Enhanced expression of aconitase raises acetic acid resistance in Acetobacter aceti

Acetobacter spp. are used for industrial vinegar production because of their high ability to oxidize ethanol to acetic acid and high resistance to acetic acid. Two-dimensional gel electrophoretic analysis of a soluble fraction of Acetobacter aceti revealed the presence of several proteins whose production was enhanced, to various extents, in response to acetic acid in the medium. A protein with an apparent molecular mass of 100 kDa was significantly enhanced in amount by acetic acid and identified to be aconitase by NH2-terminal amino acid sequencing and subsequent gene cloning. Amplification of the aconitase gene by use of a multicopy plasmid in A. aceti enhanced the enzymatic activity and acetic acid resistance. These results showed that aconitase is concerned with acetic acid resistance. Enhancement of the aconitase activity turned out to be practically useful for acetic acid fermentation, because the A. aceti transformant harboring multiple copies of the aconitase gene produced a higher concentration of acetic acid with a reduced growth lag-time.     

Non-fucosylated therapeutic antibodies: the next generation of therapeutic antibodies

Therapeutic antibody IgG1 has two N-linked oligosaccharide chains bound to the Fc region. The oligosaccharides are of the complex biantennary type, composed of a trimannosyl core structure with the presence or absence of core fucose, bisecting N-acetylglucosamine (GlcNAc), galactose, and terminal sialic acid, which gives rise to structural heterogeneity. Both human serum IgG and therapeutic antibodies are well known to be heavily fucosylated. Recently, antibody-dependent cellular cytotoxicity (ADCC), a lytic attack on antibody-targeted cells, has been found to be one of the critical effector functions responsible for the clinical efficacy of therapeutic antibodies such as anti-CD20 IgG1 rituximab (Rituxan^®) and anti-Her2/neu IgG1 trastuzumab (Herceptin^®). ADCC is triggered upon the binding of lymphocyte receptors (FcγRs) to the antibody Fc region. The activity is dependent on the amount of fucose attached to the innermost GlcNAc of N-linked Fc oligosaccharide via an α-1,6-linkage, and is dramatically enhanced by a reduction in fucose. Non-fucosylated therapeutic antibodies show more potent efficacy than their fucosylated counterparts both in vitro and in vivo, and are not likely to be immunogenic because their carbohydrate structures are a normal component of natural human serum IgG. Thus, the application of non-fucosylated antibodies is expected to be a powerful and elegant approach to the design of the next generation therapeutic antibodies with improved efficacy. In this review, we discuss the importance of the oligosaccharides attached to the Fc region of therapeutic antibodies, especially regarding the inhibitory effect of fucosylated therapeutic antibodies on the efficacy of non-fucosylated counterparts in one medical agent. The impact of completely non-fucosylated therapeutic antibodies on therapeutic fields will be also discussed.     

The composition of newly synthesized proteins in the endoplasmic reticulum determines the transport pathways of soybean seed storage proteins

Glycinin (11S) and beta-conglycinin (7S) are major storage proteins in soybean (Glycine max L.) seeds and accumulate in the protein storage vacuole (PSV). These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the PSV by vesicles. Electron microscopic analysis of developing soybean cotyledons of the wild type and mutants with storage protein composition different from that of the wild type showed that there are two transport pathways: one is via the Golgi and the other bypasses it. Golgi-derived vesicles were observed in all lines used in this study and formed smooth dense bodies with a diameter of 0.5 to several micrometers. ER-derived protein bodies (PBs) with a diameter of 0.3-0.5 microm were observed at high frequency in the mutants containing higher amount of 11S group I subunit than the wild type, whereas they were hardly observed in the mutants lacking 11S group I subunit. These indicate that pro11S group I may affect the formation of PBs. Thus, the composition of newly synthesized proteins in the ER is important in the selection of the transport pathways.     

