Comparison of the primary structure ofwaxy proteins (granule-bound starch synthase) between polyploid wheats and related diploid species

Thewaxy proteins encoded by the genomes A, B, and D in polyploid wheats and related diploid species were isolated by SDS-PAGE. The N-terminal amino acid sequences of mature proteins and V8 protease-induced fragments were determined. A total of five amino acid substitutions was detected in these sequences, which represent about 10% of the whole sequences of thewaxy proteins. A comparison of these sequences in polyploid wheats with those in related diploid species revealed the following: (i)waxy proteins encoded by the A genome of polyploid wheats were identical to that ofTriticum monococcum, (ii) thewaxy protein encoded by the B genome ofT. turgidum was identical to that ofT. searsii, but differed from those ofT. speltoides andT. longissimum by one amino acid substitution, (iii) thewaxy protein encoded by the B genome ofT. aestivum differed from that encoded by the B genome ofT. turgidum by one amino acid substitution, and (iv) thewaxy protein encoded by the D genome ofT. aestivum was identical to that ofT. tauschii.     

A simple scaling of the effectiveness of supporting mutual cooperation in donor-recipient games by various reciprocity mechanisms

In donor-recipient games (DRG), one of the sub-classes of Prisoner's Dilemma (PD), it is well-known that a game structure is described by two parameters benefit (b) and cost (c) of cooperation. By means of a series of numerical experiments, we proved that the effectiveness of supporting mutual cooperation in DRG by various reciprocity mechanisms can be expressed in a single game structural parameter, b/c. This also implies that the dilemma strength in various donor-recipient games with various reciprocity mechanisms can be evaluated only by b/c, which is consistent with the previous novel finding by Nowak. It was also discussed whether this kind of parameterization idea can be extended to general games in PD game class.     

Differential antimutagenic effects of caffeine and the protease inhibitor antipain on mutagenesis by various mutagens in Escherichia coli

The effects of caffeine (2 mg/ml) and the protease inhibitor antipain (1.75 mb/ml) in the plating agar medium on the yields of prototrophic revertants induced by 10 mutagens in E. coli uvrA^? strains were tested. Mutagenesis by 4-nitroquinoline 1-oxide was greatly diminished by both caffeine and antipain. UV mutagenesis was decreased moderately by caffeine, and greatly by antipain. X-Ray mutagenesis was decreased very slightly by both caffeine and antipain. Mutagenesis by N-hydroxyurethan was inhibited moderately by caffeine, and greatly by antipain; that by methyl methanesulfonate was inhibited moderately by both caffeine and antipain, and that by N-methyl-N′-nitro-N-nitrosoguanidine wa was not suppressed by caffeine but was inhibited moderately by antipain. Mutagenesis by ethyl methanesulfonate was inhibited greatly by caffeine, but only slightly by antipain. The antimutagenic effect of caffeine was strong on furylfuramide (AF-2) mutagenesis, moderate on that of mitomycin C (tested with B/r type strain) and negligible on that of N-methyl-N-nitrosourea. These diverse antimutagenesis patterns are briefly discussed in relation to the current idea that antipain antimutagenesis is due to inhibition of inducible error-prone repair.     

An additional copy of the adenylate cyclase-encoding gene relieves developmental defects produced by a mutation in a vegetative incompatibility-controlling gene in Podospora anserina

To identify cellular functions involved in vegetative incompatibility in filamentous fungi, we have initiated the cloning of Podospora anserina (Pa) mod genes. These genes interfere with the lethal reaction triggered by interaction between incompatible het genes. A gene (Pa AC) has been cloned by complementation of developmental defects caused by a mutation in the mod-D gene. This gene encodes a protein of 2145 amino acids (aa) that exhibits strong similarities with many adenylate cyclases (AC). About 65% aa identity has been found between the sequence of the polypeptide encoded by this Pa AC gene and the AC of Neurospora crassa. The organization of peptidic domains in the polypeptide encoded by Pa AC is closely related to that of Saccharomyces cerevisiae CYRI. Restriction-fragment-length polymorphism (RFLP) and genetic analysis have shown that Pa AC and mod-D are distinct genes     

Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris

Yeast is capable of performing posttranslational modifications, such as N- or O-glycosylation. It has been demonstrated that N-glycans play critical biological roles in therapeutic glycoproteins by modulating pharmacokinetics and pharmacodynamics. However, N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated, and hence, not completely occupied. Genomic homology analysis indicates that the Pichia pastoris oligosaccharyltransferase (OST) complex consists of multiple subunits, including OST1, OST2, OST3, OST4, OST5, OST6, STT3, SWP1, and WBP1. Monoclonal antibodies produced in P. pastoris show that N-glycan site occupancy ranges from 75–85 % and is affected mainly by the OST function, and in part, by process conditions. In this study, we demonstrate that N-glycan site occupancy of antibodies can be improved to greater than 99 %, comparable to that of antibodies produced in mammalian cells (CHO), by overexpressing Leishmania major STT3D (LmSTT3D) under the control of an inducible alcohol oxidase 1 (AOX1) promoter. N-glycan site occupancy of non-antibody glycoproteins such as recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was also significantly improved, suggesting that LmSTT3D has broad substrate specificity. These results suggest that the glycosylation status of recombinant proteins can be improved by heterologous STT3 expression, which will allow for the customization of therapeutic protein profiles.     

Effects of egt gene transfer on the development of Bombyx mori

This study investigated the effects of gain of ecdysteroid UDP-glucosyltransferase (EGT) gene function mutation on the development of the silkworm, Bombyx mori. A novel piggyBac-derived plasmid containing the egt gene from B. mori nucleopolyhedrovirus (BmNPV) driven by a heat-shock protein (hsp) 23.7 promoter, with a neomycin-resistance gene (neo) controlled by the BmNPV ie-1 promoter and a green fluorescent protein gene (gfp) under the control of the B. mori actin 3 (A3) promoter was constructed. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes and gene transfer was verified by polymerase chain reaction, dot-blot hybridization and western blotting. The hatching rate of G1 generation silkworm eggs was about 60% lower than that of normal silkworm eggs. The duration of the G1 generation larval period was extended, and the G2 generation pupal stage lasted four days longer than that in non-transgenic silkworms. The ecdysone blood level in G2 silkworms in the third instar molting stage was reduced by up to 90%. These results show that EGT suppressed transgenic silkworm molting, and that egt expression in egt-transgenic silkworms resulted in arrest of metamorphosis from pupae to moths.     

Differential epigenetic regulation of BDNF and NT-3 genes by trichostatin A and 5-aza-2′-deoxycytidine in Neuro-2a cells

To understand epigenetic regulation of neurotrophins in Neuro-2a mouse neuroblastoma cells, we investigated the alteration of CpG methylation of brain-derived neurotrophic factor (BDNF) promoter I and neurotrophin-3 (NT-3) promoter IB and that of histone modification in Neuro-2a cells. Bisulfite genomic sequencing showed that the CpG sites of BDNF promoter I were methylated in non-treated Neuro-2a cells and demethylated following 5-aza-2′-deoxycytidine (5-aza-dC) treatment. In contrast, methylation status of the NT-3 promoter IB did not change by 5-aza-dC treatment in Neuro-2a cells. Furthermore, we demonstrated that BDNF exon I-IX mRNA was induced by trichostatin A (TSA) treatment. However, NT-3 exon IB-II mRNA was not induced by TSA treatment. Chromatin immunoprecipitation assays showed that the levels of acetylated histones H3 and H4 on BDNF promoter I were increased by TSA. These results demonstrate that DNA methylation and/or histone modification regulate BDNF gene expression, but do not regulate NT-3 gene expression in Neuro-2a cells.     

