Ligation of ICAM-1 molecules inhibits target cell-induced granule exocytosis of IL-12-activated natural killer cells

The importance of cell adhesion molecules such as ICAM-1 is emphasized in cell-to-cell interactions that are critical in the generation of effective immune reactions. In this study, the involvement of ICAM-1 in natural killer (NK) cell activities was characterized in IL-12-activated human NK cells. To address the question of whether ligation of ICAM-1 molecules can modulate NK cell cytolytic activities, a 4-h (51)Cr-release assay was performed after pretreatment of NK cells with R6.5 mAb (anti-human ICAM-1 mAb). Ligation of membrane ICAM-1 molecules significantly inhibited IL-12-enhanced NK cytotoxicity against K562, and the pretreatment of neutralizing soluble ICAM-1 with R6.5 mAb blocked this inhibitory effect. The involvement of Ca(2+)-dependent granular exocytosis was evaluated. BLT esterase assay demonstrated that the ligation of ICAM-1 molecules inhibited granular exocytosis of NK cells. Additionally, the ICAM-1-mediated inhibition of Ca(2+) flux in NK cells was detected using Fluo-3AM, while the pretreatment of NK cells with R6.5 mAb did not affect conjugate formation between NK and K562 cells. Collectively, these results suggest that the signals transduced from ICAM-1 molecules might be sufficient to induce inhibitory effects on NK cells.     

Production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from Kimchi

Lactococcus lactis subsp. lactis A164 was isolated from Kimchi (Korean traditional fermented vegetables). The bacteriocin produced by strain A164 was active against closely related lactic acid bacteria and some food-borne pathogens including Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium. The antimicrobial spectrum was nearly identical to that of nisin. Bacteriocin activity was not destroyed by exposure to elevated temperatures at low pH values, but the activity was lost at high pH values. This bacteriocin was inactivated by pronase E and alpha, beta-chymotrypsin, but not by trypsin, pepsin, and alpha-amylase. Cultures of L. lactis subsp. lactis A164 maintained at a constant pH of 6.0 exhibited maximum production of the bacteriocin. It was purified to homogeneity by ammonium sulphate precipitation, sequential ion exchange chromatography, and ultrafiltration. Tricine-SDS-PAGE of purified bacteriocin gave the same molecular weight of 3.5 kDa as that of nisin. The gene encoding this bacteriocin was amplified by PCR with nisin gene-specific primers and sequenced. It showed identical sequences to the nisin gene. These results indicate that bacteriocin produced by Lactococcus lactis A164 is a nisin-like bacteriocin.     

A thermodynamic study of mesophilic, thermophilic, and hyperthermophilic L-arabinose isomerases: the effects of divalent metal ions on protein stability at elevated temperatures

To gain insight into the structural stability of homologous homo-tetrameric l-arabinose isomerases (AI), we have examined the isothermal guanidine hydrochloride (GdnHCl)-induced unfolding of AIs from mesophilic Bacillus halodurans (BHAI), thermophilic Geobacillus stearothermophilus (GSAI), and hyperthermophilic Thermotoga maritima (TMAI) using circular dichroism spectroscopy. The GdnHCl-induced unfolding of the AIs can be well described by a two-state reaction between native tetramers and unfolded monomers, which directly confirms the validity of the linear extrapolation method to obtain the intrinsic stabilities of these proteins. The resulting unfolding free energy (DeltaGU) values of the AIs as a function of temperature were fit to the Gibbs-Helmholtz equation to determine their thermodynamic parameters based on a two-state mechanism. Compared with the stability curves of BHAI in the presence and absence of Mn2+, those of holo GSAI and TMAI were more broadened than those of the apo enzymes at all temperatures, indicating increased melting temperatures (Tm) due to decreased heat capacity (DeltaGp). Moreover, the extent of difference in DeltaCp between the apo and holo thermophilic AIs is larger than that of BHAI. From these studies, we suggest that the metal dependence of the thermophilic AIs, resulting in the reduced DeltaCp, may play a significant role in structural stability compared to their mesophilic analogues, and that the extent of metal dependence of AI stability seems to be highly correlated to oligomerization.     

Simple one-step purification of nisin Z from unclarified culture broth of Lactococcus lactis subsp. lactis A164 using expanded bed ion exchange chromatography

A simple one-step purification method, using expanded bed, ion-exchange chromatography, for the fractionation of nisin Z produced by Lactococcus lactis subsp. lactis A164 was developed. The highest dynamic binding capacity (0.92) of the adsorbent was obtained at a superficial velocity of 367 cm h(-1), resulting in approx. 2.7-fold bed expansion. The range of pH for the maximum adsorption was 3-4. The isocratic elution with 0.15 M NaCl led to approx. >90% recovery. Single-step purification of nisin Z from unclarified A164 culture broth resulted in 31-fold purification with a 90% yield.     

