A Surrogate Animal Model for Screening of Ebola and Marburg Glycoprotein-Targeting Drugs Using Pseudotyped Vesicular Stomatitis Viruses.

Filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. There is no approved therapy against these deadly viruses. Antiviral drug development has been hampered by the requirement of a biosafety level (BSL)-4 facility to handle infectious EBOV and MARV because of their high pathogenicity to humans.

R409K mutation prevents acid-induced aggregation of human IgG4.

Human immunoglobulin G isotype 4 (IgG4) antibodies are suitable for use in either the antagonist or agonist format because their low effector functions prevent target cytotoxicity or unwanted cytokine secretion. However, while manufacturing therapeutic antibodies, they are exposed to low pH during purification, and IgG4 is more susceptible to low-pH-induced aggregation than IgG1. Therefore, we investigated the underlying mechanisms of IgG4 aggregation at low pH and engineered an IgG4 with enhanced stability.

Engineering a human IgG2 antibody stable at low pH.

IgG2 subclass antibodies have unique properties that include low effector function and a rigid hinge region. Although some IgG2 subclasses have been clinically tested and approved for therapeutic use, they have a higher propensity than IgG1 for aggregation, which can curtail or abolish their biological activity and enhance their immunogenicity. In this regard, acid-induced aggregation of monoclonal antibodies during purification and virus inactivation must be prevented.

Generation of Yellow Flowers of the Japanese Morning Glory by Engineering Its Flavonoid Biosynthetic Pathway toward Aurones.

Wild-type plants of the Japanese morning glory (Ipomoea nil) produce blue flowers that accumulate anthocyanin pigments, whereas its mutant cultivars show wide range flower color such as red, magenta and white. However, I. nil lacks yellow color varieties even though yellow flowers were curiously described in words and woodblocks printed in the 19th century.

A stable engineered human IgG3 antibody with decreased aggregation during antibody expression and low pH stress.

Human IgG comprises four subclasses with different biological functions. The IgG3 subclass has a unique character, exhibiting high effector function and Fab arm flexibility. However, it is not used as a therapeutic drug owing to an enhanced susceptibility to proteolysis.

Conformational effects of N-glycan core fucosylation of immunoglobulin G Fc region on its interaction with Fcγ receptor IIIa.

Antibody-dependent cellular cytotoxicity (ADCC) is promoted through interaction between the Fc region of immunoglobulin G1 (IgG1) and Fcγ receptor IIIa (FcγRIIIa), depending on N-glycosylation of these glycoproteins. In particular, core fucosylation of IgG1-Fc N-glycans negatively affects this interaction and thereby compromises ADCC activity. To address the mechanisms of this effect, we performed replica-exchange molecular dynamics simulations based on crystallographic analysis of a soluble form of FcγRIIIa (sFcγRIIIa) in complex with IgG1-Fc.

Genome sequence and analysis of the Japanese morning glory Ipomoea nil.

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I.

Comparison of biological activities of human antithrombins with high-mannose or complex-type nonfucosylated N-linked oligosaccharides.

The structure of the N-linked oligosaccharides attached to antithrombin (AT) has been shown to affect its anticoagulant activity and pharmacokinetics. Human AT has biantennary complex-type oligosaccharides with the unique feature of lacking a core fucose, which affects its biological activities by changing its heparin-binding affinity. In human plasma, AT circulates as a mixture of the α-form bearing four oligosaccharides and the β-form lacking an oligosaccharide at Asn135.

Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors.

Antibody-dependent cellular cytotoxicity (ADCC) is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG) improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa) and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr-296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1.

Spontaneous mutations of the UDP-glucose:flavonoid 3-O-glucosyltransferase gene confers pale- and dull-colored flowers in the Japanese and common morning glories.

UDP-glucose:flavonoid 3- O -glucosyltransferase is essential for maintaining proper production quantity, acylation, and glucosylation of anthocyanin, and defects cause pale and dull flower pigmentation in morning glories. The Japanese (Ipomoea nil) and the common (I. purpurea) morning glory display bright blue and dark purple flowers, respectively.

A petal-specific InMYB1 promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops.

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal-specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage.

Therapeutic activity of glycoengineered anti-GM2 antibodies against malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare and highly aggressive neoplasm that arises from the pleural, pericardial, or peritoneal lining. Although surgery, chemotherapy, radiotherapy, and combinations of these therapies are used to treat MPM, the median survival of such patients is dismal. Therefore, there is a compelling need to develop novel therapeutics with different modes of action.

Backbone (1)H, (13)C, and (15)N resonance assignments of the Fc fragment of human immunoglobulin G glycoprotein.

The Fc portion of immunoglobulin G (IgG) recruits complements and its cognate receptors, thereby promoting defensive mechanisms in the humoral immune system. These effector functions critically depend on N-glycosylation at the Fc region, which is therefore regarded as a crucial factor in the design and production of therapeutic antibodies. NMR spectroscopy plays a unique role in the characterization of conformational dynamics and intermolecular interactions of IgG-Fc in solutions.

