USE OF AN ANTIBODY AGAINST THE SOLUBLE INTERLEUKIN 2 RECEPTOR α SUBUNIT CAN MODULATE THE STABILITY AND BIODISTRIBUTION OF INTERLEUKIN-2

The authors have previously reported that the soluble serum form of the alpha subunit of the IL-2 receptor (sIL-2Rα), whose natural half-life is approximately 40 min, survived much longer in the circulation when bound by a specific antibody. In the present study, the authors evaluated the extent to which sIL-2Rα protected IL-2 in freshly collected serum using biochemical analyses, and a functional CTLL-2 assay. In particular, sIL-2Rα protected IL-2 from forming complexes with α₂-macroglobulin and from inactivation in vitro. In addition, the authors demonstrated that the anti-IL-2Rα monoclonal antibody 7G7/B6, which does not inhibit the binding of IL-2 to its binding site on sIL-2Rα, protected IL-2 from degradation and inactivation in vivo in the presence of sIL-2Rα. Both¹²⁵I-labelled and unlabelled IL-2 were injected into mice preinjected with humanized anti-Tac (hTac) or 7G7/B6 and sIL-2Rα, or sIL-2Rα alone. Using size-exclusion HPLC, ELISA, and CTLL-2 cell proliferation assays, we observed that the presence of 7G7/B6 led to formation of complexes with sIL-2Rα and increased the serum levels of IL-2 more than 3- to 40-fold those of groups receiving IL-2 alone, sIL-2Rα, or hTac. Taken as a whole, these results suggest that the complex of 7G7/B6 and sIL-2Rα not only prolongs the survival of IL-2 in vivo, but also maintains the bioactivity of IL-2. The use of antibodies against endogenous soluble receptors could increase the in vivo survival of cytokines, protect their bioactivity and thereby facilitate their clinical use in the treatment of various malignancies and AIDS.     

Eicosapentaenoic acid increases lipolysis through up-regulation of the lipolytic gene expression and down-regulation of the adipogenic gene expression in 3T3-L1 adipocytes

In this study, we investigated the lipolytic effects of eicosapentaenoic acid (EPA) in 3T3-L1 adipocytes. The differentiated 3T3-L1 adipocytes were treated in a serum-free medium with 300 muM of EPA for 3, 6, 12, and 24 h. In comparison with the control, intracellular lipid accumulation was significantly decreased by 24% at 24 h following the addition of EPA (P < 0.05). Under the same experimental conditions, there was an increase of glycerol and free fatty acids (FFAs). The mRNA level of carnitine palmitoyltransferase I-a, a component of the fatty-acid shuttle system involved in the mitochondrial oxidation of long-chain fatty acids, was also significantly elevated by EPA (P < 0.05). However, the expression of peroxisome proliferator-activated receptor-gamma and acetyl-CoA carboxylase (ACC), which are involved in adipogenesis, was significantly down-regulated by EPA (P < 0.05). These results suggest that EPA may modulate lipid metabolism by stimulation of lipolysis, which was associated with induction of lipolytic gene expression and suppression of adipogenic gene expression in 3T3-L1 adipocytes.     

Lower Ordovician sponge bioherms in the Makkol Formation, Taebaeksan Basin, mideast Korea

Summary Isolated sponge bioherms are documented from the Lower Ordovician Makkol Formation of the Taebaek Group in the Taebaeksan Basin, mideast Korea. They are formed by an association of a lithistid spongeArchaeoscyphia, a receptaculidCalathium and stromatolitic algae, and share many features with the Lower Ordovician buildups known elsewhere. These bioherms were established in an incised bottom and reached up to about 1 m in height. As the bioherms grew upward, they were more severely affected by intense wave action and frequent storms, which eventually perished the bioherms. The occurrence ofArchaeoscyphia-Calathium association suggests a close biogeographic link between Korea and North China, supporting the paleogeographic model that the Taebaeksan Basin was connected through contiguous shallow waters to North China in the early Paleozoic.     

