Chromosome aberrations in workers of nuclear-power plants

The frequencies of chromosome aberrations in 135 workers from nuclear-power plants were compared with those in 135 age-matched controls. A total of 135,000 cells was scored. The frequencies of dicentric chromosome were 1.67 × 10⁻³ in the exposed group and 0.49 × 10⁻³ in the control group and those of chromosome-type deletion were 3.33 × 10⁻³ and 1.10 × 10⁻³, respectively. The frequencies of all types of chromosome aberrations in the exposed subjects were higher than those in the control group, but no significant trend of dose-dependent increase was observed when only the exposed group were considered. Poisson regression analysis, with both exposed and control included, showed that there was a significant association of chromosome aberration with radiation dose and the duration of work, but not with age, smoking habit and alcohol intake. It was also found that recent exposure to radiation, within the last 5 years, had contributed more to the observed chromosome aberration than earlier exposure.     

Direct involvement of the small GTPase Rac in activation of the superoxide-producing NADPH oxidase Nox1

Activation of the non-phagocytic superoxide-producing NADPH oxidase Nox1, complexed with p22(phox) at the membrane, requires its regulatory soluble proteins Noxo1 and Noxa1. However, the role of the small GTPase Rac remained to be clarified. Here we show that Rac directly participates in Nox1 activation via interacting with Noxa1. Electropermeabilized HeLa cells, ectopically expressing Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner, which is abrogated by expression of a mutant Noxa1(R103E), defective in Rac binding. Superoxide production in Nox1-expressing HeLa and Caco-2 cells is decreased by depletion or sequestration of Rac; on the other hand, it is enhanced by expression of the constitutively active Rac1(Q61L), but not by that of a mutant Rac1 with the A27K substitution, deficient in binding to Noxa1. We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22(phox): the Noxa1-Noxo1 and Noxo1-p22(phox) interactions are both essential for Nox1 activity. Rac likely facilitates the membrane localization of Noxa1: although Noxa1(W436R), defective in Noxo1 binding, neither associates with the membrane nor activates Nox1, the effects of the W436R substitution are restored by expression of Rac1(Q61L). The Rac-Noxa1 interaction also serves at a step different from the Noxa1 localization, because the binding-defective Noxa1(R103E), albeit targeted to the membrane, does not support superoxide production by Nox1. Furthermore, a mutant Noxa1 carrying the substitution of Ala for Val-205 in the activation domain, which is expected to undergo a conformational change upon Rac binding, fully localizes to the membrane but fails to activate Nox1.     

Tonsil-derived mesenchymal stem cells alleviate concanavalin A-induced acute liver injury

Acute liver failure, the fatal deterioration of liver function, is the most common indication for emergency liver transplantation, and drug-induced liver injury and viral hepatitis are frequent in young adults. Stem cell therapy has come into the limelight as a potential therapeutic approach for various diseases, including liver failure and cirrhosis. In this study, we investigated therapeutic effects of tonsil-derived mesenchymal stem cells (T-MSCs) in concanavalin A (ConA)- and acetaminophen-induced acute liver injury. ConA-induced hepatitis resembles viral and immune-mediated hepatic injury, and acetaminophen overdose is the most frequent cause of acute liver failure in the United States and Europe. Intravenous administration of T-MSCs significantly reduced ConA-induced hepatic toxicity, but not acetaminophen-induced liver injury, affirming the immunoregulatory capacity of T-MSCs. T-MSCs were successfully recruited to damaged liver and suppressed inflammatory cytokine secretion. T-MSCs expressed high levels of galectin-1 and -3, and galectin-1 knockdown which partially diminished interleukin-2 and tumor necrosis factor α secretion from cultured T-cells. Galectin-1 knockdown in T-MSCs also reversed the protective effect of T-MSCs on ConA-induced hepatitis. These results suggest that galectin-1 plays an important role in immunoregulation of T-MSCs, which contributes to their protective effect in immune-mediated hepatitis. Further, suppression of T-cell activation by frozen and thawed T-MSCs implies great potential of T-MSC banking for clinical utilization in immune-mediated disease.     

Structural alteration of Escherichia coli Hsp31 by thermal unfolding increases chaperone activity

Escherichia coli Hsp31, encoded by hchA, is a heat-inducible molecular chaperone. We found that Hsp31 undergoes a conformational change via temperature-induced unfolding, generating a high molecular weight (HMW) form with enhanced chaperone activity. Although it has previously been reported that some subunits of the Hsp31 crystal structure show structural heterogeneity with increased hydrophobic surfaces, Hsp31 basically forms a dimer. We found that a C-terminal deletion (CΔ19) of Hsp31 exhibited structurally and functionally similar characteristics to that of the HMW form. Both the CΔ19 and HMW forms achieved a structure with considerably more β-sheets and less α-helices than the native dimeric form, exposing a portion of its hydrophobic surfaces. The structural alterations were determined from its spectral changes in circular dichroism, intrinsic fluorescence of tryptophan residues, and fluorescence of bis-ANS binding to a hydrophobic surface. Interestingly, during thermal transition, the dimeric Hsp31 undergoes a conformational change to the HMW species via the CΔ19 structure, as monitored with near-UV CD spectrum, implying that the CΔ19 resembles an intermediate state between the dimer and the HMW form. From these results, we propose that Hsp31 transforms itself into a fully functional chaperone by altering its tertiary and quaternary structures.     

