Sequence polymorphisms in Pvs48/45 and Pvs47 gametocyte and gamete surface proteins in Plasmodium vivax isolated in Korea

Nucleotide sequence analyses of the Pvs48/45 and Pvs47 genes were conducted in 46 malaria patients from the Republic of Korea (ROK) (n = 40) and returning travellers from India (n = 3) and Indonesia (n = 3). The domain structures, which were based on cysteine residue position and secondary protein structure, were similar between Plasmodium vivax (Pvs48/45 and Pvs47) and Plasmodium falciparum (Pfs48/45 and Pfs47). In comparison to the Sal-1 reference strain (Pvs48/45, PVX_083235 and Pvs47, PVX_083240), Korean isolates revealed seven polymorphisms (E35K, H211N, K250N, D335Y, A376T, I380T and K418R) in Pvs48/45. These isolates could be divided into five haplotypes with the two major types having frequencies of 47.5% and 20%, respectivelfy. In Pvs47, 10 polymorphisms (F22L, F24L, K27E, D31N, V230I, M233I, E240D, I262T, I273M and A373V) were found and they could be divided into four haplotypes with one major type having a frequency of 75%. The Pvs48/45 isolates from India showed a unique amino acid substitution site (K26R). Compared to the Sal-1 and ROK isolates, the Pvs47 isolates from travellers returning from India and Indonesia had amino acid substitutions (S57T and I262K). The current data may contribute to the development of the malaria transmission-blocking vaccine in future clinical trials.     

Wafer-scale nanowell array patterning based electrochemical impedimetric immunosensor

We have reported that nanowell array (NWA) can enhance electrochemical detection of molecular binding events by controlling the binding sites of the captured molecules. Using NWA biosensor based amperometric analysis, we have detected biological macromolecules such as DNA, protein or aptamers at low concentrations. In this research, we developed an impedimetric immunosensor based on wafer-scale NWA for electrochemical detection of stress-induced-phosphoprotein-1 (STIP-1). In order to develop NWA sensor through the cost-effective combination of high-throughput nanopattern, the NWA electrode was fabricated on Si wafer by krypton-fluoride (KrF) stepper semiconductor process. Finally, 12,500,000 ea nanowell with a 500 nm diameter was fabricated on 4 mm × 2 mm substrate. Next, by using these electrodes, we measured impedance to quantify antigen binding to the immunoaffinity layer. The limit of detection (LOD) of the NWA was improved about 100-fold compared to milli-sized electrodes (4 mm × 2 mm) without an NWA. These results suggest that wafer-scale NWA immunosensor will be useful for biosensing applications because their interface response is appropriate for detecting molecular binding events.     

Integrating cell‐free biosyntheses of heme prosthetic group and apoenzyme for the synthesis of functional P450 monooxygenase

Harnessing the isolated protein synthesis machinery, cell‐free protein synthesis reproduces the cellular process of decoding genetic information in artificially controlled environments. More often than not, however, generation of functional proteins requires more than simple translation of genetic sequences. For instance, many of the industrially important enzymes require non‐protein prosthetic groups for biological activity. Herein, we report the complete cell‐free biogenesis of a heme prosthetic group and its integration with concurrent apoenzyme synthesis for the production of functional P450 monooxygenase. Step reactions required for the syntheses of apoenzyme and the prosthetic group have been designed so that these two separate pathways take place in the same reaction mixture, being insulated from each other. Combined pathways for the synthesis of functional P450 monooxygenase were then further integrated with in situ assay reactions to enable real‐time measurement of enzymatic activity during its synthesis. Biotechnol. Bioeng. 2013; 110: 1193–1200. © 2012 Wiley Periodicals, Inc.     

