Characterization of halotolerant Bicosoecida and Placididea (Stramenopila) that are distinct from marine forms,and the phylogenetic pattern of salinity preference in heterotrophic stramenopiles

Recent culture‐based studies demonstrate the distinctiveness of the microbial eukaryote biota of very hypersaline environments. In contrast, microscopy‐based faunistic studies suggest that the biota of habitats of more moderate hypersalinity (60–150‰) overlaps substantially with that of marine environments, but this has barely been studied with modern techniques. To investigate the diversity and salinity tolerance range of these organisms, eight cultures of heterotrophic stramenopiles were established from (or from nearby) moderately hypersaline locations. These isolates represent five independent groups; Groups A, B and C are bicosoecids; Groups D and E belong to Placididea. One isolate (Group A) is a strain of the widespread marine species Cafeteria roenbergensis, and cannot grow above 100‰ salinity. The other isolates – Groups B–E – can all grow at 150–175‰ salinities and are probably moderate halophiles. Groups B–E all represent previously unsequenced species or even genera, although Group B is the sister group of the borderline extreme halophile Halocafeteria. The high level of novelty en countered suggests that moderately hypersaline environments may harbour a heterotrophic stramenopile biota distinct from that of marine environments. Interestingly, our new isolates are all most closely related to marine or halophilic forms, and our phylogenies show large clades defined by saline/non‐saline habitats within bicosoecids, placidomonads and related lineages. In particular, most freshwater/soil bicosoecids form one well‐supported clade. The sole major exception is Bicosoeca, which is intermixed with marine environmental sequences originally referred to as ‘MAST‐13’, which are from brackish water, not typical seawater. It seems that the freshwater/marine barrier has been crossed very few times in the evolutionary history of these heterotrophic stramenopile flagellates.     

Reorganization of horizontal cell processes in the developing FVB/N mouse retina

We investigated the morphological changes of horizontal cells after postnatal photoreceptor degeneration in the developing FVB/N mouse retina, using immunocytochemistry with anti-calbindin D-28K. From postnatal day 14 (P14) onwards, processes emerging from horizontal cells descend into the inner plexiform layer (IPL) and ramify mainly in stratum 1 of the IPL. Electron microscopy revealed that the descending processes make synaptic contacts with bipolar cells in the outer plexiform layer. Our results clearly demonstrate that loss of photoreceptor cells induces the reorganization of horizontal cell processes in the retinas of FVB/N mice as they mature.     

Characterization of a Bifidobacterium longum BORI dipeptidase belonging to the U34 family

A dipeptidase was purified from a cell extract of Bifidobacterium longum BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50 degrees C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides, p-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search of the amino acid sequence of an internal tryptic fragment against protein sequences deduced from the total genome sequence of B. longum NCC2705 revealed that it was identical to an internal sequence of the dipeptidase gene (pepD), which comprised 1,602 nucleotides encoding 533 amino acids with a molecular mass of 60 kDa, and thereby differed considerably from the 49-kDa mass of the purified dipeptidase. To understand this discrepancy, pepD was cloned into an Escherichia coli expression vector (pBAD-TOPO derivative) to generate the recombinant plasmids pBAD-pepD and pBAD-pepD-His (note that His in the plasmid designation stands for a polyhistidine coding region). Both plasmids were successfully expressed in E. coli, and the recombinant protein PepD-His was purified using nickel-chelating affinity chromatography and reconfirmed by internal amino acid sequencing. The PepD sequence was highly homologous to those of the U34 family of peptidases, suggesting that the B. longum BORI dipeptidase is a type of cysteine-type N-terminal nucleophile hydrolase and has a beta-hairpin motif similar to that of penicillin V acylase, which is activated by autoproteolytic processing.     

