B cell selection and affinity maturation during an antibody response in the mouse with limited B cell diversity

The quasi-monoclonal mouse has limited B cell diversity, whose major (approximately 80%) B cell Ag receptors are comprised of the knockin V(H) 17.2.25 (V(H)T)-encoded H chain and the lambda1 or lambda2 L chain, thereby being specific for 4-hydroxy-3-nitrophenylacetyl. The p-nitrophenylacetyl (pNP) was found to be a low affinity analog of nitrophenylacetyl. We examined affinity maturation of anti-pNP IgG by analyzing mAbs obtained from quasi-monoclonal mice that were immunized with this low affinity Ag. The results are: 1) Although V(H)T/lambda1 and V(H)T/lambda2 IgM were equally produced, V(H)T/lambda2 IgG almost exclusively underwent affinity maturation toward pNP. 2) A common mutation in complementarity-determining region 3 of V(H)T (T313A) mainly contributed to generating the specificity for pNP. 3) Because mutated V(H)T-encoded gamma-chains could form lambda1-bearing IgG in Chinese hamster ovary cells, apparent absence of V(H)T/lambda1 anti-pNP IgG may not be due to the incompatibility between the gamma-chains and the lambda1-chain, but may be explained by the fact that V(H)T/lambda1 B cells showed 50- to 100-fold lower affinity for pNP than V(H)T/lambda2 B cells. 4) Interestingly, a pNP-specific IgM mAb that shared common mutations including T313A with high affinity anti-pNP IgG was isolated, suggesting that a part of hypermutation coupled with positive selection can occur before isotype switching. Thus, even weak B cell receptor engagement can elicit an IgM response, whereas only B cells that received signals stronger than a threshold may be committed to an affinity maturation process.     

Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal-regulated kinase-dependent cascade.

Our laboratory showed that bikunin, a Kunitz-type protease inhibitor, suppresses 4beta-phorbol 12-myristate 13-acetate (PMA)- or tumor necrosis factor-alpha (TNFalpha)-induced urokinase-type plasminogen activator (uPA) expression in different cell types. In addition to its effects on protease inhibition, bikunin could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we examined whether bikunin was able to suppress the expression of uPA receptor (uPAR) mRNA and protein in a human chondrosarcoma cell line, HCS-2/8, and two human ovarian cancer cell lines, HOC-I and HRA. The present study showed that (a) bikunin suppresses the expression of constitutive and PMA-induced uPAR mRNA and protein in a variety of cell types; (b) an extracellular signal-regulated kinase (ERK) activation system is necessary for the PMA-induced increase in uPAR expression, as PD098059 and U0126, which prevent the activation of MEK1, reduce the uPAR expression; (c) bikunin markedly suppresses PMA-induced phosphorylation of ERK1/2 at the concentration that prevents uPAR expression, but does not reduce total ERK1/2 antigen level; (d) bikunin has no ability to inhibit overexpression of uPAR in cells treated with sodium vanadate; and (e) we further studied the inhibition of uPAR expression by stable transfection of HRA cells with bikunin gene, demonstrating that bikunin secretion is necessary for inhibition of uPAR expression. We conclude that bikunin downregulates constitutive and PMA-stimulated uPAR mRNA and protein possibly through suppression of upstream targets of the ERK-dependent cascade, independent of whether cells were treated with exogenous bikunin or transfected with bikunin gene.     

Determination of the Agretopic Residues of a Peptide Co-Restricted to Different Class II Isotypes,I-Au and I-Eu,and Its Application for Preparation of a Synthetic Peptide Vaccine against Influenza Virus A/Aichi/2/68

We have defined that residues 46 and 54 on a synthetic peptide composed of residues 43–58 of pigeon cytochrome c (p43–58) work as agretopes (sites bound to an MHC molecule) in I-A^b mice. Substitution of amino acid residues on these positions altered the peptide to bind with the other MHC molecules. Furthermore, by substituting the agretopic residues with a variety of amino acids, we could determine the class II binding motif for each MHC molecule. In the present study, immunogenicity of a peptide, 46R50V54A, carrying valine (V) at epitopic (site bound to TCR) position 50, arginine (R) and alanine (A) at agretopic positions 46 and 54 of the p43–58, respectively has been analyzed in B10.PL (H-2^u) mice. We found that this peptide bound to two different class II isotypes, I-A^u and I-E^u. Arginine at position 46 or alanine at position 54 of the 46R50V54A was shown to be critical for binding to I-A^u or I-E^u, respectively. Further, on the basis of this class II binding motif we could prepare potent peptide vaccines against influenza A/Aichi/2/68 virus in B10. PL mice.     

