Tumor necrosis factor receptor-associated factor 3 (TRAF3) is an adaptor protein that directly binds to a number of receptors of the tumor necrosis factor receptor (TNF-R) superfamily. Despite in vitro evidence that TRAF3 plays diverse roles in different cell types, little is known about the in vivo functions of TRAF3. To address this gap in knowledge and to circumvent the early lethal effect of TRAF3 null mutations, we generated conditional TRAF3-deficient mice. B-cell-specific Traf3(-/-) mice displayed severe peripheral B cell hyperplasia, which culminated in hyperimmunoglobulinemia and increased T-independent antibody responses, splenomegaly and lymphadenopathy. Resting splenic B cells from these mice exhibited remarkably prolonged survival ex vivo independent of B cell activating factor and showed increased amounts of active nuclear factor-kappaB2 but decreased amounts of nuclear protein kinase Cdelta. Furthermore, these mice developed autoimmune manifestations as they aged. These findings indicate that TRAF3 is a critical regulator of peripheral B cell homeostasis and may be implicated in the regulation of peripheral self-tolerance induction.
In zebrafish the cartilages of the pharynx develop during late embryogenesis and grow extensively in the larva before eventually being replaced by bone. Here we examine chondrocyte arrangements, shapes, numbers, and divisions in the young hyoid cartilages. We observe two distinct developmental phases, morphogenesis and growth. The first phase generates stereotypically oriented chondrocyte stacks that might form by intercalations among cells within the precartilage condensations. In mutants that have deformed cartilages the orientation of the stacks is changed, and we propose that their correct formation underlies the correct initial shaping of the organ. The following period of rapid, nearly isometric cartilage growth occurs by divisions of chondrocytes that are largely located near the joints, and appears to be under quite separate regulation.
An unusual xenoma-forming microsporidium was discovered in the central nervous system of moribund zebrafish from a laboratory colony in Eugene, Oregon. Infected fish were often emaciated and lethargic, and histological examination commonly revealed severe myelitis and myositis associated with the infection. Based on its structure, development, and small subunit ribosomal DNA sequence it is unique among fish microsporidia. Spores are uninucleate, ovoid to pyriform, with a prominent posterior vacuole. Spores average 5.4 x 2.7 microm with 13-16 coils of the polar filament. The microsporidium produces xenomas within the spinal cord and hindbrain of fish, and xenomas contained sporophorous vesicles with up to 16 spores. Sporoblasts and presporoblast stages (probably sporonts) are found occasionally in small aggregates dispersed randomly throughout xenomas. It clustered in the "Ichthyosporidium group" along with other fish microsporidian genera based on rDNA sequence analysis. The rDNA sequence of the zebrafish microsporidium was most similar to that of Ichthyosporidium, but showed only 12.1% similarity and therefore this microsporidium can be considered a distinct genus and species, which we have named Pseudoloma neurophilia n. g., n. sp.
We used the phosphatase substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6- chloro-4-[3H]-quinazolinone, with standard alkaline phosphatase-mediated immunohistochemical techniques, to visualize a number of antibodies that bind to adult zebrafish retinal tissue. This compound, known as the ELF (enzyme-labeled-fluorescence) phosphatase substrate, produces a precipitate that fluoresces at approximately 500-580 nm (bright yellow-green). We show that the precipitated product from the ELF phosphatase substrate has a number of characteristics that make it superior to fluorescein-labeled secondary reagents. The staining produced with the ELF substrate is much more photostable than that produced by fluorescein-labeled secondary reagents, thus allowing time to examine, focus, and photograph the ELF-labeled tissue under high magnification. Moreover, the ELF precipitate exhibits a Stokes shift of greater than 100 nm, a characteristic that has enabled us to overcome the problem of distinguishing signal from background in this autofluorescent tissue. In addition, we show that the ELF product's large Stokes shift makes the ELF substrate ideal for multicolor applications.
Current methods for detecting lacZ expression in transformed cells are limited because they require such harsh conditions that viability of the cells after detection is drastically reduced. To overcome this problem, we developed a series of new substrates for detection of lacZ expression in living cells under standard culture or physiological conditions. After incubation with these fluorogenic substrates, cultured lacZ-positive mammalian cells appear morphologically normal, continue to divide, and retain the fluorescent product. Because the product is so well retained, fluorescence intensity can be quantitatively related to the level of gene expression. We have demonstrated this correlation using transformed yeast cells bearing various plasmids, each containing the lacZ gene and a unique promoter sequence with known capabilities for promoting gene expression in yeast.
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