The Arabidopsis pex12 and pex13 mutants are defective in both PTS1- and PTS2-dependent protein transport to peroxisomes

Peroxisome biogenesis requires various complex processes including organelle division, enlargement and protein transport. We have been studying a number of Arabidopsis apm mutants that display aberrant peroxisome morphology. Two of these mutants, apm2 and apm4, showed green fluorescent protein fluorescence in the cytosol as well as in peroxisomes, indicating a decrease of efficiency of peroxisome targeting signal 1 (PTS1)-dependent protein transport to peroxisomes. Interestingly, both mutants were defective in PTS2-dependent protein transport. Plant growth was more inhibited in apm4 than apm2 mutants, apparently because protein transport was more severely decreased in apm4 than in apm2 mutants. APM2 and APM4 were found to encode proteins homologous to the peroxins PEX13 and PEX12, respectively, which are thought to be involved in transporting matrix proteins into peroxisomes in yeasts and mammals. We show that APM2/PEX13 and APM4/PEX12 are localized on peroxisomal membranes, and that APM2/PEX13 interacts with PEX7, a cytosolic PTS2 receptor. Additionally, a PTS1 receptor, PEX5, was found to stall on peroxisomal membranes in both mutants, suggesting that PEX12 and PEX13 are components that are involved in protein transport on peroxisomal membranes in higher plants. Proteins homologous to PEX12 and PEX13 have previously been found in Arabidopsis but it is not known whether they are involved in protein transport to peroxisomes. Our findings reveal that APM2/PEX13 and APM4/PEX12 are responsible for matrix protein import to peroxisomes in planta.     

Contribution of spermatozoal centrosomes to the microtubule-organizing centre in Antarctic minke whale ( Balaenoptera bonaerensis )

Using an interspecies microinsemination assay with bovine oocytes, it was examined whether centrosomes of Antarctic minke whale spermatozoa function as the microtubule-organizing centre (MTOC). Bull and rat spermatozoa were used as positive and negative controls, respectively. Vitrified-warmed bovine mature oocytes were subjected to immunostaining against alpha-tubulin 4-6 h after intracytoplasmic injection (ICSI) of 5 mM dithiothreitol-treated spermatozoa. Aster formation occurred from whale spermatozoa (33%) and bull spermatozoa (33%), but very little from rat spermatozoa (3%). Activation treatment for the microinseminated oocytes with 7% ethanol + 2 mM 6-dimethylaminopurine resulted in a similar proportion of oocytes forming a whale sperm aster (35% vs 27% in the non-treated group; 4 h after ICSI) but a significantly larger aster (ratio of aster diameter to oocyte diameter, 0.57 vs 0.30 in the non-treated group). These results indicate that the centrosome introduced into bovine oocytes by whale spermatozoa contributes to the MTOC and that assembly of the microtubule network is promoted by oocyte activation.     

Discovery of heteroaryl sulfonamides as new EP1 receptor selective antagonists

4-({2-[Isobutyl(phenylsulfonyl)amino]-5-(trifluoromethyl)phenoxy}methyl)benzoic acid (1) is a functional PGE2 antagonist selective for EP1 receptor subtype. Analogs of 1, in which the phenyl-sulfonyl moiety has been replaced with more hydrophilic heteroarylsulfonyl moieties, exhibited more optimized antagonist activity, while some of them showed in vivo antagonist activity. Structure-activity relationship (SAR) studies are also presented.     

Optimization of sulfonamide derivatives as highly selective EP1 receptor antagonists

A series of 4-[(2-{isobutyl[(5-methyl-2-furyl)sulfonyl]amino}phenoxy)methyl]benzoic acids and 4-({2-[isobutyl(1,3-thiazol-2-ylsulfonyl)amino]phenoxy}methyl)benzoic acids were synthesized and evaluated for their EP receptor affinities and EP1 receptor antagonist activities. Further structural optimization was carried out to reduce inhibitory activity against hepatic cytochrome P450 isozymes, which could represent a harmful potential drug interaction. Selected compounds were also evaluated for their binding affinities to hTP, hDP, mFP, and hIP, and for their hEP1 receptor antagonist activities. The results of structure-activity relationship studies are also presented.     

Fucosylation of N-glycans regulates the secretion of hepatic glycoproteins into bile ducts

Fucosylated alpha-fetoprotein (AFP) is a highly specific tumor marker for hepatocellular carcinoma (HCC). However, the molecular mechanism by which serum level of fucosylated AFP increases in patients with HCC remains largely unknown. Here, we report that the fucosylation of glycoproteins could be a possible signal for secretion into bile ducts in the liver. We compared oligosaccharide structures on glycoproteins in human bile with those in serum by several types of lectin blot analyses. Enhanced binding of biliary glycoproteins to lectins that recognize a fucose residue was observed over a wide range of molecular weights compared with serum glycoproteins. A structural analysis of oligosaccharides by two-dimensional mapping high performance liquid chromatography and matrix-assisted laser desorption ionization time-of flight mass spectrometry confirmed the increases in the fucosylation of biliary glycoproteins. Purification followed by structural analysis on alpha1-antitrypsin, alpha1-acid glycoprotein and haptoglobin, which are synthesized in the liver, showed higher fucosylation in bile than in serum. To find direct evidence for fucosylation and sorting signal into bile ducts, we used alpha1-6 fucosyltransferase (Fut8)-deficient mice because fucosylation of glycoproteins produced in mouse liver was mainly an alpha1-6 linkage. Interestingly, the levels of alpha1-antitrypsin and alpha1-acid glycoprotein were quite low in bile of Fut8-deficient mice as compared with wild-type mice. An immunohistochemical study showed dramatic changes in the localization of these glycoproteins in the liver of Fut8-deficient mice. Taken together, these results suggest that fucosylation is a possible signal for the secretion of glycoproteins into bile ducts in the liver. A disruption in this system might involve an increase in fucosylated AFP in the serum of patients with HCC.     