Intra- and extra-cellular organophosphorus hydrolase production with recombinant <Emphasis Type="Italic">E. coli</Emphasis> using fed-batch fermentation

A fed-batch fermentation process for the production of organophosphorus hydrolase (OPH) (EC 3.1.8.1) by E. coli pET812 was developed in this research. With batch fermentation, the maximum OPH concentrations attained by batch fermentation were as low as 4 × 10⁵ U/l because cell growth and OPH production were inhibited by a high initial concentration of glucose. To develop a fed-batch fermentation process for obtaining higher concentrations of OPH, highly concentrated glucose solution (500 g/l) was added intermittently or continuously to increase the carbon source concentration. Eventually, 3.2 × 10⁶ U/l of OPH was produced with fed-batch fermentation in 24 h. This was eight times higher than the yield with conventional batch fermentation. A total concentration of 399–441 mg of OPH was produced/l, which was four times higher than that reported when using E. coli. Nearly half (44%) of the produced OPH was secreted into the culture solution.     

Functional expression of recombinant human ribonuclease/angiogenin inhibitor in stably transformed <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> S2 cells

A recombinant plasmid harboring heterologous genes coding human ribonuclease/angiogenin inhibitor (RAI) was expressed in stably transformed Drosophila melanogaster Schneider 2 (S2) cells. Stably transformed polyclonal cell populations expressing RAI were isolated after 4 weeks of selection with hygromycin B. Recombinant RAI with a molecular weight of 50 kDa was detected in the intracellular (cell) and extracellular (medium) fractions of S2 cells. Recombinant RAI was purified from the extracellular fraction using a two-step purification scheme comprised of Ni-NTA and ion-exchange chromatography. Purified RAI migrated on SDS-PAGE as a single band in the elution fraction containing 300 mM NaCl. The ribonuclease inhibitor activity of purified RAI was measured using yeast tRNA and RNase A. Purified RAI exhibited an activity of ∼8 U μg⁻¹ for the inhibition of RNA degradation by RNase A. Cultivation of stably transformed S2 cells using HyQ^®SFX-insect MP medium increased cell growth by 79% and approximately doubled the production of recombinant RAI.     

Glucose oxidase: natural occurrence, function, properties and industrial applications

Glucose oxidase (GOX) from Aspergillus niger is a well-characterised glycoprotein consisting of two identical 80-kDa subunits with two FAD co-enzymes bound. Both the DNA sequence and protein structure at 1.9 Ǻ have been determined and reported previously. GOX catalyses the oxidation of d-glucose (C₆H₁₂O₆) to d-gluconolactone (C₆H₁₀O₆) and hydrogen peroxide. GOX is produced naturally in some fungi and insects where its catalytic product, hydrogen peroxide, acts as an anti-bacterial and anti-fungal agent. GOX is Generally Regarded As Safe, and GOX from A. niger is the basis of many industrial applications. GOX-catalysed reaction removes oxygen and generates hydrogen peroxide, a trait utilised in food preservation. GOX has also been used in baking, dry egg powder production, wine production, gluconic acid production, etc. Its electrochemical activity makes it an important component in glucose sensors and potentially in fuel cell applications. This paper will give a brief background on the natural occurrence, functions as well as the properties of glucose oxidase. A good coverage on the diverse uses of glucose oxidase in the industry is presented with a brief outline on the working principles in the various settings. Furthermore, food grade GOX preparations are relatively affordable and widely available; the readers may be encouraged to explore other potential uses of GOX. One example is that GOX-catalysed reaction generates significant amount of heat (∼200 kJ/mol), and this property has been mostly neglected in the various applications described so far.     

