Comparative studies of polyethylene glycol-modified liposomes prepared using different PEG-modification methods

To address the issue of excess polyethylene glycol (PEG)-lipid degradation observed when PEG-modified liposomes are prepared using the pH-gradient method, a concept using a novel PEG-modification method, called the post-modification method, was proposed and evaluated. To assess the proof concept, a preservation-stability study and a pharmacokinetic study were performed that compared the conventional PEG-modification method, called the pre-modification method, with the post-modification method. The results show that PEG-lipid degradation could be markedly inhibited in the post-modification method. Furthermore, the post-modification method could be used without any manufacturing process difficulties, especially with high PEG-lipid content. In addition, a higher blood circulation capability was observed in the post-modification method. Through comparative studies, it was found that the post-modification method was advantageous compared to the pre-modification method. In conclusion, the post-modification method has the potential to be a novel PEG-modification method that can achieve a higher preservation stability of PEG-lipid, a greater ease of manufacturing, and a higher blood circulation capability, especially in the manufacturing of pH-gradient liposomal products.     

Central versus peripheral impact of estradiol on the impaired glucose metabolism in ovariectomized mice on a high-fat diet

Age-related loss of ovarian function promotes adiposity and insulin resistance in women. Estrogen (E(2)) directly enhances insulin sensitivity and suppresses lipogenesis in peripheral tissues. Recently, the central actions of E(2) in the regulation of energy homeostasis are becoming clearer; however, the functional relevance and degree of contribution of the central vs. peripheral actions of E(2) are currently unknown. Therefore, we prepared and analyzed four groups of mice. 1) Control: sham-operated mice fed a regular diet, 2) OVX-HF: ovariectomized (OVX) mice fed a 60% high-fat diet (HF), 3) E2-SC: OVX-HF mice subcutaneously treated with E(2), and 4) E2-ICV: OVX-HF mice treated with E(2) intracerebroventricularly. OVX-HF mice showed increased body weight with both visceral and subcutaneous fat volume enlargement, glucose intolerance, and insulin resistance. Both E2-SC and E2-ICV equally ameliorated these abnormalities. Although the size of adipocytes and number of CD11c-positive macrophages in perigonadal fat in OVX-HF were reduced by both E(2) treatments, peripherally administered E(2) decreased the expression of TNFα, lipoprotein lipase, and fatty acid synthase in the white adipose tissue (WAT) of OVX-HF. In contrast, centrally administered E(2) increased hormone-sensitive lipase in WAT, decreased the hepatic expression of gluconeogenic enzymes, and elevated core body temperature and energy expenditure with marked upregulation of uncoupling proteins in the brown adipose tissue. These results suggest that central and peripheral actions of E(2) regulate insulin sensitivity and glucose metabolism via different mechanisms, and their coordinated effects may be important to prevent the development of obesity and insulin resistance in postmenopausal women.     

Parasiticidal activity of Haemaphysalis longicornis longicin P4 peptide against Toxoplasma gondii

The Haemaphysalis longicornis longicin P4 peptide is an active part peptide produced by longicin which displays bactericidal activity against both Gram-negative and Gram-positive bacteria and other microorganisms. In the present study, the effect of the longicin P4 peptide on the infectivity of Toxoplasma gondii parasites was examined in vitro. Tachyzoites of T. gondii incubated with longicin P4 had induced aggregation and lost the trypan blue dye exclusion activity and the invasion ability into the mouse embryonal cell line (NIH/3T3). Longicin P4 bound to T. gondii tachyzoites, as demonstrated by fluoresce microscopic analysis. An electron microscopic analysis and a fluorescence propidium iodide exclusion assay of tachyzoites exposed to longicin P4 revealed pore formation in the cellular membrane, membrane disorganization, and hollowing as well as cytoplasmic vacuolization. The number of tachyzoites proliferated in mouse macrophage cell line (J774A.1) was significantly decreased by incubation with longicin P4. These findings suggested that longicin P4 conceivably impaired parasite membranes, leading to the destruction of Toxoplasma parasites in J774A.1 cells. Thus, longicin P4 is an interesting candidate for antitoxoplasmosis drug design that causes severe toxicity to T. gondii and plays an important role in reducing cellular infection. This is the first report showing that longicin P4 causes aggregation and membrane injury of parasites, leading to Toxoplasma tachyzoite destruction.     

