Cytokeratin filaments are present in golden hamster oocytes and early embryos

Light and electron microscope methods were used to study cytokeratin expression in the recently ovulated oocytes, fertilized eggs and early embryos from the golden hamster. Two cytokeratin polypeptides (Mr 51,000 and 58,000) were detected in oocyte lysates by immunoblotting using a polyclonal antiserum to prekeratin. In the oocyte, cytokeratin occurred as patchy aggregates consisting of short anastomosing 10-nm filaments that formed tight meshworks distributed throughout the cytoplasm. After fertilization the aggregates appeared to merge, becoming larger and concentrated at the cortical region. Prominent immunofluorescent fibrils were seen interconnecting the aggregates. In the 2-, 4- and 8-cell embryos, networks of cytokeratin filaments extended throughout the cortical and perinuclear regions, while aggregates progressively disappeared.     

Translocation and induction of alpha B crystallin by heat shock in rat glioma (GA-1) cells.

Response to heat shock of alpha B crystallin expressed in rat astrocytoma GA-1 cells was analysed quantitatively using an immunoassay method. GA-1 cells contained a considerable amount of alpha B crystallin at growing phase. When the growing cells were heated at 45 degrees C, concentrations of alpha B crystallin in cell extracts were decreased to less than one-fifth of the original level within 15 min, with an increase in the insoluble fraction which was detected by immunoblotting. The low level of alpha B crystallin in the cytoplasm, that was observed for a few hours after heat shock, gradually recovered to the control level within several hours. At 10 h following heat shock (45 degrees C for 15 min), the concentration of alpha B crystallin in the soluble extract was about twice that of the control level, with little detectable amounts in the insoluble fraction. These results are additional evidence that suggest that alpha B crystallin is a small heat shock protein.     

Osteoclast precursor cells are present in the blood of preossification chick embryos

In an attempt to ascertain the time of appearance of circulating osteoclast precursor cells, we have transplanted quail bone rudiments into or onto the extraembryonic membranes of variously aged chick embryos. We observed that osteoclast precursor cells (1) are present in the embryonic circulation prior to the onset of osteogenesis, (2) differentiate precociously in response to a factor or factors present in developing bone rudiments, and (3) increase in number until about midway in embryonic life.     

Teratocarcinoma stem cells and early mouse embryos contain only a single major lamin polypeptide closely resembling lamin B

The nuclear lamina in adult mammalian somatic cells is composed of three major proteins, lamins A, B, and C. The expression of these proteins during the differentiation of teratocarcinomas and mouse embryogenesis is described. Embryos up to day 8 of gestation and embryonal carcinoma (EC) cells express only a single lamin species closely resembling, if not identical to, lamin B. Lamins A and/or C were detected in fertilized eggs, but disappear during the first 2-4 cleavage divisions, only reappearing in 8 day post-implantation embryos. These two lamins are absent from EC cells, but are strongly expressed in some of their derivatives. These results show that cells of the early mouse embryo do not have a functional requirement for lamins A and C and imply that the structural organization of the nucleus may change fundamentally during embryogenesis.     

Functional early endosomes are required for maturation of major histocompatibility complex class II molecules in human B lymphoblastoid cells.

Major histocompatibility complex (MHC) class II molecules are targeted together with their invariant chain (Ii) chaperone from the secretory pathway to the endocytic pathway. Within the endosome/lysosome system, Ii must be degraded to enable peptide capture by MHC class II molecules. It remains controversial exactly which route or routes MHC class II/Ii complexes take to reach the sites of Ii processing and peptide loading. We have asked whether early endosomes are required for successful maturation of MHC class II molecules by using an in situ peroxidase/diaminobenzidine compartment ablation technique. Cells whose early endosomes were selectively ablated using transferrin-horseradish peroxidase conjugates fail to mature their newly synthesized MHC class II molecules. We show that whereas transport of secretory Ig through the secretory pathway is virtually normal in the ablated cells, newly synthesized MHC class II/Ii complexes never reach compartments capable of processing Ii. These results strongly suggest that the transport of the bulk of newly synthesized MHC class II molecules through early endosomes is obligatory and that direct input into later endosomes/lysosomes does not take place.     

Selective expression of RAG-2 in chicken B cells undergoing immunoglobulin gene conversion

Chickens create their immunoglobulin (Ig) repertoires during B cell development in the bursa of Fabricius by intrachromosomal gene conversion. Recent evidence has suggested that Ig gene conversion may involve cis-acting DNA elements related to those involved in V(D)J recombination. Therefore, we have examined the potential role of the V(D)J recombination activating genes, RAG-1 and RAG-2, in regulating chicken Ig gene conversion. In contrast to the coexpression of RAG-1 and RAG-2 observed in mammalian B cells that undergo V(D)J recombination, chicken B cells isolated from the bursa of Fabricius express high levels of the RAG-2 mRNA but do not express RAG-1 mRNA. The developmental and phenotypic characteristics of the bursal lymphocytes and chicken B cell lines that express RAG-2 mRNA demonstrate that selective RAG-2 expression occurs specifically in B cells undergoing Ig diversification by gene conversion. These data suggest that RAG-2 plays a fundamental role in Ig-specific gene conversion.     

Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells.

The activity of ICE-like proteases or caspases is essential for apoptosis. Multiple caspases participate in apoptosis in mammalian cells but how many caspases are involved and what is their relative contribution to cell death is poorly understood. To identify caspases activated in apoptotic cells, we developed an approach to simultaneously detect multiple active caspases. Using tumor cells as a model, we have found that CPP32 (caspase 3) and Mch2 (caspase 6) are the major active caspases in apoptotic cells, and are activated in response to distinct apoptosis-inducing stimuli and in all cell lines analyzed. Both CPP32 and Mch2 are present in apoptotic cells as multiple active species. In a given cell line these species remained the same irrespective of the apoptotic stimulus used. However, the species of CPP32 and Mch2 detected varied between cell lines, indicating differences in caspase processing. The strategy described here is widely applicable to identify active caspases involved in apoptosis.     

CD8alpha+ dendritic cells selectively present MHC class I-restricted noncytolytic viral and intracellular bacterial antigens in vivo

CD8alpha(+) dendritic cells (DCs) have been shown to be the principal DC subset involved in priming MHC class I-restricted CTL immunity to a variety of cytolytic viruses, including HSV type 1, influenza, and vaccinia virus. Whether priming of CTLs by CD8alpha(+) DCs is limited to cytolytic viruses, which may provide dead cellular material for this DC subset, or whether these DCs selectively present intracellular Ags, is unknown. To address this question, we examined Ag presentation to a noncytolytic virus, lymphocytic choriomeningitis virus, and to an intracellular bacterium, Listeria monocytogenes. We show that regardless of the type of intracellular infection, CD8alpha(+) DCs are the principal DC subset that initiate CD8(+) T cell immunity.     

GABAA alpha6-containing receptors are selectively compromised in cerebellar granule cells of the ataxic mouse, stargazer

Stargazer mice fail to express the gamma2 isoform of transmembrane alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor regulatory proteins that has been shown to be absolutely required for the trafficking and synaptic targeting of excitatory AMPA receptors in adult murine cerebellar granule cells. Here we show that 30 +/- 6% fewer inhibitory gamma-aminobutyric acid, type A (GABA(A)), receptors were expressed in adult stargazer cerebellum compared with controls because of a specific loss of GABA(A) receptor expression in the cerebellar granule cell layer. Radioligand binding assays allied to in situ immunogold-EM analysis and furosemide-sensitive tonic current estimates revealed that expression of the extrasynaptic (alpha6betaxdelta) alpha6-containing GABA(A) receptor were markedly and selectively reduced in stargazer. These observations were compatible with a marked reduction in expression of GABA(A) receptor alpha6, delta (mature cerebellar granule cell-specific proteins), and beta3 subunit expression in stargazer. The subunit composition of the residual alpha6-containing GABA(A) receptors was unaffected by the stargazer mutation. However, we did find evidence of an approximately 4-fold up-regulation of alpha1betadelta receptors that may compensate for the loss of alpha6-containing GABA(A) receptors. PCR analysis identified a dramatic reduction in the steady-state level of alpha6 mRNA, compatible with alpha6 being the primary target of the stargazer mutation-mediated GABA(A) receptor abnormalities. We propose that some aspects of assembly, trafficking, targeting, and/or expression of extrasynaptic alpha6-containing GABA(A) receptors in cerebellar granule cells are selectively regulated by AMPA receptor-mediated signaling.     

Two 5S genes are expressed in chicken somatic cells.

Two 5S RNA species were detected in chicken cells. 5S I RNA has the nucleotide sequence of chicken 5S RNA previously published by Brownlee et al. (1) and 5S II RNA differs from it by 10 mutations. The secondary structure of both species is compatible with that proposed for other eukaryotic 5S RNAs. 5S II RNA represents 50-60% of 5S I RNA. Both species were found in total chicken liver and brain and were present in polysomes in the same relative proportions. Only one 5S RNA species could be detected in rat liver and HeLa cells. Chicken is the first vertebrate described so far in which two 5S RNA genes are expressed in somatic cells.     

Genes for bone morphogenetic proteins are differentially transcribed in early amphibian embryos.

