Age-Specific Profiles of Antibody Responses against Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is one of the most prevalent causative agents of lower respiratory tract infections worldwide, especially in infants around 3 to 4months old. Infants at such a young age have maternally-transferred passive antibodies against RSV but do not have active immune systems efficient enough for the control of RSV infection. In order to elucidate age-specific profiles of immune responses against RSV protection, antibody responses were examined by using blood samples in both acute and convalescent phases obtained from child patients and adult patients. In addition to the serum neutralization activity, antibody responses to the RSV fusion protein (F protein) were dissected by analyzing levels of total IgG, IgG subclasses, the binding stability, and the levels of antibody for the neutralization epitopes. It was suggested that children's antibody responses against RSV are matured over months and years in at least 5 stages based on 1) levels of the neutralization titer and IgG3 for F protein in the convalescent phase, 2) geometric mean ratios of the neutralization titers and levels of IgG1 and IgG2 for F protein in the convalescent phase compared to those levels in the acute phase, 3) the affinity maturation of IgG for F protein and the cross reactivity of IgG for RSV glycoproteins of groups A and B, 4) levels of neutralization epitope-specific IgG, and 5) augmentation of overall antibody responses due to repetitive RSV infection.

Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies.

Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins.

Control of reactive oxygen species (ROS) production through histidine kinases in Aspergillus nidulans under different growth conditions.

Sensor histidine kinases (HKs) are important factors that control cellular growth in response to environmental conditions. The expression of 15 HKs from Aspergillus nidulans was analyzed by quantitative real-time PCR under vegetative, asexual, and sexual growth conditions. Most HKs were highly expressed during asexual growth. All HK gene-disrupted strains produced reactive oxygen species (ROS). Three HKs are involved in the control of ROS: HysA was the most abundant under the restricted oxygen condition, NikA is involved in fungicide sensing, and FphA inhibits sexual development in response to red light. Phosphotransfer signal transduction via HysA is essential for ROS production control.

[Evaluation of family function in home care].

Quantitative and qualitative analyses of a caring family are needed to improve home care. We propose a three-dimensional quantitative evaluation of family functioning. The first dimension is food, clothing, and shelter; the second dimension is patient, medical, and caring conditions; and the third dimension is the caring family condition. We used the home care score and Family Adaptability and Cohesion Evaluation Scale at Kwansei Gakuin(FACESKG)IV for the quantitative evaluation of family functioning. Narrative medicine and ethnography are valuable for the qualitative evaluation of a caring family.

Rapid production of antigen-specific monoclonal antibodies from a variety of animals.

BACKGROUND: Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging. RESULTS: We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals. CONCLUSIONS: Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.

Target-selective joint polymerase chain reaction: a robust and rapid method for high-throughput production of recombinant monoclonal antibodies from single cells.

BACKGROUND: During the development of a therapeutic antibody, large numbers of monoclonal antibodies are required to screen for those that are best suited for the desired activity. Although the single cell-based immunoglobulin variable gene cloning technique is a powerful tool, the current methods remain an obstacle to the rapid production of large numbers of recombinant antibodies. RESULTS: We have developed a novel overlap extension polymerase chain reaction, the target-selective joint polymerase chain reaction (TS-jPCR), and applied it to the generation of linear immunoglobulin gene expression constructs. TS-jPCR is conducted using a PCR-amplified immunoglobulin variable gene and an immunoglobulin gene-selective cassette (Ig-cassette) that contains all essential elements for antibody expression and overlapping areas of immunoglobulin gene-specific homology. The TS-jPCR technique is simple and specific; the 3'-random nucleotide-tailed immunoglobulin variable gene fragment and the Ig-cassette are assembled into a linear immunoglobulin expression construct, even in the presence of nonspecifically amplified DNA. We also developed a robotic magnetic beads handling instrument for single cell-based cDNA synthesis to amplify immunoglobulin variable genes by rapid amplification of 5' cDNA ends PCR. Using these methods, we were able to produce recombinant monoclonal antibodies from large numbers of single plasma cells within four days. CONCLUSION: Our system reduces the burden of antibody discovery and engineering by rapidly producing large numbers of recombinant monoclonal antibodies in a short period of time.

Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells.

BACKGROUND: Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies. RESULTS: We developed a single-step cloning method, target-selective homologous recombination (TS-HR), in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector) with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into E. coli. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells. CONCLUSION: The time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique.

Bacillus subtilis pgsE (Formerly ywtC) stimulates poly-γ-glutamate production in the presence of zinc.