Genetic polymorphisms of FCGR2A encoding Fcγ receptor IIa in a Japanese population and functional analysis of the L273P variant

Fcγ receptor IIa (FcγRIIa) plays an important role in antibody-dependent cellular cytotoxicity and inflammation. Changes in FcγRIIa expression levels or activity caused by genetic polymorphisms in FCGR2A, the gene encoding FcγRIIa, may lead to differences in disease progression as well as efficacy of antibody therapeutics between individuals. In this study, we sequenced the 5′-flanking region along with all exons and their flanking regions of FCGR2A from 111 Japanese subjects. Forty-eight genetic variations were found including 12 novel ones. Beside the well-known functional 497A?>?G (H166R) polymorphism, we detected 818T?>?C (L273P) at 0.005 frequency. Since the functional significance of this polymorphism has not been revealed, we next assessed the functions of the L273P substitution by expressing wild-type and the variant proteins in human Jurkat cells. The L273P variant protein showed similar cell surface expression and IgG-binding properties as the wild-type, but it exhibited a stronger signal transduction activity based on the approximately 2-fold enhancement of tyrosine phosphorylation of FcγRIIa itself. The current results suggest that L273P could have functional significance in the antibody-dependent clinical responses through FcγRIIa.     

Drosophila Syntrophins are involved in locomotion and regulation of synaptic morphology

Syntrophin components of the dystrophin glycoprotein complex (DGC) feature multiple protein interaction domains that may act in molecular scaffolding, recruiting signaling proteins to membranes and the DGC. Drosophila Syntrophin-1 (Syn1) and Syntrophin-2 (Syn2) are counterparts of human α1/β1/β2-syntrophins and γ1/γ2-syntrophins, respectively. α1/β1/β2-syntrophins are well documented, while little is known about γ1/γ2-syntrophins. Here, we performed immunohistochemical analyses with a specific antibody to Syn2 and demonstrated predominant expression in the larval and adult central nervous system. To investigate the in vivo functions of Syn2, we have generated Drosophila Syn2 deficiency mutants. Although the Syn2 mutants exhibit no overt phenotype, the combination of Syn1 knockdown and Syn2³⁷ mutation dramatically shortened life span, synergistically reduced locomotion ability and synergistically enhanced overgrowth of neuromuscular junctions in N-ethylmaleimide sensitive factor 2 mutants. From these data we conclude that Syn1 and Syn2 are required for locomotion and are involved in regulation of synaptic morphology. In addition, the two syntrophins can at least partially compensate for each other's functions.     

Role of hydroperoxide lyase in white-backed planthopper (Sogatella furcifera Horváth)-induced resistance to bacterial blight in rice, Oryza sativa L.

A pre-infestation of the white-backed planthopper (WBPH), Sogatella furcifera Horváth, conferred resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae ( Xoo ) in rice ( Oryza sativa  L.) under both laboratory and field conditions. The infestation of another planthopper species, the brown planthopper (BPH) Nilaparvata lugens Stål, did not significantly reduce the incidence of bacterial blight symptoms. A large-scale screening using a rice DNA microarray and quantitative RT-PCR revealed that WBPH infestation caused the upregulation of more defence-related genes than did BPH infestation. Hydroperoxide lyase 2 ( OsHPL2 ), an enzyme for producing C₆ volatiles, was upregulated by WBPH infestation, but not by BPH infestation. One C₆ volatile, ( E )-2-hexenal, accumulated in rice after WBPH infestation, but not after BPH infestation. A direct application of ( E )-2-hexenal to a liquid culture of Xoo inhibited the growth of the bacterium. Furthermore, a vapour treatment of rice plants with ( E )-2-hexenal induced resistance to bacterial blight. OsHPL2 -overexpressing transgenic rice plants exhibited increased resistance to bacterial blight. Based on these data, we conclude that OsHPL2 and its derived ( E )-2-hexenal play some role in WBPH-induced resistance in rice.     

