An ex vivo method for rapid generation of monoclonal antibodies (ADLib system)

Here, we describe a protocol for using the ADLib (Autonomously Diversifying Library) system to rapidly generate specific monoclonal antibodies using DT40, a chicken B-cell line that undergoes constitutive gene conversion at both light- and heavy-chain immunoglobulin loci. We previously developed the ADLib system on the basis of our finding that gene conversion in DT40 cells was enhanced by treatment of the cells with a histone deacetylase inhibitor, trichostatin A (TSA). TSA treatment evolves a diversified library of DT40 cells (ADLib), in which each cell has different surface IgM specificity. Antigen-specific DT40 cells are selected from ADLib using antigen-conjugated magnetic beads, and their specificity can be examined by various immunological assays, using culture supernatant containing secreted IgM. The whole process from selection to screening can be completed in about 1 week. Thus, the ADLib system will accelerate biological studies, including drug discovery and design.     

Vitamin E reduces amyloidosis and improves cognitive function in Tg2576 mice following repetitive concussive brain injury

Traumatic brain injury is a well-recognized environmental risk factor for developing Alzheimer's disease. Repetitive concussive brain injury (RCBI) exacerbates brain lipid peroxidation, accelerates amyloid (Abeta) formation and deposition, as well as cognitive impairments in Tg2576 mice. This study evaluated the effects of vitamin E on these four parameters in Tg2576 mice following RCBI. Eleven-month-old mice were randomized to receive either regular chow or chow-supplemented with vitamin E for 4 weeks, and subjected to RCBI (two injuries, 24 h apart) using a modified controlled cortical impact model of closed head injury. The same dietary regimens were maintained up to 8 weeks post-injury, when the animals were killed for biochemical and immunohistochemical analyses after behavioral evaluation. Vitamin E-treated animals showed a significant increase in brain vitamin E levels and a significant decrease in brain lipid peroxidation levels. After RBCI, compared with the group on regular chow, animals receiving vitamin E did not show the increase in Abeta peptides, and had a significant attenuation of learning deficits. This study suggests that the exacerbation of brain oxidative stress following RCBI plays a mechanistic role in accelerating Alphabeta accumulation and behavioral impairments in the Tg2576 mice.     

Estimation of carbon biomass and community structure of planktonic bacteria in Lake Biwa using respiratory quinone analysis

The relationship between bacterial respiratory quinone (RQ) concentration and biomass was assessed for Lake Biwa bacterial assemblages to evaluate the utility of bacterial RQ concentration as an indicator of bacterial carbon. The biomass estimated from the RQ concentration correlated well with that from cell volume, indicating that RQ concentration is an appropriate indicator of bacterial biomass. The estimated carbon content per unit of RQ (carbon conversion factor) of bacteria was 0.67 mg C nmol RQ^?1. Bacterial carbon biomass, which was estimated from the RQ concentration using the conversion factor, ranged between 0.008 and 0.054 mg C L^?1 (average 0.025 mg C L^?1) at 5 m depth and between 0.010 and 0.024 mg C L^?1 (average 0.015 mg C L^?1) at 70 m depth. Ubiquinone-8-containing bacteria dominated the epilimnion and hypolimnion. Compared to conventional image analysis, bacterial RQ analysis is a less laborious method of simultaneously determining bacterial biomass and community.     

Intracellular uptake and inhibition of gene expression by PNAs and PNA-peptide conjugates

Peptide nucleic acids (PNAs) offer a distinct option for silencing gene expression in mammalian cells. However, the full value of PNAs has not been realized, and the rules governing the recognition of cellular targets by PNAs remain obscure. Here we examine the uptake of PNAs and PNA-peptide conjugates by immortal and primary human cells and compare peptide-mediated and DNA/lipid-mediated delivery strategies. We find that both peptide-mediated and lipid-mediated delivery strategies promote entry of PNA and PNA-peptide conjugates into cells. Confocal microscopy reveals a punctate distribution of PNA and PNA-peptide conjugates regardless of the delivery strategy used. Peptide D(AAKK)(4) and a peptide containing a nuclear localization sequence (NLS) promote the spontaneous delivery of antisense PNAs into cultured cells. The PNA-D(AAKK)(4) conjugate inhibits expression of human caveolin 1 (hCav-1) in both HeLa and primary endothelial cells. DNA/lipid-mediated delivery requires less PNA, while peptide-mediated delivery is simpler and is less toxic to primary cells. The ability of PNA-peptide conjugates to enter primary and immortal human cells and inhibit gene expression supports the use of PNAs as antisense agents for investigating the roles of proteins in cells. Both DNA/lipid-mediated and peptide-mediated delivery strategies are efficient, but the compartmentalized localization of PNAs suggests that improving the cellular distribution may lead to increased efficacy.     