Subcellular accumulation of Cu in the Antarctic bivalve<Emphasis Type="Italic"> Laternula elliptica</Emphasis> from a naturally Cu-elevated bay of King George Island

Subcellular Cu sequestration was examined in the digestive gland, kidney and gill of the Antarctic bivalve Laternula elliptica collected from a Cu-elevated bay in King George Island. Cu was associated with both the soluble cytosolic and insoluble particulate cell fractions in all three organs, but their relative contributions to Cu sequestration varied with tissue type and the total amount of Cu accumulated. Low-molecular-weight (10–13 kDa) metallothionein-like proteins were the major Cu-binding ligands in the cytosol of all three organs. Significant portions of the cytosolic Cu were also bound to proteins with different molecular weights in the kidney and gill. A strong immunological response to a metallothionein (MT) antibody confirmed the presence of MTs in all three organs. Numerous electron-dense granules, which are likely to be metal-rich, were observed in renal epithelial cells by transmission electron microscopy, suggesting that these granules also play a role in Cu sequestration.     

Secretory phospholipase A2-potentiated inducible nitric oxide synthase expression by macrophages requires NF-kappa B activation

The effect of secretory group II phospholipase A2 (sPLA2) on the expression of the inducible NO synthase (iNOS) and the production of NO by macrophages was investigated. sPLA2 by itself barely stimulated nitrite production and iNOS expression in Raw264.7 cells. However, in combination with LPS, the effects were synergistic. This potentiation was shown for sPLA2 enzymes from sPLA2-transfected stable cells or for purified sPLA2 from human synovial fluid. The effect of PLA2 on iNOS induction appears to be specific for the secretory type of PLA2. LPS-stimulated activation of iNOS was inhibited by the well-known selective inhibitors of sPLA2 such as 12-epi-scalaradial and p-bromophenacyl bromide. In contrast, the cytosolic PLA2-specific inhibitors methyl arachidonyl fluorophosphate and arachidonyltrifluoromethyl ketone did not affect LPS-induced nitrite production and iNOS expression. Moreover, when we transfected cDNA-encoding type II sPLA2, we observed that the sPLA2-transfected cells produced two times more nitrites than the empty vector or cytosolic PLA2-transfected cells. The sPLA2-potentiated iNOS expression was associated with the activation of NF-kappa B. We found that the NF-kappa B inhibitor pyrrolidinedithiocarbamate prevented nitrite production, iNOS induction, and mRNA accumulation by sPLA2 plus LPS in Raw264.7 cells. Furthermore, EMSA analysis of the activation of the NF-kappa B involved in iNOS induction demonstrated that pyrrolidinedithiocarbamate prevented the NF-kappa B binding by sPLA2 plus LPS. Our findings indicated that sPLA2, in the presence of LPS, is a potent activator of macrophages. It stimulates iNOS expression and nitrite production by a mechanism that requires the activation of NF-kappa B.     

Hsp90 Inhibitors Are Efficacious against Kaposi Sarcoma by Enhancing the Degradation of the Essential Viral Gene LANA,of the Viral Co-Receptor EphA2 as well as Other Client Proteins

Heat-shock protein 90 (Hsp90) inhibitors exhibit activity against human cancers. We evaluated a series of new, oral bioavailable, chemically diverse Hsp90 inhibitors (PU-H71, AUY922, BIIB021, NVP-BEP800) against Kaposi sarcoma (KS). All Hsp90 inhibitors exhibited nanomolar EC₅₀ in culture and AUY922 reduced tumor burden in a xenograft model of KS. KS is associated with KS-associated herpesvirus (KSHV). We identified the viral latency associated nuclear antigen (LANA) as a novel client protein of Hsp90 and demonstrate that the Hsp90 inhibitors diminish the level of LANA through proteasomal degradation. These Hsp90 inhibitors also downregulated EphA2 and ephrin-B2 protein levels. LANA is essential for viral maintenance and EphA2 has recently been shown to facilitate KSHV infection; which in turn feeds latent persistence. Further, both molecules are required for KS tumor formation and both were downregulated in response to Hsp90 inhibitors. This provides a rationale for clinical testing of Hsp90 inhibitors in KSHV-associated cancers and in the eradication of latent KSHV reservoirs.