cDNA sequence and expression of a cold-responsive gene in Citrus unshiu

A cDNA clone encoding a protein (CuCOR19), the sequence of which is similar to Poncirus COR19, of the dehydrin family was isolated from the epicarp of Citrus unshiu. The molecular mass of the predicted protein was 18,980 daltons. CuCOR19 was highly hydrophilic and contained three repeating elements including Lys-rich motifs. The gene expression in leaves increased by cold stress.     

Oxidative stress can affect the gene silencing effect of DOTAP liposome in an in vitro translation system

Oxidative stress can affect in vitro GFP expression through its control of the gene silencing effect of the liposome prepared by 1,2-dioleoyl-3-trimethyl-ammonium propane (DOTAP). The gene silencing effect of cationic DOTAP liposome in in vitro GFP expression, especially focusing on its translation process, and the effects of oxidative stress on its silencing effect were investigated. GFP expression, initiated by mRNA, was found to be thoroughly inhibited in the presence of DOTAP liposome at concentration of more than 2.5 mM, though its inhibitory effect was reduced in the presence of hydrogen peroxide. The analyses of (i) the interaction of mRNA with DOTAP, (ii) the chemical structure of DOTAP, and (iii) the membrane fluidity of DOTAP liposome imply the possible role of gene expression by the liposome membrane and stress conditions.     

Novel immobilized liposomal glucose oxidase system using the channel protein OmpF and catalase

The reactivity of immobilized glucose oxidase-containing liposomes (IGOL) prepared in our previous work (Wang et al. [2003] Biotechnol Bioeng 83:444-453) was considerably improved here by incorporating the channel protein OmpF from Escherichia coli into the liposome membrane as well as by entrapping inside the liposome's aqueous interior not only glucose oxidase (GO), but also catalase (CA), both from Aspergillus niger. CA was used for decomposing the hydrogen peroxide produced in the glucose oxidation reaction inside the liposomes. The presence of OmpF enhanced the transport of glucose molecules from the exterior of the liposomes to the interior. In a first step of the work, liposomes containing GO and CA (GOCAL) were prepared and characterized. A remarkable protection effect of the liposome membrane on CA inside the liposomes at 40 degrees C was found; the remaining CA activity at 72 h incubation was more than 60% for GOCAL, while less than 20% for free CA. In a second step, OmpF was incorporated into GOCAL membranes, leading to the formation of OmpF-embedded GOCAL (abbreviated GOCAL-OmpF). The activity of GO inside GOCAL-OmpF increased up to 17 times in comparison with that inside GOCAL due to an increased glucose permeation across the liposome bilayer, without any leakage of GO or CA from the liposomes. The optimal system was estimated to contain on average five OmpF molecules per liposome. Finally, GOCAL-OmpF were covalently immobilized into chitosan gel beads. The performance of this novel biocatalyst (IGOCAL-OmpF) was examined by following the change in glucose conversion, as well as by following the remaining GO activity in successive 15-h air oxidations for repeated use at 40 degrees C in an airlift bioreactor. IGOCAL-OmpF showed higher reactivity and reusability than IGOL, as well as IGOL containing OmpF (IGOL-OmpF). The IGOCAL-OmpF gave about 80% of glucose conversion even when the catalyst was used repeatedly four times, while the corresponding conversions were about 60% and 20% for the IGOL and IGOL-OmpF, respectively. Due to the absence of CA, IGOL-OmpF was less stable and resulted in drastically inhibited GO.     

Laser-induced Propagation and Destruction of Amyloid β Fibrils

The amyloid deposition of amyloid β (Aβ) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of β₂-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Aβ fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Aβ fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.     