Poly-γ-glutamate (PGA) is a versatile nylon-like material, and enhanced production of PGA is required for various bio-industrial applications. In this study, we first examined the effects of available sugars on the production of Bacillus subtilis PGA, and demonstrated the good applicability of pentoses (e.g., D-xylose). Then, we characterized the pgsE gene of B. subtilis, which encodes a 6.5-kDa protein of 55 amino acids (PgsE), as a genetic tool for increasing the yield of PGA without changing its structural features (e.g., polymer stereochemistry and molecular size distribution). In the presence of Zn(2+), the induction of PgsE tripled the PGA productivity of B. subtilis subsp. chungkookjang. This finding will contribute to the establishment of an improved PGA-production system.

Fluorescent labeling of peptides on solid phase.

N(alpha)-Fmoc-N(epsilon)-[(7-methoxycoumarin-4-yl)acetyl]-L-lysine (N(alpha)-Fmoc-L-Lys(Mca)-OH) 3 is conveniently prepared by benzotriazole methodology (52% over two steps). N-Acylbenzotriazoles Mca-Bt 2, N(alpha)-Fmoc-L-Lys(Mca)-Bt 4, coumarin-3-ylcarbonyl (Cc)-Bt 5, N(alpha)-Fmoc-L-Lys(Cc)-Bt 7 and N(alpha)-(Cc)-L-Lys(Fmoc)-Bt 9 enable the efficient microwave enhanced solid-phase fluorescent labeling of peptides.

N-Fmoc-protected(alpha-dipeptidoyl)benzotriazoles for efficient solid-phase peptide synthesis by segment condensation.

N-Fmoc-protected(alpha-aminoacyl)benzotriazoles 1a-d readily afford chiral N-Fmoc-protected-alpha-dipeptides 2a-f (77-89%). Compounds 2a-f are further converted into N-Fmoc-protected(alpha-dipeptidoyl)benzotriazoles 3a-f (71% average yield). Under mild microwave irradiation, 3a-f are used in solid-phase peptide segment condensation syntheses to give tri-, tetra-, penta-, hexa-, and heptapeptides (20-68%).

Microwave-assisted solid-phase peptide synthesis utilizing N-Fmoc-protected (alpha-aminoacyl)benzotriazoles.

A novel microwave-assisted solid-phase peptide synthesis utilizing N-Fmoc-protected(alpha-aminoacyl)benzotriazoles was applied in the preparation of tri-, tetra-, penta-, hexa-, and heptapeptides in 71% average crude yield.

C-aminoimidoylation and C-thiocarbamoylation of esters, sulfones, and ketones.

Esters, sulfones, and ketones were C-aminoimidoylated and C-thiocarbamoylated by benzotriazole-1-carboxamidines 8a-g and 1-(alkyl-or-arylthiocarbamoyl)benzotriazoles 9a-i, respectively. The present work represents the first systematic approach to these compound classes, the few previously known examples of which were obtained by diverse approaches.

[Central hypotensive effect involving neurotransmitters of long-term administration of taurine to stroke-prone spontaneously hypertensive rat].

BACKGROUND: Taurine is an inhibitory neurotransmitter or neuromodulator that reduces blood pressure when systemically or centrally administered. We studied the central hypotensive effects of long-term oral taurine administration. METHODS: Arterial blood pressure was measured after delivering an intracisternal injection of 100 mg x 20 microl(-1) or 200 microg x 20microl(-1) of taurine in normal saline, or 20 micro1 normal saline to anesthetized Sprague-Dawley rats. Drinking water containing 3% taurine was administered to stroke-prone spontaneously hypertensive rats (SHRSP) from the age of 4 weeks. Amino acids and monoamine neurotransmitters in the cerebrospinal fluid were measured at 8, 12, 16, 18 weeks of age in taurine treated SHRSP and normotensive Wistar Kyoto rats (WKY) and in untreated SHRSP using high performance liquid chromatography. RESULTS: Intracisternal injections of taurine caused a dose dependent decrease in arterial blood pressure. Although concentrations of taurine decreased in treated SHRSP rats in an age-related manner, the drug persistently suppressed the development of hypertension. The values of excitatory amino acids and GABA, norepinephrine, NMN, dopamine metabolites, serotonin and its metabolite were lower in taurine-treated SHRSP than those in untreated SHRSP. CONCLUSIONS: Taurine reduces blood pressure through not only direct inhibition of the cardiovascular center in the medulla, but also by reducing brain monoamine concentrations.

Synthesis of mono- and symmetrical di-N-hydroxy- and N-aminoguanidines.

Novel mono- and symmetrical di-N-hydroxy- and N-aminoguanidines were readily prepared from the reaction of diverse hydroxylamines or hydrazines with reagent classes di(benzotriazol-1-yl)methanimine 6, (bis-benzotriazol-1-yl-methylene)amines 8a,b, benzotriazole-1-carboxamidines 10a-i, benzotriazole-1-carboximidamides 11a,b, and N'-hydroxy-1H-1,2,3-benzotriazole-1-carboximidamide 18. The preparation is described for a variety of N-hydroxy- and N-aminoguanidines with different substitution patterns in good yields.