Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.     

Anaerobic digestion of secondary residuals from an anaerobic bioreactor at a brewery to enhance bioenergy generation

Many beer breweries use high-rate anaerobic digestion (AD) systems to treat their soluble high-strength wastewater. Biogas from these AD systems is used to offset nonrenewable energy utilization in the brewery. With increasing nonrenewable energy costs, interest has mounted to also digest secondary residuals from the high-rate digester effluent, which consists of yeast cells, bacteria, methanogens, and small (hemi)cellulosic particles. Mesophilic (37 °C) and thermophilic (55 °C) lab-scale, low-rate continuously-stirred anaerobic digestion (CSAD) bioreactors were operated for 258 days by feeding secondary residuals at a volatile solids (VS) concentration of ∼40 g l⁻¹. At a hydraulic retention time (HRT) of 15 days and a VS loading rate of 2.7 g VS l⁻¹ day⁻¹, the mesophilic bioreactor showed an average specific volumetric biogas production rate of 0.88 l CH₄ l⁻¹ day⁻¹ and an effluent VS concentration of 22.2 g VS l⁻¹ (43.0% VS removal efficiency) while the thermophilic bioreactor displayed similar performances. The overall methane yield for both systems was 0.21 l CH₄ g⁻¹ VS fed and 0.47–0.48 l CH₄ g⁻¹ VS removed. A primary limitation of thermophilic digestion of this protein-rich waste is the inhibition of methanogens due to higher nondissociated (free) ammonia (NH₃) concentrations under similar total ammonium (NH₄ ⁺) concentrations at equilibrium. Since thermophilic AD did not result in advantageous methane production rates or yields, mesophilic AD was, therefore, superior in treating secondary residuals from high-rate AD effluent. An additional digester to convert secondary residuals to methane may increase the total biogas generation at the brewery by 8% compared to just conventional high-rate digestion of brewery wastewater alone. JIMB-2008: BioEnergy—Special issue.     

Magnesium Supplementation did not Affect the Increasing Bone Zinc Concentration in Rats Given Excess Calcium as Carbonate

We investigated the effect of magnesium supplementation on zinc distribution in rats given excess calcium as carbonate. Rats were given a control diet (5 g/kg calcium and 0.5 g/kg magnesium), a high calcium diet (HC, 25 g/kg calcium and 0.5 g/kg magnesium) or the high calcium diet supplied with magnesium (HCM, 25 g/kg calcium and 2.5 g/kg magnesium) for 4 weeks. Calcium carbonate and magnesium oxide were used for increasing these mineral concentrations in diets. Although feed intake did not differ among the groups, the excess calcium suppressed feed efficiency, irrespective of dietary magnesium concentration. Femoral magnesium concentration was lower in the HC group than in the control and the HCM groups. Femoral zinc concentration was higher in the HC group and the HCM group than in the control group. The zinc concentration in the kidney was lower in the HC group and the HCM group than in the control group. The excess calcium did not affect zinc concentration in plasma and other tissues such as the liver, testis, and spleen, irrespective of dietary magnesium. These results suggest that the increasing bone zinc and the decreasing renal zinc do not result from magnesium insufficiency in rats given excess calcium as carbonate.     

Effect of acidic pH on the invasion efficiency and the type III secretion system of <Emphasis Type="Italic">Burkholderia thailandensis</Emphasis>

Burkholderia thailandensis is a close relative of Burkholderia pseudomallei. These organisms are very similar, but B. thailandensis is far less virulent than B. pseudomallei. Nucleotide sequencing and analysis of 14 B. thailandensis isolates revealed variation in the regions coding for the type III secreted BipD protein. The degree of B. thailandensis BipD sequence variation was greater than that found in B. pseudomallei. Western blot analysis indicated that, unlike B. pseudomallei, B. thailandensis type III secreted proteins including BipD and BopE could not be detected in the supernatant of culture medium unless induced by acidic conditions. In addition, culturing B. thailandensis under acidic growth conditions (pH 4.5) can induce the ability of this bacterium to invade human respiratory epithelial cells A549. The identification of an environmental stimulus that increases the invasion capability of B. thailandensis invasion is of value for those who would like to use this bacterium as a model to study B. pseudomallei virulence.     

Mycorrhizal detection of native and non-native truffles in a historic arboretum and the discovery of a new North American species, <Emphasis Type="Italic">Tuber arnoldianum</Emphasis> sp. nov.

During a study comparing the ectomycorrhizal root communities in a native forest with those at the Arnold Arboretum in Massachusetts (USA), the European species Tuber borchii was detected on the roots of a native red oak in the arboretum over two successive years. Since T. borchii is an economically important edible truffle native to Europe, we conducted a search of other roots in the arboretum to determine the extent of colonization. We also wanted to determine whether other non-native Tuber species had been inadvertently introduced into this 140-year-old Arboretum because many trees were imported into the site with intact soil and roots prior to the 1921 USDA ban on these horticultural practices in the USA. While T. borchii was not found on other trees, seven other native and exotic Tuber species were detected. Among the North American Tuber species detected from ectomycorrhizae, we also collected ascomata of a previously unknown species described here as Tuber arnoldianum. This new species was found colonizing both native and non-native tree roots. Other ectomycorrhizal taxa that were detected included basidiomycetes in the genera Amanita, Russula, Tomentella, and ascomycetes belonging to Pachyphlodes, Helvella, Genea, and Trichophaea. We clarify the phylogenetic relationships of each of the Tuber species detected in this study, and we discuss their distribution on both native and non-native host trees.     

