The expression of the sIgD isotype in wild-derived mice

IgD and IgM are concomitantly expressed on the surface of most mouse B lymphocytes and both molecules serve as receptor for antigen. In this communication we report that in contrast to IgM, which is expressed in a constant manner on the surface of spleen B lymphocytes of different laboratory and wild-derived mice, IgD expression is variable among the spleen cells of wild-derived mice. SPE, SEI, and SFM mice belonging to the Mus 3 subgroup show a fluorescence profile characterized by a marked diminution in the population of B lymphocytes expressing the IgD isotype; in addition, these cells have a low sIgD density on their membranes. These findings were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the iodinated membrane proteins. Polyclonal in vitro activation with lipopolysaccharide increases the frequency of surface IgD (sIgD)-bearing spleen cells and sIgD density in the SPE strain but decreases both the frequency and the density of IgD bearing cells in the BALB/c strain. This result suggests that delta gene expression is regulated differently in SPE and BALB/c mice. In addition, genetic analysis of sIgD expression in (BALB/c X SPE)F1 hybrids suggests that the proportion of sIgD-bearing cells and sIgD density are independently regulated.     

Immunocytochemical detection of Ca2+-dependent subspecies of protein kinase C in mouse embryos before and during compaction

Summary To confirm the possibility that protein kinase C is involved in compaction of mouse embryos, the presence and distribution pattern of Ca²⁺-dependent subspecies of this enzyme in mouse embryos, before and during compaction, were examined immunocytochemically with three different monoclonal antibodies. These were MC-1a, MC-2a and MC-3a, which selectively interact with the subspecies of the enzyme known as types I, II and III, respectively. Only when embryos were incubated with MC-3a, was immunofluorescence clearly detected in all cells of embryos before and during compaction. This result demonstrates the presence of type III protein kinase C in embryos before and during compaction and suggests the possibility that the type III enzyme may be involved in compaction. No marked differences were found in the distribution pattern of the type III enzyme between embryos examined before and during compaction.     

Preparation of soluble murine IL-6 receptor and anti-murine IL-6 receptor antibodies

Starting with a previously isolated cDNA clone encoding murine IL-6R, a stable transformed Chinese hamster ovary cell line constitutively expressing soluble murine IL-6R (smIL-6R) has been established. The smIL-6R was purified to homogeneity by sequential filtration and chromatography of culture medium. The smIL-6R augmented the sensitivity of M1 cells to IL-6 in their growth inhibition in a dose-response manner. Rat hybridomas producing mAb specific to murine IL-6R were also established. One of the clones, RS13, produced IgG2a isotype that was capable of inhibiting IL-6 activity. ELISA for the quantitation of smIL-6R was established, which could detect smIL-6R in a quantity as low as 1 ng/ml.     

Effect of age on bacterial translocation from the gastrointestinal tract in mice.

To clarify the effects of age on bacterial translocation from the gastrointestinal tract, mice at the age of 1, 2, 4, 6, 12, and 15 months were antibiotic-decontaminated for 4 days and then inoculated orally with streptomycin-resistant Escherichia coli C25. Mice treated with cyclophosphamide and untreated controls were tested for bacterial translocation to the mesenteric lymph nodes (MLN) 2 days later. The population levels of E. coli C25 in cyclophosphamide-treated and untreated mice were approximately 10(9.3) and 10(9.5) per gram of cecum, respectively, at each tested age. There were no significant differences in the incidence of translocation of E. coli C25 to MLN at any of the tested ages, whereas the number of E. coli C25 detected in MLN was higher in young mice than in aged mice in both the cyclophosphamide-treated and untreated groups. These findings suggest that bacterial translocation from the GI tract may be a more important problem in young animals than in aged animals.     

EBV increases phosphoinositide kinase activities in human B cells.

To determine whether EBV affects phosphoinositide kinase activities of human B cells, we compared the activities between EBV- and EBV+ human B cell lymphoma lines. The two types of human B cells contained both phosphatidylinositol (PtdIns) 4-kinase and phosphatidylinositol 4-phosphate (PtdIns(4)P) kinase activities irrespective of the presence of EBV. However, both activities were increased in EBV+ cells compared to EBV- cells. The increases were associated with neither altered Km values for substrates nor altered elution profiles in DEAE-cellulose chromatography. Furthermore, expression of a latent EBV protein, EBV nuclear Ag1 (EBNA1) in BHK cells by the transfection of EBNA1 DNA was accompanied by increased PtdIns 4-kinase and PtdIns(4)P kinase activities. These increases also were not associated with altered Km values for substrates. However, phospholipase C activity was altered in neither EBV+ cells nor in EBNA1-expressing cells. These results indicate that EBV selectively increases the two phosphoinositide kinase activities in human B cells, although the viral gene product has no intrinsic phosphoinositide kinase activity. PtdIns 4-kinase and PtdIns(4)P kinase cooperatively synthesize PtdIns 4,5-bisphosphate, the major source of 1,2-diacylglycerol and inositol 1,4,5-triphosphate, the two second messengers in transducing signals for cell activation. Such increase therefore may play a role in EBV-induced human B cell activation.     

Purification and characterization of soluble human IL-6 receptor expressed in CHO cells

An immunosorbent assay system to detect genetically engineered IL-6 receptor (IL-6R) was established, whereby soluble IL-6 receptor (sIL-6R) was detected in the culture medium when sIL-6R cDNA was transfected into COS1 cells. A stably transformed Chinese hamster ovary (CHO) cell line constitutively expressing sIL-6R has been established. The recombinant sIL-6R was purified to homogeneity by sequential filtration and chromatography of the culture medium. The recombinant sIL-6R augmented the sensitivity of M1 cells to IL-6 in growth inhibition assay in a dose-dependent manner. Furthermore, a radioisotope immunosorbent assay (RIA) utilizing recombinant sIL-6R was established which could detect IL-6 in a quantity as low as 10 ng/ml.     

Distribution of retinoids in the chick limb bud: analysis with monoclonal antibody

Retinoic acid induces anteroposterior duplicate formation in developing chick limb bud, and it may be a natural morphogen involved in limb pattern formation. Retinoic acid is produced from retinol locally in the limb bud via retinal, and thus, to elucidate the distribution of these retinoids in the limb bud seems to be important for the understanding of the morphogen formation. We produced a monoclonal antibody against the retinoids with BSA-RA (bovine serum albumin-retinoic acid) conjugate for antigen, and investigated the distribution of retinoids in the chick limb bud. The antibody predominantly bound to retinoic acid, but weakly to retinol and retinal. Retinoids appeared in the limb bud at stage 18 and were distributed through stages 20-24, when the pattern formation in distal mesoderm was in progress. Initially they were found evenly in the whole mesoderm, but disappeared gradually from core mesoderm and remained only in the region of peripheral mesoderm at stage 24. At stage 26, retinoids were detected only in ectoderm. These results support the idea that the retinoids actually play roles in limb pattern formation and suggest that the retinoids in the peripheral mesoderm are important for pattern formation. Further, the role of retinoids in epidermis development at later limb bud stages is also suggested.