Anti-inflammatory effects of Stephania tetrandra S. Moore on interleukin-6 production and experimental inflammatory disease models

Deregulation of interleukin-6 (IL-6) expression caused the synthesis and release of many inflammatory mediators. It is involved in chronic inflammation, autoimmune diseases, and malignancy. Stephania tetrandra S. Moore is a Chinese medicinal herb which has been used traditionary as a remedy for neuralgia and arthritis in China. To investigate the anti-inflammatory effects of S. tetrandra S. Moore in vitro and in vivo, its effects on the production of IL-6 and inflammatory mediators were analysed. When human monocytes/macrophages stimulated with silica were treated with 0.1-10 mug/ml S. tetranda S. Moore, the production of IL-6 was inhibited up to 50%. At these concentrations, it had no cytotoxicity effect on these cells. It also suppressed the production of IL-6 by alveolar macrophages stimulated with silica. In addition, it inhibited the release of superoxide anion and hydrogen peroxide from human monocytes/macrophages. To assess the anti-fibrosis effects of S. tetrandra S. Moore, its effects on in vivo experimental inflammatory models were evaluated. In the experimental silicosis model, IL-6 activities in the sera and in the culture supernatants of pulmonary fibroblasts were also inhibited by it. In vitro and in vivo treatment of S. tetrandra S. Moore reduced collagen production by rat lung fibroblasts and lung tissue. Also, S. tetrandra S. Moore reduced the levels of serum GOT and GPT in the rat cirrhosis model induced by CCL(4), and it was effective in reducing hepatic fibrosis and nodular formation. Taken together, these data indicate that it has a potent anti-inflammatory and antifibrosis effect by reducing IL-6 production.     

Isolation and characterization of a thermophilic lipase from Bacillus thermoleovorans ID-1

A thermophilic microorganism, Bacillus thermoleovorans ID-1, isolated from hot springs in Indonesia, showed extracellular lipase activity and high growth rates on lipid substrates at elevated temperatures. On olive oil (1.5%, w/v) as the sole carbon source, the isolate ID-1 grew very rapidly at 65 degrees C with its specific growth rate (2.50 h(-1)) and its lipase activity reached the maximum value of 520 U l(-1) during the late exponential phase and then decreased. In addition to this, isolate ID-1 could grow on a variety of lipid substrates such as oils (olive oil, soybean oil and mineral oil), triglycerides (triolein, tributyrin) and emulsifiers (Tween 20, 40). The excreted lipase of ID-1 was purified 223-fold to homogeneity by ammonium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography and Sephacryl S-200 gel filtration chromatography. As a result, the relative molecular mass of the lipase was determined to be 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme showed optimal activity at 70-75 degrees C and pH 7.5 and exhibited 50% of its original activity after 1 h incubation at 60 degrees C and 30 min at 70 degrees C and its catalytic function was activated in the presence of Ca(2+) or Zn(2+).     

1D copper(II) and zinc(II) coordination polymers containing an unusual twisted oxalate bridge

Two new copper(II) complexes, Cu(L1)(ClO₄)₂ (1), {[(μ-oxalate)Cu(L1)] · 5H₂O}_n (2), and a zinc(II) complex, {[(μ-oxalate)Zn(L2)] · 3H₂O · 0.5DMF}_n (3) (L = 3,14-dimethyl-2,6,13,17-tetraazatricyclo[14,4,0^1.18,0^7.12]docosane), have been synthesized and characterized by X-ray crystallography. In 1, the ligand conformation is planar, and the octahedral coordination about the copper(II) ion is completed by weakly interacting ions. In 2 and 3, bridging oxalate ligands coordinate to copper(II) or zinc(II) ions in an unusually twisted bis-monodentate (trans-1,1′-bicoordination) mode.

The rigidity and steric hindrance of macrocycles L1 and L2 by the introduction of two cyclohexane rings and methyl groups on a cyclam (1,4,8,11-tetraazacyclotetradecane) skeleton cause the bridging oxalate ligands to adopt such unusual geometries in 2 and 3.     

Cloning, expression and characterization of xylose isomerase, XylA, from Caldanaerobacter subterraneus subsp. yonseiensis

The xylA gene, coding for xylose isomerase, from the extreme thermophile, Caldanaerobacter subterraneus subsp. yonseiensis was cloned, sequenced, and expressed in Escherichia coli. The nucleotide sequence of the xylA gene encoded a polypeptide of 438 residues with a calculated molecular weight of 50,170 Da. The purified XylA showed high sequence homology (92% identity) with that of Thermoanaerobacter thermohydrosulfuricus. The recombinant enzyme expressed in Escherichia coli was purified by heat treatment and gel chromatography. The purified enzyme was thermostable with optimal activity at 95°C. The enzyme required divalent cations including Zn²⁺ for its maximal activity and thermostability.     

Radiosensitization by celastrol is mediated by modification of antioxidant thiol molecules

The radiosensitizing effects of naturally occurring triterpenes were investigated in human lung cancer cells. Several quinone methide-containing triterpenes (QMTs) enhanced the cytotoxic effect of ionizing radiation (IR) and of these QMTs, celastrol (CE) had the greatest enhancing effect on IR-induced cell death in vitro. Additionally, the quinone methide moiety of CE was shown to be essential for CE-mediated radiosensitization; in contrast, dihydrocelastrol (DHCE), does not contain this moiety. Reactive oxygen species (ROS) production by IR was augmented in combination with CE, which was responsible for CE-mediated radiosensitization. CE induced the thiol reactivity and inhibited the activities of antioxidant molecules, such as thioredoxin reductase and glutathione. In vivo, nude mouse xenografting data also revealed that tumor growth delay was greater in mice treated with CE plus IR, compared with those treated with CE or IR alone. When DHCE, instead of CE, was combined with IR, tumor growth delay was similar to that in IR alone-treated mice. These results demonstrate that CE synergistically enhances the effects of IR and suggest the novel anticancer therapeutic use of CE in combination with radiation therapy.