The unique hydrogen bonded water in the reduced form of<Emphasis Type="Italic"> Clostridium pasteurianum</Emphasis> rubredoxin and its possible role in electron transfer

Rubredoxin is a small iron-sulfur (FeS₄) protein involved in oxidation–reduction reactions. The side chain of Leu41 near the iron-sulfur center has two conformations, which we suggested previously serve as a gate for a water molecule during the electron transfer process. To establish the role of residue 41 in electron transfer, an [L41A] mutant of Clostridium pasteurianum rubredoxin was constructed and crystallized in both oxidation states. Despite the lack of the gating side chain in this protein, the structure of the reduced [L41A] rubredoxin reveals a specific water molecule in the same position as observed in the reduced wild-type rubredoxin. In contrast, both the wild-type and [L41A] rubredoxins in the oxidized state do not have water molecules in this location. The reduction potential of the [L41A] variant was ~50 mV more positive than wild-type. Based on these observations, it is proposed that the site around the S of Cys9 serves as a port for an electron acceptor. Lastly, the Fe–S distances of the reduced rubredoxin are expanded, while the hydrogen bonds between S of the cysteines and the backbone amide nitrogens are shortened compared to its oxidized counterpart. This small structural perturbation in the Fe(II)/Fe(III) transition is closely related to the small energy difference which is important in an effective electron transfer agent.     

Purification and characterization of heparin lyase I from Bacteroides stercoris HJ-15

Heparin lyase I was purified to homogeneity from Bacteroides stercoris HJ-15 isolated from human intestine, by a combination of DEAE-Sepharose, gel-filtration, hydroxyapatite, and CM-Sephadex C-50 column chromatography. This enzyme preferred heparin to heparan sulfate, but was inactive at cleaving acharan sulfate. The apparent molecular mass of heparin lyase I was estimated as 48,000 daltons by SDS-PAGE and its isoelectric point was determined as 9.0 by IEF. The purified enzyme required 500 mM NaCl in the reaction mixture for maximal activity and the optimal activity was obtained at pH 7.0 and 50 degrees C. It was rather stable within the range of 25 to 50 degrees C but lost activity rapidly above 50 degrees C. The enzyme was activated by Co(2+) or EDTA and stabilized by dithiothreitol. The kinetic constants, K(m) and V(max) for heparin were 1.3 10(-5) M and 8.8 micromol/min.mg. The purified heparin lyase I was an eliminase that acted best on porcine intestinal heparin, and to a lesser extent on porcine intestinal mucosa heparan sulfate. It was inactive in the cleavage of N-desulfated heparin and acharan sulfate. In conclusion, heparin lyase I from Bacteroides stercoris was specific to heparin rather than heparan sulfate and its biochemical properties showed a substrate specificity similar to that of Flavobacterial heparin lyase I.     

Allergen-induced proteolytic cleavage of annexin-1 and activation of cytosolic phospholipase A2 in the lungs of a mouse model of asthma

To identify proteins that might play an important role in allergen-induced asthma, we analyzed lung extracts prepared from allergen (ovalbumin)-challenged animals in a mouse model of this condition. The combination of two-dimensional gel electrophoresis and mass spectrometry revealed that annexin-1, a 37 kDa anti-inflammatory protein that inhibits the activity of cytosolic phospholipase A(2) (cPLA(2)), was down-regulated by allergen challenge in the lungs of ovalbumin-sensitized mice. Immunoblot analysis showed that this effect of ovalbumin challenge was attributable to proteolytic cleavage of annexin-1. The ovalbumin-induced degradation of annexin-1 was blocked by pretreatment of mice with the antioxidant N-acetylcysteine (NAC) or with sodium selenite, both of which have previously been shown to exert anti-inflammatory effects in this asthma model. Ovalbumin challenge also both increased the expression of cPLA(2) in lung tissue and reduced the extent of the interaction between cPLA(2) and annexin-1, and these effects were inhibited by NAC or selenite. Moreover, the concentrations of cysteinyl leukotrienes in bronchoalveolar lavage fluid and of leukotriene B(4) in lung tissue were increased by ovalbumin challenge in a NAC- or selenite-sensitive manner. Together, these results suggest that allergen-induced oxidative stress results in proteolysis of annexin-1 and consequent up-regulation of cPLA(2) activity and leukotriene production in this mouse model of asthma, and that the anti-inflammatory effects of selenite may provide a basis for the development of new antiasthmatic drugs.     

Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A

Protein phosphatase 2A (PP2A) holoenzymes consist of a catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits that recruit the AC dimer to substrates. PP2A is required for chromosome segregation, but PP2A's substrates in this process remain unknown. To identify PP2A substrates, we carried out a two-hybrid screen with the regulatory B/PR55 subunit. We isolated a human homolog of C. elegans HCP6, a protein distantly related to the condensin subunit hCAP-D2, and we named this homolog hHCP-6. Both C. elegans HCP-6 and condensin are required for chromosome organization and segregation. HCP-6 binding partners are unknown, whereas condensin is composed of the structural maintenance of chromosomes proteins SMC2 and SMC4 and of three non-SMC subunits. Here we show that hHCP-6 becomes phosphorylated during mitosis and that its dephosphorylation by PP2A in vitro depends on B/PR55, suggesting that hHCP-6 is a B/PR55-specific substrate of PP2A. Unlike condensin, hHCP-6 is localized in the nucleus in interphase, but similar to condensin, hHCP-6 associates with chromosomes during mitosis. hHCP-6 is part of a complex that contains SMC2, SMC4, kleisin-beta, and the previously uncharacterized HEAT repeat protein FLJ20311. hHCP-6 is therefore part of a condensin-related complex that associates with chromosomes in mitosis and may be regulated by PP2A.     

Autoantibody to DNA binding protein B as a novel serologic marker in systemic sclerosis

Systemic sclerosis is a systemic disease that is characterized by tissue fibrosis, small-vessel vasculopathy, and an autoimmune response associated with autoantibodies. We performed serological analysis of cDNA expression library (SEREX) to identify autoantibodies associated with systemic sclerosis. We identified 4 clones that react with sera of patients with SSc but not with those of healthy donors. These clones are phosphoglycerate mutase, centromere autoantigen C, U1 small nuclear ribonucleoprotein, and DNA binding protein B (dbpB). We chose to study autoantibody to DNA binding protein B. Immunoreactivity against recombinant dbpB was detected in 40.5% (15/37) of patients with SSc, 14.6% (6/41) of patents with systemic lupus erythematosus, 6.7% (1/15) of patients with rheumatoid arthritis, 0% (0/12) of patients with Sjogren syndrome, and 5.9% (1/17) of patients with polymyositis/dermatomyositis. The frequency of anti-dbpB was significantly higher in the SSc patients (15/37, 40.5%) compared to the healthy controls (3/41, 7.3%, p=0.0005 by chi(2) test). Eleven patients (11/20, 55%) with the diffuse cutaneous type of SSc had anti-dbpB and 4 patients (4/17, 23.5%) with the limited cutaneous type had anti-dbpB. The presence of anti-dbpB was significantly associated with the diffuse cutaneous type (p=0.00003 by chi(2) test). This is the first report to suggest that autoantibody to dbpB can be used as a serologic marker of systemic sclerosis.     

Unrestrained Spindle Elongation during Recovery from Spindle Checkpoint Activation in cdc15-2 Cells Results in Mis-Segregation of Chromosomes

During normal metaphase in Saccharomyces cerevisiae, chromosomes are captured at the kinetochores by microtubules emanating from the spindle pole bodies at opposite poles of the dividing cell. The balance of forces between the cohesins holding the replicated chromosomes together and the pulling force from the microtubules at the kinetochores result in the biorientation of the sister chromatids before chromosome segregation. The absence of kinetochore–microtubule interactions or loss of cohesion between the sister chromatids triggers the spindle checkpoint which arrests cells in metaphase. We report here that an MEN mutant, cdc15-2, though competent in activating the spindle assembly checkpoint when exposed to Noc, mis-segregated chromosomes during recovery from spindle checkpoint activation. cdc15-2 cells arrested in Noc, although their Pds1p levels did not accumulate as well as in wild-type cells. Genetic analysis indicated that Pds1p levels are lower in a mad2Δ cdc15-2 and bub2Δ cdc15-2 double mutants compared with the single mutants. Chromosome mis-segregation in the mutant was due to premature spindle elongation in the presence of unattached chromosomes, likely through loss of proper control on spindle midzone protein Slk19p and kinesin protein, Cin8p. Our data indicate that a slower rate of transition through the cell division cycle can result in an inadequate level of Pds1p accumulation that can compromise recovery from spindle assembly checkpoint activation.     

Fishing gears involved in entanglements of minke whales (Balaenoptera acutorostrata) in the East Sea of Korea

Entanglement of marine mammals in fishing gear is a global issue. It is considered a significant threat to minke whales (Balaenoptera acutorostrata) in the East Sea of Korea. A total of 214 entanglements of minke whales in this area between 2004 and 2007 were used to investigate types and parts of fishing gears involved in entanglements. The majority of entanglements were mainly caused by three types of fishing gears: set nets, pots, and gill nets (n= 207, 96.7%). Other entanglements were associated with bottom trawls, purse seines, and trawls. A total of 65 entanglements were attributed to the main and branch lines of fishing gears. The most common body part of minke whales which attached to fishing gears was the mouth (n= 63, 30.4%). Most entanglements took place within 10 nmi from land (n= 179, 86.5%), and between 10 and 220 m of water depth. The mean length of entangled minke whales in set nets was significantly smaller than that of whales in pots and gill nets samples (P < 0.001). Also, the mean body length of minke whales that entangled in the coastal area and shallow waters was significantly shorter than that of whales in the offshore area and deep waters (P < 0.001). This information can be used as fundamental data to conserve and manage this population of minke whales in the East Sea of Korea, and also to modify fishing gear to reduce entanglements. Future studies should focus on investigating the impact of these entanglements on the population and the effectiveness of mitigation measures to reduce entanglements of minke whales in this area.