Dhrs3 Protein Attenuates Retinoic Acid Signaling and Is Required for Early Embryonic Patterning

All-trans-retinoic acid (atRA) is an important morphogen involved in many developmental processes, including neural differentiation, body axis formation, and organogenesis. During early embryonic development, atRA is synthesized from all-trans-retinal (atRAL) in an irreversible reaction mainly catalyzed by retinal dehydrogenase 2 (aldh1a2), whereas atRAL is converted from all-trans-retinol via reversible oxidation by retinol dehydrogenases, members of the short-chain dehydrogenase/reductase family. atRA is degraded by cytochrome P450, family 26 (cyp26). We have previously identified a short-chain dehydrogenase/reductase 3 (dhrs3), which showed differential expression patterns in Xenopus embryos. We show here that the expression of dhrs3 was induced by atRA treatment and overexpression of Xenopus nodal related 1 (xnr1) in animal cap assay. Overexpression of dhrs3 enhanced the phenotype of excessive cyp26a1. In embryos overexpressing aldh1a2 or retinol dehydrogenase 10 (rdh10) in the presence of their respective substrates, Dhrs3 counteracted the action of Aldh1a2 or Rdh10, indicating that retinoic acid signaling is attenuated. Knockdown of Dhrs3 by antisense morpholino oligonucleotides resulted in a phenotype of shortened anteroposterior axis, reduced head structure, and perturbed somitogenesis, which were also found in embryos treated with an excess of atRA. Examination of the expression of brachyury, not, goosecoid, and papc indicated that convergent extension movement was defective in Dhrs3 morphants. Taken together, these studies suggest that dhrs3 participates in atRA metabolism by reducing atRAL levels and is required for proper anteroposterior axis formation, neuroectoderm patterning, and somitogenesis.     

WUSCHEL-RELATED HOMEOBOX4 Is Involved in Meristem Maintenance and Is Negatively Regulated by the CLE Gene FCP1 in Rice

The shoot apical meristem is the ultimate source of the cells that constitute the entire aboveground portion of the plant body. In Arabidopsis thaliana, meristem maintenance is regulated by the negative feedback loop of WUSCHEL-CLAVATA (WUS-CLV). Although CLV-like genes, such as FLORAL ORGAN NUMBER1 (FON1) and FON2, have been shown to be involved in maintenance of the reproductive meristems in rice (Oryza sativa), current understanding of meristem maintenance remains insufficient. In this article, we demonstrate that the FON2-LIKE CLE PROTEIN1 (FCP1) and FCP2 genes encoding proteins with similar CLE domains are involved in negative regulation of meristem maintenance in the vegetative phase. In addition, we found that WUSCHEL-RELATED HOMEOBOX4 (WOX4) promotes the undifferentiated state of the meristem in rice and that WOX4 function is associated with cytokinin action. Consistent with similarities in the shoot apical meristem phenotypes caused by overexpression of FCP1 and downregulation of WOX4, expression of WOX4 was negatively regulated by FCP1 (FCP2). Thus, FCP1/2 and WOX4 are likely to be involved in maintenance of the vegetative meristem in rice.     

The resistances of Micrococcus radiodurans to killing and mutation by agents which damage DNA

The resistance of Micrococcus radiodurans to the lethal and mutagenic action 3f ultraviolet (UV) light, ionising (γ) radiation, mitomycin C (MTC), nitrous acid (NA), hydroxylamine (HA), N-methyl-N′-nitro-N-nitrosoguanidine (NG), ethylmethanesulphonate (EMS) and β-propiolactone (βPL) has been compared with that of Escherichia coli B/r.M. radiodurans was much more resistant than E. coli B/r to the lethal effects of UV light (by a factor of 33), γ-radiation (55), NG (15) and NA (62), showed intermediate resistance to MTC (4) and HA(7), but was sensitive to EMS (1) and βPL (2). M. radiodurans was very resistant to mutagens producing damage which can be repaired by a recombination system, indicating that it possesses an extremely efficient recombination repair mechanism.Both species were equally sensitive to mutation to trimethoprim resistance by NG, but M. radiodurans was more resistant the E. coli B/r to the other multagens tested, being non-mutable by UV light, γ-radiation, MTC and HA, and only slightly sensitive to mutation by NA, EMS, and βPL. The resistance of M. radiodurans to mutation by UV-light, γ-radiation and MTC is consistent with an hypothesis that recombination repair in M. radiodurans is accurate since these mutagens may depend on an “error-prone” recombination system for their mutagenic effect in E. coli B/r. However, because M. radiodurans is also resistant to mutagens such as HA and EMS, which are mutagenic in E. coli in the absence of an “error-prone” system, we propose that all the mutagens tested may have a common mode of action in E. coli B/r, but that this mutagenic pathway is missing in M. radiodurans.     