Methods for reducing nonspecific interaction in antibody-antigen assay via atomic force microscopy

We developed a method to measure the rupture forces between antibody and antigen by atomic force microscopy (AFM). Previous studies have reported that in the measurement of antibody–antigen interaction using AFM, the specific intermolecular forces are often obscured by nonspecific adhesive binding forces between antibody immobilized cantilever and substrate surfaces on which antigen or nonantigen are fixed. Here, we examined whether detergent and nonreactive protein, which have been widely used to reduce nonspecific background signals in ordinary immunoassay and immunoblotting, could reduce the nonspecific forces in the AFM measurement. The results showed that, in the presence of both nonreactive protein and detergent, the rupture forces between anti-ferritin antibodies immobilized on a tip of cantilever and ferritin (antigen) on the substrate could be successfully measured, distinguishing from nonspecific adhesive forces. In addition, we found that approach/retraction velocity of the AFM cantilever was also important in the reduction of nonspecific adhesion. These insights will contribute to the detection of specific molecules at nanometer scale region and the investigation of intermolecular interaction by the use of AFM.     

Screening of wild accessions resistant to gray mold (Botrytis cinerea Pers.) in Lycopersicon

Tomato gray mold (Botrytis cinerea Pers.) is a common disease worldwide, and often causes serious production loss by infecting leaves, stems, flowers and fruits. Presently, no resistant cultivars are available. To find new breeding materials for gray mold resistance, assessment for resistance of the leaflet and stem in six tomato cultivars, 44 wild tomato accessions and a Solanum lycopersicoides accession was performed. Although no correlation was observed (r=−0.127^ns) between resistance of the leaflet and the stem, L. peruvianum LA2745, L. hirsutum LA2314 and L. pimpinellifolium LA1246 showed high resistance both in the leaflet and in the stem. Particularly, in the leaves of LA2745, no lesions were observed even more than two weeks after the inoculation with conidia, and F1s between a cultivated tomato and LA2745 also showed high resistance as observed in LA2745. From these results, LA2745 is thought to be a promising material for breeding gray-mold resistant cultivars.     

Thyroid hormonal activity of the flame retardants tetrabromobisphenol A and tetrachlorobisphenol A

The thyroid hormonal-disrupting activity of the flame retardants tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) was examined and compared with that of bisphenol A, a typical estrogenic xenobiotic. TBBPA and TCBPA, halogenated derivatives of bisphenol A, markedly inhibited the binding of triiodothyronine (T(3); 1 x 10(-10) M) to thyroid hormone receptor in the concentration range of 1 x 10(-6) to 1 x 10(-4) M, but bisphenol A did not. The thyroid hormonal activity of TBBPA and TCBPA was also examined using rat pituitary cell line GH3 cells, which grow and release growth hormone (GH) depending on thyroid hormone. TBBPA and TCBPA enhanced the proliferation of GH3 cells and stimulated their production of GH in the concentration range of 1 x 10(-6) to 1 x 10(-4) M, while bisphenol A was inactive. TBBPA, TCBPA, and bisphenol A did not show antagonistic action, i.e., these compounds did not inhibit the hormonal activity of T(3) to induce growth and GH production of GH3 cells. TBBPA and TCBPA, as well as bisphenol A, enhanced the proliferation of MtT/E-2 cells, whose growth is estrogen-dependent. These results suggest that TBBPA and TCBPA act as thyroid hormone agonists, as well as estrogens.     

Nitrogen-flow in the rotifer Brachionus rotundiformis and its significance in mass cultures