Stabilization of quaternary structure and activity of bovine liver catalase through encapsulation in liposomes

Bovine liver catalase was encapsulated in an aqueous phase of the phospholipid vesicle (liposome) to improve the stability of its tetrameric structure and activity. The catalase-containing liposomes (CALs) prepared were 30, 50 and 100 nm in mean diameters (CAL₃₀, CAL₅₀ and CAL₁₀₀, respectively). The CAL₁₀₀ included the types I, II and III based on the amounts of catalase encapsulated. The CAL₃₀, CAL₅₀ and CAL₁₀₀-I contained one catalase molecule per liposome, and the CAL₁₀₀-II and CAL₁₀₀-III on average 5.2 and 17 molecules, respectively. The storage stability of catalase in either CAL system was significantly increased compared to that of free catalase at 4 °C in a buffer of pH 7.4. At 55 °C, free catalase was much more deactivated especially with decreasing its concentration predominantly due to enhanced dissociation of catalase into subunits while it was so done at excessively high enzyme concentration mainly due to enhanced formation of catalase intermolecular aggregates. Among the three types of CAL₁₀₀, the CAL₁₀₀-II showed the highest thermal stability, indicating that an excess amount of catalase in the CAL₁₀₀-III was also disadvantageous to maintain an active form of the catalase even in liposome. In the CAL₁₀₀-III, however, the stability of catalase was significantly improved compared to that of free catalase at the same concentration. The CAL thermal stability was little affected by the liposome size as observed in the CAL₃₀, CAL₅₀ and CAL₁₀₀-I. An intrinsic tryptophan fluorescence of the catalase recovered from the CAL₁₀₀-II thermally treated at 55 °C revealed that a partially denatured catalase molecule was stabilized through its hydrophobic interaction with liposome membrane. This interaction depressed not only dissociation of catalase into subunits but also formation of an inactive intermolecular aggregate between the catalase molecules in a liposome. Furthermore, either type of CAL₁₀₀ showed a higher stability than free catalase in the successive decompositions of 10 mM H₂O₂ at 25 °C mainly because the H₂O₂ concentration was kept low inside liposomes due to the permeation barrier of the lipid membrane to H₂O₂.     

Liposomes destabilize tRNA during heat stress

Biomembranes play an important role in cellular response to heat stress. In this study, we focus on the interaction between liposomes and tRNA. Upon heat treatment we determined circular dichroism spectra of tRNA in presence of liposomes prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and cholesterol (Ch). To compare thermal stability, midpoint temperature (T_m) of tRNA was calculated from normalized θ₂₀₈. Addition of POPC/Ch liposomes decreased the T_m value of tRNA from 48°C to 38°C. We conclude that POPC/Ch liposomes interact with tRNA and destabilize its conformation under heat stress.     

Characterization of feline serum ferritin-binding proteins: the presence of a novel ferritin-binding protein as an inhibitory factor in feline ferritin immunoassay

Ferritin-binding proteins (FBPs) such as anti-ferritin antibody, α-2-macroglobulin, apolipoprotein B are expected to interact with circulating ferritin to eliminate it from circulation. However, we found that feline serum more strongly inhibits the detection of canine liver ferritin by immunoassay than its apoferritin; putative FBPs probably conceal ferritin epitopes detected by anti-ferritin antibodies. After complex formation between affinity-purified FBPs and canine liver ferritin, co-immunoprecipitates of the complex by anti-bovine spleen ferritin antibody were found to contain autoantibodies (IgG, IgM, and IgA) to ferritin by immunoblot analysis with antibodies specific for feline IgG, IgM, and IgA. On the other hand, affinity-purified samples did not show any inhibitory effect in the ferritin immunoassay. This result shows that feline serum has another FBP, which inhibits ferritin immunoassays, but not anti-ferritin autoantibody. A feline FBP was partially purified from feline serum by (NH₄)₂SO₄ fractionation (33–50%), gel filtration chromatography, and anion exchange chromatography. After binding of the partially purified sample with canine liver ferritin coupled-Sepharose gel, the FBP was separated and purified from complexes formed in a native-PAGE gel. SDS–PAGE analysis showed that the purified FBP is a homomultimer composed of 31 kDa monomeric subunits connected by intermolecular disulfide bonds. Detection of feline liver ferritin by immunoassay was inhibited by FBP in a dose-dependent manner. The purified protein molecules appeared to be conglomerate of pentraxin-like molecules by its electron micrographic appearance. These results demonstrate that feline serum contains a novel FBP as inhibitory factor of ferritin immunoassay with different molecular properties from those of other mammalian FBPs, in addition to auto-antibodies (IgG, IgM, and IgA) to ferritin.     