Sonoporation is an efficient tool for intracellular fluorescent dextran delivery and one-step double-crossover mutant construction in Fusobacterium nucleatum

Studies of microorganisms are often hindered by a lack of effective genetic tools. One such example is Fusobacterium nucleatum, a gram-negative anaerobe associated with various human infections, including those causing periodontal disease and preterm birth. The first double-crossover allelic-exchange mutant in F. nucleatum was recently constructed using sonoporation, a novel ultrasound-mediated intracellular delivery method, demonstrating potential for bacterial gene transfection. To better unveil its mechanism, the current study examines the factors affecting the outcome of sonoporation. Delivery of Texas Red-conjugated dextran into F. nucleatum by sonoporation was at least twice as efficient as that by electroporation, and sonoporation was nonbactericidal, unlike electroporation. The delivery efficiency was affected by the acoustic pressure amplitude, the duty cycle, and the quantity of microbubbles used to initiate cavitation but not by the pulse repetition frequency of ultrasound application. To examine the involvement of homologous recombination in sonoporation-mediated mutant construction, the highly conserved recA gene, which carried most of the consensus residues, including the P loop, was identified in F. nucleatum, and a double-crossover recA mutant of F. nucleatum 12230, US1610, was constructed by sonoporation. The mutant exhibited increased sensitivity to UV exposure compared with that of the wild type, indicating that the RecA function in F. nucleatum was conserved. Interestingly, US1610 was also sensitive to ultrasound treatment, suggesting the likely involvement of RecA in postsonoporation repair and survival. Since sonoporation has consistently generated one-step double-crossover mutants in F. nucleatum by use of intact suicide plasmids, this technology may be developed into an efficient tool for streamlining mutant construction in bacteria.     

Pharmacokinetics of 125I-GST-TatdMt, a recombinant fusion protein possessing potent anti-obesity activity, after intravenous, nasal, oral, and subcutaneous administration

This study first reports the absorption kinetics of GST-TatdMt, a recombinant Tat protein possessing potent anti-obesity activity, in rats after nasal, s.c., and p.o. administration. GST-TatdMt was over-expressed in E. coli, purified, and radioiodinated using the IODO-GEN method. The radioiodinated 125I-GST-TatdMt was administered to rats by nasal, s.c., and oral routes at doses of 7.3 microg (420.7 nCi), 146.5 microg (8413.8 nCi), and 146.5 microg (8413.8 nCi), respectively. For the determination of absolute bioavailability, 125I-GST-TatdMt was also given to rats by i.v. injection (73.2 microg, 4206.9 nCi). Following administration by extravascular routes, the systemic absorption of radioactivity was prolonged, with Cmax being attained within 4.2-8.0 h. The absolute bioavailability calculated as dose-normalized AUC(extravascular)/AUC(i.v.) was 98.0, 75.8, and 87.1% after nasal, s.c., and oral administration, respectively. The majority of administered radioactivity was excreted in urine (57.5-64.7%), with fecal excretion being less (2.5-12.7%). The distribution of 125I-GST-TatdMt to various tissues was also determined at 4 and 72 h after s.c. injection. The findings of this study suggest that this protein may be absorbed into the systemic circulation when given by extravascular administration.     

Synergistic effect of TGFbeta-3 on chondrogenic differentiation of rabbit chondrocytes in thermo-reversible hydrogel constructs blended with hyaluronic acid by in vivo test

In this study, a hydrogel composite, based on the thermo-reversible hydrogel of p(NiPAAm-co-AAc) and hyaluronic acid (HA) was used as an injectable cell and growth factor carrier for cartilage tissue engineering applications. Rabbit chondrocytes were embedded in blended hydrogel composites co-encapsulated with the transforming growth factor beta-3 (TGFbeta-3). The blended hydrogel with the embedded chondrocytes and HA co-encapsulating unloaded growth factors and those with the thermo-reversible hydrogel were used as the controls to examine the effects of TGFbeta-3 on neocartilage formation. The blended hydrogel loaded with TGFbeta-3 embedded with chondrocytes were injected subcutaneously into the nude mice. The mice were monitored for 8 weeks after the injection. Both the differentiation and level of cartilage-specific ECM production were significantly higher in the presence of HA and growth factor than in the control without the growth factor. The level of cartilage associated ECM proteins was examined by immunohistochemical staining (collagen types II and X) as well as by Safranin-O and Alcian blue (GAG) staining. The results showed the potential application of blended hydrogel mixed with the growth factor to neocartilage formation.     