N‐glycan maturation is crucial for cytokinin‐mediated development and cellulose synthesis in Oryza sativa

To explore the physiological significance of N‐glycan maturation in the plant Golgi apparatus, gnt1, a mutant with loss of N‐acetylglucosaminyltransferase I (GnTI) function, was isolated in Oryza sativa. gnt1 exhibited complete inhibition of N‐glycan maturation and accumulated high‐mannose N‐glycans. Phenotypic analyses revealed that gnt1 shows defective post‐seedling development and incomplete cell wall biosynthesis, leading to symptoms such as failure in tiller formation, brittle leaves, reduced cell wall thickness, and decreased cellulose content. The developmental defects of gnt1 ultimately resulted in early lethality without transition to the reproductive stage. However, callus induced from gnt1 seeds could be maintained for periods, although it exhibited a low proliferation rate, small size, and hypersensitivity to salt stress. Shoot regeneration and dark‐induced leaf senescence assays indicated that the loss of GnTI function results in reduced sensitivity to cytokinin in rice. Reduced expression of A‐type O. sativa response regulators that are rapidly induced by cytokinins in gnt1 confirmed that cytokinin signaling is impaired in the mutant. These results strongly support the proposed involvement of N‐glycan maturation in transport as well as in the function of membrane proteins that are synthesized via the endomembrane system.     

Asymmetric synthesis and effect of absolute stereochemistry of YCZ-2013, a brassinosteroid biosynthesis inhibitor

The four stereoisomers of 2RS,4RS-1-[[2-(2,4-dichlorophenyl)-4-(2-(2-propenyloxy)phenoxymethyl)-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole (YCZ-2013), a novel brassinosteroid biosynthesis inhibitor, were prepared. The diastereomers of 2RS,4R-5 and 2RS,4S-5 were prepared by using the corresponding optically pure R and S toluene-4-sulfonic acid 2,3-dihydroxypropyl ester (R-4,S-4). The enatiomerically and diastereomerically pure acetonide (5) was obtained by a method involving diastereoselective crystallisation of the tosylate salt, followed by re-equilibration with the mother liquor and chromatography. The optical purity of four target compounds (YCZ-2013) was confirmed by chiral high-performance liquid chromatography (HPLC) and NMR. The effects of these stereoisomers on Arabidopsis stem elongation indicated that the cis isomers of 2S,4R-YCZ-2013 and 2R,4S-YCZ-2013 exhibited potent inhibitory activity with IC₅₀ values of approximately 24 ± 3 and 24 ± 2 nM, respectively. The IC₅₀ values of the trans isomers of 2S,4S-YCZ-2013 and 2R,4R-YCZ-2013 are approximately 1510 ± 50 and 3900 ± 332 nM, respectively. Co-application of brassinolide (10 nM), the most potent BR, and GA₃ (1 μM) to Arabidopsis seedlings grown in the dark with 2R,4S-YCZ-2013 and 2S,4R-YCZ-2013 revealed that brassinolide recovered the induced dwarfism of Arabidopsis seedlings, whereas GA₃ showed no effect.     

Evolution of temperature responses in the Cladophora vagabunda complex and the C. albida/sericea complex (Chlorophyta)

Differentiation in temperature responses (survival and growth) was investigated among isolates of two tropical to temperate green algal lineages: the Cladophora vagabunda complex and the C. albida/sericea complex. The results were analysed in relation to published data on 18S rRNA and ITS sequence divergence, which have shown that the overall degree of genetic divergence is similar in the two lineages but that very different patterns of radiation have occurred. In the C. vagabunda complex, the two main clades in the well-resolved phylogenetic tree differed mainly in their tolerance to low temperatures. Within-clade variation was no stronger in the Atlantic/Pacific than in the all-Pacific clade. In the C. albida/sericea complex, six distinctive ITS types indicated early radiation. Although distinctive differences were found between some of these types, the thermal responses of others were very similar, indicating physiological stasis. In both lineages there was evidence for some adaptation to local temperature regimes but phylogenetic constraints were generally more important. Isolates with the same ITS sequences showed similar temperature responses even though collected from different climate zones. Evidence was found for a physiological trade-off between growth at high and at low temperatures in the C. albida/sericea complex, whereas, in the C. vagabunda complex, one clade showed more eurythermal growth responses than the other. In the C. vagabunda complex, which is the ancestral lineage of the C. albida/sericea complex, major differentiation was found in cold tolerance but not in heat tolerance, whereas the reverse pattern was found in the derived C. albida/sericea complex. These findings suggest that an acquisition of cold tolerance preceded the loss of heat tolerance during adaptation to colder climates.     