Direct interaction of post-synaptic density-95/Dlg/ZO-1 domain-containing synaptic molecule Shank3 with GluR1 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor

A class of scaffolding protein containing the post-synaptic density-95/Dlg/ZO-1 (PDZ) domain is thought to be involved in synaptic trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors during development. To clarify the molecular mechanism of AMPA receptor trafficking, we performed a yeast two-hybrid screening system using the cytoplasmic tail of the GluR1 subunit of AMPA receptor as a bait and identified a synaptic molecule, Shank3/ProSAP2, as a GluR1 subunit-interacting molecule. Shank3 is a PDZ domain-containing multidomain protein and is predominantly expressed in developing neurons. Using the glutathione S-transferase pull-down assay and immunoprecipitation technique we demonstrated that the GluR1 subunit directly binds to the PDZ domain of Shank3 via its carboxyl terminal PDZ-binding motif. We raised anti-Shank3 antibody to investigate the expression of Shank3 in cortical neurons. The pattern of Shank3 immunoreactivity was strikingly punctate, mainly observed in the spines, and closely matched the pattern of post-synaptic density-95 immunoreactivity, indicating that Shank3 is colocalized with post-synaptic density-95 in the same spines. When Shank3 and the GluR1 subunit were overexpressed in primary cortical neurons, they were also colocalized in the spines. Taken together with the biochemical interaction of Shank3 with the GluR1 subunit, these results suggest that Shank3 is an important molecule that interacts with GluR1 AMPA receptor at synaptic sites of developing neurons.     

Applications of yeast cell-surface display in bio-refinery

The dependency on depleting natural resources is a challenge for energy security that can be potentially answered by bioenergy. Bioenergy is derived from starchy and lignocellulosic biomass in the form of bioethanol or from vegetable oils in the form of biodiesel fuel. The acid and enzymatic methods have been developed for the hydrolysis of biomass and for transesterifiaction of plant oils. However, acid hydrolysis results in the production of unnatural compounds which has adverse effects on yeast fermentation. Recent advancements in the yeast cell surface engineering developed strategies to genetically immobilize amylolytic, cellulolytic and xylanolytic enzymes on yeast cell surface for the production of fuel ethanol from biomass. This review gives an insight in to the recent technological developments in the production of bioenergy, i.e, bioethanol using surface engineered yeast.     

A simple method for preserving environmental DNA in water samples at ambient temperature by addition of cationic surfactant

Environmental DNA (eDNA) analysis is a powerful tool within ecology for the study of the distribution or abundance of aquatic species, although the simplification of water sampling is required for enabling light and fast field sampling to expand further application of eDNA analysis. Here, certain candidate chemicals belonging to the group of cationic surfactants were examined for their effectiveness as preservatives for eDNA water samples by simply adding the chemicals to water samples to suppress the degradation of eDNA. The quaternary ammonium compound benzalkonium chloride (BAC) at a final concentration of 0.01% was effective to retain 92% of eDNA derived from the bluegill sunfish Lepomis macrochirus in an 8-h incubation test at ambient temperature, which assumed a transportation of water samples in 1-day field sampling during the daytime. Meanwhile, eDNA in water samples without BAC retained only 14% of the initial eDNA. Moreover, an additional long-term incubation test (up to 10 days) revealed BAC-treated samples retained ~70 and 50% of bluegill DNA compared to the initial amount after 1- and 10-day incubation at ambient temperature, respectively. Meanwhile, eDNA in naïve samples reduced to 20% after 1-day incubation and reached undetectable levels after 10 days. Up to now, many eDNA studies have adopted on-site filtration followed by filter fixation, which requires many pieces of equipment. Addition of BAC can protect eDNA in water samples with less effort and equipment resulting in an increase of measurement accuracy of the eDNA quantity and detection probability of rare species by preventing the disappearance of rare sequences in water samples.     

On the Biology of the Colombian Fluted Scale, <Emphasis Type="Italic">Crypticerya multicicatrices</Emphasis> Kondo &amp; Unruh (Hemiptera: Monophlebidae)

The Colombian fluted scale Crypticerya multicicatrices Kondo & Unruh (Hemiptera: Monophlebidae) is a polyphagous insect pest native to continental Colombia and reported on 148 species, mainly palms (Arecaceae); mango, Mangifera indica (Anacardiaceae); and leguminous trees and shrubs (Fabaceae). The biology and ecological parameters of C. multicicatrices were investigated using as a host plant Caesalpinia pluviosa var. peltophoroides (Fabaceae) under two environmental conditions: semifield (max temp., 31.5?±?3.0°C; max. RH, 78.9?±?6.0%) and a glass house (max. temp., 37.1?±?5.1°C, max. RH, 67.0?±?6.2%) in the premises of Corpoica, Palmira Research Station. The duration of the different developmental stages was highly different under both temperature conditions. First- and third-instar nymphs, the period of ovisac growth of the adult, and the duration of the life cycle were significantly higher in the semifield conditions compared to the glass house conditions. In contrast, the second-instar nymph stage was shorter in the semifield conditions. The duration of the fourth-instar stage (adult) and the reproductive period were the same in both environments. As for the evaluated ecological parameters, a great difference was found between the two environmental conditions, where the net reproductive rate (R _o) and mean generation time (T) were significantly higher in the semifield conditions than in the glass house conditions. The obtained biological and ecological information is an essential tool for finding strategies to control this insect pest. This is the first detailed study on the biology of a species in the genus Crypticerya Cockerell.