Adsorption of molecular oxygen by polymer covalently bonded co(II) porhyrin complex in toluene

Reaction betwenn molecular oxygen and polystyrene covalently bonded Co(II) protoporphyrin IX complex, which was prepaired by the incorporation of a cobaltous ion into the metal-free porphyrin polymer, was studied in the presence of N-ethylimidazole by measuring visible absorption and electron spin resonance spectra. It was found that the complex forms a monomeric oxygen adduct reversibly at low temperature dependent on oxygen pressure. In the presence of molecular oxygen, a new electron spin resonance signal due to the oxygen complex at g_iso=2.02 shows no superhyperfine splitting structure in fluid toluene solution even at ?80 °C, but it was observed in frozen toulene glass solution at ?120°C, The oxygen adducts of the complexes between C0(II) protoporphyrin IX dimethyl ester and N-ethylimidazole and copoly(styrene-N-vinylimidazole) showed eight resolved superhyperfine splitting at ?40 and ?60°C, respectively. The polymer covalently bonded Co(II) complex with N-ethylimidazole was oxidized at room temperature under oxygen atmosphere. It was suggested that a Co(II) porphyrin–oxygen adduct with an axial ligand may be oxidised monomolecularly at high temperature.     

Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2.

Two closely related genes coding for NELL proteins (NELL1 and NELL2) have been cloned by the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C betaI (PKCbetaI) as bait. The rat NELL proteins show about 55% identity with each other and contain several protein motifs assigned to a secretion signal peptide, an NH(2)-terminal thrombospondin-1 (TSP-1)-like module, five von Willebrand factor C domains, and six epidermal growth factor-like domains; the NELL proteins share many protein motifs with TSP-1. The NELL proteins expressed in COS-7 cells are homotrimeric glycoproteins and possess heparin-binding activity. Furthermore, while NELL1 and NELL2 show distinct subcellular localization in cytoplasm, they both are partially secreted into the culture medium of COS-7 cells. Although the NELL1 mRNA is faintly expressed in adult neural cells, the NELL2 mRNA is expressed abundantly, particularly in the pyramidal cells of rat hippocampus, showing neuronal high plasticity. During mouse embryogenesis, expression of the NELL2 mRNA is initiated 7-11 days postcoitum, simultaneously with neural plate formation. These results strongly suggest that the NELL2 protein, similar to but not identical with TSP-1, is involved in the growth and differentiation of neural cells. Additionally, the NELL1 and NELL2 mRNAs were found to be expressed abundantly in Burkitt's lymphoma Raji cells and colorectal adenocarcinoma SW480 cells, respectively. Thus, it is likely that the NELL proteins also participate in the growth, differentiation, and oncogenesis of cancer cell lines.     

Phosphoinositide regulates dynamic movement of the S4 voltage sensor in the second repeat in two-pore channel 3

The two-pore channels (TPCs) are voltage-gated cation channels consisting of single polypeptides with two repeats of a canonical 6-transmembrane unit. TPCs are known to be regulated by various physiological signals such as membrane voltage and phosphoinositide (PI). The fourth helix in the second repeat (second S4) plays a major role in detecting membrane voltage, whereas the first repeat contains a PI binding site. Therefore, each of these stimuli is detected by a unique repeat to regulate the gating of the TPC central pore. How these various stimuli regulate the dynamic structural rearrangement of the TPC molecule remain unknown. Here, we found that PI binding to the first repeat in TPC3 regulates the movement of the distally located second S4 helix, showing that the PI-binding signal is not confined to the pore gate but also transmitted to the voltage sensor. Using voltage clamp fluorometry, measurement of gating charges, and Cys-accessibility analysis, we observed that PI binding significantly potentiates the voltage dependence of the movement of the second S4 helix. Notably, voltage clamp fluorometry analysis revealed that the voltage-dependent movement of the second S4 helix occurred in two phases, of which the second phase corresponds to the transfer of the gating charges. This movement was observed in the voltage range where gate-opening occurs and was potentiated by PI. In conclusion, this regulation of the second S4 helix by PI indicates a tight inter-repeat coupling within TPC3, a feature which might be conserved among TPC family members to integrate various physiological signals.     

