their distribution in the pars distalis of Japanese long-fingered bat, Miniopterus schreibersii fuliginosus, collected at various stages of the reproductive cycle. Six distinct cell types have been identified in the pars distalis by the unlabeled immunoperoxidase technique and by the ABC method. Growth hormone (GH) and prolactin (PRL) cells were immunostained with antisera against chicken GH and ovine PRL. The GH-immunoreactive cells were round or oval orangeophilic cells distributed throughout the pars distalis with prominent aggregation in the posterolateral region. The PRL cells were pleomorphic carminophilic cells that occurred in small groups within the central and dorsocaudal regions of the pars distalis. They were sparsely distributed in the central region of the pars distalis in the hibernating bats, but increased significantly in the pregnant and lactating bats. The adrenocorticotropic (ACTH) cells were large round or polygonal amphophilic cells in the rostroventral and ventrolateral regions of the pars distalis. The thyrotropic (TSH) cells were small rounded or polygonal and distributed mainly in the ventrolateral region of the pars distalis. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) cells were identified immunocytochemically with antisera against the specific beta subunits of ovine LH and rat FSH. There were two populations of LH and FSH cells, one aggregated in the zona tuberalis and the other scattered singly throughout the rest of the pars distalis. The aggregated cells were immunoreactive with both antisera directed to LH and FSH, while scattered cells were reactive solely with antiserum to either LH or FSH and exhibited seasonal variations. In females, the proportional volume of the pars distalis occupied by LH cells was significantly reduced during pregnancy and lactation. No evidence of involution was observed in pars distalis cells except for PRL cells in males or females during hibernation.     

A hypotetraploid human T lymphoid cell line established by cell fusion.

A human T lymphoid cell line was established by cell hybridization technique from peripheral blood leucocytes of a patient with Sezary syndrome. The cells beared the surface antigens of human T lymphocyte specificity as demonstrated by immune cytolysis tests, but did not form E rosettes with sheep red blood cells. Isozyme patterns of enzymes in this line such as lactate dehydrogenase, glucose 6-phosphate dehydrogenase and esterase were of human type. The line had 79 chromosomes in modal number. This case supports the proposal that the production of tetraploids is favourable for establishment of cell lines.     

Affinity chromatography of trypsin and related enzymes. V. Basic studies of quantitative affinity chromatography.

A detailed study of the quantitative affinity chromatography of trypsin [EC 3.4.21.4] is reported here. Frontal chromatography using an enzyme solution of very low concentration on an affinity adsorbent gave the dissociation constant of the enzyme-immobilized ligand complex (Kd). Kd values determined under various conditions enabled us to discuss in detail the interaction of trypsin and affinity adsorbents (mainly Gly-Gly-Arg Sepharose). The pH dependence of Kd was consistent with that of the interaction of trypsin and product-type compounds. The effects of changes in temperature, ionic strength, dielectric constant, etc., were also studied. The Ki values of soluble competitive inhibitors can be determined by analysis of their effects on the elution volume of the enzyme. The values obtained were in good agreement with those obtained by kinetic analysis. The present method proved to be useful as a general procedure to investigate the interaction of a protein and a specific ligand.     

Sequential action of ATP-dependent subunit conformational change and interaction between helical protrusions in the closure of the built-in lid of group II chaperonins

ATP drives the conformational change of the group II chaperonin from the open lid substrate-binding conformation to the closed lid conformation to encapsulate an unfolded protein in the central cavity. The detailed mechanism of this conformational change remains unknown. To elucidate the intra-ring cooperative action of subunits for the conformational change, we constructed Thermococcus chaperonin complexes containing mutant subunits in an ordered manner and examined their folding and conformational change abilities. Chaperonin complexes containing wild-type subunits and mutant subunits with impaired ATP-dependent conformational change ability or ATP hydrolysis activity, one by one, exhibited high protein refolding ability. The effects of the mutant subunits correlate with the number and order in the ring. In contrast, the use of a mutant lacking helical protrusion severely affected the function. Interestingly, these mutant chaperonin complexes also exhibited ATP-dependent conformational changes as demonstrated by small angle x-ray scattering, protease digestion, and changes in fluorescence of the fluorophore attached to the tip of the helical protrusion. However, their conformational change is likely to be transient. They captured denatured proteins even in the presence of ATP, whereas addition of ATP impaired the ability of the wild-type chaperonin to protect citrate synthase from thermal aggregation. These results suggest that ATP binding/hydrolysis causes the independent conformational change of the subunit, and further conformational change for the complete closure of the lid is induced and stabilized by the interaction between helical protrusions.     

Random replication and random assortment model for plasmid incompatibility in bacteria

To understand the incompatibility between two related plasmids, both of which replicate in an autonomous state under a common control mechanism, we have developed a model that assumes a random choice mechanism for replication of plasmid copies and their random assortment into daughter cells upon cell division. Segregation kinetics by this model is analyzed mathematically and the number of generations required for segregation is calculated as a function of plasmid copy number per cell. The results obtained offer enough quantitative data to make our model reasonably realistic.