IL-4 pretreatment selectively enhances cytokine and chemokine production in lipopolysaccharide-stimulated mouse peritoneal macrophages

Although well recognized for its anti-inflammatory effect on gene expression in stimulated monocytes and macrophages, IL-4 is a pleiotropic cytokine that has also been shown to enhance TNF-alpha and IL-12 production in response to stimulation with LPS. In the present study we expand these prior studies in three areas. First, the potentiating effect of IL-4 pretreatment is both stimulus and gene selective. Pretreatment of mouse macrophages with IL-4 for a minimum of 6 h produces a 2- to 4-fold enhancement of LPS-induced expression of several cytokines and chemokines, including TNF-alpha, IL-1alpha, macrophage-inflammatory protein-2, and KC, but inhibits the production of IL-12p40. In addition, the production of TNF-alpha by macrophages stimulated with IFN-gamma and IL-2 is inhibited by IL-4 pretreatment, while responses to both LPS and dsRNA are enhanced. Second, the ability of IL-4 to potentiate LPS-stimulated cytokine production appears to require new IL-4-stimulated gene expression, because it is time dependent, requires the activation of STAT6, and is blocked by the reversible protein synthesis inhibitor cycloheximide during the IL-4 pretreatment period. Finally, IL-4-mediated potentiation of TNF-alpha production involves specific enhancement of mRNA translation. Although TNF-alpha protein is increased in IL-4-pretreated cells, the level of mRNA remains unchanged. Furthermore, LPS-stimulated TNF-alpha mRNA is selectively enriched in actively translating large polyribosomes in IL-4-pretreated cells compared with cells stimulated with LPS alone.     

The highly conserved Gln49 and Ser50 of mammalian connexin43 are present in chick connexin43 and essential for functional gap junction channels

In an attempt to compare the regulation of chick connexin43 channels to those of mammalian connexin43, we found that the nucleotide sequence reported for chick connexin43 differs from that of the chick connexin gene by two codons that had been entered as histidine49 (H49) and valine50 (V50) (accession no. M29003), but are in fact glutamine49 (Q49) and serine50 (S50). Neuro2A cells were transfected with corrected wild-type (Q49/S50) chick connexin43 (accession no. AF233738), the double-replacement Q49H/S50V connexin43, or the single replacement of Q49H or S50V. All clones had gap junctions in membrane based on immunocytochemistry and immunoblots of the triton-resistant membrane fraction. Wild-type transfectants had three conductance states with a predominant channel conductance of 85 ±5 pS. Cells producing the Q49H-Cx43 or the double-replacement Q49H/S50V-Cx43 protein had no detectable connexin43 channels. In contrast, cells expressing S50V-Cx43 gap junctions had channels with reduced conductances (75 ±8 pS) compared to wild-type controls. Low or high pH of the bathing solution had no effect on the Q49H-Cx43 channels. We conclude that glutamine49 is important for channel function, and replacement of this residue with histidine most likely distorts secondary structure of the first extracellular loop, possibly by changing the orientation of conserved cysteines, and this inhibits channel function. The S50V substitution may also cause similar but less severe structural changes.     

Germinal vesicle material is essential for nucleus remodeling after nuclear transfer

Successful cloning by nuclear transfer has been reported with somatic or embryonic stem (ES) cell nucleus injection into enucleated mouse metaphase II oocytes. In this study, we enucleated mouse oocytes at the germinal vesicle (GV) or pro-metaphase I (pro-MI) stage and cultured the cytoplasm to the MII stage. Nuclei from cells of the R1 ES cell line were injected into both types of cytoplasm to evaluate developmental potential of resulting embryos compared to MII cytoplasmic injection. Immunocytochemical staining revealed that a spindle started to organize 30 min after nucleus injection into all three types of cytoplasm. A well-organized bipolar spindle resembling an MII spindle was present in both pro-MI and MII cytoplasm 1 h after injection with ES cells. However, in the mature GV cytoplasm, chromosomes were distributed throughout the cytoplasm and a much bigger spindle was formed. Pseudopronucleus formation was observed in pro-MI and MII cytoplasm after activation treatment. Although no pronucleus formation was found in GV cytoplasm, chromosomes segregated into two groups in response to activation. Only 8.1% of reconstructed embryos with pro-MI cytoplasm developed to the morula stage after culture in CZB medium. In contrast, 53.5% of embryos reconstructed with MII cytoplasm developed to the morula/blastocyst stage, and 5.3% of transferred embryos developed to term. These results indicate that GV material is essential for nucleus remodeling after nuclear transfer.     

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.     

Cultured gill epithelia as models for the freshwater fish gill

We review recent progress in the development of models for the freshwater teleost gill based on reconstructed flat epithelia grown on permeable filter supports in primary culture. Methods are available for single-seeded insert (SSI) preparations consisting of pavement cells (PVCs) only from trout and tilapia, and double-seeded insert (DSI) preparations from trout, containing both PVCs (85%) and mitochondria-rich cells (MRCs, 15%), as in the intact gill. While there are some quantitative differences, both SSI and DSI epithelia manifest electrical and passive permeability characteristics typical of intact gills and representative of very tight epithelia. Both preparations withstand apical freshwater exposure, exhibiting large increases in transepithelial resistance (TER), negative transepithelial potential (TEP), and low rates of ion loss, but there is only a small active apical-to-basolateral "influx" of Cl(-) (and not of Na(+)). Responses to various hormonal treatments are described (thyroid hormone T3, prolactin, and cortisol). Cortisol has the most marked effects, stimulating Na(+),K(+)-ATPase activity and promoting active Na(+) and Cl(-) influxes in DSI preparations, and raising TER and reducing passive ion effluxes in both epithelia via reductions in paracellular permeability. Experiments using DSI epithelia lacking Na(+) uptake demonstrate that both NH(3) and NH(4)(+) diffusion occur, but are not large enough to account for normal rates of branchial ammonia excretion, suggesting that Na(+)-linked carrier-mediated processes are important for ammonia excretion in vivo. Future research goals are suggested.     

Gene content and organization of an 85-kb DNA segment from the genome of the phytopathogenic mollicute Spiroplasma kunkelii

Spiroplasma kunkelii, the causative agent of corn stunt disease in maize ( Zea mays L.), is a helical, cell wall-less prokaryote assigned to the class Mollicutes. As part of a project to sequence the entire S. kunkelii genome, we analyzed an 85-kb DNA segment from the pathogenic strain CR2-3x. This genome segment contains 101 ORFs and two tRNA genes. The majority of the ORFs code for predicted proteins that can be assigned to respective clusters of orthologous groups (COGs). These COGs cover diverse functional categories including genetic information storage and processing, cellular processes, and metabolism. The most notable gene cluster in this genome segment is a super-operon capable of encoding 24 ribosomal proteins. The organization of genes in this operon reflects the unique evolutionary position of the spiroplasma. Gene duplications, domain rearrangements, and frameshift mutations in the segment are interpreted as indicators of phase variation in the spiroplasma. To our knowledge, this is the first analysis of a large genome segment from a plant pathogenic spiroplasma.Communicated by W. Goebel