It��s All About Change: The Antigen-driven Initiation of B-Cell Receptor Signaling

B-cell responses are initiated by the binding of foreign antigens to the clonally distributed B-cell receptors (BCRs) resulting in the triggering of signaling cascades that activate a variety of genes associated with B-cell activation. Although we now understand the molecular nature of the signaling pathways in considerable detail what remains only poorly understood are the mechanisms by which the information that antigen has bound to the BCR ectodomain is transduced across the B-cell membrane to the BCR cytoplasmic domains to trigger signaling. To a large part this gap in knowledge is because of the paucity of techniques to temporally and spatially resolve changes in the behavior of the BCR that occur within several seconds of antigen binding. With the advent of new live-cell imaging technologies we are gaining our first clear views of the events that lead up to the triggering of BCR signaling cascades. These events may provide potential new targets for therapeutic intervention in disease involving hyper or chronic activation of B cells.Specific, high-affinity antibody responses are the result of processes based on clonal selection (reviewed in Rajewsky 1996). In the absence of antigen, individuals generate a B-cell repertoire in which each B cell expresses a single heavy and light chain gene, the product of somatic recombination of variable and constant region gene segments. Self-reactive B cells are removed from the repertoire and when antigen enters the immune system it selects those B cells expressing BCR’s with highest affinity for the antigen. Under the influence of both T cell and innate immune system regulation the antigen-selected B cells are induced to differentiate into short-lived antibody producing cells or enter germinal centers where they undergo the molecularly linked processes of somatic hypermutation and isotype switching. Antigen selection within the germinal centers results in high-affinity memory B cells expressing isotype switched BCRs. These memory B cells account, in large part, for the high titered, high affinity IgG antibody responses observed upon re-exposure to antigen. Thus, we presume that B cells are capable of initiating responses to the universe of foreign antigens to which individuals are exposed and do so through mechanisms that are sensitive to the affinity of the BCR for antigen and by which isotype switched BCRs are more effective. Until recently, the events by which the binding of antigen to the BCRs triggered signaling remained largely unknown due in a large part to the paucity of experimental approaches that were able to provide the spatial and temporal resolution necessary to capture the earliest events that follow the binding of antigens to BCRs that result in triggering the B cell’s signaling cascades. The conventional biochemical techniques that were used so successfully to describe the components of the BCR signaling cascades were too slow to study early events and could not provide spatial information. The application of new live-cell imaging technologies that allow resolution of single molecules over a timeframe of several seconds to the study of antigen-induced B-cell responses is providing the first views of these processes. Here we review progress in understanding the initiation of the BCR signaling using live-cell imaging technologies and how this new knowledge may explain in part the mechanisms that underlie hyper or chronic activation of B cells in autoimmunity and in B-cell cancers.     

The structure of the coiled-coil domain of Ndel1 and the basis of its interaction with Lis1, the causal protein of Miller-Dieker lissencephaly

Ndel1 and Nde1 are homologous and evolutionarily conserved proteins, with critical roles in cell division, neuronal migration, and other physiological phenomena. These functions are dependent on their interactions with the retrograde microtubule motor dynein and with its regulator Lis1--a product of the causal gene for isolated lissencephaly sequence (ILS) and Miller-Dieker lissencephaly. The molecular basis of the interactions of Ndel1 and Nde1 with Lis1 is not known. Here, we present a crystallographic study of two fragments of the coiled-coil domain of Ndel1, one of which reveals contiguous high-quality electron density for residues 10-166, the longest such structure reported by X-ray diffraction at high resolution. Together with complementary solution studies, our structures reveal how the Ndel1 coiled coil forms a stable parallel homodimer and suggest mechanisms by which the Lis1-interacting domain can be regulated to maintain a conformation in which two supercoiled alpha helices cooperatively bind to a Lis1 homodimer.     

Increasing production in Korean shrimp farms with white-spot syndrome virus PCR-negative brood stock

White-spot syndrome virus (WSSV) is a devastating, infectious virus affecting shrimp. Although sensitive techniques involving PCR have been developed to assist farmers in screening shrimp (brood stock) for WSSV prior to stocking ponds, such practices have not yet been applied in Korea. Despite the rationality of implementing screening, there has been some doubt as to whether the stocking of WSSV-PCR-negative fry epidemiologically decreases white-spot disease outbreaks. Here, we report a retrospective analysis of data from shrimp farms in the western coast of Korea where WSSV-PCR-negative brood stocks were used to stock rearing ponds. A total of 366 shrimp from Heuksan Island were sampled for WSSV with PCR. Of the tested shrimp, 7.2% (28 brood stocks) were identified as WSSV positive; only WSSV-PCR-negative shrimp were used for brood stocks. Total unit production (final shrimp production/ the area of the ponds) was higher, at 1.96, in ponds where WSSV-PCR-negative shrimp were used, as compared with 1.02 in other ponds in Korea in 2004. This retrospective analysis of WSSV in Korea may be useful to the shrimp aquaculture industry, suggesting a testable hypothesis that may contribute to the eventual control of WSSV outbreaks.     