A new NF-κB inhibitor based on the amino-epoxyquinol core of DHMEQ

The amino-epoxyquinols 6a and 6b were synthesized as soluble derivatives of an NF-κB inhibitor DHMEQ (1). In spite of the opposite configuration from 1, 6b rather than 6a affected the deactivation of NF-κB, based on NO secretion and MALDI-TOF MS analysis. It was indicated that 6b inhibited the activation by different manner from that of 1.     

Herbivore-Specific,Density-Dependent Induction of Plant Volatiles: Honest or “Cry Wolf” Signals?

Plants release volatile chemicals upon attack by herbivorous arthropods. They do so commonly in a dose-dependent manner: the more herbivores, the more volatiles released. The volatiles attract predatory arthropods and the amount determines the probability of predator response. We show that seedlings of a cabbage variety (Brassica oleracea var. capitata, cv Shikidori) also show such a response to the density of cabbage white (Pieris rapae) larvae and attract more (naive) parasitoids (Cotesia glomerata) when there are more herbivores on the plant. However, when attacked by diamondback moth (Plutella xylostella) larvae, seedlings of the same variety (cv Shikidori) release volatiles, the total amount of which is high and constant and thus independent of caterpillar density, and naive parasitoids (Cotesia vestalis) of diamondback moth larvae fail to discriminate herbivore-rich from herbivore-poor plants. In contrast, seedlings of another cabbage variety of B. oleracea (var. acephala: kale) respond in a dose-dependent manner to the density of diamondback moth larvae and attract more parasitoids when there are more herbivores. Assuming these responses of the cabbage cultivars reflect behaviour of at least some genotypes of wild plants, we provide arguments why the behaviour of kale (B. oleracea var acephala) is best interpreted as an honest signaling strategy and that of cabbage cv Shikidori (B. oleracea var capitata) as a “cry wolf” signaling strategy, implying a conflict of interest between the plant and the enemies of its herbivores: the plant profits from being visited by the herbivore''s enemies, but the latter would be better off by visiting other plants with more herbivores. If so, evolutionary theory on alarm signaling predicts consequences of major interest to students of plant protection, tritrophic systems and communication alike.     

Production of ω3 fatty acids in marine cyanobacterium Synechococcus sp. strain NKBG 15041c via genetic engineering

Omega-3 fatty acids (ω3 FAs) have attracted attention because they have various health benefits for humans. Fish oils are currently major sources of ω3 FAs, but a sustainable supply of ω3 FAs based on fish oils is problematic because of the increasing demand. In this study, the production potential of a genetically engineered marine cyanobacterium, Synechococcus sp. strain NKBG 15041c, was examined as an alternative source of ω3 FAs. A change in fatty acid composition of this cyanobacterium was successfully induced by the expression of a heterologous Δ6-desaturase, and the transformants synthesized stearidonic acid, which the wild type cannot produce. As a result of optimization of culture conditions, maximal contents of stearidonic acid and total ω3 FAs reached 12.2 ± 2.4 and 118.1 ± 3.5 mg/g, respectively. The maximal ω3 FA productivity was 4.6 ± 0.7 mg/(L⋅day). These are the highest values of the contents of stearidonic acid and ω3 FAs in genetically engineered cyanobacteria reported thus far. Therefore, genetically engineered Synechococcus sp. strain NKBG 15041c may be a promising sustainable source of ω3 fatty acids.