Analysis of gene-trap Ds rice populations in Korea

Insertional mutagen-mediated gene tagging populations have been essential resources for analyzing the function of plant genes. In rice, maize transposable elements have been successfully utilized to produce transposant populations. However, many generations and substantial field space are required to obtain a sufficiently sized transposant population. In rice, the japonica and indica subspecies are phenotypically and genetically divergent. Here, callus cultures with seeds carrying Ac and Ds were used to produce 89,700 lines of Dongjin, a japonica cultivar, and 6,200 lines of MGRI079, whose genome is composed of a mixture of the genetic backgrounds of japonica and indica. Of the more than 3,000 lines examined, 67% had Ds elements. Among the Ds-carrying lines, 81% of Dongjin and 63% of MGRI079 contained transposed Ds, with an average of around 2.0 copies. By examining more than 15,000 lines, it was found that 12% expressed the reporter gene GUS during the early-seedling stage. GUS was expressed in root hairs and crown root initials at estimated frequencies of 0.78% and 0.34%, respectively. The 5,271 analyzed Ds loci were found to be randomly distributed over all of the rice chromosomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sung Han Park, Nam Soo Jun, Chul Min Kim are contributed equally to this paper     

Glycyrrhizin protection against 3-morpholinosydnonime-induced mitochondrial dysfunction and cell death in lung epithelial cells

The present study was designed to assess the preventive effect of licorice compounds glycyrrhizin and 18beta-glycyrrhetinic acid against mitochondrial damage and cell death in lung epithelial cells exposed to 3-morpholinosydnonime, a donor of nitric oxide and superoxide. Treatment of lung epithelial cells with 3-morpholinosydnonime resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. Treatment of glycyrrhizin and 18beta-glycyrrhetinic acid attenuated the 3-morpholinosydnonime-induced mitochondrial damage, formation of reactive oxygen species and GSH depletion and revealed a maximal inhibitory effect at 10 and 1 muM, respectively; beyond these concentrations the inhibitory effect declined. Melatonin, carboxy-PTIO, rutin and uric acid reduced the 3-morpholinosydnonime-induced cell death. The results show that glycyrrhizin and 18beta-glycyrrhetinic acid seem to prevent the toxic effect of 3-morpholinosydnonime against lung epithelial cells by suppressing the mitochondrial permeability transition that leads to the release of cytochrome c and activation of caspase-3. The preventive effect may be ascribed to the inhibitory action on the formation of reactive oxygen species and depletion of GSH. The findings suggest that licorice compounds seem to prevent the nitrogen species-mediated lung cell damage.     

Detoxification of hydrogen peroxide maintains the water transport activity in figleaf gourd (Cucurbita ficifolia) root system exposed to low temperature

The figleaf gourd ( Cucurbita ficifolia Bouché) root system has the ability to take up water and nutrients at low soil temperatures, and in the present paper, we attempt to reveal some of the molecular mechanisms behind this low-temperature tolerance. Exposure of figleaf gourd root system to low temperature induced accumulation of H₂O₂ along the plasma membrane but not in the cytoplasm. H⁺-ATPase (EC 3.6.1.35) activity of isolated root plasma membranes and root hydraulic conductivity ( Lp_r ) were largely insensitive to externally applied H₂O₂. However, using bromocresol purple, it was shown that the acidification of the medium surrounding the root was strongly inhibited with low temperature- and H₂O₂-treated roots. Addition of catalase (EC 1.11.1.6) to the root medium during low-temperature exposure led to a recovery of H⁺-efflux along the root surface and increased Lp_r , demonstrating the importance of an H₂O₂ detoxification system in the root cells. Additional evidence for an increased Lp_r was obtained by the Fenton reaction wherein a warming of the solution increased the activity of the detoxification system. All available evidence suggests that the ability of figleaf gourd root system to maintain a low level of H₂O₂ in the cytoplasm and to detoxify reactive oxygen species is related to the maintenance of water transport activity at low temperatures.