The synthesis,characterization, and preliminary biological evaluation of 1-β-D-arabinofuranosylcytosine-5′-diphosphate-L-1,2-dipalmitin

Recently, 1-β-$\text{D}$-arabinofuranosylcytosine-5′-diphosphate-$\text{DL}$-1,2-dipalmitin (VIa) was reported to inhibit the growth of L51784 cells in mice and of human colon carcinoma HCT-15 cells, also in mice. This paper describes the synthesis of a single diastereomer by conversion of 1-β-$\text{D}$-arabinofuranosylcytosine 5′-monophosphate (II) to the nucleoside 5′-phosphomorpholidate (III), followed by reaction with $\text{L}$-α-dipalmitoylphosphatidic acid (IV) to give 1-β-$\text{D}$-arabinofuranosylcytosine-5′-diphosphate-$\text{L}$-1,2-dipalmitin (V) in good yield. The separation of the product is described and its characterization by chromatography, elemental analysis, and spectroscopic methods. The lipophilic nature of V renders it insoluble in aqueous media and a method of sample preparation utilizing sonication techniques is described which provides a clear solution suitable for biological evaluation. In addition, the ability of V to inhibit the $\text{in}\text{vitro}$ growth of L1210 cells and of mouse myeloma MPC 11 cells is desscribed and compared with 1-β-$\text{D}$-arabinofuranosylcytosine (I) and other lipophilic prodrugs of I.     

The stability of L-ATC hydrolase participating in L-cysteine production

Summary In the production of L-cysteine from D,L-ATC stability of the relevant enzymes produced byPseudomonas sp. was tested, and strategies to improve the stability of L-ATC hydrolase were investigated in view of water activity and ionic strength. Among the three enzymes which participate in L-cysteine production, i.e., ATC racemase, L-ATC hydrolase, and S-carbamyl-L-cysteine hydrolase, L-ATC hydrolase is the least stable. Various mixtures of salts and sorbitol were added to adjust the water activities of the tested solutions. As water activity decreased from 0.93 to 0.80, the stability of L-ATC hydrolase was sharply enhanced. In the absence of sorbitol the stability of L-ATC hydrolase increased in proportion to ionic strength. Even though enzyme stability was not good at a low ionic strength, it was enhanced by lowering water activity with addition of sorbitol. The half life of L-ATC hydrolase in sorbitol-salt mixtures increased by tenfold to twentyfold compared to that of a control.     

Serum IgE levels in rats infected with Paragonimus westermani]

Paragonimus westermani is a common fluke in Korea. The present study aimed to determine serum total IgE and specific IgG levels in experimental paragonimiasis of rats. Each Wistar rat was inoculated orally with 20-25 metacercariae of P. westermani from Cambaroides similis. Before and after infection (1, 2, 3, 4, 6, 8 weeks) of P. westermani, the blood was collected from the retro-orbital venous plexus of rats and kept serum at -70 degrees C. Serum total IgE and specific IgG levels were determined by the capture and conventional enzyme-linked immunosorbent assay, respectively. The results were as follows; 1. Serum IgE values were increased to 0.18 +/- 0.042 at 2 weeks, 0.28 +/- 0.151 at 4 weeks and 0.43 +/- 0.055 at 8 weeks after infection. The absorbances of non-infected rats ranged 0.07 +/- 0.021-0.12 +/- 0.025. 2. Specific IgG values were slightly increased at 3 weeks (0.20 +/- 0.032) and gradually increased up to 8 weeks (0.31 +/- 0.067) after infection. The absorbances of non-infected rats ranged 0.11 +/- 0.035-0.18 +/- 0.019. The present results suggested that P. westermani could elevate serum IgE and specific IgG antibodies in Wistar rats which were not a good definitive host.     

Determination of kinetic parameters related to the piasmid instability: for the recombinant fermentation under repressed condition

The plasmid stability under the repressed state of cloned gene was studied theoretically as well as experimentally using recombinant E. coli K12DeltaH1Deltatrp/pPLc23trpA1 as a "host-vector" model system. The important kinetic parameters studied were the plasmid loss rate (theta) describing the rate at which the plasrnid-harboring cells lose plas-mids and the plasmid-free cells are generated per unit time and the difference in growth rates (Delta) between the two genotypes. These parameters were carefully defined, studied, and compared with other key kinetic parameters involved in the recombinant fermentation to further our understanding of metabolism of recombinants. The ratio of the concentration of plasmid-free cells to plasmid-harboring cells (Omega) was introduced, and the mathematical model was derived and used for the determination of the kinetic parameters associated with plasmid instability. These methods developed based on the theoretical considerations were tested experimentally. The results of these methods were compared, and the best method was selected and recommended. The effect of temperature and dilution rate on kinetic parameters theta and Delta were also studied in continuous culture, in order to provide some practical information related to the operation and control of recombinant fermentation processes.     

Environment-mediated accumulation of diacyl lipoproteins over their triacyl counterparts in Staphylococcus aureus

Bacterial lipoproteins are believed to exist in only one specific lipid-modified structure, such as the diacyl form or the triacyl form, in each bacterium. In the case of Staphylococcus aureus, recent extensive matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis revealed that S. aureus lipoproteins exist in the α-aminoacylated triacyl form. Here, we discovered conditions that induce the accumulation of diacyl lipoproteins that lack α-aminoacylation in S. aureus. The accumulation of diacyl lipoproteins required a combination of conditions, including acidic pH and a post-logarithmic-growth phase. High temperatures and high salt concentrations additively accelerated the accumulation of the diacyl lipoprotein form. Following a post-logarithmic-growth phase where S. aureus MW2 cells were grown at pH 6, SitC lipoprotein was found almost exclusively in its diacyl structure rather than in its triacyl structure. This is the first report showing that the environment mediates lipid-modified structural alterations of bacterial lipoproteins.