Liposome membrane can act like molecular and metal chaperones for oxidized and fragmented superoxide dismutase

A mechanism for liposome-recruited activity of oxidized and fragmented superoxide dismutase (Fr.-SOD) [Tuan LQ, Umakoshi H, Shimanouchi T, Kuboi R. Liposome-recruited activity of oxidized and fragmented superoxide dismutase. Langmuir 2008;24:350–4] was further investigated, focusing on the secondary structure of Fr.-SOD. Liposome membrane was found to assist the conformational change of Fr.-SOD and reactivate the enzymatic activity, like molecular and metal chaperones. The loss of SOD activity and its secondary structure was observed during 6 h oxidation in 2 mM hydrogen peroxide. The contents of the α-helix and β-sheet structures in the oxidized and fragmented SOD (2 μM) were increased only in the presence of 10 μM Cu²⁺ and Zn²⁺ together, or in the presence of 2 mM POPC liposomes. The mixture of all of these elements (fragmented SOD and POPC liposomes with Cu²⁺ and Zn²⁺) gave not only the increase of the α-helix and β-sheet contents but also the mediation of the high SOD-like activity.     

Covalent immobilization of unilamellar liposomes in gel beads for chromatography

For immobilized (proteo)liposome chromatography, unilamellar liposomes were covalently bound within gel beads that had been activated by CNBr, N-hydroxysuccinimide, tresyl, or chloroformate. Liposomes composed of phosphatidylcholine (PC) and 2 mol% of amino-containing lipid (phosphatidylethanolamine-caproylamine) were immobilized in the activated gels at 5-35 micromol lipid/ml gel and yields of 11-70%. The highest immobilized amount was found in chloroformate-activated TSK G6000PW gel, which contains large pore size (>100 nm). Liposomes composed of PC alone could also be attached to the chloroformate-activated gels at 33-42 micromol/ml gel and yields of 58-65%, probably by crosslinking of the phosphate moiety of phospholipid with the active group of the adsorbent. Liposomes prepared by various phospholipids with or without amino-containing lipids can generally be immobilized in the chloroformate-activated gels. The covalently bound liposomes were characterized by their high stability, unilamellarity, permeability of the membranes, and drug-membrane partition properties. A stable membrane phase was constructed for chromatographic experiments to be performed under extreme elution conditions.     

Enhancement of apparent substrate selectivity of proteinase K encapsulated in liposomes through a cholate-induced alteration of the bilayer permeability

Proteinase K-containing liposomes with highly selective membrane permeability properties were prepared. The selectivity obtained was with respect to the two substrate molecules added to the external aqueous phase of the liposomes: acetyl-L-Ala-Ala-Ala-p-nitroanilide (Ac-AAA-pNA) and succinyl-L-Ala-Ala-Ala-p-nitroanilide (Suc-AAA-pNA). The liposome-forming lipid used was POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and modulation of the membrane permeability was achieved using the detergent cholate. Proteinase K-containing mixed liposomes (PKCL) were prepared by adding cholate to preformed proteinase K-containing POPC liposomes (PKL) at a defined effective cholate/POPC molar ratio in the liposomal bilayer membrane R(e). Proteinase K was kept inside PKCL with a negligible amount of leakage into the bulk aqueous phase at R(e) < or = 0.30. At higher R(e), leakage of proteinase K was pronounced, even under conditions where POPC/cholate mixed liposomes seemed to be still intact (0.30 < R(e) < or = 0.39). At R(e) < or = 0.30, the reactivity of proteinase K in the PKCL measured with the externally added substrate Ac-AAA-pNA increased with increasing R(e), while the reactivity measured with Suc-AAA-pNA remained low, regardless of the R(e) value. This showed that externally added Ac-AAA-pNA molecules permeated the liposomal membrane more easily than Suc-AAA-pNA by modulating the membrane with cholate. Consequently, Ac-AAA-pNA was hydrolyzed in PKCL with considerably higher apparent substrate selectivity in comparison with the cases of proteinase K in PKL and free proteinase K (without liposomal encapsulation). The results obtained clearly demonstrate that the prepared PKCL can be utilized as a kind of nano-scaled bioreactor system which can take up a particular target substrate with high apparent substrate selectively from the external phase of the liposomes. Inside the liposomes, the target substrate is then converted into the corresponding products.