We have previously demonstrated that activin, a member of the TGF-beta family, has a potent mesoderm-inducing activity in Xenopus embryos. In the course of screening for activin-related genes from Xenopus, we have cloned cDNAs for Xenopus homologue of BMP-2, -4 and -7. Northern blot analysis revealed that these BMP genes are maternally encoded and differentially regulated after fertilization. Alkaline phosphatase-inducing assay using the recombinant BMP proteins has shown that at least BMP-2 and -4 have similar activity to mammalian counterparts.     

Proteins reacting with anti-spectrin antibodies are present in Chlamydomonas cells.

It was found either in Western-blot analysis or in indirect immunofluorescence microscopy that cells of the alga Chlamydomonas reinhardtii contain polypeptides cross-reacting with antibodies directed against red blood cell spectrin. The protein could also be detected by immunoprecipitation with anti-spectrin antibodies. C. reinhardtii cells contain distinct polypeptide chains reacting with antibodies directed against either α- or β-spectrin subunits. This protein was extracted from the cells with low ionic strength solution but was not with nonionic detergent.     

A T cell receptor V alpha region selectively expressed in CD4+ cells.

The peripheral TCR V beta repertoire is strongly influenced by the processes of negative selection (deletion) and positive selection in the thymus. In order to investigate whether such selection events influence the V alpha repertoire, we have produced an anti-V alpha 11 mAb. This antibody was made by immunization with a chimeric TCR:Ig protein containing V alpha 11 in place of the VH of an IgG2a, lambda Ig. This scheme optimizes the specificity of immunization and facilitates the screening procedure. The antibody recognizes a panel of V alpha 11-expressing T cell clones. Analysis of mouse strains indicates that the antibody recognizes V alpha 11 only in mice of the C57 background. The expression of the epitope on peripheral T cells is strongly biased to the CD4+ subset, suggesting positive selection of V alpha 11 on class II MHC molecules. In some strain comparisons, the percentage of V alpha 11-expressing T cells in the CD4+ subset was elevated in I-E+ relative to I-E- strains. These data suggest that V alpha 11 can differentially influence the selection of T cells into the CD4+/CD8+ subsets.     

Multiple growth factor mRNAs are expressed in chicken adipocyte precursor cells.

We have examined the expression of growth factor genes in primary cultures of chicken adipocyte precursors. RNA was extracted from proliferating and differentiated cells, reversed transcribed and amplified by PCR using gene specific primers. The identity of the PCR products was confirmed by restriction mapping. We show, for the first time, constitutive expression of TGF-beta 2, TGF-beta 3, TGF-beta 4 and bFGF genes in chicken adipocyte precursors. We also detect GH-independent, but differentiation-dependent IGF-I gene expression. The synthesis and action of these growth factors supports the hypothesis that they act as autocrine and/or paracrine regulators of adipocyte precursor cell proliferation and differentiation.     

Antigen receptor engagement selectively induces macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta chemokine production in human B cells

We show herein that B cell Ag receptor (BCR) triggering, but not stimulation by CD40 mAb and/or IL-4, rapidly induced the coordinated expression of two closely related T cell chemoattractants, macrophage inflammatory protein-1 beta (MIP-1 beta) and MIP-1 alpha, by human B cells. Naive, memory, and germinal center B cells all produced MIP-1 alpha/beta in response to BCR triggering. In contrast to MIP-1 alpha/beta, IL-8, which is spontaneously produced by germinal center B cells but not by naive and memory B cells, was not regulated by BCR triggering. Culturing follicular dendritic cell-like HK cells with activated B cells did not regulate MIP-1 alpha/beta production, but it did induce production of IL-8 by HK cells. Microchemotaxis assays showed that CD4+CD45RO+ T cells of the effector/helper phenotype actively migrated along a chemotactic gradient formed by BCR-stimulated B cells. This effect was partially blocked by anti-MIP-1 beta and anti-CC chemokine receptor 5 Ab, but not by anti-MIP-1 alpha Ab suggesting that MIP-1 beta plays a major role in this chemoattraction. Since maturation of the B cell response to a peptide Ag is mostly dependent on the availability of T cell help, the ability of Ag-stimulated B cells to recruit T cells via MIP-1 alpha/beta, may represent one possible mechanism enabling cognate interactions between rare in vivo Ag-specific T and B cells.     

Subunits of laminin are differentially synthesized in mouse eggs and early embryos

Synthesis of laminin by mouse preimplantation embryos was examined by specific immunoprecipitation of [³⁵S]methionine-labeled cell lysates. The three polypeptide subunits of laminin, A, B₁, and B₂, are not necessarily synchronously expressed during development. Oocytes and eggs synthesize only one immunoprecipitable polypeptide, which comigrates with the intracellular B₁ chains made by PYS cells and contains N-linked oligosaccharide residues which are sensitive to endo-β-N-acetylglucosaminidase H. From the 4- to 8-cell stage, only B₁ and B₂ polypeptides are synthesized, whereas from the 16-cell stage onwards all three laminin polypeptides are made.