Fast compaction of alpha-lactalbumin during folding studied by stopped-flow X-ray scattering

To monitor the fast compaction process during protein folding, we have used a stopped-flow small-angle X-ray scattering technique combined with a two-dimensional charge-coupled device-based X-ray detector that makes it possible to improve the signal-to-noise ratio of data dramatically, and measured the kinetic refolding reaction of alpha-lactalbumin. The results clearly show that the radius of gyration and the overall shape of the kinetic folding intermediate of alpha-lactalbumin are the same as those of the molten globule state observed at equilibrium. Thus, the identity between the kinetic folding intermediate and the equilibrium molten globule state is firmly established. The present results also suggest that the folding intermediate is more hydrated than the native state and that the hydrated water molecules are dehydrated when specific side-chain packing is formed during the change from the molten globule to the native state.     

Safety of intradermal injection of monosodium urate crystals as a vaccine carrier in volunteers

Monosodium urate (MSU) crystals are known to induce gouty arthritis, but also evoke specific cell immunity and work as an adjuvant by delivering several kinds of binding proteins, including idiotypic cancer vaccine peptides into dendritic cells. To investigate the potency of MSU crystals as a cancer vaccine carrier in vivo, this preclinical study examined whether intradermal injection of MSU crystals was safe for healthy adults. Subjects comprised 12 volunteers. Four different dose levels of MSU crystals were injected as follows: 2 μg (n = 3), 20 μg (n = 3), 200 μg (n = 3), or 2000?μg (n = 3). At 24 hours after administration, documented erythema was seen around the injection site in a dose-dependent manner, particularly in all adults with MSU dose ≥200 μg. However, redness was limited to the grade I level of the National Cancer Institute toxicity criteria. Serum uric acid levels did not show any change before and after injection. Moreover, neither gouty arthritis nor tophi developed in any volunteers, indicating that intradermal injection of MSU crystals did not induce systemic inflammation at the doses that evoked significant local inflammation. These findings suggest that intradermal injection of MSU crystals is fundamentally safe and should be made available for clinical trials using MSU-crystal-conjugated cancer vaccines.     

An FXPRLamide neuropeptide induces seasonal reproductive polyphenism underlying a life-history tradeoff in the tussock moth

The white spotted tussock moth, Orgyia thyellina, is a typical insect that exhibits seasonal polyphenisms in morphological, physiological, and behavioral traits, including a life-history tradeoff known as oogenesis-flight syndrome. However, the developmental processes and molecular mechanisms that mediate developmental plasticity, including life-history tradeoff, remain largely unknown. To analyze the molecular mechanisms involved in reproductive polyphenism, including the diapause induction, we first cloned and characterized the diapause hormone-pheromone biosynthesis activating neuropeptide (DH-PBAN) cDNA encoding the five Phe-X-Pro-Arg-Leu-NH(2) (FXPRLa) neuropeptides: DH, PBAN, and α-, β-, and γ-SGNPs (subesophageal ganglion neuropeptides). This gene is expressed in neurosecretory cells within the subesophageal ganglion whose axonal projections reach the neurohemal organ, the corpus cardiacum, suggesting that the DH neuroendocrine system is conserved in Lepidoptera. By injection of chemically synthetic DH and anti-FXPRLa antibody into female pupae, we revealed that not only does the Orgyia DH induce embryonic diapause, but also that this neuropeptide induces seasonal polyphenism, participating in the hypertrophy of follicles and ovaries. In addition, the other four FXPRLa also induced embryonic diapause in O. thyellina, but not in Bombyx mori. This is the first study showing that a neuropeptide has a pleiotropic effect in seasonal reproductive polyphenism to accomplish seasonal adaptation. We also show that a novel factor (i.e., the DH neuropeptide) acts as an important inducer of seasonal polyphenism underlying a life-history tradeoff. Furthermore, we speculate that there must be evolutionary conservation and diversification in the neuroendocrine systems of two lepidopteran genera, Orgyia and Bombyx, in order to facilitate the evolution of coregulated life-history traits and tradeoffs.