20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol,a metabolite of ginseng,inhibits colon cancer growth by targeting TRPC channel-mediated calcium influx

Abnormal regulation of Ca²⁺ mediates tumorigenesis and Ca²⁺ channels are reportedly deregulated in cancers, indicating that regulating Ca²⁺ signaling in cancer cells is considered as a promising strategy to treat cancer. However, little is known regarding the mechanism by which Ca²⁺ affects cancer cell death. Here, we show that 20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol (20-GPPD), a metabolite of ginseng saponin, causes apoptosis of colon cancer cells through the induction of cytoplasmic Ca²⁺. 20-GPPD decreased cell viability, increased annexin V-positive early apoptosis and induced sub-G1 accumulation and nuclear condensation of CT-26 murine colon cancer cells. Although 20-GPPD-induced activation of AMP-activated protein kinase (AMPK) played a key role in the apoptotic death of CT-26 cells, LKB1, a well-known upstream kinase of AMPK, was not involved in this activation. To identify the upstream target of 20-GPPD for activating AMPK, we examined the effect of Ca²⁺ on apoptosis of CT-26 cells. A calcium chelator recovered 20-GPPD-induced AMPK phosphorylation and CT-26 cell death. Confocal microscopy showed that 20-GPPD increased Ca²⁺ entry into CT-26 cells, whereas a transient receptor potential canonical (TRPC) blocker suppressed Ca²⁺ entry. When cells were treated with a TRPC blocker plus an endoplasmic reticulum (ER) calcium blocker, 20-GPPD-induced calcium influx was completely inhibited, suggesting that the ER calcium store, as well as TRPC, was involved. In vivo mouse CT-26 allografts showed that 20-GPPD significantly suppressed tumor growth, volume and weight in a dose-dependent manner. Collectively, 20-GPPD exerts potent anticarcinogenic effects on colon carcinogenesis by increasing Ca²⁺ influx, mainly through TRPC channels, and by targeting AMPK.     

Epigallocatechin-3-gallate prevents systemic inflammation-induced memory deficiency and amyloidogenesis via its anti-neuroinflammatory properties

Neuroinflammation has been known to play a critical role in the pathogenesis of Alzheimer's disease (AD) through amyloidogenesis. In a previous study, we found that systemic inflammation by intraperitoneal (ip) injection of lipopolysaccharide (LPS) induces neuroinflammation and triggers memory impairment. In this present study, we investigated the inhibitory effects of epigallocatechin-3-gallate (EGCG) on the systemic inflammation-induced neuroinflammation and amyloidogenesis as well as memory impairment. ICR mice were orally administered with EGCG (1.5 and 3 mg/kg) for 3 weeks, and then the mice were treated by ip injection of LPS (250 μg/kg) for 7 days. We found that treatment of LPS induced memory-deficiency-like behavior and that EGCG treatment prevented LPS-induced memory impairment and apoptotic neuronal cell death. EGCG also suppressed LPS-induced increase of the amyloid beta-peptide level and the expression of the amyloid precursor protein (APP), β-site APP cleaving enzyme 1 and its product C99. In addition, we found that EGCG prevented LPS-induced activation of astrocytes and elevation of cytokines including tumor necrosis factor-α, interleukin (IL)-1β, macrophage colony-stimulating factor, soluble intercellular adhesion molecule-1 and IL-16, and the increase of inflammatory proteins, such as inducible nitric oxide synthase and cyclooxygenase-2, which are known factors responsible for not only activation of astrocytes but also amyloidogenesis. In the cultured astrocytes, EGCG also inhibited LPS-induced cytokine release and amyloidogenesis. Thus, this study shows that EGCG prevents memory impairment as well as amyloidogenesis via inhibition of neuroinflammatory-related cytokines released from astrocytes and suggests that EGCG might be a useful intervention for neuroinflammation-associated AD.