Reversible switching of immunoglobulin hypermutation machinery in a chicken B cell line

A chicken B lymphoma line, DT40, hypermutates immunoglobulin (Ig) genes spontaneously during culture. Thus, cultured DT 40 cells constitute a useful Ig library for screening antibodies (Abs) in vitro. To fix desirable Ig mutants by stopping hypermutation or to resume mutation for further improvement of Ab affinity, activation-induced cytidine deaminase (AID), a key enzyme responsible for the Ig mutation machinery, must be switched on or off. To this end, we generated a DT40 line whose one AID allele was disrupted, and the other allele was replaced by the loxP-flanked AID construct. In this engineered cell line designated as DT40-SW, AID expression could be switched reversibly by tamoxifen-regulated Cre recombinase. Devices were also introduced to discriminate between the "AID-ON" and the "AID-OFF" cells by GFP expression and puromycin resistance, respectively. Starting from a single DT40-SW cell, Ig gene repertoire was efficiently diversified during culture only when AID expression was on.     

Characterization of plant Aurora kinases during mitosis

The Aurora kinase family is a well-characterized serine/threonine protein kinase family that regulates different processes of mitotic events. Although functions of animal and yeast Aurora kinases have been analyzed, plant aurora kinases were not identified and characterized. We identified three Aurora kinase orthologs in Arabidopsis thaliana and designated these as AtAUR1, AtAUR2, and AtAUR3. These AtAURs could phosphorylate serine 10 in histone H3, in vitro. Dynamic analyses of GFP-fused AtAUR proteins revealed that AtAUR1 and AtAUR2 localized at the nuclear membrane in interphase and located in mitotic spindles during cell division. AtAUR1 also localized in the cell plates. AtAUR3 showed dot-like distribution on condensed chromosomes at prophase and then localized at the metaphase plate. At late anaphase, AtAUR3 is evenly localized on chromosomes. The localization of AtAUR3 during mitosis is very similar to that of phosphorylated histone H3. Interestingly, an overexpression of AtAUR3 induces disassembly of spindle microtubules and alteration of orientation of cell division. Our results indicate that plant Aurora kinases have different characters from that of Aurora kinases of other eukaryotes.†These authors equally contributed to this work     

Role of prostaglandin E2 receptor subtypes in ovarian follicle growth in the rat in vivo. Correlation with interleukin-8 and neutrophils

This study was conducted to elucidate the role of three of prostaglandin E2 (PGE2) receptor subtype (EP2, EP3, and EP4) agonists in the process of follicular growth. The influence of these agonists on ovarian expression of intimately related factors to follicle development (neutrophils and interleukin-8 (IL-8)) was also investigated. Immature female Wistar rats were injected once with these agonists and killed 48 hours later. Another group of rats were injected pregnant mare serum gonadotrophin. For evaluation of follicle growth, morphometric assessment of antral and ovulatory follicles was performed in serial ovarian sections. The study demonstrated that, EP2 and EP4 agonists showed the maximum follicle counts and diameters versus the control. EP2 and EP4 agonists mimicked PMSG induced follicle growth. Injection of the three agonists induced neutrophil infiltration into theca layer. EP4 agonist showed the most intense ovarian neutrophil accumulation. In addition, dense ovarian IL-8 expression was observed only after EP4 agonist injection. CONCLUSIONS: Our data suggests that: 1) EP2 and EP4 receptors are the key PGE2 receptors engaged in follicle growth. 2) Ovarian IL-8 expression and neutrophil infiltration are chiefly mediated via the EP4 receptor. EP2 and EP4 receptor agonists may be candidates for promising reagents that induce follicle maturation in clinical or agricultural fields. This knowledge could provide numerous targets for manipulation of fertility.