Topochemical distribution of lignin and hydroxycinnamic acids in sugar-cane cell walls and its correlation with the enzymatic hydrolysis of polysaccharides

Background  

Lignin and hemicelluloses are the major components limiting enzyme infiltration into cell walls. Determination of the topochemical distribution of lignin and aromatics in sugar cane might provide important data on the recalcitrance of specific cells. We used cellular ultraviolet (UV) microspectrophotometry (UMSP) to topochemically detect lignin and hydroxycinnamic acids in individual fiber, vessel and parenchyma cell walls of untreated and chlorite-treated sugar cane. Internodes, presenting typical vascular bundles and sucrose-storing parenchyma cells, were divided into rind and pith fractions.     

Marker-assisted breeding of a photoperiod-sensitive male sterile <Emphasis Type="Italic">japonica</Emphasis> rice with high cross-compatibility with <Emphasis Type="Italic">indica</Emphasis> rice

The incomplete fertility of japonica × indica rice hybrids has inhibited breeders’ access to the substantial heterotic potential of these hybrids. As hybrid sterility is caused by an allelic interaction at a small number of loci, it is possible to overcome it by simple introgression at the major sterility loci. Here we report the use of marker-assisted backcrossing to transfer into the elite japonica cv. Zhendao88 a photoperiod-sensitive male sterility gene from cv. Lunhui422S (indica) and the yellow leaf gene from line Yellow249 (indica). The microsatellite markers RM276, RM455, RM141 and RM185 were used to tag the fertility genes S5, S8, S7 and S9, respectively. Line 509S is a true-breeding photoperiod-sensitive male sterile plant, which morphologically closely resembles the japonica type. Genotypic analysis showed that the genome of line 509S comprises about 92% japonica DNA. Nevertheless, hybrids between line 509S and japonica varieties suffer from a level of hybrid sterility, although the line is highly cross-compatible with indica types, with the resulting hybrids expressing a significant degree of heterosis. Together, these results suggest that segment substitution on fertility loci based on known information and marker-assisted selection are an effective approach for utilizing the heterosis of rice inter-subspecies.     

Synthesis of Cell Coat in Normal and Transformed Cells

THE surface of transformed cells has been a focus of considerable attention recently because some of the properties which distinguish these cells from their precursors, such as decreased cell adhesiveness, altered cell orientation and loss of contact and density dependent inhibition^1–3, may relate to changes on their surface. A common feature of vertebrate cells is the cell coat, a glycoprotein structure surrounding the plasma membrane⁴. Electron microscopy has revealed that transformed cells have a thicker coat than normal cells⁵ and we have now found that coat synthesis in cells transformed by an oncogenic DNA virus and in cells transformed by a chemical carcinogen occurs faster than in normal controls whereas only in the virus-transformed cells is the coat significantly thicker.     

Production and characterization of intergeneric somatic hybrids between <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> and their backcrossing progenies

Alien chromosome addition lines have been widely used for identifying gene linkage groups, assigning species-specific characters to a particular chromosome and comparing gene synteny between related species. In plant breeding, their utilization lies in introgressing characters of agronomic value. The present investigation reports the production of intergeneric somatic hybrids Brassica napus (2n = 38) + Orychophragmus violaceus (2n = 24) through asymmetric fusions of mesophyll protoplasts and subsequent development of B. napus-O. violaceous chromosome addition lines. Somatic hybrids showed variations in morphology and fertility and were mixoploids (2n = 51–67) with a range of 19–28 O. violaceus chromosomes identified by genomic in situ hybridization (GISH). After pollinated with B. napus parent and following embryo rescue, 20 BC₁ plants were obtained from one hybrid. These exhibited typical serrated leaves of O. violaceus or B. napus-type leaves. All BC₁ plants were partially male fertile but female sterile because of abnormal ovules. These were mixoploids (2n = 41–54) with 9–16 chromosomes from O. violaceus. BC₂ plants showed segregations for female fertility, leaf shape and still some chromosome variation (2n = 39–43) with 2–5 O. violaceus chromosomes, but mainly containing the whole complement from B. napus. Among the selfed progenies of BC₂ plants, monosomic addition lines (2n = 39, AACC + 1O) with or without the serrated leaves of O. violaceus or female sterility were established. The complete set of additions is expected from this investigation. In addition, O. violaceus plants at diploid and tetraploid levels with some variations in morphology and chromosome numbers were regenerated from the pretreated protoplasts by iodoacetate and UV-irradiation. Z. Zhao and T. Hu make equal contributions to this work.     

Nigericin inhibits epithelial ovarian cancer metastasis by suppressing the cell cycle and epithelial−mesenchymal transition

Epithelial ovarian cancer (EOC) has the highest mortality among various types of gynecological malignancies. Most patients die of metastasis and recurrence due to cisplatin resistance. Thus, it is urgent to develop novel therapies to cure this disease. CCK-8 assay showed that nigericin exhibited strong cytotoxicity on A2780 and SKOV3 cell lines. Flow cytometry indicated that nigericin could induce cell cycle arrest at G0/G1 phase and promote cell apoptosis. Boyden chamber assay revealed that nigericin could inhibit migration and invasion in a dose-dependent manner by suppressing epithelial–mesenchymal transition (EMT) in EOC cells. These effects were mediated, at least partly, by the Wnt/β-catenin signaling pathway. Our results demonstrated that nigericin could inhibit EMT during cell invasion and metastasis through the canonical Wnt/β-catenin signaling pathway. Nigericin may prove to be a novel therapeutic strategy that is effective in patients with metastatic EOC.