The nitrogen budget in the rotifer Brachionus rotundiformis wasmeasured by the stable-isotope technique. The budget was estimatedusing the difference in the turnover time between egestion andexcretion. The rotifer was fed on the algae Nannochloropsiswhich was labeled with ¹⁵N as a tracer. The turnover time ofegestion and excretion were 20 min and 2.5 hours, respectively. Where77% of the ingested nitrogen was egested, and of the assimilated23%, 18% were devoted to growth and 5% to excretion.As for the unassimilated nitrogen egested as faeces, it recycled tothe rotifer through bacteriovory. When the algae provided as foodwere almost fully consumed, bacteriovory became dominant. Thethreshold occurred when the concentration of algae in the culture wasbetween 1.5 and 0.5 million cells of Nannochloropsis per ml. Ina chemostat operated with un-limited food condition, bacterialnitrogen corresponding to 20% of algal feeding, was consumed by therotifer.In a semi-continuous mass culture where food condition was limited,bacteriovory was more effective in supporting the rotiferreproduction. It contributed to the extremely high nitrogen recoveryfrom the provided foods (algae and oil-yeast) to the harvestedrotifers. The rapid and large nitrogen outflow from rotifersaccelerated the propagation of edible bacteria and can explain thestrange paradox observed in the culture; daily supply of foods didnot cover the sum of growth and excretion.It is not too exaggerated to state that the rotifer mass culture issupported by bacteria. The future strategy for maintenance of masscultures should consider this aspect.     

Interplant communication: airborne methyl jasmonate is essentially converted into JA and JA-Ile activating jasmonate signaling pathway and VOCs emission

Methyl jasmonate (MeJA) was identified as an airborne signal involved in mediating interplant defense response communications over a decade ago. However, how MeJA activates plant defense systems and what becomes of the compound after it has done so has, thus far, remained unknown. To investigate this, Achyranthes bidentata plants were exposed to deuterated methyl jasmonate (d₂MeJA) followed by absolute quantification of metabolic products of d₂MeJA, and emissions of volatile organic compound (VOC) as defensive markers. We found that d₂MeJA was metabolized mainly into deuterated jasmonic acid (d₂JA) and jasmonoyl isoleucine (d₂JA-Ile), and to a much lesser extent, deuterated jasmonoyl leucine (d₂JA-Leu). Increases in d₂JA-Ile/Leu and also endogenous JA-Ile/Leu were tightly co-related with, and significantly influenced the pattern and amount of, VOC emissions. The amount of accumulated d₂JA-IIe was 13.1-fold higher than d₂JA-Leu, whereas the amounts of JA-IIe and JA-Leu accumulated were almost identical. This study demonstrates that exogenous MeJA activates defensive systems (such as VOC emissions) in receiver plants by essentially converting itself into JA and JA-IIe and initiating a signal transduction leading to VOC emissions and induction of endogenous JA-IIe and JA-Leu, which in turn cause further amplification of VOC emissions.     

Endogenous Dopamine Maintains Synchronous Oscillation of Intracellular Calcium in Primary Cultured-Mouse Midbrain Neurons

We demonstrated synchronous oscillation of intracellular Ca2+ in cultured-mouse mid-brain neurons. This synchronous oscillation was thought to result from spontaneous and synchronous neural bursts in a synaptic neural network. We also examined the role of endogenous dopamine in neural networks showing synchronous oscillation. Immunocytochemical study revealed a few tyrosine hydroxylase (TH)-positive dopaminergic neurons, and that cultured neurons expressed synaptophysin and synapsin I. Western blot analyses comfirmed synaptophysin, TH, and 2 types of dopamine receptor (DR), D1R and D2R expression. The synchronous oscillation in midbrain neurons was abolished by the application of R(-)-2-amino-5-phosphonopentanoic acid (AP-5) as an N-methyl-D-aspartate receptor (NMDAR) antagonist. This result suggests that the synchronous oscillation in midbrain neurons requires glutamatergic transmissions, as was the case in previously reported cortical neurons. SCH-12679, a D1R antagonist, inhibited synchronous oscillation in midbrain neurons, while raclopride, a D2R antagonist, induced a transient increase of intracellular Ca2+ and inhibited synchronous oscillation. We consider that endogenous dopamine maintains synchronous oscillation of intracellular Ca2+ through D1R and D2R, and that these DRs regulate intracellular Ca2+in distinctly different ways. Synchronous oscillation of midbrain neurons would be a useful tool for in vitro researches into various neural disorders directly or indirectly caused by dopaminergic neurons.