Sulfated chitooligosaccharides as prolyl endopeptidase inhibitor

Prolyl endopeptidase (PEP, EC 3.4.21.26) is a proline-specific endopeptidase with a serine-type mechanism, which digests small peptide-like hormones, neuroactive peptides, and various cellular factors. PEP has been involved in neurodegenerative disorders, therefore, the discovery of PEP inhibitors can revert memory loss caused by amnesic compounds. In this study, we prepared hetero-chitooligosaccharides (COSs) with different molecular sizes using ultrafiltration (UF) membrane reactor system from hetero-chitosan with different degrees of deacetylation (DD; 90%, 75% and 50% deacetylation), and synthesized sulfated COSs (SCOSs). PEP inhibitory activities of SCOSs were evaluated and the results showed that 50% deacetylated SCOSs (50-SCOSs) exhibited higher inhibitory activities than those of 90% and 75% deacetylated SCOSs (90-SCOSs and 75-SCOSs). Among the 50-SCOSs (50-SCOS I, 5000–10,000 Da; 50-SCOS II, 1000–5000 Da; 50-SCOS III, below 1000 Da), 50-SCOS II possessed the highest inhibitory activity and IC₅₀ value was 0.38 mg/ml. Kinetics studies with 50-SCOS II indicated a competitive enzyme inhibition with a K_i value of 0.78 mg/ml. It was concluded that the 50-SCOS II may be useful for PEP inhibitor and for developing a new type PEP inhibitor from carbohydrate based materials.     

Hypoglycemic effects of exopolysaccharides produced by mycelial cultures of two different mushrooms Tremella fuciformis and Phellinus baumii in ob/ob mice

The anti-diabetic activities of the exopolysaccharides (EPS) produced by submerged mycelial culture of two different mushrooms, Tremella fuciformis and Phellinus baumii, in ob/ob mice were investigated. All the animals were randomly divided into three groups with seven animals in each group: The control group received 0.9% NaCl solution; the diabetic groups were treated with EPS from T. fuciformis (Tf EPS) and P. baumii (Pb EPS) at the level of 200 mg/kg body weight using an oral zoned daily for 52 days. The plasma glucose levels in the EPS-fed mice were substantially reduced by about 52% (Tf EPS) and 32% (Pb EPS), respectively, as compared to control mice. The results of oral glucose tolerance test (OGTT) revealed that both EPS-fed groups significantly increased the glucose disposal after 52 days of EPS treatments. Furthermore, higher food efficiency ratios and reduced blood triglyceride levels were observed in the EPS-treated groups. Because peroxisome proliferator-activated receptor gamma (PPAR-γ) is indeed a key regulator of insulin action, we investigated the expression pattern of adipose tissue PPAR-γ messenger RNA (mRNA) and plasma levels of PPAR-γ. It was revealed that PPAR-γ was significantly activated in response to EPS treatments. The results suggested that both EPS exhibited considerable hypoglycemic effect and improved insulin sensitivity possibly through regulating PPAR-γ-mediated lipid metabolism. Our results indicated that two mushroom-derived EPS might be developed as potential oral hypoglycemic agents or functional foods for the management of non-insulin-dependent diabetes mellitus.     

Expression, purification, and antibody binding activity of human papillomavirus 16 L1 protein fused to maltose binding protein

Genetic human papillomavirus type 16 L1 (HPV16 L1) has been widely studied for cervical cancer vaccine development. For the enzyme-linked immunosorbent assay (ELISA) screening of these vaccines, HPV16 L1 protein, which is required as a coating protein, has previously been expressed from costly and laborious recombinant baculovirus-infected insect cells. For a novel HPV16 L1 expression system characterized by a high yield of soluble form with simple purification steps, we have cloned and expressed two different types of HPV16 L1, both fused to maltose binding protein (MBP) or glutathione-S-transferase (GST) in Escherichia coli. The yield of soluble HPV16 L1 was influenced by the cultivation temperature. The yield of soluble form in the total MBP-fused HPV16 L1 protein (MBP-HPV16 L1) was 35% at 37 degrees C, but increased to 85% at 22 degrees C. Among the fusion partners, MBP provided higher yields of total and soluble HPV16 L1 than did GST. MBP-HPV16 L1 showed a 4.9-fold higher yield of the soluble form over insoluble inclusion bodies under optimized culture conditions. The soluble form of MBP-HPV16 L1 was purified via MBP affinity chromatography in a recovery yield of 9.7%. After fusion with MBP, HPV16 L1 showed binding activity to HPV16 L1-specific monoclonal antibody comparable to HPV16 L1 from the insect cells in ELISA tests. These results demonstrate that the use of MBP as a fusion partner may generate a high yield of soluble HPV16 L1 under optimized temperature conditions, and that MBP-fused HPV16 L1 might be applied further in evaluations of the immune responses of HPV16 L1-based cervical cancer vaccines.     

Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library

Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.