OmpA-Mediated Biofilm Formation Is Essential for the Commensal Bacterium Sodalis glossinidius To Colonize the Tsetse Fly Gut

Many bacteria successfully colonize animals by forming protective biofilms. Molecular processes that underlie the formation and function of biofilms in pathogenic bacteria are well characterized. In contrast, the relationship between biofilms and host colonization by symbiotic bacteria is less well understood. Tsetse flies (Glossina spp.) house 3 maternally transmitted symbionts, one of which is a commensal (Sodalis glossinidius) found in several host tissues, including the gut. We determined that Sodalis forms biofilms in the tsetse gut and that this process is influenced by the Sodalis outer membrane protein A (OmpA). Mutant Sodalis strains that do not produce OmpA (Sodalis ΔOmpA mutants) fail to form biofilms in vitro and are unable to colonize the tsetse gut unless endogenous symbiotic bacteria are present. Our data indicate that in the absence of biofilms, Sodalis ΔOmpA mutant cells are exposed to and eliminated by tsetse''s innate immune system, suggesting that biofilms help Sodalis evade the host immune system. Tsetse is the sole vector of pathogenic African trypanosomes, which also reside in the fly gut. Acquiring a better understanding of the dynamics that promote Sodalis colonization of the tsetse gut may enhance the development of novel disease control strategies.     

Gγ1, a Downstream Target for the hmgcr-Isoprenoid Biosynthetic Pathway, Is Required for Releasing the Hedgehog Ligand and Directing Germ Cell Migration

The isoprenoid biosynthetic pathway leading from the production of mevalonate by HMGCoA reductase (Hmgcr) to the geranylation of the G protein subunit, Gγ1, plays an important role in cardiac development in the fly. Hmgcr has also been implicated in the release of the signaling molecule Hedgehog (Hh) from hh expressing cells and in the production of an attractant that directs primordial germ cells to migrate to the somatic gonadal precursor cells (SGPs). The studies reported here indicate that this same hmgcr→Gγ1 pathway provides a novel post-translational mechanism for modulating the range and activity of the Hh signal produced by hh expressing cells. We show that, like hmgcr, gγ1 and quemao (which encodes the enzyme, geranylgeranyl diphosphate synthetase, that produces the substrate for geranylation of Gγ1) are components of the hh signaling pathway and are required for the efficient release of the Hh ligand from hh expressing cells. We also show that the hmgcr→Gγ1 pathway is linked to production of the germ cell attractant by the SGPs through its ability to enhance the potency of the Hh signal. We show that germ cell migration is disrupted by the loss or gain of gγ1 activity, by trans-heterozygous combinations between gγ1 and either hmgcr or hh mutations, and by ectopic expression of dominant negative Gγ1 proteins that cannot be geranylated.     

Glutathione initiates the development of Dictyostelium discoideum through the regulation of YakA

Reduced glutathione (GSH) is an essential metabolite that performs multiple indispensable roles during the development of Dictyostelium. We show here that disruption of the gene (gcsA¯) encoding γ-glutamylcysteine synthetase, an essential enzyme in GSH biosynthesis, inhibited aggregation, and that this developmental defect was rescued by exogenous GSH, but not by other thiols or antioxidants. In GSH-depleted gcsA¯ cells, the expression of a growth-stage-specific gene (cprD) was not inhibited, and we did not detect the expression of genes that encode proteins required for early development (cAMP receptor, carA/cAR1; adenylyl cyclase, acaA/ACA; and the catalytic subunit of protein kinase A, pkaC/PKA-C). The defects in gcsA¯ cells were not restored by cAMP stimulation or by cAR1 expression. Further, the expression of yakA, which initiates development and induces the expression of PKA-C, ACA, and cAR1, was regulated by the intracellular concentration of GSH. Constitutive expression of YakA in gcsA¯ cells (YakA^OE/gcsA¯) rescued the defects in developmental initiation and the expression of early developmental genes in the absence of GSH. Taken together, these findings suggest that GSH plays an essential role in the transition from growth to development by modulating the expression of the genes encoding YakA as well as components that act downstream in the YakA signaling pathway.