The MET1b gene encoding a maintenance DNA methyltransferase is indispensable for normal development in rice.

While Arabidopsis bears only one MET1 gene encoding the DNA methyltransferase that is mainly responsible for maintaining CG methylation after DNA replication, rice carries two MET1 genes, MET1a and MET1b, expressed in actively replicating and dividing cells, and MET1b is more abundantly expressed than is MET1a. A met1a null mutant displayed no overt phenotypes, implying that MET1b must play a major role in the maintenance DNA methylation. Here, we employed two met1b null mutants, generated by homologous recombination-mediated knock-in targeting and insertion of endogenous retrotransposon Tos17.

A chalcone isomerase-like protein enhances flavonoid production and flower pigmentation.

Flavonoids are major pigments in plants, and their biosynthetic pathway is one of the best-studied metabolic pathways. Here we have identified three mutations within a gene that result in pale-colored flowers in the Japanese morning glory (Ipomoea nil). As the mutations lead to a reduction of the colorless flavonoid compound flavonol as well as of anthocyanins in the flower petal, the identified gene was designated enhancer of flavonoid production (EFP).

A mutable albino allele in rice reveals that formation of thylakoid membranes requires the SNOW-WHITE LEAF1 gene.

Active DNA transposons are important tools for gene functional analysis. The endogenous non-autonomous transposon, nDart1-0, in rice (Oryza sativa L.) is expected to generate various transposon-insertion mutants because nDart1-0 elements tend to insert into genic regions under natural growth conditions.

Homologous recombination-mediated gene targeting in the liverwort Marchantia polymorpha L.

The liverwort Marchantia polymorpha is an emerging model organism on account of its ideal characteristics for molecular genetics in addition to occupying a crucial position in the evolution of land plants. Here we describe a method for gene targeting by applying a positive/negative selection system for reduction of non-homologous random integration to an efficient Agrobacterium-mediated transformation system using M. polymorpha sporelings.

TAWAWA1, a regulator of rice inflorescence architecture, functions through the suppression of meristem phase transition.

Inflorescence structures result from the activities of meristems, which coordinate both the renewal of stem cells in the center and organ formation at the periphery. The fate of a meristem is specified at its initiation and changes as the plant develops. During rice inflorescence development, newly formed meristems acquire a branch meristem (BM) identity, and can generate further meristems or terminate as spikelets.

Activation and epigenetic regulation of DNA transposon nDart1 in rice.

A large part of the rice genome is composed of transposons. Since active excision/reintegration of these mobile elements may result in harmful genetic changes, many transposons are maintained in a genetically or epigenetically inactivated state. However, some non-autonomous DNA transposons of the nDart1-3 subgroup, including nDart1-0, actively transpose in specific rice lines, such as pyl-v which carries an active autonomous element, aDart1-27, on chromosome 6.

A nonfucosylated variant of the anti-HIV-1 monoclonal antibody b12 has enhanced FcγRIIIa-mediated antiviral activity in vitro but does not improve protection against mucosal SHIV challenge in macaques.

Eliciting neutralizing antibodies is thought to be a key activity of a vaccine against human immunodeficiency virus (HIV). However, a number of studies have suggested that in addition to neutralization, interaction of IgG with Fc gamma receptors (FcγR) may play an important role in antibody-mediated protection. We have previously obtained evidence that the protective activity of the broadly neutralizing human IgG1 anti-HIV monoclonal antibody (MAb) b12 in macaques is diminished in the absence of FcγR binding capacity.

A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny.

Genes that promote DNA methylation and demethylation in plants have been characterized mainly in Arabidopsis. Arabidopsis DNA demethylation is mediated by bi-functional DNA enzymes with glycosylase activity that removes 5-methylcytosine and lyase activity that nicks double-stranded DNA at an abasic site. Homologous recombination-promoted knock-in targeting of the ROS1a gene, the longest of six putative DNA demethylase genes in the rice genome, by fusing its endogenous promoter to the GUS reporter gene, led to reproducibly disrupted ROS1a in primary (T(0)) transgenic plants in the heterozygous condition.

Structural basis for improved efficacy of therapeutic antibodies on defucosylation of their Fc glycans.

Removal of the fucose residue from the N-glycans of the Fc portion of immunoglobulin G (IgG) results in a dramatic enhancement of antibody-dependent cellular cytotoxicity (ADCC) through improved affinity for Fcγ receptor IIIa (FcγRIIIa). Here, we present the 2.2-Å structure of the complex formed between nonfucosylated IgG1-Fc and a soluble form of FcγRIIIa (sFcγRIIIa) with two N-glycosylation sites.

Examination of transpositional activity of nDart1 at different stages of rice development.

As a useful tool to elucidate gene functions, a rice transposon tagging line has been developed using an active endogenous DNA transposon, nDart1. It was highly desirable to evaluate the transposition timing and frequency of the nDart1 elements during rice development to facilitate the generation of an efficient mutant isolation system. Comparison of the detected new insertions at different stages of rice development by transposon display analysis demonstrated that the last heading tiller carry a higher number of nDart1 elements than the main culm.